Immunomodulating complexes and their therapeutic uses - Patent Application 20070122997

Abstract

The present invention relates to a molecular complex consisting of at least one ligand of a sulfated sugar of the glycosaminoglycan family bound to at least one ligand of a surface molecule of an antigen-presenting cell or a NK or NKT cell for use as an immunomodulator, in particular for the immunotherapy of cancer and infectious diseases.

Description

[Technical Field]

[0001] The present invention is in the field of regulatory immunotherapy and relates to a molecular complex consisting of at least one ligand for a sulfated sugar of the glycosaminoglycan family and at least one ligand for another surface molecule of antigen-presenting cells or NK or NKT cells, wherein said ligands are bound to each other, for use as an immunomodulatory agent, in particular in the immunotherapy of cancer and infectious diseases. [Background technology]

[0002] The majority of therapeutic approaches are based on drugs that can directly target disease-causing cells or cells exhibiting a non-physiological condition. However, disease processes can be accelerated by immune system dysfunction. In particular, immunosuppressive mechanisms have been observed during tumor progression (Non-Patent Document 1) or infection with infectious pathogens (Non-Patent Document 2). These observations have led to the development of immunotherapeutic agents that can re-establish the proper function of immune defenses, specifically by modulating the activity of T lymphocytes or NK or NKT cells, to more effectively control disease processes. The goal of these regulatory immunotherapies is to re-establish or inhibit the function of a large portion of the lymphocyte repertoire, particularly the CD4+ T, CD8+ T, or regulatory T repertoire. In this respect, it differs from vaccine immunotherapy, whose goal is to induce a limited number of lymphocytes to respond to specific antigens.

[0003] To develop immunotherapeutic drugs, it is necessary to identify targets that play a key role in regulating immune responses. Throughout the 1990s, it was demonstrated that two molecules, PD-1 and CTLA-4, expressed on the surface of T lymphocytes, can induce inhibitory signals that downregulate the activity of these cells, thereby modulating specific immune defense mechanisms (Non-Patent Documents 3-5). These two molecules were named immune checkpoint inhibitors, immune checkpoint inhibitors, or ICP inhibitors.

[0004] The molecular mechanisms underlying the activity of these ICP inhibitors have been studied. In particular, it has been observed that PD-1 and CTLA-4 interact with ligands expressed on the surface of antigen-presenting cells (APCs) or tumor cells. These ligands are designated PD-L1 and B7, respectively. PD-1 / PD-L1 or CTLA-4 / B7 interactions subsequently mediate inhibitory signals from T lymphocytes, resulting in different immunosuppressive mechanisms. Furthermore, antibodies (Abs) specific for PD-1, PD-L1, or CTLA-4 can block the PD-1 / PD-L1 or CTLA-4 / B7 interaction, removing the inhibition and allowing T lymphocytes to reactivate (Non-Patent Documents 6, 7). Some of these immunomodulatory antibodies have been shown to limit the growth of various cancers (e.g., melanoma, lung cancer) and significantly extend patient life expectancy. They are now commonly used in humans as antitumor immunotherapeutic agents (Non-Patent Documents 8, 9). They are also envisaged for other therapeutic areas, in particular for the treatment of infectious diseases (Non-Patent Document 10).

[0005] However, these initial inhibitory anti-ICPs have the disadvantage of only working in 10–30% of cancer patients (Non-Patent Document 11). Thus, numerous research groups are working to discover new immunomodulatory compounds that are more effective and / or can be used in combination with the above-mentioned immunotherapies. Since the identification of several novel ICP inhibitors, research has focused on selecting compounds that can bind to these ICPs or their ICP ligands to reactivate T lymphocytes, thereby neutralizing ICP / ICP-ligand binding. The discovery of ICP activators expressed on the surface of T cells has led to other research focused on selecting agonist ligands for these ICPs (Non-Patent Documents 12–14).

[0006] To immunomodulate immune responses as effectively as possible, it has been necessary to select therapeutic molecules that can bind to ICP ligands that are selectively expressed on the surface of effector immune cells, such as ICPs or T lymphocytes, NK or NKT cells, or Ag-presenting cells (APCs). This expression selectivity allows for the restriction of diffusion of the molecules to non-immune cells, thereby enhancing therapeutic efficacy and reducing the risk of side effects.

[0007] The glycocalyx is composed of proteoglycans, glycoproteins containing one or more unbranched glycosaminoglycan (GAG) chains. Among the latter, the family of heparan sulfate proteoglycans (HSPGs) corresponds to proteins related to sulfated GAGs: heparan sulfate (HS). HSPGs play a central role in numerous biological processes (cell proliferation, cell adhesion, inflammation, coagulation, and cell penetration by pathogenic microorganisms, especially viruses and parasites) and are found on the surface of most mammalian cells and in the extracellular matrix (Non-Patent Document 15). Therefore, this ubiquitous expression means that HSPGs and their HS domains cannot be considered relevant immunomodulatory targets. Furthermore, the ability of HS ligands to induce immune cell activation has yet to be elucidated. [Non-Patent Document 1] Galon J. and D. Bruni, Nat. Rev. Drug Discov., 2019 18(3):197-218 [Non-patent document 2] Wherry EJ and M. Kurachi, Nature Rev. Immunol., 2015, 15(8):486-499 [Non-patent document 3] Ishida, Y., Agata, Y., Shibahara, K., and Honjo, T. (1992) EMBO J., 11(11), 3887-3895. [Non-patent document 4] Freeman et al. (2000). J Exp Med, 192(7), 1027-1034 [Non-patent document 5] Krummel MF, Allison JP, J.Exp.Med.,1995 August 1;182(2):459-65 [Non-patent document 6] Hodi, FS et al., (2003), 100(8), 4712-4717 [Non-Patent Document 7] Iwai, Y. et al., Int. Immunol. (2005) 17(2), 133-144PNAS [Non-patent document 8] Adachi K. and K. Tamada, Cancer Sci., 2015;106(8):945-50 [Non-Patent Document 9] Riley RS et al., Nat. Rev. Drug Discov. 2019 18(3):175-196 [Non-Patent Document 10] Rao M. et al., Int. J. Dis., 2017;56:221-228 [Non-Patent Document 11] Pitt JM et al. Immunity. 2016 June 21;44(6):1255-69 [Non-Patent Document 12] Mahoney KM. et al., Nat. Rev. Drug Discov., 2019, 14:561-584 [Non-Patent Document 13] De Sousa Linhares A., Front.Immunol.2018, 31;9:1909. [Non-Patent Document 14] Granier C. et al., ESMO Open, 2017, July 3;2(2):e000213 [Non-Patent Document 15] Dreyfuss et al., Annals of the Brazilian Academy of Sciences, 2009, 81, 409-429 Summary of the Invention [Means for solving the problem]

[0008] During their work, the inventors first found that heparan sulfate (HS), a ligand for sulfated sugars of the GAG ​​family, was unable to activate dendritic cells in vitro. They then found that a molecular complex containing this ligand and a ligand for a surface molecule of antigen-presenting cells (APCs) enhanced the immunomodulatory effects of the HS ligand. Indeed, as indicated by increased secretion of the cytokines IL-6 and IL-12, this molecular complex more potently induced APCs to activate subsequent T lymphocytes. The present inventors have shown that the association between the ligands can be produced in the form of covalent or non-covalent complexes, particularly in the form of fusion proteins, and have further found that the immunomodulatory properties of these complexes allow for better control of disease processes, particularly slowing tumor progression.

