Immunomodulatory complexes and their therapeutic uses

Abstract

This invention relates to molecular complexes consisting of at least one ligand of a sulfated sugar of the glycosaminoglycan family linked to at least one ligand of a surface molecule of an antigen-presenting cell or NK or NKT cell, which are used as immunomodulatory drugs, particularly in the immunotherapy of cancer and infectious diseases.

Description

Technical Field

[0001] This invention pertains to the field of immunomodulatory therapy. The invention relates to a molecular complex comprising at least one ligand of a sulfated sugar of the glycosaminoglycan family and at least one ligand of another surface molecule of antigen-presenting cells, NK cells, or NKT cells, the ligands being bonded to each other, which is used as an immunomodulatory agent, particularly in the immunotherapy of cancer and infectious diseases. Background Technology

[0002] Most therapeutic approaches are based on drugs that act directly on the targets that cause disease or cells exhibiting a non-physiological state. However, immune system dysfunction can exacerbate disease progression, particularly during tumor progression (Galon J. and D. Bruni, Nat. Rev. Drug Discov., 2019 18(3):197-218) or during infection with infectious pathogens (Wherry EJ and M. Kurachi, Nature Rev. Immunol., 2015, 15(8):486-499). These observations have led to the hypothesis of developing immunotherapeutic drugs that can re-establish adequate immune defenses, particularly by modulating the activity of T lymphocytes or NK or NKT cells, to enable the immune system to more effectively control the disease process. The aim of these regulatory immunotherapies is to re-establish or suppress the function of most lymphocyte repositories, particularly CD4+ T, CD8+ T, or regulatory T lymphocyte repositories. In this respect, they differ from vaccine immunotherapy, which aims to induce a limited number of lymphocytes corresponding to specific cells containing Ag(s) in the vaccine.

[0003] To develop immunotherapeutic drugs, it is necessary to pre-identify targets that play a key role in controlling the immune response. In the 1990s, molecules expressed on the surface of T lymphocytes (named PD-1 and CTLA-4, respectively) were shown to induce inhibitory signals that downregulate the activity of these cells, and thus modulate some immune defense mechanisms (Ishida, Y., Agata, Y., Shibahara, K., & Honjo, T. (1992). EMBO J., 11(11), 3887-3895.; Freeman, et al. (2000). J Exp Med, 192(7), 1027-1034.; Krummel MF, Allison JP, J. Exp. Med., 19950801; 182(2):459-65). These two molecules were named immune checkpoints, immune checkpoint inhibitor molecules, or ICP inhibitors.

[0004] The molecular mechanisms responsible for the activity of these ICP inhibitors have been investigated. In particular, PD-1 and CTLA-4 have been observed to interact with ligands expressed on the surface of antigen-presenting cells (APCs) or tumor cells. These ligands have been named PD-L1 and B7, respectively. The PD-1 / PD-L1 or CTLA-4 / B7 interaction then mediates inhibitory signals from T lymphocytes, resulting in different immunosuppressive mechanisms. Furthermore, it has been demonstrated that PD-1, PD-L1, or CTLA-4 specific antibodies (Abs) can block the PD-1 / PD-L1 or CTLA-4 / B7 interaction, thereby potentially removing inhibition and reactivating T lymphocytes (Hodi, F. Set et al., PNAS. (2003), 100(8), 4712-4717; Iwai, Y. et al., Int. Immunol., (2005), 17(2), 133-144). Some of these immunomodulatory antibodies have been shown to limit the growth of various cancers (melanoma, lung cancer, etc.) and significantly prolong patients' life expectancy. They are now commonly used as antitumor immunotherapies in humans (Adachi K. and K. Tamada, Cancer Sci., 2015; 106(8):945-50; Riley RS et al., Nat. Rev. Drug Discov. 2019 18(3):175-196). They are also envisioned for use in other therapeutic areas, particularly for treating infectious diseases (Rao M. et al., Int. J. Infect. Dis., 2017; 56:221-228). However, a drawback of these first-generation inhibitory anti-ICP antibodies is that they are only effective in 10% to 30% of cancer patients (Pitt JM et al., Immunity. 20160121; 44(6):1255-69). Therefore, many research groups aim to discover novel immunomodulatory compounds that are more effective and / or can be used in combination with the aforementioned immunotherapies. Since several novel ICP inhibitors have been identified, research has focused on selecting compounds that can bind to these ICPs or their ICP ligands, thereby neutralizing ICP / ICP ligand binding to reactivate T lymphocytes. The discovery of ICP activators expressed on the surface of T cells has led other studies to focus on selecting agonist ligands for these ICPs (Mahoney KM.Etal.,Nat.Rev.Drug Discov.,2019,14:561-584; De Sousa Linhares A.,Front.Immunol.,2018,31;9:1909; Granier C.et al.,ESMO Open,20170703;2(2):e000213).

[0005] To modulate immune responses as effectively as possible, therapeutic molecules or Ag-presenting cells (APCs) that bind to ICP or ICP-ligands selectively expressed on the surface of effector immune cells, such as T lymphocytes and NK or NKT cells, have been sought. This is because such expression selectivity can limit the diffusion of molecules to non-immune cells. This leads to enhanced therapeutic efficacy and reduced risk of side effects.

[0006] Glycocalyxes are specifically composed of proteoglycans, which are glycoproteins containing one or more unbranched glycosaminoglycan (GAG) chains. Among the latter, the heparan sulfate proteoglycan (HSPG) family corresponds to proteins associated with sulfated GAGs: heparan sulfate (HS). HSPGs (which play a central role in many biological processes—cell proliferation, cell adhesion, inflammation, coagulation, and cellular penetration by pathogens, particularly viruses and parasites) are found on the surface of most mammalian cells and in the extracellular matrix.

[0007] (Dreyfuss et al., Annuals of the Brazilian Academy of Sciences, 2009, 81, 409-429). Therefore, this ubiquitous expression implies that HSPGs and their HS domains are not considered to represent relevant immunomodulatory targets. Furthermore, the ability of HS ligands to induce immune cell activation has not yet been determined. Summary of the Invention

[0008] In their work, the inventors first discovered that ligands of sulfated sugars from the GAG ​​family (heparan sulfate (HS)) could not activate dendritic cells in vitro. They then observed that molecular complexes containing this ligand and antigen-presenting cell (APC) surface ligands enhanced the immunomodulatory effects of the heparan sulfate (HS) ligand. In fact, as indicated by increased secretion of cytokines IL-6 and IL-12, this molecular complex induced APCs even more strongly and subsequently activated T lymphocytes. The inventors have demonstrated that association between ligands can occur in the form of covalent or non-covalent complexes, particularly as fusion proteins. They also observed that the immunomodulatory properties of this complex can better control disease processes, especially slowing tumor progression.

[0009] These results are surprising because, although described as capable of acting as a receptor or co-receptor, HSPG has never been shown to enhance ligand-mediated activation by receptors specifically expressed on the surface of APCs. Finally, the molecular complex targeting ubiquitously expressed HS can diffuse into the extracellular matrix or into cells of no interest to the disease. Therefore, theoretically, it cannot modulate the activity of APCs and large numbers of T lymphocytes to the point of being sufficient to produce immunotherapeutic effects.

[0010] The inventors also discovered that the results obtained using APC can be transferred to other cells of the innate immune response, NK and NKT cells. Therefore, the inventors have demonstrated that ligands on the surface molecules of NK or NKT cells cannot induce these cells on their own, but become capable of increasing the number of activated cells when bound in a molecular complex to a sulfated sugar ligand of the GAG ​​family.

[0011] Therefore, one subject of the present invention is a molecular complex used as an immunomodulatory drug, preferably an immunostimulatory drug, said complex comprising at least one ligand (first ligand or L1) of a sulfated sugar of the glycosaminoglycan family and at least one ligand (second ligand or L2) of another surface molecule of APC, NK, or NKT cells, said ligands being bonded to each other, and said complex not containing a specific antigen of the disease to be treated.

[0012] According to a preferred embodiment of the present invention, the first ligand is a heparan sulfate-binding peptide selected from the group consisting of: peptides derived from HIV Tat protein, which at least include the basic region Tat49-57 (SEQ ID NO: 3) such as peptides Tat 49-57 (SEQ ID NO: 3), Tat 37-57 (SEQ ID NO: 8) and Tat 22-57C(22-37)S (SEQ ID NO: 9); R7 to R11 polyarginine peptides; and peptides derived from the R domain of diphtheria toxin, which include the R domain of diphtheria toxin (SEQ ID NO: 5) or fragments DT453-467 (SEQ ID NO: 7) that at least include the heparan sulfate-binding region.

[0013] According to a preferred embodiment of the present invention, the second ligand targeting the APC surface molecules is selected from the group consisting of: C-type lectin receptors, membrane-bound immunoglobulins, immunoglobulin homeostasis receptors, and immune checkpoint (ICP) molecules.

[0014] According to a preferred embodiment of the present invention, the second ligand targeting the surface molecules of NK or NKT cells is selected from the group consisting of: NKG2D, NKp30, NKp44, NKp46, NKp80, Ly49H receptor, KIR, NKG2A receptor, and ICP PD-1, CTLA-4, TIM-3, TIGIT, and LAG-3.

[0015] The second ligand is preferably selected from the group consisting of: (i) antibodies against the surface molecules of APCs or NK or NKT cells and fragments thereof containing at least an antibody-binding site; (ii) immunoglobulins, preferably IgG, and fragments thereof containing at least an Fc region; and (iii) proteins and protein fragments that bind to the Fc and / or Fab regions of antibodies, particularly Staphylococcus aureus protein A, its BB fragment (SEQ ID NO: 1), and its ZZ derivative (SEQ ID NO: 2).

[0016] According to a preferred embodiment of the invention, the complex is in the form of an oligomer or a mixture of a monomer and an oligomer.

[0017] According to a preferred embodiment of the invention, the complex is composed of a fusion protein between a first and a second ligand. A particularly preferred class of complexes according to the invention is composed of a fusion protein comprising a first ligand and a second ligand, wherein the first ligand is selected from: Tat 49-57 (SEQ ID NO: 3), Tat 37-57 (SEQ ID NO: 8), Tat22-57C(22-37)S (SEQ ID NO: 9), the R domain of diphtheria toxin (SEQ ID NO: 5), or fragment DT453-467 (SEQ ID NO: 7), and the second ligand is selected from: the BB fragment of protein A (SEQ ID NO: 1) or its ZZ derivative (SEQ ID NO: 2).

[0018] According to other preferred embodiments of the invention, the second ligand is an antibody or antibody fragment, and the first ligand forms a fusion protein with an immunoglobulin-binding element, preferably Staphylococcus aureus protein A, its BB fragment (SEQ ID NO: 1) or its ZZ derivative (SEQ ID NO: 2). Preferably, the fusion protein comprises a first ligand and an immunoglobulin-binding element, wherein the first ligand is selected from Tat49-57 (SEQ ID NO: 3), Tat37-57 (SEQ ID NO: 8), Tat22-57C(22-37)S (SEQ ID NO: 9), the R domain of diphtheria toxin (SEQ ID NO: 5), or fragment DT453-467 (SEQ ID NO: 7), and the immunoglobulin-binding element is selected from the BB fragment of protein A (SEQ ID NO: 1) or its ZZ derivative (SEQ ID NO: 2). In a particularly preferred class of complexes according to the invention, the immunoglobulin-binding element is the BB fragment of protein A (SEQ ID NO: 1), and the fusion protein is complexed with a second ligand, the second ligand being composed of a complete immunoglobulin. In another type of complex, which is more particularly preferred according to the invention, the fusion protein is complexed with a second ligand selected from anti-RFcγI, II and / or III; anti-DEC-205; anti-DC-SIGN; anti-CD74; anti-CD275; anti-CD335; anti-CD336; anti-CD56; anti-CTLA-4; anti-PD-L1; anti-OX40 antibody or fragments of the aforementioned antibodies that at least contain an antibody-binding site.

[0019] According to a preferred embodiment of the invention, the complex is used as an immunostimulatory drug, preferably for activating antigen-presenting cells, particularly dendritic cells or monocytes; for activating NK or NKT cells; and / or for activating the secretion of cytokines IL-6 and / or IL-12.

[0020] Another subject of the invention is a composition used as an immunomodulatory drug, preferably an immunostimulatory drug, comprising at least one molecular complex according to the invention, and at least one pharmaceutically acceptable carrier, carrier substance, and / or adjuvant. The adjuvant is preferably CpG oligodeoxynucleotide, polyinosinic acid, or a mixture of CpG oligodeoxynucleotide and polyinosinic acid, and / or the carrier substance is nanoparticles.

[0021] According to a preferred embodiment of the present invention, the composition comprises at least one other therapeutic agent, preferably at least one immune checkpoint inhibitor, preferably anti-PD-1, anti-PD-L1 or anti-CTLA-4.

[0022] According to a preferred embodiment of the present invention, the composition is used for immunotherapy of cancer or infectious diseases.

[0023] Invention Disclosure Content

[0024] One subject of the present invention is a molecular complex for use as an immunomodulatory drug, preferably an immunostimulatory drug, said complex comprising at least one ligand (first ligand) of a sulfated sugar of the glycosaminoglycan family and at least one ligand (second ligand) of a surface molecule of another APC, NK, or NKT cell, said ligands being bonded to each other, and said complex not containing a specific antigen of the disease to be treated.

[0025] This complex can induce immune system cells to facilitate the triggering of an immune response. Therefore, when spleen cells are incubated with these complexes, activation of dendritic cells is observed, which become capable of secreting IL-6 and IL-12 cytokines. Furthermore, the induction of dendritic cells (DCs) can be achieved with the second ligand, an effect not triggered when they are not bound, such as the dissociated form of the APC ligand called ZZ. Binding of the HS ligand can also enhance the effect of the ligand, which is capable of activating APCs in the dissociated state, such as nonspecific polyclonal human antibodies. Binding of the HS ligand with NK or NKT cell ligands can also activate these cell types, a capability it lacks in the dissociated form. Finally, the immunomodulatory complexes according to the invention, preferably the immunostimulatory complexes, can induce therapeutic effects, particularly antitumor effects, which are superior to those obtained with anti-ICP (anti-PD1) antibodies, especially when the composition contains an adjuvant.

[0026] definition

[0027] "Innate immune cells" refers to dendritic cells, NK and NKT cells, granulocytes (mast cells, neutrophils, eosinophils and basophils) and phagocytes (monocytes, macrophages, neutrophils, etc.).

[0028] Antigen-presenting cells (APCs) are cells designed to present antigens (Ags). They are cells that express one or more major histocompatibility complex (MHC) class I and II molecules (in humans, HLA class I and II molecules) and are capable of presenting Ags to CD4+ and CD8+ T lymphocytes specific to that Ag. Ag-presenting cells can specifically include dendritic cells (DCs), monocytes, macrophages, B lymphocytes, lymphoblast cell lines, and genetically modified human or animal cell lines that express MHC class I and II molecules, particularly HLA I and HLA II molecules.

[0029] "NK cells" refers to granular lymphocytes that express the molecules CD56 and CD16 and have cytotoxic activity that does not require prior exposure to Ag.

[0030] "NKT cells" refers to granular lymphocytes that express NK cell markers, particularly the molecules CD56 and CD16, and T lymphocyte markers, particularly the molecule CD3. These cells possess cytotoxic activity that does not require prior exposure to Ag.

[0031] "APC surface molecules" refers to molecules expressed on the surface of Ag-presenting cells.

[0032] "Surface molecules of NK or NKT cells" refers to molecules expressed on the surface of NK or NKT cells.

[0033] "APC-specific surface molecules" refers to molecules that are primarily expressed on Ag-presenting cells, meaning they are expressed on a very limited number of cells other than APCs. Therefore, this is a molecule with high-level expression specificity for APCs.