[0009] These results are surprising because, although HSPGs have been described as capable of acting as receptors or coreceptors, it has not been demonstrated that they can enhance ligand-mediated activation of receptors specifically expressed on the surface of APCs. Finally, molecular complexes targeting ubiquitously expressed HS may spread to the extracellular matrix or to untargeted disease cells. Thus, theoretically, they should not be able to modulate the activity of a large repertoire of APCs and T lymphocytes to produce a sufficiently efficient immunotherapeutic effect.

[0010] The present inventors also found that the results obtained with APCs can be transferred to other cells of the innate immune response, such as NK and NKT cells. Thus, the present inventors found that, although these cells alone cannot be induced, the number of activated NK or NKT cells can be increased when their surface molecule ligands bind to sulfated sugar ligands of the GAG ​​family in a molecular complex.

[0011] Thus, a subject of the present invention is a molecular complex for use as an immunomodulator, preferably an immunostimulant, which consists of at least one ligand of a sulfated sugar of the glycosaminoglycan family (first ligand or L1) and at least one ligand of a surface molecule of an antigen-presenting cell or of an NK or NKT cell (second ligand or L2), said ligands binding to each other, and said complex being a molecular complex that does not contain a specific antigen of the disease of interest.

[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 the HIV Tat protein containing at least the basic region Tat49-57 (SEQ ID NO: 3), such as peptides Tat49-57 (SEQ ID NO: 3), Tat37-57 (SEQ ID NO: 8), and Tat22-57C(22-37)S (SEQ ID NO: 9), which contain a heparan sulfate-binding region; polyarginine peptides R7 to R11; and a peptide derived from the R domain of diphtheria toxin (SEQ ID NO: 5) or at least a fragment of said domain, DT453-467 (SEQ ID NO: 7).

[0013] According to a preferred embodiment of the present invention, the second ligand targets a surface molecule of an APC selected from the group consisting of a C-type lectin receptor, a membrane-bound immunoglobulin, a receptor for the constant region of an immunoglobulin, and an immune checkpoint (ICP) molecule.

[0014] According to a preferred embodiment of the invention, the second ligand targets a surface molecule of an NK or NKT cell selected from the group consisting of NKG2D, NKp30, NKp44, NKp46, NKp80, Ly49H receptor, KIR, NKG2A receptor, ICP PD-1, CTLA-4, TIM-3, TIGIT, LAG-3.

[0015] The second ligand is preferably selected from the group consisting of: (i) antibodies directed against surface molecules of APCs or NK or NKT cells and fragments thereof comprising at least the paratope; (ii) immunoglobulins, preferably IgG, and fragments thereof comprising at least the Fc region; and (iii) proteins and protein fragments that bind to the Fc and / or Fab region of antibodies, in particular protein A of Staphylococcus aureus, 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 monomers and oligomers.

[0017] According to a preferred embodiment of the invention, the complex consists of a fusion protein between a first and a second ligand. A particularly preferred type of complex according to the invention consists of a fusion protein comprising a first ligand 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 a second ligand selected from the BB fragment of protein A (SEQ ID NO: 1) or its ZZ derivative (SEQ ID NO: 2).

[0018] According to another preferred embodiment 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) and its ZZ derivative (SEQ ID NO: 2). Preferably, the fusion protein comprises a first ligand 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 an immunoglobulin-binding element selected from the BB fragment of protein A (SEQ ID NO: 1) or its ZZ derivative (SEQ ID NO: 2). In one particularly preferred complex according to the invention, the immunoglobulin-binding element is the BB fragment of protein A (SEQ ID NO: 1), and the fusion protein forms a complex with a second ligand consisting of a whole immunoglobulin. In another particularly preferred class of conjugates according to the invention, the fusion protein is conjugated to a second ligand selected from anti-RFcγI, II and / or III, anti-DEC-205, anti-DC-SIGN, anti-CD74, anti-CD275, anti-CD-335, anti-CD-336, anti-CD56, anti-CTLA-4, anti-PD-L1, anti-OX40 antibodies, or fragments of the above antibodies comprising at least the paratope.

[0019] According to a preferred embodiment of the present invention, the complex is for use as an immunostimulant, preferably for activating antigen-presenting cells, in particular dendritic cells or monocytes, for activating NK or NKT cells and / or for activating the secretion of the cytokines IL-6 and / or IL-12.

[0020] Another subject of the present invention is a composition for use as an immunomodulator, preferably an immunostimulant, comprising at least one molecular complex according to the invention and at least one pharmaceutically acceptable carrier, carrier substance and / or adjuvant, the adjuvant preferably being CpG oligodeoxynucleotides, polyinosinic:polycytidylic acid or a mixture of CpG oligodeoxynucleotides and polyinosinic:polycytidylic acid, and / or the carrier substance being 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-PDL-1 or anti-CTLA4.

[0022] According to a preferred embodiment of the invention, the composition is for use in the immunotherapy of cancer or infectious diseases. DETAILED DESCRIPTION OF THE INVENTION

[0023] DISCLOSURE OF THE INVENTION The subject of the present invention is a molecular complex for use as an immunomodulator, preferably an immunostimulant, which consists of at least one ligand of a sulfated sugar of the glycosaminoglycan family (first ligand) and at least one ligand of a surface molecule of APC or NK or NKT cells (second ligand), said ligands binding to each other, and said complex being a molecular complex that does not contain a specific antigen of the disease of interest.