[0034] "Surface molecules specific to NK or NKT cells" refers to molecules that are primarily expressed on NK or NKT cells, meaning they are expressed on a very small number of cells other than NK or NKT cells. Therefore, this is a molecule with high-level expression specificity to NK or NKT cells.

[0035] "Glycosaminoglycans (GAGs)" refer to linear polysaccharides composed of repeating disaccharides, typically containing a hexosamine (glucosamine (GIcN) or galactosamine (GaIN)) and another sugar (glucuronic acid (GIcA), iduronic acid (IdoA), galactose (Gal)). Glucosamine is N-sulfated (GlcNS) or N-acetylated (GlcNac). Galactosamine is always N-acetylated (GalNac). Sulfated glycosaminoglycans are particularly simple polymers of GlcA, such as chondroitin sulfate, and copolymers containing residues of GlcA and / or IdoA and / or Gal, such as heparin, heparan sulfate, dermatan sulfate, and keratin sulfate. GAG chains can be covalently bonded to proteins (proteoglycans) expressed on the surface of mammalian cells and / or secreted into the extracellular matrix. The first ligand according to the invention binds to sulfated sugars of the glycosaminoglycan family, which are expressed on the surface of mammalian cells, including, in particular, APC and NK or NKT cells.

[0036] "Ab" refers to immunoglobulins (IgG, IgM, IgA, IgD, IgE). The term Ab indicates a specific Ab, meaning an Ab targeting a specific molecule x (anti-molecule xAb), particularly an Ab targeting the surface molecules of APC (anti-APC surface molecule antibody). The term immunoglobulin refers to a non-specific antibody.

[0037] "Individual" refers to a human or animal, preferably a human or an individual.

[0038] The term "ligand" of a molecule refers to any reagent that can bind to the molecule with a sufficiently high affinity to form a stable complex in vitro and in vivo.

[0039] "APC ligand" refers to the ligand of the surface molecules of Ag-presenting cells.

[0040] "NK or NKT cell ligand" refers to the ligand of molecules on the surface of NK or NKT cells. "Heparan sulfate ligand" refers to a reagent that binds heparin with an optical density signal at least equal to ZZ-Tat. 22-57C(22-37)S In the ELISA assay of Example 1, 50% of the signal was measured when incubated at 100 nM and pH 7.2.

[0041] "Antigen" refers to an antigen that can be specifically recognized by the immune system, especially by the immune system's antibodies and cells (B lymphocytes, CD4+ T lymphocytes, CD8+ T lymphocytes), and can induce a specific immune response.

[0042] "Specific antigens of the disease to be treated" refers to antigens that induce a specific immune response against the disease to be treated. The specific immune response to the disease to be treated includes the production of antibodies and / or the induction of cytotoxic T cell responses (activation of CD8+ T lymphocytes) or helper T cell responses (activation of CD4+ T lymphocytes) leading to pathogens or tumor cells of the disease to be treated.

[0043] "Immunomotor" means an agent capable of controlling an immune response and, in particular, upregulating or downregulating the relative responses of different populations or subpopulations of immune cells, such as T and B lymphocytes, APCs, NK or NKT cells. The term immunomodulator encompasses the terms immunosuppressants and immunostimulants. "Immunostimulant" means an agent capable of upregulating (i.e. activating) the relative responses of different populations or subpopulations of immune cells, such as T and B lymphocytes, APCs, NK or NKT cells. The action of an immunomodulator, preferably an immunostimulant, is exerted across a broad range of cell populations. It is independent of the presence of a specific antigen of the disease to be treated, unlike vaccines which require the presence of a specific antigen of the disease to be treated and induce a specific immune response against said antigen. While the use of immunogens or vaccines is limited to diseases containing a specific antigen of that disease, the immunomodulatory complex (preferably an immunostimulant complex) according to the invention is independent of the presence of a specific antigen of the disease to be treated and can be used for the immunotherapy of a variety of diseases, such as cancer and infectious diseases.

[0044] The term "peptide" refers to a natural or synthetic amino acid sequence that may be modified. The use of the term peptide is independent of the size of the amino acid sequence.

[0045] The molecular complex according to the invention comprises at least two ligands of a surface molecule of an APC, NK, or NKT cell that is bonded to each other: a first ligand, called L1, which targets sulfated GAG, and a second ligand, called L2, which targets another surface molecule of the APC. The molecular complex according to the invention may contain one or more L1 ligands and one or more L2 ligands bonded to each other. According to some preferred embodiments of the invention, the molecular complex comprises one L1 ligand bonded to one L2 ligand.

[0046] The molecular complexes according to the invention, preferably immunostimulatory complexes, do not contain specific antigens of the disease to be treated, such as specific vaccine antigens of the disease to be treated. If one of the ligands of the complex according to the invention contains a specific antigen of the disease to be treated, then treatment of the disease with said complex is excluded from the invention.

[0047] The ligands for APC, NK, or NKT cells are natural, recombinant, or synthetic molecules or complexes of molecules that, in nature, are proteins (proteins, peptides, polypeptides), or lipids, carbohydrates, nucleic acids, or mixtures (glycolipids, glycoproteins, lipoproteins). According to some preferred embodiments of the invention, the first and second ligands are proteins, polypeptides, or peptides, hereinafter referred to as "peptides," which are preferably recombinant or synthetic. In suitable expression systems, recombinant proteins, polypeptides, or peptides are advantageously produced in prokaryotic or eukaryotic cells, particularly suitable for the production of therapeutic proteins. For example, recombinant proteins, polypeptides, or peptides can be produced in *Escherichia coli*, HEK, or CHO cells.

[0048] The first and second ligands associate or bond with each other in any suitable manner. They can bind directly or covalently or nonvalently through a linker, thereby forming a molecular complex. The molecular complex or complex consists of two or more ligands and optionally a suitable linker.

[0049] In particular, the covalent association or binding of ligands is achieved through covalent chemical coupling (formation of covalent conjugates) or through the construction of fusion proteins (genetic fusion).

[0050] Immunomodulatory complexes, preferably immunostimulatory complexes, are in monomeric, oligomeric, or mixed forms (mixtures of monomers and oligomers). According to some preferred embodiments of the invention, the complexes are in oligomeric or mixed forms.

[0051] According to some preferred embodiments of the invention, the immunomodulatory molecular complex, preferably the immunostimulatory complex, consists of a fusion protein between a first ligand (L1) and a second ligand (L2). The amino acid sequences of L1 and L2 are fused directly or via a suitable peptide spacer in a suitable order. Depending on the respective sizes of the amino acid sequences of L1 and L2, they are either fused at their ends (the N-terminus of one sequence is fused to the C-terminus of the other sequence), or one sequence is inserted into the other at a suitable site that does not adversely affect the binding of the ligand to its receptor expressed on the surface of APCs, NK, or NKT cells.

[0052] Non-covalent bonds are generated, particularly through adsorption on nanoparticles. They can also be obtained using molecules (linkers or binding elements) with high specific affinity for L1 or L2. The linker covalently binds to one of the ligands to non-covalently bind to the other ligand. When one of the ligands is an antibody (Ab), the linker specifically binds to proteins or protein fragments that bind to the Fc and / or Fab regions of immunoglobulins, as described in application FR2759296. Such immunoglobulin binding elements particularly include Staphylococcus aureus protein A, its BB fragment (SEQ ID NO: 1), and its ZZ derivative (SEQ ID NO: 2), the first two proteins binding to both the Fc and Fab regions of immunoglobulins, while ZZ binds only to the Fc region. For example, when one of the ligands is an Ab or antibody fragment, the immunoglobulin binding element covalently binds to the other ligand (covalent chemical coupling or fusion protein). The linker can also bind partners that are coupled to L1 and L2, respectively; for example, the linker is streptavidin, which binds biotinylated ligands L1 and L2 (L1-biotin / streptavidin / biotin-L2). The affinity of the linker for its partner in the L1-L2 complex is sufficient to prevent it from immediately dissociating from the complex in vivo.

[0053] According to some preferred embodiments of the immunomodulatory complex and the immunostimulatory complex of the present invention, one of the ligands is an antibody or antibody fragment and the other ligand is an immunoglobulin-binding element, preferably a protein of Staphylococcus aureus protein A, its BB fragment (SEQ ID NO: 1) or its ZZ derivative (SEQ ID NO: 2) to form a fusion protein. Preferably, the second ligand is an antibody or antibody fragment and the first ligand is an immunoglobulin-binding element, preferably a protein of Staphylococcus aureus protein A, its BB fragment (SEQ ID NO: 1) or its ZZ derivative (SEQ ID NO: 2) to form a fusion protein.

[0054] The immunomodulatory properties of the complex according to the invention are preferably assessed by conventional immunological tests known to those skilled in the art, such as those described in the examples. The immunostimulatory properties are assessed, in particular, by analyzing the amplification and / or activation of different populations or subsets of immune cells, such as CD4+ T lymphocytes, CD8+ T lymphocytes and B lymphocytes, monocytes, dendritic cells, including conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs), NK cells, and NKT cells. Specifically, amplification is analyzed using flow cytometry with markers suitable for the different cell populations to be analyzed. Immune cell activation can be analyzed by detecting activation markers (CD69) and / or maturation markers (CD86) using flow cytometry, or by conventional assays (such as ELISA) by measuring secreted cytokines, particularly IL-6 and IL-12, in the extracellular culture medium. As described above, the immunomodulatory effect of the molecular complex according to the invention is preferably independent of the presence of specific antigens of the disease to be treated.

[0055] The molecular complex according to the invention, preferably an immunostimulatory complex, binds at least one sulfated sugar of the glycosaminoglycan family expressed on the surface of APCs, NKs, or NKTs (sulfated GAGs), and binds another surface molecule of APCs, NKs, or NKTs, particularly a surface molecule specific to APCs, NKs, or NKTs. The sulfated GAG targeted by the first ligand is preferably heparan sulfate, chondroitin sulfate, dermatan sulfate, or keratin sulfate, with heparan sulfate being preferred. The sulfated GAG ligand (first ligand) can be derived from mammalian cells or pathogenic microorganisms, particularly viruses (adenovirus, cytomegalovirus, HIV, Sindbis virus), bacteria (Mycobacterium bovis, Bordetella pertussis), parasites (Leishmania sp.), or toxins; in particular, it is a molecular complex binding a molecule or fragment of heparin and / or heparan sulfate. Such ligands are specifically described in “Heparan Binding Proteins”, H. Edward Conrad, Academic Press, San Diego and London and Dreyfuss et al., Annuals of the Brazilian Academy of Sciences, 2009, 81, 409-429 (see Table 2 for details). Examples of these ligands are not limited to: endogenous GAG ligands (thrombin, urokinase, porphyrin, fibroblast growth factor, etc.), HIV Tat protein (SEQ ID NO: 10) and fragments thereof, particularly those containing only the Tat basic region (Tat 49-57 (SEQ ID NO: 3)) or the Tat basic region and central region (core; Tat 38-48 (SEQ ID NO: 4)); dodecahedrons derived from adenovirus pentagons (Vivès et al., Virology, 2004, 321:332-340); HIV envelope proteins or the V3 region of such proteins (Roderiquez et al., J. Virol., 1995, 69, 2233-); and the envelope glycoprotein of Sindbis virus (Byrnes, APet). Griffin, DE, J.Virol., 1998, 2, 7349-7356) and the R domain of diphtheria toxin (DTR or DTRBD; fragments 382 to 535 of DT (SEQ ID NO: 5)); Lobeck et al., Infection and Immunity, 1998, 66, 418-423).Also mentioned are cell-penetrating peptides (CPPs) that bind heparin, heparan sulfate and / or chondroitin sulfate, such as peptides highly enriched with basic residues, particularly arginine, including peptides derived from the basic region of HIVTat protein (Tat 49-57) and polyarginine (R7 to R11) peptides, as well as basic / amphiphilic peptides, such as peptides derived from the homologous domain of antennal foot protein (penetratin; fragments 43-58 (SEQ ID NO: 6)).

[0056] Alternatively, the first ligand is a natural or recombinant Ab targeting sulfated GAG, preferably heparin, heparan sulfate, or chondroitin sulfate, or a fragment of the Ab containing at least an antibody-binding site (Ag-binding domain), such as Fab, Fab', F(ab')2, Fv or single-chain Fv (scFv), Fabc fragments, and Fab fragments containing a portion of the Fc domain. Such Abs are described in Thompson et al., J. Biol. Chem., 2009, 284, 35621-35631 and van Kuppevelt et al., J. Biol. Chem., 1998, 273, 12960-12966. The Ab or Ab fragment is preferably human or humanized.

[0057] According to the present invention, in a preferred embodiment of the molecular complex, the first ligand is a heparan sulfate-binding peptide selected from the group consisting of: peptides derived from HIV Tat protein, comprising at least the basic region Tat 49-57 (SEQ ID NO: 3), such as peptides Tat 49-57 (SEQ ID NO: 3), Tat 37-57 (SEQ ID NO: 8), and Tat 22-57C(22-37)S (SEQ ID NO: 9); R7 to R11 polyarginine peptides; and peptides derived from the R domain of diphtheria toxin, comprising the R domain of diphtheria toxin (SEQ ID NO: 5) or fragments DT453-467 (SEQ ID NO: 7) comprising at least the heparan sulfate-binding region. The first ligand is preferably a heparan sulfate-binding peptide selected from the group consisting of peptides derived from HIVTat protein, including at least the basic region Tat 49-57 (SEQ ID NO: 3), particularly the peptide Tat 22-57C(22-37)S (SEQ ID NO: 9), and a peptide containing the diphtheria toxin R domain (SEQ ID NO: 5).

[0058] In an advantageous embodiment of the molecular complex of the invention, preferably an immunostimulatory molecular complex, when the complex contains the R domain of diphtheria toxin (DTR: SEQ ID NO: 5), the domain is generated in mammalian cells, particularly in the form of a fusion protein having a second ligand or immunoglobulin binding element, to form an oligomer.

[0059] Other molecules expressed on the surface of APCs that are targeted by the second ligand are also ubiquitous surface molecules, rather than sulfated GAGs; surface molecules that are expressed in large quantities on innate or adaptive immune cells (including APCs), i.e., surface molecules specific to innate or adaptive immune cells (including APCs); or a surface molecule that is expressed in large quantities on APCs, especially on dendritic cells, i.e., surface molecules specific to APCs, especially to dendritic cells. Among these surface molecules that are highly expressed on APCs, particularly on dendritic cells, the following may be particularly involved: class II MHC molecules, especially the α and β chains and γ chains or constant chains (li, li fragments or CD74) of MHCII molecules; surface immunoglobulins or membrane-bound immunoglobulins; integrins, such as CD11c and MAC1, particularly transferrin receptors; C-type lectin receptors, such as mannose receptor (CD206), DEC-205 (CD205), CD206, DC-SIGN (CD209), LOX1, Dectin-1 (β-glucan receptor), Dectin-2, Clec9A, Clec12A, DCIR2, FIRE and CIRE; receptors for the constant regions of immunoglobulins (FcR or RFc), particularly FcγR, such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16); the TNF receptor superfamily, such as CD40; and complement receptors. Among the surface molecules that are expressed in large quantities on APCs, there may be immune checkpoint (ICP) molecules and their ligands (ICP-ligands) expressed on APCs, such as, but not limited to, PDL1, PDL2, CD155, CD80, CD86, CD40, OX40L, ICOSL (CD275), and CD70 (Wykes MN, Nat. Rev. Immunol., 2018, 18:91-104).

[0060] Other molecules expressed on the surface of NK or NKT cells and targeted by the second ligand are also ubiquitous surface molecules, not sulfated GAGs; surface molecules that are essentially expressed on immune cells in an innate or adaptive response, including NK or NKT cells, i.e., surface molecules specific to immune cells in an innate or adaptive response, including NK or NKT cells; or surface molecules that are essentially expressed on NK or NKT cells, i.e., surface molecules specific to NK or NKT cells. Among these surface molecules that are essentially expressed on NK or NKT cells, the following can be specifically mentioned: NKG2D, NKp30, NKp44, NKp46, NKp80, CD56, CD16, KIR, NKG2A receptor, and ICP PD-1, CTLA-4, TIM-3, TIGIT, LAG-3, and OX40.

[0061] According to the molecular complex of the invention, a preferred embodiment of the immunostimulatory molecular complex, the second ligand targets molecules expressed substantially on APCs and particularly on dendritic cells, i.e., surface molecules specific to APCs and particularly to dendritic cells, preferably selected from the group consisting of: type C lectin receptors, membrane-bound immunoglobulins, and immunoglobulin homeostasis receptors.

[0062] According to the molecular complex of the invention, and other advantageous embodiments of the preferred immunostimulatory molecular complex, the second ligand targets surface molecules of APCs selected from the group consisting of: type C lectin receptors, membrane-bound immunoglobulins, immunoglobulin constant region receptors (RFc or FcR), and immune checkpoint (ICP) molecules and their ligands (ICP ligands).

[0063] According to an advantageous embodiment of the invention, the second ligand targets surface molecules expressed substantially on NK or NKT cells, i.e., surface molecules specific to NK or NKT cells, preferably selected from the group consisting of: NKG2D, NKp30, NKp44, NKp46, NKp80, CD56, CD16, KIR, NKG2A receptor, and ICP PD-1, CTLA-4, TIM-3, TIGIT, LAG-3, and OX40; preferably NKp44 (CD336), NKp46 (CD335), NCAM (CD56), CTLA-4, and OX40.

[0064] The second ligand is specifically selected from carbohydrates that bind to type C lectin receptors; immunoglobulins and fragments thereof containing constant regions that bind FcRs; proteins or protein fragments that bind the Fc and / or Fab regions of membrane-bound immunoglobulins, as described in application FR2759296, particularly Staphylococcus aureus protein A, its BB fragment (SEQ ID NO: 1), and ZZ and its derivatives (SEQ ID NO: 2), preferably rZZ (SEQ ID NO: 2). Optionally, the second ligand is an antibody against these surface molecules of APC, NK, or NKT cells, or a fragment containing at least an antibody-binding site (Ag-binding domain), such as Fab, Fab', F(ab')2, Fv or single-chain Fv (scFv), Fabc fragments, and Fab fragments containing a portion of the Fc domain.

[0065] Antibodies or antibody fragments are specifically targeted at immune cell-specific surface molecules of innate or adaptive responses, including APCs, NK and NKT cells, such as unrestricted PDL1, PDL2, CD155, CD80, CD86, CD40, OX40L, ICOSL (CD275), CD70; PDL1, PDL2, CD155, CD80, CD86, CD40, OX40L, ICOSL (CD275), CD70, PD-1, CTLA-4, TIM-3, TIGIT, LAG-3; preferably PDL1 and ICOSL (CD275) or PDL1, ICOSL (CD275), CTLA-4 and OX40.

[0066] Antibodies or antibody fragments may also target surface molecules specific to APCs, particularly dendritic cells, such as anti-RFcγ (I, II, and / or III) or anti-C-type lectin receptor antibodies, particularly anti-DEC-205 or anti-DC-SIGN (CD209); anti-DEC-205, anti-DC-SIGN (CD209), or anti-invariant chain II (CD74). Antibodies can be agonists or antagonists of said surface molecules; for example, antibodies are agonists that activate surface molecules or antagonists that inhibit surface molecules. The antibodies or antibody fragments are preferably human or humanized. Such antibodies are well known to those skilled in the art and are commercially available.

[0067] According to the molecular complex, preferably an immunostimulatory molecular complex, the second ligand is selected from the group consisting of: (i) antibodies against the surface molecules of antigen-presenting cells or NK or NKT cells and fragments thereof containing at least an antibody-binding site, such as Fab, Fab', F(ab')2, Fv, scFv, Fabc, and Fab fragments having at least a portion of an Fc domain; (ii) immunoglobulins, preferably IgG, and fragments thereof containing at least an Fc region; (iii) proteins and protein fragments that bind to the Fc and / or Fab regions of antibodies, particularly Staphylococcus aureus protein A, its BB fragment (SEQ ID NO: 1), and its ZZ derivative (SEQ ID NO: 2).

[0068] The antibodies against the surface molecules of the antigen-presenting cells are preferably selected from anti-RFcγ (I, II and / or III), anti-DC-SIGN (CD209), anti-DEC-205, anti-CD206, and anti-CD40 antibodies; anti-RFcγ (I, II and / or III), anti-DC-SIGN (CD209), anti-DEC-205, anti-CD206, anti-CD40, anti-invariant chain Ii (CD74), and anti-ICOSL (CD275) antibodies; preferably, anti-DC-SIGN (CD209) and anti-DEC-205 antibodies or anti-DC-SIGN (CD209), anti-DEC-205, anti-invariant chain Ii (CD74), and anti-ICOSL (CD275) antibodies.

[0069] The antibodies targeting the surface molecules of NK or NKT cells are preferably selected from anti-CD16, anti-CD56, anti-CD335, anti-CD336, anti-NKp30, anti-NKG2D, anti-NKp80, anti-Ly49H, anti-NKG2A, anti-PD-1, anti-CTLA-4, anti-TIM-3, anti-TIGIT, anti-LAG-3, and anti-OX40 antibodies; preferably, anti-NCAM (CD56), anti-Nkp46 (CD335), anti-Nkp44 (CD336), anti-CTLA-4, and anti-OX40 antibodies.

[0070] Other ligands for the molecular complex, preferably other ligands for the immunostimulatory molecular complex, are advantageously selected from sulfated GAG ligands and ligands for APC surface molecules as defined above.

[0071] The first preferred type of immunostimulatory complex according to the invention comprises a sulfated GAG ligand peptide (first ligand) as defined above and a protein or protein fragment that binds to the Fc and / or Fab regions of immunoglobulins, such as the BB fragment of protein A from Staphylococcus aureus and its ZZ derivative (second ligand), preferably in the form of a fusion protein comprising the first and second ligands. A particularly preferred type of complex comprises a fusion protein comprising: a first ligand and a second ligand, wherein the first ligand is selected from: Tat 49-57 (SEQ ID NO: 3), Tat 37-57 (SEQ ID NO: 8), Tat 22-57C(22-37)S (SEQ ID NO: 9), the R domain of diphtheria toxin (DTR: SEQ ID NO: 5), or fragment DT453-467 (SEQ ID NO: 7), and the second ligand is selected from: the BB fragment of protein A (SEQ ID NO: 1) or its ZZ derivative (SEQ ID NO: 2), preferably its ZZ derivative (SEQ ID NO: 2). The first and second ligands are fused directly or via a suitable spacer peptide. In this first type of particularly preferred complex, the second ligand is preferably located at the N-terminus of the fusion protein, and the first ligand is located at the C-terminus of the fusion protein; the first and second ligands are separated by a suitable spacer peptide, particularly selected from SEQ ID NO: 21 to 23. More preferably, the complex comprises one of the sequences SEQ ID NO: 12, 14, 16, 18, and 20. When the complex comprises the R domain of diphtheria toxin (DTR: SEQ ID NO: 5), this domain is advantageously generated in mammalian cells, particularly in the form of a fusion protein with the second ligand, to form an oligomer.

[0072] The second preferred type of immunostimulatory complex according to the invention comprises: (i) a sulfated GAG ligand peptide (first ligand) as defined above, covalently bonded to an immunoglobulin-binding element as defined above, particularly a protein or protein fragment binding to the Fc and / or Fab regions of Ab, preferably binding only to the Fab region, such as protein A and its BB fragment from Staphylococcus aureus, preferably in the form of a fusion protein comprising the first ligand and the immunoglobulin-binding element, and (ii) an immunoglobulin, preferably IgG, or a fragment comprising at least the Fc region (second ligand). Full-strength immunoglobulin is preferred, preferably full-strength IgG. The first ligand and the immunoglobulin-binding element are fused directly or via a suitable spacer peptide. The second type of particularly preferred complex comprises a fusion protein containing a sequence selected from Tat 49-57 (SEQ ID NO: 3), Tat 37-57 (SEQ ID NO: 8), Tat 22-57C(22-37)S (SEQ ID NO: 9), the R domain of diphtheria toxin (DTR: SEQ ID NO: 5) or fragment DT453-467 (SEQ ID NO: 7), and an immunoglobulin-binding element (SEQ ID NO: 1) containing a BB fragment of protein A, said fusion protein being complexed with an intact immunoglobulin. In this second type of particularly preferred complex, the immunoglobulin-binding element (BB) is preferably located at the N-terminus of the fusion protein, and the first ligand is located at the C-terminus of the fusion protein; the immunoglobulin-binding element (BB) and the first ligand are separated by a suitable spacer peptide, particularly selected from SEQ ID NO: 21 to 23. More preferably, the complex comprises one of the sequences SEQ ID NO: 16 or 18. When the complex contains the R domain of diphtheria toxin (DTR: SEQ ID NO: 5), the domain is advantageously produced in mammalian cells, particularly in the form of a fusion protein with an immunoglobulin-binding element, to form an oligomer.

[0073] The third preferred type of immunostimulatory complex according to the invention comprises: a sulfated GAG ligand peptide (first ligand) as defined above, and an Ab selected from the group consisting of: anti-RFcγ (I, II and / or III) Ab, anti-DEC-205 Ab, anti-DC-SIGN (CD209) Ab, anti-invariant chain II (CD74) Ab, anti-ICOSL (CD275) Ab, anti-NKp46 (CD335) Ab, anti-NKp44 (CD336) Ab, anti-NCAM (CD56) Ab, anti-CTLA-4 Ab, anti-PDL1 Ab, anti-OX40 Ab, and fragments of the above-mentioned Abs containing at least an antibody-binding site. Preferably, the sulfated GAG ligand peptide (first ligand) as defined above is covalently bonded to an immunoglobulin-binding element as defined above, particularly a protein or protein fragment that binds to the Fc and / or Fab regions of immunoglobulins, such as the BB fragment of protein A of Staphylococcus aureus and its ZZ derivative, preferably in the form of a fusion protein of the first ligand and the immunoglobulin-binding element. The first ligand and the immunoglobulin binding element are fused directly or via a suitable spacer peptide. The third type of particularly preferred complex consists of a fusion protein comprising: a first ligand and an immunoglobulin-binding element, wherein the first ligand is selected from: Tat 49-57 (SEQ ID NO: 3), Tat 37-57 (SEQ ID NO: 8), Tat 22-57C(22-37)S (SEQ ID NO: 9), the R domain of diphtheria toxin (DTR: SEQ ID NO: 5) or fragment DT453-467 (SEQ ID NO: 7), and the immunoglobulin-binding element comprises a BB fragment of protein A (SEQ ID NO: 1) or its ZZ derivative (SEQ ID NO: 2), and the fusion protein is complexed with an anti-RFcγ (I, II and / or III), anti-DEC-205, anti-DC-SIGN, anti-CD74, anti-CD275, anti-CD335, anti-CD336, anti-CD56, anti-CTLA-4, anti-PDL1, anti-OX40 antibody, or a fragment of the aforementioned antibody containing at least an antibody binding site. In this third particularly preferred complex, the immunoglobulin-binding element (BB or ZZ) is preferably located at the N-terminus of the fusion protein, and the first ligand is located at the C-terminus of the fusion protein; the immunoglobulin-binding element (BB or ZZ) and the first ligand are separated by a suitable spacer peptide, particularly selected from SEQ ID NO: 21 to 23. More preferably, the complex comprises one of the sequences SEQ ID NO: 12, 14, 16, 18, or 20. When the complex comprises the R domain of diphtheria toxin (DTR: SEQ ID NO: 5), this domain is advantageously produced in mammalian cells, particularly in the form of a fusion protein having an immunoglobulin-binding element, to form oligomers.Preferably, the complex comprises an antibody against DEC-205, DC-SIGN, CD74, CD275, CD335, CD336, CD56, CTLA-4, PDL1, or OX40.

[0074] According to a preferred embodiment of the invention, the complex is used as an immunostimulant, preferably for activating antigen-presenting cells, particularly dendritic cells or monocytes, for activating NK or NKT cells, and / or for activating the secretion of cytokines IL-6 and / or IL-12.

[0075] Another subject of the present invention is an immunomodulatory composition, preferably an immunostimulatory composition, comprising at least one immunomodulatory complex according to the invention, preferably an immunostimulatory complex, and at least one pharmaceutically acceptable carrier, carrier substance, and / or adjuvant.

[0076] Pharmaceutically acceptable carriers are those that are routinely used.

[0077] The adjuvant is a commonly used adjuvant for humoral and / or cellular immunity in immunotherapy. The adjuvant is advantageously selected from the group consisting of: oily emulsions, minerals, bacterial extracts, saponins, aluminum hydroxide, monophospholipid A, squalene, and TLR ligands, particularly oligodeoxynucleotides (CpG oligodeoxynucleotides) containing at least one CpG sequence, which are TLR9 ligands; or polyinosinic acid (poly(I):poly(C) or polyI:C), which are TLR3 ligands. According to a preferred embodiment of the invention, the composition comprises at least one adjuvant, preferably CpG oligodeoxynucleotides, polyinosinic acid, or a mixture of CpG oligodeoxynucleotides and polyinosinic acid.

[0078] The carrier material is one of those conventionally used. These include, in particular, monolayer or multilayer liposomes, ISCOMs, virions (virus-like particles), saponin micelles, saccharides (poly(lactide-co-glycolic acid)), or gold-containing solid microspheres, as well as nanoparticles. According to a preferred embodiment of the invention, the composition comprises at least one carrier material, such as nanoparticles or mixtures of nanoparticles.

[0079] The immunomodulatory compositions according to the invention, preferably immunostimulatory compositions, comprise complexes or mixtures of different complexes containing two or more ligands, said complexes optionally being covalently or non-covalently bonded to each other, and / or incorporated into or on the surface of particles (such as liposomes, virions or nanoparticles).

[0080] According to specific embodiments of the invention, the composition comprises a polynucleotide or mixture of polynucleotides encoding an APC, NK, or NKT ligand, which is a protein, polypeptide, or peptide. The polynucleotide consists of a recombinant, synthetic, or semi-synthetic nucleic acid that can be expressed in a host cell to which the composition is administered. The nucleic acid can be DNA, RNA, particularly mRNA, a mixed nucleic acid (DNA / RNA), and can be modified. For example, the composition comprises a polynucleotide containing a sequence encoding a fusion protein, the fusion protein containing at least sequences encoding first and second ligands, suitably fused within a frame, and optionally a sequence encoding a linker as defined above. Alternatively, the composition comprises a mixture of polynucleotides comprising at least a first polynucleotide encoding a first ligand sequence and a second polynucleotide encoding a second ligand sequence, the first or second polynucleotide also containing a sequence encoding a linker as defined above. Preferably, the polynucleotide is inserted into one or more expression vectors containing suitable transcriptional and / or translational regulatory sequences (promoters, transcriptional activators, transcriptional terminators, polyadenylation signals) for expressing the first and second ligands, and optionally other ligands in the individual to which the composition is administered. Many vectors that can be used for treatment are known in themselves. Viral vectors (adenovirus, retrovirus, lentivirus, AAV) and non-viral vectors (naked DNA), particularly plasmids, can be used, with the target sequence pre-inserted. Alternatively, the polynucleotide can be mRNA, preferably modified. The use of mRNA in treatment is well known to those skilled in the art (e.g., reviewed by Drew Weissman, Expert Reviews of Vaccines, 201410, 1-17, doi:10.1586 / 14760584.2015.973859).