[0024] Such complexes are preferably capable of inducing cells of the immune system to elicit an immune response. Thus, when splenocytes are incubated with these complexes, dendritic cell activation is observed, allowing the dendritic cells to secrete IL-6 and IL-12 cytokines. Furthermore, DC induction can be achieved through the use of a second ligand that cannot induce this type of effect without its association, such as an isolated form of an APC ligand called ZZ. Binding of an HS ligand can enhance the effects of ligands capable of activating APCs in an isolated state, such as nonspecific polyclonal human Abs. Binding of an HS ligand to a ligand for NK or NKT cells can also activate these cell types, but lacks this ability in their isolated form. Finally, the immunomodulatory complexes, preferably immunostimulatory complexes, according to the present invention can induce therapeutic effects, particularly antitumor effects superior to those achieved with anti-ICP (anti-PD1) Abs, especially when the composition contains an adjuvant.

[0025] (definition) By "immune cells of the innate response" is meant dendritic cells, NK and NKT cells, granulocytes (mast cells, neutrophils, eosinophils, basophils) and phagocytes (monocytes, macrophages, neutrophils, etc.).

[0026] An "antigen-presenting cell" (APC) is a cell that presents an Ag, expressing one or more class I and class II molecules of the major histocompatibility complex (MHC) (in humans, class I and class II HLA molecules), and is capable of presenting the Ag to CD4+ and CD8+ T lymphocytes specific for that Ag. By Ag-presenting cells, we mean, inter alia, dendritic cells (DCs), monocytes, macrophages, B lymphocytes, lymphoblastoid cell lines, and genetically modified human or animal cell lines that express class I and class II molecules of the MHC, in particular HLA1 and HLA2 molecules.

[0027] "NK cells" refers to granular lymphocytes that express the molecules CD56 and CD16 and have cytotoxic activity without the need for prior exposure to Ag.

[0028] "NKT cells" refer to granular lymphocytes that express NK cell markers, particularly the molecules CD56 and CD16, and T lymphocyte markers, particularly the molecule CD3. These cells have cytotoxic activity that does not require prior exposure to Ag.

[0029] "Surface molecule of APC" means a molecule expressed on the surface of an Ag-presenting cell.

[0030] "NK or NKT cell surface molecule" means a molecule expressed on the surface of an NK or NKT cell.

[0031] An "APC-specific surface molecule" refers to a molecule that is expressed substantially on Ag-presenting cells, i.e., on a very limited number of cells other than APCs. Thus, this is a molecule that has a high level of APC expression specificity.

[0032] By "NK or NKT cell-specific surface molecule" is intended to mean a molecule that is expressed substantially on NK or NKT cells, i.e., a molecule that is expressed on a very limited number of cells other than NK or NKT cells, and thus is a molecule that has high levels of NK or NKT cell expression specificity.

[0033] "Glycosaminoglycan (GAG)" refers to a linear polysaccharide consisting of repeating disaccharides that always contain a hexosamine (glucosamine (GlcN) or galactosamine (GalN)) and another sugar (glucuronic acid (GlcA), iduronic acid (IdoA), or galactose (Gal)). Glucosamine is either N-sulfated (GlcNS) or N-acetylated (GlcNac). Galactosamine is always N-acetylated (GalNac). Sulfated glycosaminoglycans are, in particular, simple polymers of GlcA, such as chondroitin sulfate, and copolymers containing GlcA and / or IdoA and / or Gal residues, such as heparin, heparan sulfate, dermatan sulfate, and keratan sulfate. GAG chains can be covalently attached to proteins (proteoglycans) that are expressed on the surface of mammalian cells and / or excreted in the extracellular matrix. The first ligand according to the present invention binds to sulfated sugars of the glycosaminoglycan family expressed on the surface of mammalian cells, particularly APCs and NK or NKT cells.

[0034] "Ab" refers to immunoglobulin (IgG, IgM, IgA, IgD, IgE). The term Ab refers to a specific Ab, i.e., an Ab against a specific molecule x (anti-molecule xAb), particularly a surface molecule of an APC (anti-APC surface molecule Ab). The term immunoglobulin refers to a non-specific Ab.

[0035] "Individual" is intended to mean a human or animal, preferably a human individual.

[0036] A "ligand" of a molecule is intended to mean any agent capable of binding to that molecule with sufficient affinity to form a stable complex in vitro and in vivo.

[0037] "APC ligand" is intended to mean a ligand of a surface molecule of an Ag-presenting cell.

[0038] "NK or NKT cell ligand" is intended to mean a ligand of a surface molecule of an NK or NKT cell. "Heparan sulfate ligand" refers to ZZ-Tat 22-57c(22-37)s is intended to mean an agent that binds to heparin in the ELISA assay of Example 1 with an optical density signal that is at least 50% of the signal measured when incubated at 100 nM, pH 7.2.

[0039] "Antigen" is intended to mean any substance that can be specifically recognized by the immune system, in particular by antibodies and cells of the immune system (B lymphocytes, CD4+ T lymphocytes, CD8+ T lymphocytes) and that is capable of inducing a specific immune response.

[0040] "Specific antigen of a disease of interest" is intended to mean an antigen that induces an immune response specifically directed against the disease of interest, said specific immune response against the disease of interest comprising the production of antibodies and / or the induction of a cytotoxic T cell response (activation of CD8+ T lymphocytes) or a helper T cell response (activation of CD4+ T lymphocytes) directed against an antigen of a pathogen or an antigen of a tumor cell that is the cause of the disease of interest.

[0041] The term "immunomodulator" is intended to mean an agent capable of regulating immune responses, particularly up-regulating or down-regulating 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 immunosuppressant and immunostimulator. The term "immunostimulator" refers to an agent capable of up-regulating, 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 effects of immunomodulators, preferably immunostimulators, are exerted across a broad cellular repertoire. Unlike vaccines, which require the presence of a specific antigen of a target disease, immunomodulators induce specific immune responses against antigens independently of the presence of the specific antigen of the target disease. While the use of immunogens or vaccines is limited to diseases containing the specific antigen of the disease, the immunomodulatory, preferably immunostimulatory, complexes of the present invention can be used for immunotherapy of many diseases, such as cancer and infectious diseases, independent of the presence of the specific antigen of the target disease.

[0042] "Peptide" is intended to mean a sequence of optionally modified natural or synthetic amino acids. The term peptide is used without regard to the size of the amino acid sequence.

[0043] The molecular complex of the present invention comprises at least two ligands of surface molecules of APCs, NK cells, or NKT cells bound to each other: a first ligand designated L1 that targets sulfated GAGs and a second ligand designated L2 that targets another surface molecule of APCs. The molecular complex of the present invention may comprise one or more L1 ligands and one or more L2 ligands bound to each other. According to a preferred embodiment of the present invention, the molecular complex is composed of one L1 ligand bound to one L2 ligand.