[0081] According to a specific embodiment of the invention, the composition comprises cells modified with the composition according to the invention. For example, the cells are modified with polynucleotides, mixtures of polynucleotides, or carriers as defined above, or loaded with ligand complexes as defined above. The cells are particularly natural antigen-presenting cells (such as dendritic cells), or artificial antigen-presenting cells (such as exogenous cells derived from dendritic cells), or vesicles derived from cells expressing ligands of the molecular complex according to the invention. For example, the cells are antigen-presenting cells of the individual to be treated, particularly ex vivo modified dendritic cells prior to re-administration to the individual (ex vivo cell therapy).

[0082] The immunomodulatory composition may also contain at least one other therapeutic agent, particularly an anticancer agent, an anti-infective agent, another immunomodulatory agent, or a disease-specific vaccine antigen, said vaccine antigen being advantageously bound to a carrier substance or contained in a suitable carrier. According to a preferred embodiment of the invention, the composition further contains at least one immune checkpoint inhibitor (Marin-Acevedo J. et al., 2018, Hematol. Oncol., 11:39), such as, but not limited to, anti-PD-1, anti-PDL-1, or anti-CTLA4, particularly an antibody, preferably a monoclonal antibody, against molecular PD-1, PDL-1, or CTLA4, preferably human molecular hPD-1, hPDL-1, or hCTLA4. The composition according to the invention advantageously contains anti-PD-1, particularly a monoclonal anti-PD-1 antibody, preferably anti-hPD-1. According to other preferred embodiments of the invention, the composition further contains at least one disease-specific vaccine antigen, preferably bound to a carrier substance or contained in a suitable carrier.

[0083] An immunomodulatory composition, preferably an immunostimulatory composition, comprises an effective dose of a complex, polynucleotide, carrier, or cell sufficient to induce an immune response capable of producing a therapeutic effect on the disease to be treated, i.e., alleviating the symptoms of the disease. This particularly relates to reducing the consequences of the action of a pathogen (infectious or non-infectious) or reducing tumor growth in an individual treated with the composition. The dose is determined and adjusted according to factors of the subject, such as age, sex, and weight. The immunomodulatory composition according to the invention, preferably an immunostimulatory composition, is a pharmaceutical form suitable for the selected administration. The composition is typically administered according to a conventional immunotherapy regimen at a dose and duration sufficient to induce an effective immune response against the disease to be treated. Administration can be intratumoral, subcutaneous, intramuscular, intravenous, intradermal, intraperitoneal, oral, sublingual, rectal, vaginal, intranasal, inhaled, or transdermal. The composition is a pharmaceutical form suitable for the selected administration.

[0084] Isolated or inserted polynucleotides into plasmid vectors are introduced into the individual to be treated using physical methods (such as electroporation) or by binding them to any substance capable of passing through the plasma membrane, such as transport proteins, e.g., nanotransporters, liposomes, lipids, or cationic polymers. Furthermore, these methods can be advantageously combined, for example, using liposome-associated electroporation.

[0085] The immunomodulatory compositions according to the invention, preferably immunostimulatory compositions, are used for immunotherapy, particularly for antitumor or anti-infective immunotherapy. The immunomodulatory compositions according to the invention, preferably immunostimulatory compositions, are used preventively or therapeutically, i.e., for the prevention of an individual's disease or for the treatment of an individual suffering from a disease. According to a preferred embodiment of the invention, the compositions are used therapeutically, i.e., for the treatment of an individual suffering from a disease. They can be used in combination with other treatments, whether therapeutic or surgical, particularly with other therapeutic agents as defined above. The compositions according to the invention and other therapeutic agents can be administered simultaneously, separately, or sequentially.

[0086] According to a preferred embodiment of the present invention, the composition is used to treat cancer. Cancer is any type of cancer that may benefit from immunotherapy, such as, but not limited to: breast cancer, colon cancer, prostate cancer, esophageal cancer, stomach cancer, lung cancer, ENT (ear, nose, and throat) cancer, skin cancer, ovarian cancer, uterine cancer, brain cancer, liver cancer, or kidney cancer.

[0087] According to other preferred embodiments of the invention, the composition is used to treat infectious diseases, particularly those in which the infectious agent remains in the body. Infectious diseases are any type of infectious disease that may benefit from immunotherapy (Wykes MN et al., Nat. Rev. Immunol., 2018, 18:91-104), such as, but not limited to, viral, bacterial, fungal, or parasitic infections, particularly HIV, HBV, HCV, Mycobacterium tuberculosis, or Plasmodium falciparum infections. When the complex contains Tat or a Tat fragment as defined above, the composition is used to treat infectious diseases other than HIV infection (AIDS). When the complex contains DTR or a DTR fragment as defined above, the composition is used to treat infectious diseases other than diphtheria (infections caused by Corynebacterium diphtheriae).

[0088] The immunomodulatory composition according to the invention, preferably the immunostimulatory composition, is used for conventional treatment or cell therapy, or a combination of both.

[0089] Cell therapy involves preparing antigen-presenting cells, particularly dendritic cells, or NK or NKT cells, using standard protocols, including isolating peripheral blood mononuclear cells (PBMCs) from the patient to be treated and culturing the dendritic cells, NK or NKT cells in the presence of the molecular complex, polynucleotide, and carrier as defined above. In a second step, the antigen-presenting cells, NK or NKT cells loaded with the molecular complex or modified with the polynucleotide or carrier are re-injected into the patient.

[0090] Another subject of the present invention is an immunotherapy method, particularly an antitumor or anti-infective method, characterized in that it comprises administering an immunomodulatory composition, preferably an immunostimulatory composition, to an individual by any suitable means as defined above.

[0091] Another subject of the present invention is the use of immunomodulatory compositions, preferably immunostimulatory compositions as defined above, in the preparation of medicaments intended for use in immunotherapy, preferably in antitumor or anti-infective immunotherapy.

[0092] The ligand complexes for APC, NK, or NKT cells according to the present invention are prepared using conventional techniques known to those skilled in the art, namely:

[0093] Ligands for surface molecules of APC, NK, or NKT cells can be produced chemically or by expressing recombinant DNA in suitable eukaryotic or prokaryotic cell systems. According to the Fmoc technique originally described by Merrifield et al. (J. Am. Chem. Soc., 1964, 85, 2149-), peptides or proteins can be synthesized in the solid phase and purified by reversed-phase high-performance liquid chromatography. Peptides and proteins can be generated from the corresponding cDNA and cloned into suitable eukaryotic or prokaryotic expression vectors; peptides or proteins generated in cells modified with recombinant vectors can be purified by any suitable method, particularly by affinity chromatography. Abs targeting surface molecules of APC, NK, or NKT cells are well known and commercially available. For example, non-limitingly, anti-CD205 (#555831); anti-CD206 (#555952); anti-CD209 (#551186); and anti-HLA-DR (#555556) Abs are available from BECTON-DICKINSON, while anti-CD56 Abs are available from Biolegend (#304622), and anti-CD335 (AM31284AF-N) and anti-CD336 (#AM50346PU-N) Abs are available from Origene. Alternatively, monoclonal Abs can be produced using conventional techniques known to those skilled in the art. For example, according to The technique described by et al. Milstein (Nature, 1975, 256, 495-497) generates monoclonal antibodies by fusing B lymphocytes (from animals immunized with surface molecules of APCs) and hybridomas obtained from myeloma; the hybridomas are cultured in vitro, particularly in fermenters, or produced in vivo as ascites; alternatively, the monoclonal antibodies are produced through genetic engineering, as described in patent US4,816,567. Humanized Abs are produced using common methods, such as those described in international application WO98 / 45332. The Ab fragment is generated from cloned V... H and V LThe RNA is produced from the region, hybridoma mRNA, or splenic lymphocytes from an immunized animal; for example, Fv, scFv, or Fab fragments are expressed on the surface of filamentous phages according to the technique of Winter et Milstein (Nature, 1991, 349, 293-299); after several selection steps, Ag-specific Ab fragments are isolated and expressed in a suitable expression system using conventional techniques for cloning and expressing recombinant DNA. The Abs or fragments thereof as defined above are purified using conventional techniques known to those skilled in the art, such as affinity chromatography.

[0094] The covalent binding of a first ligand (L1) to a second ligand (L2) on the surface molecules of APCs, NK, or NKT cells can be achieved by constructing a fusion protein, in which the nucleotide sequences encoding L1 and L2 are fused directly or by means of nucleotide sequences encoding suitable spacer peptides within a suitable sequence frame. Depending on the respective sizes of the amino acid sequences of L1 and L2, they are either fused at their ends (the N-terminus of one sequence is fused to the C-terminus of the other), or one sequence is inserted into the other at a suitable site that does not adversely affect the binding of the ligand to its receptor expressed on the surface of APCs, NK, or NKT cells. Alternatively, the ligands can be covalently coupled in any suitable manner. Coupling is achieved through reactive groups that are initially present or pre-introduced into the ligand. Coupling can particularly occur at amino acid residues whose side chains contain reactive functions. Among these amino acids may be polar amino acids with functions including: OH [serine (S), threonine (T), or tyrosine (Y)], -SH [cysteine ​​(C)], -NH2 [lysine (K), or arginine (R)], -COOH [aspartic acid (D), or glutamic acid (E)], and polar amino acids having side chains functionalized by the addition of reactive functions, particularly chloroacetyl or bromoacetyl groups reacting with thiols or hydrazide groups reacting with aldehydes. The ligands are coupled by any suitable means; these methods, known to those skilled in the art, particularly include coupling using homobifunctional reagents such as glutaraldehyde or dithiobis(succinimide propionate)). Preferably, coupling is generated using heterobifunctional reagents, particularly m-maleimide-benzoyl-N-hydroxysuccinimide (SMCC) or sulfonyl-SMCC, each containing a maleimide group capable of reacting with free thiols. In this case, SMCC is pre-covalently bound to the amine functional group present on the ligand. Simultaneously, another heterobifunctional reagent (such as N-succinimidyl-S-acetylthioacetate containing a thioester group that can be cleaved by hydroxylamine, or succinimidyl-pyridyl-dithiopropionate containing a disulfide bridge that can be reduced under mild conditions) is bound to the amine functional group of a second chaperone, which is one of the ligands. The second chaperone is then treated with hydroxylamine or with a reducing agent to release the thiol. The thiolated compound is then incubated with a compound incorporating maleimide, and coupling is achieved through the reaction of the thiohydroxy group with the maleimide group. This type of covalent coupling is specifically described in Léonetti et al., J.Exp.Med., 1999, 189, 1217-1228. Alternatively, the thiol group already present on one of the compounds can be released and then coupled to another compound pre-modified with SMCC. This method, often used to couple Abs to ligands, is specifically described in Ishikawa et al., J. Immunoassay, 1983, 4, 209-327.

[0095] - A non-covalent complex is prepared by contacting a second ligand (L2) with a first ligand (L1) under conditions that allow the two ligands to interact. This interaction may involve a linker, particularly a protein or peptide, that has a high specific affinity for one of the chaperones (L1 or L2) of the complex. In particular, the linker's affinity for that chaperone in the complex is sufficient to prevent it from immediately dissociating from the complex in vivo. When one of the ligands is an immunoglobulin, the linker is an immunoglobulin-binding element as described in application FR2759296. For example, an immunoglobulin-binding element is covalently bonded to L1 to form a non-covalent complex with L2.

[0096] - The polynucleotides according to the invention are obtained using conventional methods known per se, according to standard protocols known to those skilled in the art. For example, they can be obtained by amplifying nucleic acid sequences by PCR or RT-PCR, by screening genomic DNA libraries through hybridization with homologous probes, or by full or partial chemical synthesis. Recombinant vectors are constructed and introduced into host cells using conventional recombinant DNA and genetic engineering methods known per se.

[0097] Unless otherwise stated, the present invention is practiced using conventional methods of immunology, cell culture, cell biology, molecular biology, and recombinant DNA known to those skilled in the art. Attached Figure Description

[0098] Other features, details, and advantages of the invention will become apparent from the following detailed description of exemplary embodiments of the invention, and from an analysis of the accompanying drawings, wherein:

[0099] Figure 1

[0100] [ Figure 1 The molecular weight and degree of homogeneity of the molecular complex produced in *E. coli* or HEK cells are shown. Protein ZZ-DTRBD was analyzed by electrophoresis under denaturing conditions. HEK ZZ-DTRBD coli BB-DTRBD HEK BB-DTRBD coli and ZZ-Tat 22-57C(22-37)S Proteins were deposited in the presence of molecular weight markers. After electrophoretic migration, the proteins were stained with Coomassie blue.

[0101] Figure 2

[0102] [ Figure 2 The image shows the binding of various molecular complexes to heparin. Proteins ZZfree and ZZ-DTRBD are also mentioned.HEK ZZ-DTRBD coli BB-DTRBD HEK BB-DTRBD coli and ZZ-Tat 22-57C(22-37)S A series of dilutions were incubated in microtiter plate wells at pH 7.2, pre-adsorbed with rabbit IgG. After 4 hours, the plates were washed and biotinylated heparin was added. After 30 minutes of incubation, the binding of heparin to the plate was detected using streptavidin coupled with peroxidase and the enzyme's substrate (ABTS). When ZZ-Tat 22-57C(22-37)S When incubated on a microtiter plate at 100 nM and pH 7.2, heparin binding is considered significant when the optical density signal is at least 50% of the measured signal.

[0103] Figure 3

[0104] [ Figure 3 The image shows the fusion protein ZZ-DTRBD in a mouse spleen cell population. HEK The cell type that is bound. In the presence or absence of a fixed amount of ZZ-DTRBD. HEK Spleen cells were incubated at 100 nM. A series of fluorescent Abs specific to CD11b+ pDCs, cDCs, and CD8+ cDCs were then added (A); CD4+ T lymphocytes, CD8+ T lymphocytes, B lymphocytes, and monocytes (B). After 30 minutes at 4°C, the cells were washed and analyzed by flow cytometry. ZZ-DTRBD in the relevant subsets was shown. HEK Percentage of cells bonded.

[0105] Figure 4

[0106] [ Figure 4 [Displays ZZ-DTRBD] HEK Inducing spleen cells to secrete IL-6 and IL-12. A fixed amount of ZZ, DTRBD, or ZZ-DTRBD was administered. HEK (1 μM) was incubated with mouse spleen cells. After 24 hours, the supernatant was collected and the presence of IL-6 and IL-12 was assessed by enzyme immunoassay.

[0107] Figure 5

[0108] [ Figure 5 [Displays ZZ-Tat] 22-57C(22-37)S Inducing spleen cells to secrete IL-6 and IL-12. Fixed amounts of ZZ and Tat were used. CY49-57 ZZ+Tat CY49-57 Or ZZ-Tat 22-57C(22-37)S(1 μM) was incubated with mouse spleen cells. After 24 hours, the supernatant was collected and the presence of IL-6 and IL-12 was assessed by enzyme immunoassay.

[0109] Figure 6

[0110] [ Figure 6 [Displays protein ZZ-DTRBD] HEK BB-DTRBD HEK Induces spleen cells to secrete more than ZZ-DTRBD coli and BB-DTRBD coli Larger amounts of IL-6 and IL-12. Fixed amounts of ZZ-DTRBD HEK ZZ-DTRBD coli BB-DTRBD HEK and BB-DTRBD coli (1 μM) was incubated with mouse spleen cells. After 24 hours, the supernatant was collected and the presence of IL-6 and IL-12 was assessed by enzyme immunoassay.