[0044] The molecular complexes according to the present invention, preferably immunostimulatory complexes, do not contain a specific antigen of a disease of interest, such as a vaccine antigen specific to the disease of interest. If one of the ligands of the complexes according to the present invention contains a specific antigen of a disease of interest, treatment of the disease with the complex is excluded from the invention.

[0045] The ligands for APCs, NK or NK cells are natural, recombinant, or synthetic molecules or complexes of molecules, which may be natural proteins (proteins, peptides, polypeptides), or natural lipids, carbohydrates, nucleic acids, or mixtures (glycolipids, glycoproteins, lipoproteins). According to a preferred embodiment of the present invention, the first and second ligands are preferably recombinant or synthetic proteins, polypeptides, or peptides, referred to as "peptides." Recombinant proteins, polypeptides, or peptides are advantageously produced in prokaryotic or eukaryotic cells in suitable expression systems, particularly suited for the production of therapeutic proteins. For example, recombinant proteins, polypeptides, or peptides can be produced in E. coli, HEK, or CHO cells.

[0046] The first and second ligands are associated or bound to each other by any suitable means. They may be covalently or non-covalently linked, either directly or via a linking group, thereby forming a molecular complex. A molecular complex or complex consists of two or more ligands and any suitable linker.

[0047] Covalent association or binding of the ligands is produced inter alia by covalent chemical coupling (formation of a covalent bond) or by the formation of a fusion protein (gene fusion).

[0048] The immunomodulating complexes, preferably immunostimulating complexes, are in monomeric, oligomeric or mixed form (mixture of monomers and oligomers). According to certain preferred embodiments of the invention, the complexes are in oligomeric or mixed form.

[0049] According to a preferred embodiment of the present invention, the immunomodulatory molecule complex, preferably the immunostimulatory complex, comprises a fusion protein between a first ligand (L1) and a second ligand (L2). The amino acid sequences of L1 and L2 are fused in an appropriate order, either directly or via a suitable peptide spacer. Depending on the size of each of the L1 and L2 amino acid sequences, they can be fused at their ends (the N-terminus of one sequence fused to the C-terminus of the other), or one of the sequences can be inserted into the other sequence at an appropriate site that does not adversely affect the binding of the ligand to its receptor expressed on the surface of APCs, NK, or NKT cells.

[0050] Non-covalent binding is particularly achieved by adsorption to nanoparticles. It can also be achieved using molecules (linkers or binding elements) with high and specific affinity for L1 or L2. The linker is covalently bound to one ligand for non-covalent binding to another ligand. When one of the ligands is an antibody (Ab), the linker is a protein or protein fragment that binds to the Fc and / or Fab regions of immunoglobulins, as described in patent application FR2759296. Such immunoglobulin-binding elements include, in particular, Staphylococcus aureus protein A, its BB fragment (SEQ ID NO: 1) and its ZZ derivative (SEQ ID NO: 2), where the first two proteins bind to the Fc and Fab regions of immunoglobulins, and ZZ binds only to the Fc region. For example, when one of the ligands is an Ab or antibody fragment, the immunoglobulin-binding element is covalently bound to the other ligand (covalent chemical coupling or fusion protein). The linker can also bind to a partner that binds to L1 and L2, respectively, e.g., the linker is streptavidin that binds to biotinylated ligands L1 and L2 (L1-biotin / streptavidin / biotin-L2), where the affinity of the linker for the partner in the L1-L2 complex is sufficient so that it does not rapidly dissociate from the complex in vivo.

[0051] According to a particularly preferred embodiment of the immunomodulatory complex of the invention, which is preferably an immunostimulatory complex, one of the ligands is an antibody or antibody fragment and the other 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 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).

[0052] The immunomodulatory, preferably immunostimulatory, properties of the complexes of the present invention are evaluated by conventional immunological tests known to those skilled in the art, such as those described in the Examples. The immunostimulatory properties are evaluated by analyzing the expansion and / or activation of different populations or subpopulations of immune cells, particularly 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. Expansion is analyzed using flow cytometry, particularly with markers appropriate for the various cell populations being analyzed. Immune cell activation can be analyzed by detecting markers of activation (CD69) and / or maturation (CD86) by flow cytometry or conventional assays, such as assays of cytokines secreted in the extracellular medium, particularly IL-6 and IL-12, or ELISA. As mentioned above, the immunomodulatory, preferably immunostimulatory, effect of the molecular complexes of the present invention is independent of the presence of specific antigens of the disease of interest.

[0053] The molecular complex, preferably an immunostimulatory complex, according to the present invention binds to at least one sulfated sugar of the glycosaminoglycan family expressed on the surface of APCs, NKs, or NKTs (sulfated GAGs) and another surface molecule of APCs, NKs, or NKTs, particularly a surface molecule specific for APCs, NKs, or NKTs. The sulfated GAG targeted by the first ligand is preferably heparan sulfate, chondroitin sulfate, dermatan sulfate, or keratan sulfate, preferably heparan sulfate. The sulfated GAG ligand (first ligand) may 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, and is particularly a molecular complex of a molecule or fragment thereof that binds to heparin and / or heparan sulfate. Such ligands are described in particular in "Heparan Binding Proteins" by H. Edward Conrad, Academic Press, San Diego and London and by Dreyfuss et al., Annals of the Brazilian Academy of Sciences, 2009, 81, 409-429 (see especially Table II).Examples of these ligands include, but are not limited to, endogenous GAG ligands (thrombin, urokinase, vitronectin, fibroblast growth factor, etc.), the HIV Tat protein (SEQ ID NO: 10) and fragments thereof, in particular fragments containing only the Tat basic region (Tat49-57 (SEQ ID NO: 3)) or the Tat basic region and the central region (core; Tat38-48 (SEQ ID NO: 4)); the dodecahedron from the adenovirus penton (Vives et al., Virology, 2004, 321:332-340); the HIV envelope protein or the V3 region of this protein (Roderiquez et al., J. Virol., 1995, 69, 2233-), Sindbis These include the envelope glycoproteins of the HIV virus (Byrnes, AP and Griffin, DE, J. Virol., 1998, 2, 7349-7356) and the diphtheria toxin domain (DTR or DTRBD; fragment 382-535 of DT (SEQ ID NO: 5); Lobeck et al., Infection and Immunity, 1998, 66, 418-423). Other examples of cell-penetrating peptides (CPPs) that bind to heparin, heparan sulfate, and / or chondroitin sulfate include the above-mentioned peptides derived from the HIV Tat protein (Tat49-57), as well as peptides highly rich in basic residues, particularly arginine, including basic / amphipathic peptides (R11), such as polyarginine (R7-R11) peptides and peptides derived from the homeodomain of the antennapedia protein (penetratin; fragment 43-58 (SEQ ID NO: 6)).

[0054] Alternatively, the first ligand is a natural or recombinant Ab against a sulfated GAG, preferably heparin, heparan sulfate, or chondroitin sulfate, or a fragment of this Ab containing at least the paratope (Ag-binding domain), such as Fab, Fab', F(ab')2, Fv, or single-chain Fv (scFv), Fabc fragments, and Fab fragments containing part 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.

[0055] According to an advantageous embodiment of the molecular complex of the present invention, preferably the immunostimulatory molecular complex, the first ligand is a peptide Tat49-57 (SEQ ID NO: 3), Tat37-57 (SEQ ID NO: 8) and Tat22-57C, which contain a heparan sulfate binding domain. (22-37)S The first ligand is preferably a heparan sulfate-binding peptide selected from the group consisting of a peptide derived from the HIV Tat protein containing at least the basic region Tat49-57 (SEQ ID NO: 3), such as (SEQ ID NO: 9), a polyarginine peptide of R7 to R11, and a peptide containing the R domain of diphtheria toxin (SEQ ID NO: 5) or at least a fragment of said domain, DT453-467 (SEQ ID NO: 7). The first ligand is preferably a heparan sulfate-binding peptide selected from the group consisting of a peptide derived from the HIV Tat protein containing at least the basic region Tat49-57 (SEQ ID NO: 3), in particular the peptide Tat22-57C(22-37)S (SEQ ID NO: 9), and a peptide containing the R domain of diphtheria toxin (SEQ ID NO: 5).

[0056] In an advantageous embodiment of the molecular complex of the invention, preferably the immunostimulatory molecular complex, when the complex comprises the R domain of diphtheria toxin (DTR: SEQ ID NO: 5), the domain is then produced in mammalian cells, in particular in the form of a fusion protein with a second ligand or in the form of an immunoglobulin binding element for forming oligomers.

[0057] Other molecules expressed on the surface of APCs that are targeted by the second ligand can be ubiquitous surface molecules other than sulfated GAGs, surface molecules expressed substantially on immune cells of the innate or adaptive response, including APCs, i.e., surface molecules specific to immune cells of the innate or adaptive response, including APCs; or surface molecules expressed substantially on APCs, and particularly on dendritic cells, i.e., surface molecules specific to APCs and particularly dendritic cells. Among the surface molecules substantially expressed on these APCs, particularly dendritic cells, can be mentioned class II MHC molecules, particularly the α and β chains and γ chain or invariant chain of MHC II (Ii, Ii fragment or CD74); surface immunoglobulins or membrane-bound immunoglobulins; in particular CD11c and MAC1, transferrin receptors, C-type lectin receptors, particularly integrins such as mannose receptor (CD206), DEC-205 (CD205), CD206, DC-SIGN (CD209), LOX1, Dectin-1 (beta-glucan receptor), Dectin-2, Clec9A, Clec12A, DCIR2, FIRE, and CIRE; receptors for the constant region of immunoglobulins (FcR or RFc), particularly FcγRs such as FcγRI (CD64), FcγRII (CD32) and FcγRII (CD16); the TNF receptor superfamily, such as CD40; and complement receptors. Among the surface molecules that are substantially expressed on APCs are immune checkpoint (ICP) molecules and their ligands (ICP-ligands) that are expressed on APCs, including but not limited to PDL1, PDL2, CD155, CD80, CD86, CD40, OX40L, ICOSL (CD275), and CD70 (Wykes MN, Nat. Rev. Immunol., 2018, 18:91-104).

[0058] Other molecules expressed on the surface of NK or NKT cells that are targeted by the second ligand can be ubiquitous surface molecules other than sulfated GAGs, surface molecules substantially expressed on immune cells of the innate or adaptive response, including NK or NKT cells, i.e., surface molecules specific to immune cells of the innate or adaptive response, including NK or NKT cells, or surface molecules substantially expressed on NK or NKT cells, i.e., surface molecules specific to NK or NKT cells. Among these surface molecules substantially expressed on NK or NKT cells, NKG2D, NKp30, NKp44, NKp46, NKp80, CD56, CD16, KIR, NKG2A receptor, ICP PD-1, CTLA-4, TIM-3, TIGIT, LAG-3, and OX40 can be mentioned in particular.

[0059] According to an advantageous embodiment of the molecular complex of the present invention, preferably the immunostimulatory molecular complex, the second ligand targets a molecule substantially expressed on APCs and particularly dendritic cells, i.e., a surface molecule specific to APCs and particularly dendritic cells, preferably a molecule selected from the group consisting of C-type lectin receptors, membrane-bound immunoglobulins, and receptors for the constant region of immunoglobulins.

[0060] According to an advantageous embodiment of the molecular complex of the present invention, preferably the immunostimulatory molecular complex, and according to another advantageous embodiment of the molecular complex, the second ligand targets a surface molecule of an APC selected from the group consisting of a C-type lectin receptor, a membrane-bound immunoglobulin, a receptor for the constant region of immunoglobulin (RFc or FcR), an immune checkpoint (ICP) molecule and its ligand (ICP ligand).

[0061] According to an advantageous embodiment of the invention, the second ligand targets a surface molecule substantially expressed on NK or NKT cells, i.e. a surface molecule specific for NK or NKT cells, preferably a surface molecule selected from the group consisting of NKG2D, NKp30, NKp44, NKp46, NKp80, CD56, CD16, KIR, NKG2A receptor, and the ICPs PD-1, CTLA-4, TIM-3, TIGIT, LAG-3, OX40; preferably a surface molecule selected from the group consisting of NKp44 (CD336), NKp46 (CD335), NCAM (CD56), CTLA-4 and OX40.

[0062] The second ligand is selected in particular from C-type lectin receptors, immunoglobulins and fragments thereof containing a constant region that binds to FcR, proteins or protein fragments that bind to the Fc and / or Fab regions of membrane-bound immunoglobulins described in application FR2759296, in particular saccharides that bind to Staphylococcus aureus protein A, its BB fragment (SEQ ID NO: 1) and its ZZ derivative (SEQ ID NO: 2), preferably rZZ (SEQ ID NO: 2). Alternatively, the second ligand is an antibody against these surface molecules of APC, NK, or NKT cells, or a fragment of this antibody containing at least the paratope (Ag-binding domain), such as Fab, Fab', F(ab')2, Fv, or single-chain Fv (scFv), Fabc fragment, and Fab fragment containing part of the Fc domain.