[0111] Figure 7

[0112] [ Figure 7 [Displays ZZ-DTRBD] HEK Rat dendritic cells were induced to secrete IL-6 and IL-12. Dendritic cells were then isolated by magnetic sorting. A fixed amount of ZZ-DTRBD was then used in the presence or absence of the cells. HEK Dendritic cells were incubated at 0.6 μM. After 24 hours, the supernatant was collected and the presence of IL-6 and IL-12 was assessed by enzyme immunoassay.

[0113] Figure 8

[0114] [ Figure 8 [Displays protein ZZ-DTRBD] HEK and ZZ-Tat 22-57C(22-37)S It can induce dendritic cell expansion in mice. This is effective in the absence or presence of a fixed amount of ZZ-DTRBD. HEK (5 nmol per mouse) or ZZ-Tat 22-57C(22-37)S (At 10 nmol per mouse, the three groups of mice were injected with PBS buffer three days apart). Twenty-four hours after the last injection, the animals were euthanized, the spleen was removed, and the spleen cells were labeled with a fluorescent Ab mixture to distinguish cDC-CD8+ (CD11c) cells. 高 B220-CD8 + CD11b - )(A), cDC-CD11b+(CD11chighB220- CD8 - CD11b + (B) and pDC(CD11c) int CD317 + CD11b + ).

[0115] [ Figure 8 [C] shows the cells analyzed by flow cytometry. The number of positive cells is shown as a percentage of the total number of viable spleen cells. Statistical analysis was performed using the Kruskall-Wallis test (*p<0.05).

[0116] Figure 9

[0117] [ Figure 9 [Displays ZZ-DTRBD] HEK Human dendritic cells (DCs) isolated in vitro were induced to secrete IL-6 and IL-12. Under a fixed dose of ZZ-DTRBD... HEK DCs isolated from healthy human donors were incubated in the presence of 1 μM. After 24 hours, the supernatant was collected to assess the presence of cytokines IL-6 and IL-12 by enzyme immunoassay.

[0118] Figure 10

[0119] [ Figure 10 The results showed that mice injected with colorectal cancer cells subsequently received a separate injection of ZZ-DTRBD. HEK Tumor growth was slowed when injected in combination with the adjuvant mixture. A: Sixteen C57BL / 6 mice were subcutaneously injected with 0.5M MC38 cells. Three days later, eight mice were injected with the adjuvant mixture CpG-B1018 / PolyI:C (30 μg of each adjuvant) and eight were not injected (control). A second injection was performed three days later.

[0120] B: Eighteen C57BL / 6 mice were subcutaneously injected with 0.5M MC38 cells. Three days later, six mice were injected with ZZ-DTRBD in PBS buffer. HEK Six other mice were injected with ZZ-DTRBD in PBS buffer containing an adjuvant mixture of CpG-B1018 / Polyl:C (30 μg of each adjuvant). HEK (2 nmol per mouse) and 6 uninjected (control). Two more injections were given three days apart. Tumor growth was monitored by measuring the tumor using a caliper. Each injection is indicated by an arrow.

[0121] Figure 11

[0122] [ Figure 11The results showed that mice injected with colorectal cancer cells subsequently injected with ZZ-DTRBD... HEK ZZ-Tat 22-57C(22-37)S Tumor growth was slowed when combined with the adjuvant mixture. Thirty-two C57BL / 6 mice were subcutaneously injected with 0.5 M MC38 cells. Three days later, a group of eight mice were injected with ZZ-DTRBD in 50 μl PBS containing the adjuvant mixture CpG-B1018 / Polyl:C (30 μg of each adjuvant). HEK One group received 0.96 nmol of anti-PD-1 antibody (50 μl, 1.3 nmol per mouse). A third group received ZZ-Tat 22-57C(22-37)S (0.96 nmol per mouse) in 50 μl PBS containing the adjuvant mixture CpG-B1018 / Polyl:C (30 μg of each adjuvant). The last group of eight mice received no injection (control group). Two more injections were administered three days apart. Tumor growth was monitored using a caliper. Each injection is indicated by an arrow.

[0123] [ Figure 11 A] Tumor growth dynamics.

[0124] [ Figure 11 B] Tumor growth dynamics.

[0125] [ Figure 11 C] Survival rate of treated animals.

[0126] Figure 12

[0127] [ Figure 12 The molecular complex ZZ-Tat is displayed. 22-57C22-37)S Anti-DEC205 / ZZ-Tat 22-57C22-37)S and lgG / ZZ-Tat 22-57C22-37)S The formation of this substance increased the proportion of activated monocytes and CD4+ T lymphocytes induced in vitro. A fixed concentration (0.1 μM) of anti-DEC205 / ZZ-Tat... 22-57C22-37)S lgG / ZZ-Tat 22-57C22-37)S Anti-DEC205 / ZZ, IgG / ZZ, and anti-DEC205 antibodies were incubated with human PBMCs. A fixed concentration (1 μM) of ZZ-Tat was added. 22-57C22-37)S Alternatively, ZZ was incubated with human PBMCs. After 24 hours, cells were collected and labeled using fluorescent Ab. The number of activated monocytes (CD69) in the live PBMC population was assessed using the marker CD69. 3- CD1 4+ The proportion of ).

[0128] [ Figure 12A] Assessing the live CD4+ T lymphocyte population (CD69) by measuring CD69 molecule expression. 3+ CD 4+ The proportion of activated CD4+ T lymphocytes in the cell.

[0129] [ Figure 12 B] Cells were analyzed by flow cytometry.

[0130] Figure 13

[0131] [ Figure 13 [This image shows a molecular complex targeting the receptor expressed on dendritic cells (anti-CD74 / ZZ-Tat)] 22-57C22-37)S Anti-CD209 / ZZ-Tat 22-57C22-37)S and anti-CD275 / ZZ-Tat 22-57C22-37)S Induces PBMC secretion of IL-6. This occurs in the presence or absence of free forms of Ab, ZZ, or ZZ-Tat. 22-57C(22-37)S Human PBMCs were incubated with molecular complexes targeting molecules CD74(A), CD209(B), and CD275(C), respectively. After 24 hours, the supernatant was collected and the presence of IL-6 was assessed by enzyme immunoassay.

[0132] Figure 14

[0133] [ Figure 14 [This image shows a molecular complex targeting the CD335 receptor expressed by NK and NKT cells (anti-CD335 / ZZ-Tat)] 22-57C22-37 The proportion of NK and NKT cells induced by S increases. This is observed in the presence or absence of free forms of Ab, ZZ, or ZZ-Tat. 22-57C(22-37)S and molecular complex anti-CD335 / ZZ-Tat 22-57C22-37)S PBMCs were incubated under anti-CD335 / ZZ conditions. After 18 hours, cell counting was performed to assess the expression of the co-stimulatory molecule CD69 on the surface of NK cells (A) and NKT cells (B).

[0134] Figure 15

[0135] [ Figure 15 The results showed an increased proportion of NKT cells activated by molecular complexes targeting two receptors expressed by NK and NKT cells, respectively. This was observed in the presence or absence of free forms of Ab, ZZ, or ZZ-Tat. 22-57C(22-37)S And molecular complex anti-CD56 / ZZ-Tat 22-57C22-37)S Anti-CD56 / ZZ, Anti-CD336 / ZZ-Tat 22-57C22-37)SPBMCs were incubated with anti-CD336 / ZZ agents. After 18 hours, cell counting was performed to assess the expression of the co-stimulatory molecule CD69 on the surface of NKT cells.

[0136] Figure 16

[0137] [ Figure 16 [This image shows a molecular complex targeting the receptor expressed on NK cells (anti-CD56 / ZZ-Tat)] 22-57C22-37)S Anti-CD335 / ZZ-Tat 22-57C22-37)S and anti-CD336 / ZZ-Tat 22-57C22-37)S) Induces human PBMCs to secrete IL-6. This occurs in the presence or absence of free forms of Ab, ZZ, or ZZ-Tat. 22-57C(22-37)S PBMCs were incubated with molecular complexes targeting molecules CD56(A), CD335(B), and CD336(C), respectively. After 24 hours, the supernatant was collected and the presence of IL-6 was assessed by enzyme immunoassay.

[0138] Figure 17

[0139] [ Figure 17 The image shows the presence of an anti-ICP antibody (anti-CTLA-4 / ZZ-Tat). 22-57C22-37)S Anti-PD-L1 / ZZ-Tat 22-57C22-37)S and Anti-OX40 / ZZ-Tat 22-57C22-37)S) The molecular complex induces human PBMCs to secrete IL-6. This occurs in the presence or absence of free forms of Ab, ZZ, or ZZ-Tat. 22-57C(22-37)S PBMCs were incubated with molecular complexes targeting molecules CTLA-4(A), PD-L1(B), and OX40(C), respectively. After 24 hours, the supernatant was collected and the presence of IL-6 was assessed by enzyme immunoassay. Detailed Implementation

[0140] Example 1 The expression, purification, biochemical characterization, and heparan sulfate binding capacity of the fusion protein in Escherichia coli or HEK cells were studied.

[0141] Materials and methods

[0142] Production of different molecular complexes

[0143] The fusion protein ZZ-DTRBD in Escherichia coli (referred to as ZZ-DTRBD) coli The expression of this protein was previously described in the publications of Lobeck et al. (Infection and Immunity, 1998, 66, 418-423). The fusion protein ZZ-DTRBDcoli (SEQ ID NO: 12) is encoded by a polynucleotide having the sequence SEQ ID NO: 11. The fusion protein ZZ-Tat 22-57C(22-37)S Expression in *E. coli* was achieved using a similar protocol to that described by Knittel et al. in their publication of the ZZOVATat 22-57S fusion protein (Vaccine, 2016, 34(27):3093-3101). The fusion protein ZZ-Tat... 22-57C(22-37)Scoli (SEQ ID NO: 20) is encoded by a polynucleotide having the sequence SEQ ID NO: 19. For expression in eukaryotic cells, it was expressed using a nucleotide encoding ZZ-DTRBD in the presence of PEI (0.5 mg / ml). HEK pCDNA3.4 plasmid (obtained by transfecting 400 μg of DNA preparation from maxiprep) was transfected into HEK cells (in 250 ml of 293F free-form medium at 2.5 x 10⁻⁶ ppm). 6 (cells / ml). The plasmid contains a polynucleotide with the sequence SEQ ID NO: 13, which encodes the fusion protein ZZ-DTRBD. HEK (SEQ ID NO: 14). The cells were then incubated at 37°C with stirring for 24 hours. Then, 250 ml of Ex-Cell medium was added. After incubation at 37°C with stirring for 4 days, the culture supernatant was collected, filtered under aseptic conditions, and a mixture of protease inhibitors was added.

[0144] After expression of the three proteins, the supernatants were diluted 1 / 2 in 0.1% PBS-Tween and then purified by passing the molecules through an IgG agarose gel column (IgG agarose gel 6 Fastflow #17-0969-02, Amersham) via immunoaffinity complex. The acidity of the fusion proteins eluted from the column was neutralized in 1M Tris-HCl buffer, pH 8. Derived from *E. coli*, ZZ-DTRBD coli and ZZ-Tat 22-57C(22-37)S The fusion protein was purified using a single S5 / 50 cation exchange column (GE Healthcare) for a second purification cycle. The column was equilibrated with 0.05 M phosphate-citrate buffer (pH 5.5) for the purification of ZZ-DTRBD. coli The column was equilibrated with 0.05M phosphate-citrate buffer at pH 4 for ZZ-Tat. 22-57C(22-37)S Purification of the fusion protein ZZ-DTRBD. coli and ZZ-Tat 22-57C(22-37)S The protein was then eluted with a linear gradient of 0 to 1 M NaCl. Finally, the protein was concentrated in PBS and stored at -20°C until use. Molecular complex BB-DTRBDHEK and BB-DTRBD coli It is based on the ZZ-DTRBD HEK and ZZ-DTRBD coli The same protocol was used to produce the fusion protein BB-DTRBD. HEK (SEQ ID NO: 18) is encoded by the polynucleotide sequence SEQ ID NO: 17. Fusion protein BB-DTRBD coli (SEQ ID NO: 16) is encoded by the polynucleotide sequence SEQ ID NO: 15.

[0145] Analysis of the molecular complex ZZ-DTRBD by gel electrophoresis HEK ZZ-DTRBDcoli, BB-DTRBD HEK and BB- DTRBD coli molecular weight and uniformity Protein ZZ-DTRBD under denaturing conditions HEK ZZ-DTRBD coli BB-DTRBD HEK and BB-DTRBD coli Molecular weight markers were deposited on an SDS-PAGE 4-12% gel and then subjected to electrophoretic migration. After migration, the presence of protein bands was revealed using Coomassie blue staining.

[0146] The binding of molecular complexes to heparin

[0147] To evaluate the binding of the molecular complex to heparan sulfate, heparin, a sulfated sugar representing the heparan sulfate family, was used.

[0148] Interactions were assessed using an enzyme immunoassay. For this purpose, a series of microtiter plates were pre-adsorbed with rabbit IgG (1 μg / 100 μl / well, 0.1 M phosphate buffer, pH 7.2) and then saturated with a buffer containing 0.3% bovine serum albumin (200 μl / well, 0.1 M phosphate buffer, pH 7.2). Another set of microtiter plates was saturated with a buffer containing 0.3% bovine serum albumin (300 μl / well, 0.1 M phosphate buffer, pH 7.2). Both series of plates were then washed, and the protein ZZ-DTRBD was serially diluted (in 0.1 M phosphate buffer, pH 7.4, containing 0.1% bovine serum albumin). HEK ZZ-DTRBD coli BB-DTRBD HEK and BB-DTRBD coli and ZZ-Tat 22-57C(22-37)SFree ZZ was deposited in the wells. After incubation at ambient temperature for 4 hours, the plate was washed and 100 μl of heparin-biotin (1 μM) was added to each well. After incubation at ambient temperature for 1 hour, the plate was washed and 100 μl of streptavidin conjugated with peroxidase (1 / 2000 dilution) was added. After incubation for 30 minutes, the plate was washed and the substrate (ABTS) was added. After incubation for 30 minutes, staining was measured at 414 nm. To eliminate non-specific binding with albumin, the light signal measured on the adsorbed plate with only bovine serum albumin was subtracted from the signal measured on the plate with adsorbed IgG. When ZZ-Tat 22-57C(22-37)S When incubated on a microtiter plate at 100 nM and pH 7.2, heparin binding was considered significant when the optical density signal was greater than or equal to 50% of the measured signal. Results

[0149] The inventors previously constructed a fusion protein called ZZ-DTRBD, which contains a double ZZ domain derived from Staphylococcus aureus protein A and a DTRBD domain derived from diphtheria toxin (Lobeck et al., Infection and Immunity, 1998, 66, 418-423). ZZ can bind to the Fc region of immunoglobulins in a similar manner to protein A. As for DTRBD, it binds to the diphtheria toxin receptor and also has a heparan sulfate binding site in the 453-467 region (Knittel et al. J. Immunol., 2015, 194(8):3601-11; Knittel et al. Vaccine, 2016, 34(27):3093-3101). These properties enable ZZ-DTRBD to target different cell types carrying surface immunoglobulins and heparan sulfate proteoglycans through interaction with heparan sulfate.

[0150] Similarly, the inventors constructed a fusion protein called BB-DTRBD by replacing the sequence encoding ZZ with a sequence called BB. The BB protein, corresponding to the dual-domain BB protein (like ZZ), is derived from Staphylococcus aureus protein A, but has specific characteristics of binding the Fc region and the immunoglobulin Fab region (Jansson 1998 FEMS Immunol. and Med. Microbiol., Léonetti et al. 1999, J. Exp. Med., 189, 1217-28).