[0063] The antibody or antibody fragment is directed against a surface molecule, in particular a surface molecule, specific for immune cells of the innate or adaptive response, including APCs, NK or NKT cells, such as, but not limited to, 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.

[0064] The antibody or antibody fragment can also be directed against a surface molecule specific for APCs, particularly dendritic cells, such as anti-RFc gamma (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 11 (CD74). The antibody can be an agonist or antagonist of the surface molecule; for example, the antibody can be an agonist of an activator surface molecule or an antagonist of an inhibitor surface molecule. The antibody or antibody fragment is preferably human or humanized. Such antibodies are well known to those skilled in the art and are commercially available.

[0065] According to an advantageous embodiment of the molecular complex, preferably the immunostimulatory molecular complex, the second ligand is selected from the group consisting of: (i) antibodies against these surface molecules of antigen-presenting cells or NK or NKT cells or fragments thereof comprising at least the paratope, such as Fab, Fab', F(ab')2, Fv or scFv, Fab fragments comprising the Fabc and part of the Fc domain, (ii) immunoglobulins, preferably IgG, and fragments thereof comprising at least the Fc region, and (iii) proteins and protein fragments that bind to the Fc and / or Fab region of antibodies, in particular Staphylococcus aureus protein A, its BB fragment (SEQ ID NO: 1) and its ZZ derivative (SEQ ID NO: 2).

[0066] The antibody against the surface molecule of an antigen-presenting cell is preferably selected from anti-RFc gamma (I, II, and / or III), anti-DC-SIGN (CD209), anti-DEC-205, anti-DC-SIGN (CD209), anti-DEC-205, anti-CD206, anti-CD40 antibody, anti-invariant chain 11 (CD74) and anti-ICOSL (CD275) antibody; preferably, anti-DC-SIGN (CD209) and anti-DEC-205 antibody or anti-DC-SIGN (CD209), anti-DEC-205, anti-invariant chain 11 (CD74), and anti-ICOSL (CD275) antibody.

[0067] The antibody against the surface molecule of NK or NKT cells is 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.

[0068] The additional ligand of the molecular complex, preferably of the immunostimulatory molecular complex, is advantageously selected from the sulfated GAG ligands and ligands of APC surface molecules as defined above.

[0069] A first type of preferred immunostimulatory complex according to the present invention consists of a sulfated GAG ligand peptide (first ligand) that binds to the Fc and / or Fab regions of an immunoglobulin, such as the BB fragment of Staphylococcus aureus protein A and its ZZ derivative (second ligand), as defined above, preferably in the form of a fusion protein containing the first and second ligands. A particularly preferred first type of complex consists of a fusion protein containing a first ligand 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 (DTR: SEQ ID NO:5) or fragment DT453-467 (SEQ ID NO:7), and a second ligand 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 at the N-terminus of the fusion protein and the first ligand is at the C-terminus of the fusion protein, the first and second ligands being separated by a suitable spacer peptide, in particular selected from SEQ ID NOs: 21 to 23. More preferably, the complex comprises one of the sequences of SEQ ID NOs: 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 produced in mammalian cells to form oligomers, in particular in the form of a fusion protein with the second ligand.

[0070] A second type of preferred immunostimulatory complex according to the present invention comprises (i) a sulfated GAG ligand peptide (first ligand) covalently linked to an immunoglobulin-binding element as defined above, in particular a protein or protein fragment that binds only to the Fc and / or Fab regions of an Ab, preferably the Fab region such as the protein A and BB fragments of Staphylococcus aureus, preferably in the form of a fusion protein comprising the first ligand and the immunoglobulin-binding element, the first ligand being covalently linked to the immunoglobulin-binding element, and (ii) a complex with an immunoglobulin, preferably an IgG, or a fragment comprising at least the Fc region (second ligand), preferably a whole immunoglobulin, preferably a whole IgG. The first ligand and the immunoglobulin-binding element are fused directly or via a suitable spacer peptide. A second type of particularly preferred complex consists of a fusion protein containing a first ligand 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 (DTR; SEQ ID NO: 5) or fragment DT453-467 (SEQ ID NO: 7), and an immunoglobulin-binding element comprising the BB fragment of protein A (SEQ ID NO: 1), wherein the fusion protein is complexed with a whole immunoglobulin. In this second type of particularly preferred complex, the immunoglobulin-binding element (BB) is preferably at the N-terminus of the fusion protein and the first ligand is at the C-terminus; the immunoglobulin-binding element (BB) and the first ligand are separated by a suitable spacer peptide, in particular selected from SEQ ID NOs: 21-23. More preferably, the complex comprises one of the sequences set forth in SEQ ID NOs: 16 or 18. When the complex comprises the R domain of diphtheria toxin (DTR: SEQ ID NO: 5), this domain is advantageously produced in mammalian cells to form oligomers, particularly in the form of a fusion protein with a second ligand.

[0071] A third type of preferred immunostimulatory complex according to the present invention consists of a sulfated GAG ligand peptide (first ligand) as defined above that binds to an Ab selected from the group consisting of anti-RFc gamma (I, II, and / or III) Ab, anti-DEC-205 Ab, anti-DC-SIGN (CD209) Ab, anti-invariant chain 11 (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 Abs that contain at least the paratope. Preferably, the sulfated GAG ligand peptide (first ligand) as defined above binds to an immunoglobulin binding element as defined above, in particular the Fc and / or Fab region of an immunoglobulin such as the BB region of Staphylococcus aureus protein A and its ZZ derivatives, preferably in the form of a fusion protein comprising the first ligand and the immunoglobulin binding element, which are fused directly or via a suitable spacer peptide. A third type of particularly preferred conjugate comprises a fusion protein containing a first ligand 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 (DTR; SEQ ID NO:5) or its fragment DT453-467 (SEQ ID NO:7), and an immunoglobulin-binding element comprising the BB fragment of protein A (SEQ ID NO:1) or its ZZ derivative (SEQ ID NO:2), which is conjugated to anti-RFc gamma (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, or anti-OX40 antibodies, or fragments of the aforementioned antibodies containing at least the paratope. This third type of conjugate is particularly conjugated with a suitable spacer peptide selected from SEQ ID NOs:21-23. More preferably, the complex comprises one of the sequences SEQ ID NO: 12, 14, 16, 18 or 20.When the conjugate comprises the R domain of diphtheria toxin (DTR: SEQ ID NO: 5), this domain is advantageously produced in mammalian cells to form oligomers, particularly in the form of a fusion protein with a second ligand. Preferably, the conjugate comprises an anti-DEC-205, anti-DC-SIGN, anti-CD74, anti-CD275, anti-CD335, anti-CD336, anti-CD56, anti-CTLA-4, anti-PDL1 or anti-OX40 antibody.