[0151] The inventors also constructed a fusion protein called ZZ-Tat 22-57C(22-37)S It contains a double ZZ domain derived from Staphylococcus aureus protein A and a Tat domain derived from HIV transactivator of transcription. 22-57C(22-37)S(WO 2011 / 092675, and Knittel et al. Vaccine, 2016, 34(27): 3093-3101) possesses a heparan sulfate binding site. These properties enable ZZ-Tat 22-57C(22-37)S It can target different cell types carrying surface immunoglobulins and heparan sulfate proteoglycans through interaction with HS.

[0152] The inventors used different expression systems to recombine the expression of ZZ-DTRBD, BB-DTRBD, and ZZ-Tat. 22-57C(22-37)S。 The complex expressed in E. coli is called ZZ-DTRBD. coli BB-DTRBD coli and ZZ-Tat 22-57C(22-37)S The complex expressed in HEK cells is called ZZ-DTRBD. HEK and BB-DTRBD HEK After expression, the complex was purified using a column containing IgG-laden gel. Next, the protein ZZ-DTRBD was analyzed. coli BB-DTRBD coli and ZZ-Tat 22-57C(22-37)S Ion exchange chromatography was performed to remove contaminating LPS. Finally, some characteristics of these complexes were evaluated by gel electrophoresis. Figure 1 As can be seen from this, ZZ-DTRBD coli and BB-DTRBD coli The dominant band, with a molecular weight of approximately 35 kDa, close to the theoretical weight of the molecule (32230), indicates that this band corresponds to a monomer. A minor band, corresponding to the degradation product, is also present in the complex of these two molecules. ZZ-DTRBD HEK and BB-DTRBD HEK The migration of ZZ-DTRBD in several bands, primarily distributed between 35 and 150 kDa, indicates that expression in HEK cells results in a heterogeneous mixture of monomeric and oligomeric forms. The molecular complex ZZ-Tat 22-57C(22-37)S Migration within the main band reflects the homogeneity of the purified protein.

[0153] The molecular weight of approximately 25 kDa is slightly larger than the calculated theoretical molecular weight (MW = 19145). However, Tat and its derivatives tend to migrate aberrantly (Kittiworakarn et al., etc.), strongly suggesting that the fusion protein ZZ-Tat expressed and purified in E. coli is not suitable for this purpose. 22-57C(22-37)S It is basically composed of monomers.

[0154] Next, the inventors investigated the ability of free ZZ and five molecular complexes to bind heparin, a sulfated polysaccharide representing the heparan sulfate family. Enzyme immunoassay failed to detect a significant optical signal from free ZZ. Figure 2 Conversely, for ZZ-DTRBD coli BB-DTRBD HEK BB-DTRBD coli ZZ-DTRBD HEK and ZZ-Tat 22-57C(22-37) The increase in optical density based on the incubation dose was measured. Therefore, this data indicates that the DTRBD domain or Tat... 22-57C(22-37)S The presence of the structural domains enables the molecular complex to bind heparin. Furthermore, due to the fusion compound ZZ-DTRBD HEK and BB-DTRBD HEK Contains oligomers (see) Figure 1 This data indicates that the presence of oligomers does not disrupt the interactions.

[0155] Example 2 : Fusion protein ZZ-DTRBD HEK Able to bind to different types of cells in the mouse immune system

[0156] Materials and methods

[0157] ZZ-DTRBD's binding with immune system cells

[0158] Spleen cells from C57BI / 6 mice were 10 x 10 6 Cells / ml were resuspended in PBS buffer, 0.5% BSA, and 2mM EDTA. 100 μl of cell suspension was placed in 96-well round-bottom plates. The presence or absence of ZZ-DTRBD was considered. HEKAt 100 nM, add 100 μl of buffer and 0.5% BSA 2 mM EDTA to each well. Incubate the mixture at 4 °C for 30 min, then wash twice in PBS 0.5% BSA 2 mM EDTA. Add 2 μg / well of rabbit IgG to the cells, incubate at 4 °C for 20 min, then wash twice with qs 200 μl PBS 0.5% BSA 2 mM EDTA. Resuspend the cells in 50 μl of labeled buffer (PBS 0.5% BSA 2 mM EDTA) containing a mixture of different Abs from BioLegend. Incubate the mixture in the dark at 4 °C for 20 min, then wash the spleen cells twice with qs 200 μl PBS 0.5% BSA 2 mM EDTA. Then fix the cells at ambient temperature for 30 min. Therefore, first add 100 μl of 4% PFA buffer, then add 100 μl of PBS, 0.5% BSA, and 2 mM EDTA, and then apply the solution to the BD FACSAria. TM Detected on a cytometer.

[0159] Mixture 1 of dendritic cells (in duplicate): B220-FITC (#103206, 1 / 200), CD11c-PE-Cy7 (#117318, 1 / 200), CD317-APC (#127016, 1 / 100), CD8a-PerCP-Cy5.5 (#100734, 1 / 200), CD11b-APC-Cy7 (#101226, 1 / 800), Donkey anti-rabbit-BV421 (#406410, 1 / 100), Live Dead Aqua (ThermoFischer #L34966, 1 / 1000).

[0160] Mixture of monocytes and T / B lymphocytes 2 (two copies): B220-FITC (#103206, 1 / 200), CD19-BV650 (#115541, 1 / 100), CD3-APC-Cy7 (#100222, 1 / 100), CD4-BV605 (#100451, 1 / 200), NK1.1-PE-Cy7 (#108714, 1 / 100), CD8a-PerCP-Cy5.5 (#100734, 1 / 200), CD11b-APC (#101212, 1 / 800), Ly6C-PE (#128007, 1 / 800), Donkey anti-rabbit-BV421 (#406410, 1 / 100), Live Dead Aqua(ThermoFischer#L34966,1 / 1000).

[0161] result

[0162] To assess the ability of the molecular complex to bind to immune system cells, ZZ-DTRBD was incubated in the presence of mouse spleen cells. HEK Therefore, it can be observed that this fusion protein preferentially binds to cDC-CD8+ cells, which are specific APCs. Figure 3 A). It also preferentially interacts with monocytes, which can also be used as APCs (A). Figure 3 (B) Finally, it binds less to lymphocytes. Therefore, this data suggests that the fusion protein preferentially targets APCs.

[0163] Example 3 : Fusion protein ZZ-DTRBD HEK and ZZ-Tat 22-57C(22-37)S In vitro, immune system cells were induced to secrete IL-6 and IL-12.

[0164] Materials and methods

[0165] C57BL / 6 mouse spleen cells were used at a rate of 2 x 10 6 Cells / ml were resuspended in 10% FCS 1% penicillin / streptomycin RPMI medium. The presence or absence of the molecular complex (ZZ, DTRBD, ZZ-DTRBD) was considered. HEK Or ZZ-Tat 22-57C(22-37)S In this case, 100 μl of cell suspension was distributed into 96-well plates and incubated with a final concentration of 1 μM. After 24 hours of incubation, the supernatant was collected for ELISA assay of cytokines IL-6 and IL-12 according to the manufacturer’s instructions (R&D#DY406-05 and #DY419).

[0166] result

[0167] The inventors wanted to know if the molecular complex could induce the activation of immune system cells. Since cell activation leads to cytokine secretion, they decided to evaluate the presence of two cytokines in the supernatant produced by incubating mouse spleen cells with the molecular complex and different control proteins in vitro. The first cytokine was IL-6, as it represents an inflammatory cytokine important for initiating an immune response. The second cytokine was IL-12, as it is crucial for inducing cellular immune responses. The inventors found that these two cytokines, when used with ZZ-DTRBD... HEK The increased concentration in the supernatant produced during incubation indicates that this molecular complex induced the activation of spleen cells. Figure 4 Conversely, they did not find an increase in the number of cytokines in the supernatant resulting from incubation with free ZZ or free DTRBD, suggesting that the dual domains for Ig binding and the heparan sulfate-binding domain alone cannot activate immune cells. Therefore, these results demonstrate that ZZ-DTRBD... HEK It can induce the activation of immune system cells, and indicates that the binding of the dual Ig binding domain and the DTRBD HS binding domain is absolutely essential for this effect.

[0168] The inventors conducted an evaluation of ZZ-Tat based on principles similar to those described in the previous paragraph. 22-57C(22-37)S The ability to activate immune system cells and participate in the binding of the ZZ and Tat domains of heparan sulfate are absolutely essential for the stimulatory effect. For this purpose, they specifically used a compound called Tat. CY49-57 The peptide, containing the basic Tat region, represents the interaction between Tat and its derivatives and heparan sulfate. They used ZZ and Tat, respectively. CY49-57 ZZ+Tat CY49-57 and ZZ-Tat 22-57C(22-37)S Mouse spleen cells were incubated. Then, they assessed the presence of IL-6 and IL-12 in the supernatant. They compared these findings with those of ZZ-Tat. 22-57C(22-37)S These two cytokines were found in the supernatant produced during incubation, but not in the presence of ZZ and Tat. CY49-57 ZZ+Tat CY49-57 These two cytokines were not found in the supernatant produced during incubation. Figure 5 Therefore, these results indicate that ZZ-Tat 22-57C(22-37)S It can induce the activation of immune system cells, and the free forms of ZZ and Tat CY49-57 The absence of this feature indicates that the association between the dual Ig binding domain and the basic Tat region responsible for HS binding is absolutely necessary for this effect.

[0169] Example 4The molecular complex i) can still trigger the secretion of IL-6 and IL-12 by immune system cells when its ZZ domain is replaced by BB, ii) has enhanced stimulatory ability when it is composed of oligomeric forms.

[0170] Materials and methods

[0171] C57BL / 6 mouse spleen cells were used at a rate of 2 x 10 6 Cells / ml were resuspended in 10% FCS 1% penicillin / streptomycin RPMI medium. Different molecular complexes (ZZ-DTRBD) were tested in the presence or absence of these complexes. HEK BB-DTRBD HEK In the case of ZZ-DTRBDcoli and BB-DTRBDcoli, 100 μl of cell suspension was distributed in 96-well plates and incubated at 1 μM. After 24 hours of incubation, the supernatant was collected for ELISA assay of cytokines IL-6 and IL-12, according to the manufacturer's instructions (R&D#DY406-05 and #DY419).

[0172] result

[0173] The work described in Example 3 was performed using two molecular complexes containing ZZ double domains. This double domain, which binds to the Fc region of Ab, can therefore target Ab located on the surface of APCs. The inventors then wanted to know whether molecular complexes that could co-target the Fc and Fab regions of Ig could also activate immune system cells. To this end, they focused on the BB double domain derived from Staphylococcus aureus protein A, which has an Fc region capable of binding Ab in the same manner as ZZ, and unlike ZZ, it also has a Fab region of Ab (Jansson B. et al., Fems Immunol. Med. Microbiol. 1998, 20:69-78). They prepared the complex BB-DTRBD described in Example 1. HEK and BB-DTRBD coli and with ZZ-DTRBD HEK and ZZ-DTRBD coli A comparison was made to show how cytokine secretion was induced in vitro when cytokines IL-6 and IL-12 were incubated with mouse spleen cells.

[0174] The inventors noted that ZZ-DTRBD and BB-DTRBD were expressed in oligomeric forms in HEK cells (see [link]). Figure 1 ), and when expressed in E. coli, they are in monomeric form. This work can also assess whether the oligomeric state affects the ability to induce the immune system.

[0175] The inventors observed the use of ZZ-DTRBD HEK The supernatant produced during incubation contained more BB-DTRBD than when used. HEK The supernatant produced during incubation contains a larger amount of cytokines. Figure 6 Similarly, using ZZ-DTRBD coli The supernatant produced during incubation was compared with that produced by BB-DTRBD. coli The supernatant produced during incubation contained more cytokines. These results indicate that the molecular complex containing BB can still induce cytokine secretion in vitro, but to a lesser extent than the molecular complex containing ZZ. Therefore, these data suggest that the activation effect can be mediated by molecular complexes containing domains capable of binding to different sites on the Ab.

[0176] Comparing the levels of cytokines in the supernatant based on the cell type used to produce the molecular complex (i.e., HEK vs. E. coli) allows for the demonstration of differences in stimulatory efficacy depending on the production type. In fact, using a complex derived from HEK cells (ZZ-DTRBD) HEK BB-DTRBD HEK The supernatant produced by incubation showed higher levels of IL-6 and IL-12 than the complex (ZZ-DTRBD) expressed in E. coli. coli and BB-DTRBD coli The supernatant produced during incubation ( Figure 6 Since the proteins expressed in HEK cells are mainly oligomers (see...), Figure 1 The inventors deduced that when molecular complexes are highly oligomerized, the immune system is induced more effectively.

[0177] Example 5 : Isolated dendritic cells in ZZ-DTRBD HEK It secretes IL-6 and IL-12 when incubated in the presence of these substances.

[0178] Materials and methods

[0179] C57BL / 6J mice were euthanized, and their spleens were recovered in RPMI medium (10% FCS, 1% penicillin / streptomycin). The spleens were perfused with 3 ml of collagenase D (2 mg / ml) in HBSS and 0.5% BSA, and then incubated at 37°C for 30 min. Spleen cells were recovered, and dendritic cells (DCs) were magnetically sorted according to the manufacturer's instructions (Miltenyi Biotec #130-100-875). The DCs were centrifuged at 390 x g for 5 min at 4°C, and then centrifuged at 2 x 10⁻⁶ cm⁻¹. 6Cells / ml were resuspended in RPMI medium, 10% FCS, and 1% penicillin / streptomycin. 100 μl of cell suspension (200,000 cells) was placed in a 96-well plate and incubated on a ZZ-DTRBD plate. HEK Add 100 μl of RPMI medium containing 10% FCS and 1% penicillin / streptomycin (final 0.6 μM) if the presence or absence of these media is present. Incubate the cells at 37°C for 24 hours, then collect the supernatant according to the manufacturer's instructions (R&D #DY406-05 and #DY419) for ELISA assay of cytokines IL-6 and IL-12.

[0180] result

[0181] The establishment of immune defense mechanisms depends on the cooperation between different cell companions. Among them, dendritic cells (DCs) represent APCs that play a central role. This is because they help activate other cell types through direct interactions or through the cytokines they secrete. To assess whether ZZ-DTRBD induces cytokine secretion by DCs, DCs were purified from mouse spleen cells C57BI / 6. These DCs were then incubated for 24 hours in the presence or absence of ZZ-DTRBD, and the supernatant was collected to determine the presence of IL-6 and IL-12. Figure 7 ). Using ZZ-DTRBD HEK The amount of IL-6 present in the supernatant produced by incubation was approximately 5 times higher than the amount of IL-6 found in the supernatant produced by incubation without the molecular complex. (This was observed using ZZ-DTRBD.) HEK A significant amount of IL-12 (approximately 8700 pg / ml) was also detected in the supernatant produced during incubation, but almost none was detected in the supernatant produced in the absence of the molecular complex. Therefore, these data suggest that ZZ-DTRBD induces the secretion of IL-6 and IL-12 by mouse dendritic cells, which may contribute to the establishment of immune defense mechanisms.

[0182] Example 6 Proteins ZZ-DTRBD and ZZ-Tat 22-57C(22-37)S It can induce dendritic cells in mice.