[0072] According to a preferred embodiment of the present invention, the complexes are used as immunostimulants, preferably for activating antigen-presenting cells, in particular dendritic cells or monocytes, for activating NK or NKT cells and / or for activating the secretion of the cytokines IL-6 and / or IL-12.

[0073] Another subject of the present invention is an immunomodulatory composition comprising at least one immunomodulatory complex, preferably an immunostimulatory complex, according to the invention, and at least one pharmaceutically acceptable carrier, carrier substance and / or adjuvant.

[0074] The pharmaceutically acceptable carriers are those conventionally used.

[0075] Adjuvants are conventionally used in immunotherapy to stimulate humoral and / or cellular immunity. The adjuvant is advantageously selected from the group consisting of oil emulsions, mineral substances, bacterial extracts, saponin, alumina hydroxide, monophosphoryl lipid A, squalene, TLR ligands, particularly specific oligodeoxynucleotides (CpG oligodeoxynucleotides) that are TLR9 ligands containing at least one CpG sequence, or polyinosinic:polycytidylic acid (poly(I):poly(C) or poly:C) that is a TLR3 ligand. According to a preferred embodiment of the present invention, the composition comprises at least one adjuvant, preferably CpG oligodeoxynucleotides, polyinosinic:polycytidylic acid, or a mixture of CpG oligodeoxynucleotides and polyinosinic:polycytidylic acid.

[0076] Carrier substances are those conventionally used: certain unilamellar or multilamellar liposomes, ISCOMs, virosomes (virus-like particles), saponin micelles, solid microspheres of sugars (poly(lactide-co-glycolide)) or gold of auriferous nature, and nanoparticles. According to a preferred embodiment of the present invention, the composition comprises at least one carrier substance, for example, a nanoparticle or a mixture of nanoparticles.

[0077] The immunomodulatory composition, preferably the immunostimulatory composition, according to the present invention comprises a complex or a mixture of different complexes comprising two or more ligands, the complexes optionally being linked to each other by covalent or non-covalent bonds and / or incorporated into or on the surface of particles such as liposomes, virosomes or nanoparticles.

[0078] According to certain embodiments of the present invention, the composition is a polynucleotide or mixture of polynucleotides encoding an APC, NK, or NKT ligand, which may be a protein, polypeptide, or peptide. The polynucleotide consists of a recombinant, synthetic, or semi-synthetic nucleic acid that can be expressed in the cells of a host to which the composition is administered. The nucleic acid may be DNA, RNA, particularly mRNA, or a mixed nucleic acid (DNA / RNA), and may be modified. For example, the composition comprises a polynucleotide comprising a sequence encoding a fusion protein containing at least the coding sequences of a first and a second ligand, suitably fused in 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 comprising a sequence encoding a first ligand and a second polynucleotide comprising a sequence encoding a second ligand, wherein the first or second polynucleotide also comprises a sequence encoding a linker as defined above. The polynucleotide is preferably inserted into one or more expression vectors containing appropriate transcriptional and / or translational regulatory sequences (promoter, transcriptional activator, transcription terminator, polyadenylation signal) for the in vivo expression of the first and second ligands, and optionally other ligands, in the individual to whom the composition is administered. Numerous vectors that can be used for therapy are known per se. In particular, viral vectors (adenovirus, retrovirus, lentivirus, AAV) and non-viral vectors (naked DNA), particularly plasmid viruses, into which the desired sequence has been previously inserted can be used. Alternatively, the polynucleotide is mRNA, preferably modified. The use of mRNA in therapy is well known to those skilled in the art (e.g., Drew Weissman, Expert Reviews of Vaccines, 2014, October, 1-17, doi:10.1586 / 14760584.2015.973859).

[0079] According to a specific embodiment of the present invention, the composition comprises cells modified with the composition of the present invention. For example, the cells are modified with the polynucleotide, mixture of polynucleotides, or vector defined above, or carry the ligand complex defined above. The cells are, in particular, natural antigen-presenting cells such as dendritic cells, or artificial antigen-presenting cells such as dendritic cell-derived exosomes or cell-derived vesicles expressing the ligand of the molecular complex of the present invention. For example, the cells are antigen-presenting cells, in particular dendritic cells, of the subject individual that are modified ex vivo (ex vivo cell therapy) before being re-administered to the individual.

[0080] The immunomodulatory composition may further comprise at least one other therapeutic agent, particularly an anti-cancer agent, an anti-infective agent, or other immunomodulatory agent or vaccine antigen specific to the target disease, preferably associated with a carrier substance or included in a suitable vector. According to a preferred embodiment of the present invention, the composition further comprises at least one immune checkpoint inhibitor (Marin-Acevedo J. et al., 2018, Hematol. Oncol., 11:39), including, but not limited to, an antibody, preferably a monoclonal antibody, against anti-PD-1, anti-PDL-1, or anti-CTLA4, particularly against the molecules PD-1, PDL-1, or CTLA4, preferably targeting the human molecules hPD-1, hPDL-1, or hCTLA4. The composition according to the present invention advantageously comprises an anti-PD-1 antibody, particularly a monoclonal anti-PD-1 antibody, preferably an anti-hPD-1 antibody. According to another preferred embodiment of the present invention, the composition further comprises at least one vaccine antigen specific to the target disease, preferably associated with a carrier substance or included in a suitable vector.

[0081] The immunomodulatory composition, preferably the immunostimulatory composition, contains an effective dose of complexes, polynucleotides, vectors, or cells sufficient to produce a therapeutic effect against the subject's disease, i.e., to induce an immune response capable of alleviating the symptoms of the disease. This is particularly relevant in the context of alleviating the consequences of the action of a pathogen (infectious or non-infectious) or reducing tumor growth in the individual treated with the composition. The dosage is determined and adjusted based on factors such as the subject's age, sex, and weight. The immunomodulatory composition, preferably the immunostimulatory composition, according to the present invention is in a pharmaceutical form suitable for the selected administration. The composition is typically administered according to conventional immunotherapy protocols, at a dose and for a duration sufficient to induce an effective immune response against the subject's disease. Administration can also be by intratumoral, subcutaneous, intramuscular, intravenous, intradermal, intraperitoneal, oral, sublingual, rectal, vaginal, intranasal, inhalation, or transdermal application. The composition is in a dosage form suitable for the selected administration.

[0082] Polynucleotides isolated or inserted into a plasmid vector can be introduced into a subject using physical methods such as electroporation, or by associating them with substances that allow them to cross cell membranes, such as transporters, e.g., nanotransporters, liposomes, lipids, or cationic polymers. Furthermore, these methods can be advantageously combined, e.g., electroporation associated with liposomes can be used.