[0183] Materials and methods

[0184] Whether ZZ-DTRBD exists or not HEK (5 nmol per mouse) or ZZ-Tat 22-57C(22-37)SAt a dose of 10 nmol per mouse, eight C57BL / 6 mice in three groups were injected three times, three days apart, with each injection consisting of 100 μl of PBS. Twenty-four hours after the last injection, the animals were euthanized, and the spleens were collected to recover splenocytes. The cells were resuspended in 50 μl of labeling buffer (PBS + 0.5% BSA + 2 mM EDTA) containing a mixture of different Abs. The cells were then incubated in the dark at 4°C for 20 min, followed by two washes in PBS + 0.5% BSA + 2 mM EDTA. The cells were then fixed in 100 μl of 4% PFA buffer at 4°C for 20 min, followed by washes in PBS + 0.5% BSA + 2 mM EDTA. Finally, they were resuspended in 200 μl of PBS + 0.5% BSA + 2 mM EDTA and then subjected to BDFACSAria. TM Flow cytometry was used for analysis. Cells were analyzed by flow cytometry. cDC-CD8+ was identified as CD11c. high B220 - CD8 + CD11b - cDC-CD11b + Identified as CD11c high B220 - CD8 - CD11b + Furthermore, pDC was identified as CD11c. int CD317 + CD11b + .

[0185] The following Ab mixtures were used for dendritic cell phenotypic analysis: B220-FITC (#103206, 1 / 200), CD11c-PE-Cy7 (#117318, 1 / 200), CD317-APC (#127016, 1 / 100), CD11b-APC-Cy7 (#101226, 1 / 800), CD8a-PerCP-Cy5.5 (#100734, 1 / 200), and Live Dead Violet (ThermoFischer #L34964, 1 / 1000).

[0186] result

[0187] Because the molecular complex preferentially binds to APCs and induces cytokine secretion in vitro, the inventors wanted to know whether the complex would induce the amplification of APCs in mice. To evaluate this aspect, the inventors tested the complex in the presence or absence of ZZ-DTRBD. HEK Or ZZ-Tat 22-57C(22-37)SIn this case, three groups of eight C57BI / 6 mice were injected with PBS solution. These animals were then euthanized, and their spleens were sampled to assess the frequency of different dendritic cell subsets in the spleen cells. Figure 8 As shown, three mouse DC populations (i.e., DC-CD8+, DC-CD11b+, and pDC) were present in the spleen of mice injected with PBS only. However, the frequencies of these three cell types decreased after injection of ZZ-DTRBD. HEK Or ZZ-Tat 22-57C(22-37)S The number of dendritic cells increased significantly in the animal group. Therefore, these data indicate that the molecular complex can induce dendritic cell proliferation in vivo. Since these cells are central to initiating the immune response, these results strongly suggest that the complex can therefore promote the induction of immune response mechanisms.

[0188] Example 7 ZZ-DTRBD HEK In vitro induction of human dendritic cells to secrete IL-6 and IL-12

[0189] Materials and methods

[0190] Isolation of human dendritic cells and incubation for studying IL-6 and IL-12 secretion

[0191] The leukocyte-platelet layer bags were diluted to 1 / 2 in AIMV medium and incubated overnight at ambient temperature with stirring. Then, 15 ml of Histopaque medium was added to four leucosep tubes, along with 4 x 25 ml of diluted blood. The tubes were centrifuged at 1000 x g for 15 minutes at ambient temperature. Loops of peripheral blood mononuclear cells (PBMCs) were recovered and washed in calcium- or magnesium-free PBS with 2 mM EDTA. PBMCs were centrifuged at 150 x g for 10 minutes at ambient temperature, then erythrocyte lysis buffer (8.3 mg / ml NH4Cl, 0.84 mg / ml NaHCO3, 0.1 mM EDTA) was added and incubated at 4°C for 10 minutes. 40 ml of PBS was added, and the cells were centrifuged at 150 x g for 10 minutes at ambient temperature. The DCs were then classified according to the manufacturer's instructions (Miltenyi Biotec #130-091-379). Centrifuge the DCs at 390 x g for 5 minutes at 4 °C, then centrifuge at 2 x 10⁻⁶ g. 6 Cells / ml were resuspended in RPMI medium 10% FCS 1% penicillin / streptomycin. 100 μl of sorted DCs (200,000 cells) were placed in 96-well plates, with ZZ-DTRBD present or absent. HEKAdd 100 μl of RPMI medium containing 10% FCS and 1% penicillin / streptomycin at a final concentration of 1 μM. Incubate the cells at 37°C for 24 hours, and then collect the supernatant according to the manufacturer's instructions (R&D#DY206-05 and #DY1270-05) for ELISA assay of cytokines IL-6 and IL-12.

[0192] result

[0193] To evaluate ZZ-DTRBD HEK The presence of IL-6 and IL-12 in the culture supernatant of these cells was assessed after 24 hours of incubation to determine whether DCs derived from healthy human donors could be induced to secrete them in vitro. Figure 9 As shown, in the presence of ZZ-DTRBD HEK The amount of IL-6 in the supernatant produced by incubation under the fusion protein condition was found to be approximately 10 times higher than that in the supernatant produced by incubation in the absence of the fusion protein. IL-12 was found in ZZ-DTRBD HEK The supernatant produced by incubating DCs in the presence of the molecular complex was abundant (2400 pg / ml), while the amount produced by incubation in the absence of the molecular complex was very low (8 pg / ml). Therefore, all these data indicate that ZZ-DTRBD induces increased secretion of IL-6 and IL-12 from human DCs. Thus, ZZ-DTRBD may contribute to the establishment of immune defense mechanisms related to the secretion of these cytokines in humans.

[0194] Example 8 ZZ-DTRBD and ZZ-Tat 22-57C(22-37)S Slow down the growth process of colorectal tumors.

[0195] Materials and methods

[0196] Study on the effect of adjuvant mixture CpG / Polyl:C on tumor growth of mouse colorectal cancer cell lines

[0197] Eight C57BL / 6 mice in two groups were subcutaneously injected into their paws with 0.5 M MC38 cells. Three and six days later, the mice were either not injected (control) or injected with an adjuvant mixture of CpG-B1018 / PolyI:C (30 μg of each adjuvant). Tumor growth was monitored by measuring the tumor size using a caliper. The mice were euthanized after reaching the cessation criteria.

[0198] The study investigated the injection of the molecular complex ZZ-DTRBD with or without adjuvant. HEK colorectal cancer in mice Effects on cell line tumor growth Six C57BL / 6 mice in three groups were subcutaneously injected into their paws with 0.5 M MC38 cells. Three, six, and nine days later, in the absence or presence of CpG-B1018+PolyI:C, the mice were either not injected (control) or injected with ZZ-DTRBD. HEK(2 nmol per mouse). (30 μg of each adjuvant per mouse). Mice were monitored individually for 15 days prior to euthanasia (tumors were measured using a caliper).

[0199] Comparison of anti-PD-1Ab and molecular complex ZZ-DTRBD HEK and ZZ-Tat 22-57C(22-37)S Mouse colorectal cancer cells The influence of tumor growth Four groups of eight C57BL / 6 mice were subcutaneously injected with 0.5M MC38 cells into their paws. Three, six, and nine days later, in the presence of CpG-B1018+PolyI:C (30 μg of each adjuvant per mouse), the mice were either not injected (control) or injected with anti-PD-1 antibody (Euromedex#BE0146-100MG, clone RMP1-14) and ZZ-DTRBD. HEK Or ZZ-Tat 22-57C(22-37)S (0.96 nmol per mouse). Mice were monitored individually over time (tumors were measured using a caliper). They were euthanized after reaching the stopping criteria.

[0200] result

[0201] To assess whether the molecular complex could influence tumor growth, the study was conducted in a homologous mouse cancer model. This model was based on a colon tumor cell line called MC38 and C57BI / 6 mice. Cancer induction was achieved by injecting 500,000 MC38 cells into each mouse.

[0202] First, the effect of the adjuvant mixture CpG1018 / polyl:C on tumor growth was evaluated. Figure 10 As shown in Figure A, tumor growth was almost unaffected in the mice group injected with the CpG1018 / polyI:C mixture compared to the untreated (control) group. Therefore, this data indicates that the adjuvant mixture alone cannot slow the growth of this colorectal tumor.

[0203] Secondly, the efficacy of ZZ-DTRBD was evaluated by injection alone or in the presence of the adjuvant mixture CpG1018 / polyI:C. HEK Its impact on tumor growth. For example... Figure 10 As shown in Figure B, compared with the untreated (control) group, the adjuvant-free ZZ-DTRBD injected on days 8 and 10... HEK Tumor growth slowed in the group treated with ZZ-DTRBD. HEK The effect was even greater in the group treated with the CpG1018 / polyl:C adjuvant mixture. In fact, in this group, tumor growth was slowed throughout the entire observation period (day 6 to day 15) in the animals. Therefore, all these data indicate that ZZ-DTRBD HEKIt has an effect on the growth of the MC38 cell line, and this effect is amplified when it is mixed with the adjuvant CpG1018 / polyl:C.

[0204] In another series of experiments, the effects of the following treatment on MC38 tumor growth in C57BI / 6 mice were compared: ZZ-DTRBD HEK / CpG1018 / polyl:C、ZZ-Tat 22-57C(22-37)S / CpG1018 / polyl:C, anti-PD-1 antibody. For example... Figure 11 As shown in A and 11B, anti-PD-1Ab treatment had no significant effect on tumor growth compared to the control group. Conversely, injections of ZZ-DTRBD... HEK / CPG1018 / polyl:C and ZZ-Tat 22-57C(22-37)S Mice with / CpG1018 / polyl:C exhibited reduced tumor growth. This slowed tumor growth led to increased survival in both groups of animals. Figure 11 C).

[0205] Example 9 CD4+ activated monocytes and lymphocytes can target surface molecules DEC205 and Fc, respectively. γ Induced by the molecular complex of the receptor.

[0206] Materials and methods

[0207] To prepare the molecular complex targeting DEC205, an anti-DEC205 antibody (BioLegend; clone NLDC-145, reference number BLE138202) was used. This was done in the absence or presence of ZZ-Tat. 22-57C22-37)S In this case, incubation was performed for 24 hours in RPMI medium without FCS at a fixed concentration (0.2 μM per molecule). The ability of the ZZ region to bind to the Fc region of the anti-DEC205Ab allows for the formation of a non-covalent molecular complex, termed anti-DEC205 / ZZ-Tat. 22-57C22-37)S Using the same method, a complex is formed between the ZZ molecule and the anti-DEC205Ab, termed anti-DEC205 / ZZ.

[0208] To prepare targeted FC γ The receptor molecular complex was synthesized using a nonspecific human polyclonal antibody. This antibody, along with ZZ-Tat, was used. 22-57C22-37)S Alternatively, ZZ can be incubated together, following the same protocol used for anti-DEC205 Ab. This allows the formation of a non-covalent molecular complex, termed IgG / ZZ-Tat. 22-57C22-37)S And IgG / ZZ complex.

[0209] Then, the human PBMCs prepared as described in Example 7 were incubated in the absence or presence of the following compound: anti-DEC205 / ZZ-Tat 22-57C22-37)S IgG / ZZ-Tat 22-57C22-37)S , anti-DEC205 / ZZ, IgG / ZZZZ-DTRBD HEK In addition to anti-DEC205 Ab and free IgG (final concentration of each compound was 0.1 μM). In these experiments, PBMCs were also reacted with ZZ-Tat at a concentration of 1 μM. 22-57C22-37)S Cells were incubated with free ZZ protein. After 24 hours, cells were collected and labeled with fluorescent antibodies to identify monocytes (CD14, anti-CD14-BV605, Biolegend), CD4+ T lymphocytes (CD4, anti-CD4-PerCP-Cy5.5, Biolegend), and CD69 molecules (anti-CD69-BV785, Biolegend). After 30 minutes of incubation, PBMCs were fixed with a solution containing 4% paraformaldehyde and then analyzed by flow cytometry.

[0210] result

[0211] The preceding examples demonstrate that molecular complexes targeting Abs can effectively induce certain immune response mechanisms and effectively slow tumor growth. Abs located on the surface of APCs are the molecules targeted by these complexes. However, APCs express a large number of other molecules that can also represent targets of the molecular complexes according to the invention. Therefore, the inventors wanted to know whether molecular complexes targeting HSs and receptors, rather than immunoglobulins located on the surface of APCs, could also activate these cells. To evaluate this aspect, they chose to evaluate two types of receptors. The first is the protein DEC205, an agglutinin selectively expressed in humans by monocytes and certain dendritic cell populations (Kato M. et al. 2006, Int. Immunol., 18:857-869). The second is Fc. γ Receptors, in most forms, are expressed by monocytes and certain dendritic cell populations.

[0212] The inventors then prepared two molecular complexes that could target these receptors separately. To target the DEC205 protein, they used a monoclonal antibody specific to that receptor and combined it with the fusion protein ZZ-Tat. 22-57C22-37)S They named this molecular complex anti-DEC205 / ZZ-Tat. 22-57C22-37)S To target Fc receptors, they used nonspecific human polyclonal IgG Ab, which interacts with these receptors through its Fc domain. They were used in this IgG and ZZ-Tat... 22-57C22-37)SA molecular complex is formed between them, called IgG / ZZ-Tat 22-57C22-37)S Therefore, these two molecular complexes described above possess the property of being transmitted via ZZ-Tat. 22-57C22-37)S The Tat (22-57C22-37)S domain binds to the APC receptor and heparan sulfate. In these molecular complexes, the two proteins are non-covalently bound via their respective Fc and ZZ domains. Therefore, ZZ can no longer target the immunoglobulin located on the surface of the APC because it has already interacted with Ab. The inventors also prepared two complexes, named anti-DEC205 / ZZ and IgG / ZZ, respectively, which do not contain Tat. 22-57C22-37)S The region is used to bind heparan sulfate as a control in subsequent activation experiments.

[0213] Since the DEC205 molecule and Fc receptor are expressed by monocytes, the inventors then wanted to know whether the molecular complex could activate this subset of APCs. To this end, they tested the anti-DEC205 / ZZ-Tat at a fixed concentration (0.1 μM) in the absence or presence of the anti-DEC205 / ZZ-Tat. 22-57C22-37)S lgG / ZZ-Tat 22-57C22-37)S PBMCs were incubated with anti-DEC205 / ZZ, IgG / ZZ, and anti-DEC205 / IgG. They were also incubated with 1 μM ZZ-Tat. 22-57C22-37)S and free ZZ protein to evaluate ZZ-Tat 22-57C22-37)S The effects were performed at the same concentrations found to activate cells as in Examples 3, 4, and 5. After 24 hours, the proportions of activated monocytes and CD4+ T lymphocytes were assessed. From... Figure 12 As can be seen from A, ZZ does not change the proportion of activated monocytes in PBMCs, while ZZ-Tat 22-57C22-37)S The significant increase indicates that this molecular complex can activate human APCs. Anti-DEC205 antibody (free or contained in the anti-DEC205 / ZZ complex) reduced the proportion of activated cells. Conversely, in the anti-DEC205 / ZZ-Tat 22-57C22-37)S In the presence of the complex, the proportion of activated monocytes increases. Similar behavior was observed when CD4+ T lymphocytes were considered. Figure 12 (B). Therefore, this data suggests that Abs that target APCs but do not have the ability to activate them may become able to induce them when contained in molecular complexes, which also makes it possible to target HS.

[0214] Analysis of monocyte activation status after incubation with free human IgG showed that this ab increased the proportion of activated monocytes. IgG / ZZ did not significantly increase the proportion of activated cells. In contrast, when PBMCs were incubated with the molecular complex IgG / ZZ-Tat... 22-57C22-37)SWhen incubated together, the number of activated monocytes more than doubled. Similar behavior was observed when CD4+ T lymphocytes were considered. Figure 12 B). Therefore, this data indicates that targeting the Fc surface of APC... γ The receptor's Ab can activate them, but the activation effect is increased when the Ab is contained in a molecular complex, thus also targeting HS.

[0215] Interestingly, these results showed a combined increase in the proportion of activated monocytes and T lymphocytes, indicating that the molecular complex induced several cytokines that play a central role in immune defense mechanisms.

[0216] Example 10: A molecular complex targeting dendritic cells induces immune system cells to secrete IL-6 in vitro.

[0217] Materials and methods

[0218] To prepare the molecular complex targeting dendritic cells (DCs), three antibodies specific to the molecules expressed by DCs were used. The first antibody (BD reference number 555538) is called the anti-CD74 antibody, which targets the CD74 molecule. The second antibody (BD reference number 551186) is called the anti-CD209 antibody, which targets the CD209 molecule. The third antibody (BD reference number 552501) is called the anti-CD275 antibody, which targets the CD275 molecule.

[0219] Whether ZZ-Tat exists or not 22-57C22-37)S In the case of ZZ (5 nM per molecule), each of the three Abs (30 nM per molecule) was incubated in RPMI medium with 5% human AB serum at 4°C for 24 hours. The ability of the ZZ region to bind to the Fc region of these Abs allows for the formation of different non-covalent molecular complexes, termed anti-CD74 / ZZ-Tat. 22-57C22-37)S Anti-CD209 / ZZ-Tat 22-57C22-37)S Anti-CD275 / ZZ-Tat 22-57C22-37)S Anti-CD74 / ZZ, anti-CD209 / ZZ and anti-CD275 / ZZ.

[0220] Human PBMCs in 5x10 6 Cells / ml were resuspended in RPMI medium with 5% human AB serum. The presence or absence of free Ab, ZZ, or ZZ-Tat was considered. 22-57C(22-37)S In the case of molecular complexes, 100 μl of cell suspension was distributed into 96-well plates. After incubation for 24 hours, the supernatant was collected for ELISA assay of cytokine IL-6 according to the manufacturer's instructions (R&D#DY406-05).

[0221] result

[0222] The inventors wanted to know whether a molecular complex targeting three molecules expressed by dendritic cells (DCs) could induce activation of immune system cells. Since cell activation can lead to cytokine secretion, the inventors decided to evaluate the presence of IL-6 in vitro. They used ZZ-Tat 22-57C22-37)S This cytokine was found in the supernatant produced by incubation, but not in the supernatant produced by incubation with ZZ. No antibody was found in the supernatant produced by incubation of PBMCs with free anti-CD74Ab. Figure 13 A) indicates that this Ab cannot induce activation in its free form. The inventors observed that when PBMCs were incubated with the anti-CD74 / ZZ complex, IL-6 secretion increased, indicating that the ZZ dual domain can achieve activation mediated by this Ab. Figure 13 A). However, using anti-CD74 / ZZ-Tat 22-57C22-37)S At the same time, the secretion of this cytokine is even stronger, indicating that the addition of the Tat region allows for the binding of HSPGs, thereby increasing cell activation mediated by this complex.

[0223] The inventors observed that free anti-CD209 and anti-CD275Abs could induce IL-6 secretion when incubated with PBMCs. Figure 13 B and 13C). This cytokine secretion was also observed when these two antibodies were conjugated with ZZ (anti-CD209 / ZZ and anti-CD275 / ZZ), respectively. However, when these two antibodies were conjugated with ZZ-Tat... 22-57C22-37)S This secretion increases upon recombination, suggesting that activation capacity increases when the molecularly targeted complex can bind to HSPGs.

[0224] Example 11 The molecular complex targeting the CD335 receptor expressed by NK and NKT cells increased the proportion of NK and NKT cells activated in vitro.

[0225] Materials and methods

[0226] To prepare a molecular complex targeting CD335 molecules on the surface of NK and NKT cells, an Ab (Origene AM31284AF-N) called anti-CD335Ab was used. This was done in the absence or presence of ZZ-Tat. 22-57C22-37)S In the case of ZZ (5 nM per molecule), the Ab was incubated at 30 nM for 5 hours at 37°C in RPMI medium and 5% human AB serum. The ability of the ZZ region to bind to the Fc region of these Abs allows for the formation of two molecular complexes, named anti-CD335 / ZZ-Tat. 22-57C22-37)S And anti-CD335 / ZZ.

[0227] Human PBMC at 5x10 6 Cells / ml were resuspended in RPMI medium with 5% human AB serum. The presence or absence of anti-CD335Ab, ZZ, or ZZ-Tat was considered. 22-57C(22-37)S In the presence of molecular complexes, 100 μl of cell suspension was distributed in 96-well plates. After 18 hours, cells were collected and labeled with fluorescent Abs to enable the identification of NK cells (CD56+, BioLegend reference number 362510, diluted 1 / 100), NKT cells (CD56+, BioLegend reference number 362510; CD3+ cells; Miltenyi reference number 130-113-136, diluted 1 / 200), and CD69 molecules (BioLegend reference number 310932, diluted 1 / 100). After 30 minutes of incubation, PBMCs were fixed with a solution containing 4% paraformaldehyde and then analyzed by flow cytometry.

[0228] result

[0229] The inventors wanted to know whether a molecular complex targeting CD335 molecules expressed on the surface of NK and NKT cells could induce activation of these two cell types. To evaluate this, they used anti-CD335 antibodies in isolated form or contained within the molecular complex. They incubated PBMCs with and without different mixtures. Since NK and NKT cells play a crucial role in immune defense mechanisms, they assessed the proportion of activated NK and NKT cells after 18 hours of incubation by monitoring the expression of the co-stimulatory molecule CD69.

[0230] like Figure 14 As observed, ZZ, isolated anti-CD335Ab, and the anti-CD335 / ZZ complex did not increase the proportion of CD69-expressing NK(A) or NKT(B) cells in PBMCs. Using free ZZ-Tat... 22-57C22-37)S Different behaviors were observed. In fact, this molecular complex did not increase the proportion of NKT cells expressing CD69, but it did increase the percentage of NK cells expressing this tag, indicating that ZZ-Tat... 22-57C(22-37)S The binding complex of ZZ and HS ligands can increase NK cell activation. This molecular complex is anti-CD335 / ZZ-Tat. 22-57C(22-37)S It has an effect on the activation of two cell subsets. In fact, it increases the proportion of NK and NKT cells expressing CD69. Therefore, all these data suggest that anti-CD335Ab does not have the ability to activate these two cell types, but when it is included in a molecular complex, it can become capable of inducing them, which also makes it possible to target HS.

[0231] Example 12The molecular complexes targeting CD56(A) and CD336(B) molecules expressed by NK and NKT cells, respectively, increased the proportion of NKT cells induced to activate in vitro.

[0232] Materials and methods

[0233] To prepare molecular complexes targeting CD56 and CD336 molecules on the surface of NK and NKT cells, Ab anti-CD56 (Biolegend reference number 304622) targeting CD56 and Ab anti-CD336 (Origene AM50346PU-N) targeting CD336 were used. In ZZ-Tat 22-57C22-37)S With or without ZZ (5 nM per molecule), these Abs were incubated at 30 nM in RPMI medium with 5% human AB serum at 37°C for 5 hours. The ability of the ZZ region to bind to the Fc region of these Abs allows for the formation of four molecular complexes, termed anti-CD56 / ZZ-Tat. 22-57C22-37)S Anti-CD56 / ZZ, Anti-CD336 / ZZ-Tat 22-57C22-37)S And anti-CD336 / ZZ.

[0234] Human PBMC at 5x10 6 Cells / ml were resuspended in RPMI medium with 5% human AB serum. The isolates were prepared in the presence or absence of anti-CD56Ab, anti-CD336Ab, anti-CD336Ab, ZZ, or ZZ-Tat. 22-57C(22-37)S In the case of non-covalent molecular complexes, 100 μl of cell suspension was dispensed into 96-well plates. After 18 hours, cells were collected and labeled with the fluorescent Ab described in Example 11, which enabled the identification of NKT cells (CD56+CD3+) and CD69 molecules. After 30 minutes of incubation, PBMCs were fixed with a solution containing 4% paraformaldehyde and then analyzed by flow cytometry.

[0235] result

[0236] The inventors wanted to know whether a molecular complex of the targeted expression molecules CD56 and CD336 could induce NKT cell activation. To evaluate this, they used anti-CD56 and anti-CD336 antibodies in isolated forms or contained within the molecular complex. They incubated PBMCs with and without different mixtures. After 18 hours of incubation, they assessed the proportion of activated NKTs by monitoring the expression of the co-stimulatory molecule CD69.

[0237] like Figure 15 As shown, ZZ, ZZ-Tat 22-57C22-37)SAnti-CD56 antibody, isolated anti-CD336 antibody, and anti-CD336 / ZZ complex did not increase the proportion of CD69-expressing NKT cells in PBMCs. The molecular complex anti-CD56 / ZZ-Tat... 22-57C22-37)S ( Figure 15 A) Anti-CD336 / ZZ-Tat 22-57C22-37)S ( Figure 15 B) Different behaviors were observed. In fact, an increase in the proportion of CD69-expressing NKT cells was observed when these two molecular complexes were incubated with PBMCs. Therefore, all these data suggest that isolated anti-CD56Ab and anti-CD336Ab do not have the ability to activate NKT cells, but when they are contained in a molecular complex that also targets HS, they can become capable of inducing them.

[0238] Example 13 A molecular complex targeting NK and NKT cells induces immune system cells to secrete IL-6 in vitro.

[0239] Materials and methods

[0240] To prepare molecular complexes targeting NK and NKT cells, three antibodies described in Examples 10 and 11 were used. These antibodies were used in the absence or presence of ZZ-Tat. 22-57C22-37)S In the case of ZZ (5 nM per molecule), each of the three Abs (30 nM per molecule) was incubated in 5% human AB serum in RPMI medium for 24 hours. The ability of the ZZ region to bind to the Fc region of these Abs allows for the formation of different non-covalent molecular complexes, respectively named anti-CD56 / ZZ-Tat. 22-57C22-37)S Anti-CD335 / ZZ-Tat 22-57C22-37)S Anti-CD336 / ZZ-Tat 22-57C22-37)S Anti-CD56 / ZZ, anti-CD335 / ZZ and anti-CD336 / ZZ.

[0241] Human PBMCs in 5x10 6 Cells / ml were resuspended in RPMI medium with 5% human AB serum. The presence or absence of free Ab, ZZ, or ZZ-Tat was considered. 22-57C(22-37)S In the case of molecular complexes, 100 μl of cell suspension was distributed into 96-well plates. After incubation for 24 hours, the supernatant was collected for ELISA assay of cytokine IL-6 according to the manufacturer's instructions (R&D#DY406-05).

[0242] result

[0243] The inventors wanted to know if a molecular complex targeting NK and NKT cells could induce activation of immune system cells. Since cell activation leads to cytokine secretion, the inventors decided to evaluate the presence of IL-6 in vitro. They used ZZ-Tat 22-57C22-37)S This cytokine was found in the supernatant produced by incubation, but not in the supernatant produced by incubation with ZZ. They did not find any IL-6 in the supernatant produced by incubation of PBMCs with free anti-CD56 and anti-CD335 Ab. Figure 16 The results (A and 16B) indicate that these two proteins cannot be induced for activation when in their free form. Instead, they detected cytokines in the supernatant produced by incubation with anti-CD336 antibody. Figure 16 C) indicates that the Ab (in its free form) has stimulatory activity. The inventors observed that, for the three antibodies, when PBMCs reacted with anti-CD56 / ZZ-Tat... 22-57C22-37)S ( Figure 16 A) Anti-CD335 / ZZ-Tat 22-57C22-37)S ( Figure 16 B) and anti-CD336 / ZZ-Tat 22-57C22-37)S complex ( Figure 16 C) During incubation, IL-6 secretion increases. Therefore, this data suggests that molecular complexes targeting NK cells and HSPGs can induce effective activation of immune cells.

[0244] Example 14 A molecular complex targeting immune checkpoints (ICPs) induces the secretion of IL-6 by immune system cells in vitro.

[0245] Materials and methods

[0246] To prepare the molecular complex targeting ICPs, three antibodies were used that are considered to be molecularly specific to ICPs. The first antibody (BioXCell reference number BE0190) is called the anti-CTLA-4 antibody, which targets the CTLA-4 molecule. The second antibody (BioXCell reference number BE0285) is called the anti-PD-L1 antibody, which targets the PD-L1 molecule. The third antibody (research reference number MAB10542) is called the anti-OX40 antibody, which targets the OX40 molecule.

[0247] Whether ZZ-Tat exists or not 22-57C22-37)S In the case of ZZ (5 nM per molecule), each of the three Abs (30 nM per molecule) was incubated in RPMI medium with 5% human AB serum at 4°C for 24 hours. The ability of the ZZ region to bind to the Fc region of these Abs allows for the formation of different non-covalent molecular complexes, referred to as anti-CTLA-4 / ZZ-Tat. 22-57C22-37)SAnti-PD-L1 / ZZ-Tat 22-57C22-37)S Anti-OX40 / ZZ-Tat 22-57C22-37)S Anti-CTLA-4 / ZZ, anti-PD-L1 / ZZ and anti-OX40 / ZZ.

[0248] Human PBMCs in 5x10 6 Cells / ml were resuspended in RPMI medium with 5% human AB serum. The presence or absence of free Ab, ZZ, or ZZ-Tat was considered. 22-57C(22-37)S In the case of molecular complexes, 100 μl of cell suspension was distributed into 96-well plates. After incubation for 24 hours, the supernatant was collected for ELISA assay of cytokine IL-6 according to the manufacturer's instructions (R&D#DY406-05).

[0249] result

[0250] The inventors wanted to know whether molecular complexes targeting ICPs could induce activation of immune system cells. Since cell activation leads to cytokine secretion, the inventors decided to evaluate the presence of IL-6 in vitro. They found none in the supernatants produced by incubating PBMCs with three different free Abs or free ZZs, indicating that these compounds do not induce activation in their free form. Figure 17 A, 17B, and 17C). In contrast, they are using ZZ-Tat 22-57C22-37)S This cytokine was found in the supernatant produced during incubation, demonstrating the presence of dual Ig binding domains and Tat. 22-57C22-37)S The association of the HS-binding domain can activate cells in the system. However, the inventors observed that when PBMCs were treated with anti-CTLA-4 / ZZ-Tat... 22-57C22-37)S ( Figure 17 A) Anti-PD-L1 / ZZ-Tat 22-57C22-37)S ( Figure 17 B) and Anti-OX40 / ZZ-Tat 22-57C22-37)S complex ( Figure 17 C) During incubation, IL-6 secretion further increased. In contrast, IL-6 was absent in the supernatant produced by incubation with anti-CTLA-4 / ZZ, anti-PD-L1 / ZZ, or anti-OX40 / ZZ, indicating that the dual ZZ domains did not contribute to these Ab-mediated activation. Therefore, all these data suggest that molecular complexes targeting ICPs and HSPGs can induce the activation of immune cells.

Claims

1. Use of a composition comprising at least one fusion protein whose amino acid sequence is represented by SEQ ID NO: 14 or SEQ ID NO: 20, and at least one pharmaceutically acceptable carrier and / or adjuvant, for the manufacture of an immunostimulatory medicament for the immunotherapy of colorectal cancer.

2. Use of a composition according to claim 1, wherein the adjuvant is a CpG oligodeoxynucleotide, a polyinosinic acid-polycytidylic acid or a mixture of a CpG oligodeoxynucleotide and a polyinosinic acid-polycytidylic acid, and / or the carrier is a nanoparticle.

3. Use of a composition according to any one of the preceding claims, wherein the composition further comprises at least one other therapeutic agent.

4. Use of a composition according to claim 3, wherein the at least one therapeutic agent is at least one immune checkpoint inhibitor.

5. Use of a composition according to claim 4, wherein the at least one immune checkpoint inhibitor is an anti-PD-1, an anti-PDL-1 or an anti-CTLA4. ​ ​

Images