[0083] The immunomodulatory composition, preferably the immunostimulatory composition, according to the present invention is used in immunotherapy, particularly antitumor or anti-infective immunotherapy. The immunomodulatory composition, preferably the immunostimulatory composition, according to the present invention is used prophylactically or curatively, i.e., for the prevention of disease in an individual or for the treatment of an individual suffering from a disease. According to a preferred embodiment of the present invention, the composition is used curatively, i.e., to treat an individual suffering from a disease. It can be used in combination with other treatments, whether therapeutic or surgical, and in combination with other therapeutic agents as defined above, the composition according to the present invention and the other therapeutic agents can be administered simultaneously, separately, or sequentially.

[0084] According to a preferred embodiment of the present invention, the composition is for use in the treatment of cancer, which may be any type of cancer that may benefit from immunotherapy, including but not limited to cancer of the breast, colon, prostate, esophagus, stomach, lung, ENT (ear, nose, throat), skin, ovary, uterus, brain, liver, and kidney.

[0085] According to another preferred embodiment of the present invention, the composition is used to treat infectious diseases, particularly those in which infectious agents remain in the body. The infectious disease can be any type of infection that may benefit from immunotherapy (Wykes MN et al., Nat. Rev. Immunol., 2018, 18:91-104), including, but not limited to, viral, bacterial, fungal, or parasitic infections, particularly infections caused by HIV, HBV, HCV, Mycobacterium tuberculosis, or Plasmodium falciparum. 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 (infection caused by Corynebacterium diphtheriae or Corynebacterium diphtheriae).

[0086] The immunomodulatory compositions, preferably immunostimulatory compositions, according to the present invention are used by conventional therapy, or by cell therapy, or by a combination of the two approaches.

[0087] Cell therapy involves the preparation of antigen-presenting cells, particularly dendritic cells, NK cells, or NKT cells, by isolating peripheral blood mononuclear cells (PBMCs) from a patient and culturing the dendritic cells, NK cells, or NKT cells in the presence of the molecular complexes, polynucleotides, or vectors defined above. In a second step, the antigen-presenting cells, NK cells, or NKT cells loaded with the molecular complexes or modified by the polynucleotides or vectors are reinfused into the patient.

[0088] Another subject of the present invention is immunotherapy, in particular an antitumor or anti-infection method, characterized in that it comprises administering to an individual, by any suitable means as defined above, an immunomodulatory composition, preferably an immunostimulatory composition, as defined above.

[0089] Another subject of the present invention is the use of an immunomodulatory composition, preferably an immunostimulatory composition, as defined above, for preparing a medicament intended for immunotherapy, preferably antitumor or anti-infectious immunotherapy.

[0090] The complexes of APC, NK, or NKT cell ligands according to the present invention can be produced by conventional techniques known to those skilled in the art, i.e., the ligands for APC, NK, or NKT cell surface molecules can be produced by chemical synthesis or by expression of recombinant DNA in suitable eukaryotic or prokaryotic cell systems. Peptides or proteins can be synthesized on solid phase according to the Fmoc technique first described by Merrifield et al. (J. Am. Chem. Soc., 1964, 85, 2149-) and purified by reverse-phase high-performance liquid chromatography. Polypeptides and proteins can be produced from the corresponding cDNA cloned into a suitable eukaryotic or prokaryotic expression vector, and the polypeptides or proteins produced in cells engineered with the recombinant vector can be purified by any suitable means, particularly affinity chromatography. Antibodies against APC, NK, or NKT cell surface molecules are well known and commercially available. For example, but not limited to, anti-CD205 (#555831); anti-CD206 (#555952); anti-CD209 (#551186); and anti-HLA-DR (#555556) Abs are available from BECTON-DICKINSON, anti-CD56A (#304622) is available from Biolegend, and anti-CD335 (AM31284AF-N) and anti-CD336 (#AM50346PU-N) Abs are available from Origene. Alternatively, monoclonal antibodies can be produced by conventional techniques known to those skilled in the art. For example, monoclonal Abs are produced from hybridomas obtained by fusing B lymphocytes from animals immunized with APC surface molecules with myelomas according to the technique of Koehler and Milstein (Nature, 1975, 256, 495-497), and the hybridomas are cultured in vitro, particularly in fermenters, or produced in vivo in the form of ascites. Alternatively, monoclonal Abs are produced by genetic engineering, as described in U.S. Pat. No. 4,816,567. Humanized Abs are produced by conventional methods, such as those described in International Application WO 98 / 45332.Ab fragments were cloned from the mRNA of hybridomas or splenic lymphocytes of immunized animals. H and V L For example, Fv, scFv, or Fab fragments are expressed on the surface of filamentous phage according to the technique of Winter and Milstein (Nature, 1991, 349, 293-299). After several selection steps, Ab fragments specific for the Ag are isolated and expressed in a suitable expression system using conventional techniques for recombinant DNA cloning and expression. The Ab or its fragments are purified by conventional techniques known to those skilled in the art, such as affinity chromatography.

[0091] Covalent linkage of a first ligand (L1) to a second ligand (L2) on the surface of APCs or NK or NKT cells can be achieved by constructing a fusion protein in which nucleotide sequences encoding L1 and L2 are fused in frame in the appropriate order, either directly or via a nucleotide sequence encoding a suitable spacer peptide. Depending on the size of each of the L1 and L2 amino acid sequences, they can be fused at their termini (the N-terminus of one sequence is fused to the C-terminus of the other), or one sequence can be inserted into the other sequence at an appropriate site that does not adversely affect binding of the ligand to its receptor expressed on the surface of APCs or NK or NKT cells. Alternatively, the ligands can be covalently linked by any suitable means. Coupling can be achieved via reactive groups that are either present initially or previously introduced into the ligand. Coupling can be achieved particularly with amino acid residues whose side chains contain reactive functional groups. Among these amino acids, polar amino acids containing the following functional groups can be mentioned: Ligands are typically functionalized with reactive functional groups such as -OH [serine (S), threonine (T), or tyrosine (Y)], -SH [cysteine ​​(C)], -NH [lysine (K) or arginine (R)], -COOH [aspartic acid (D) or glutamic acid (E)], and polar amino acids with side chains functionalized by adding reactive functional groups, particularly chloroacetyl or bromoacetyl groups that react with thiols or hydrazine groups that react with aldehydes. The ligands are coupled by any suitable means. These means, known to those skilled in the art, include, inter alia, coupling using homobifunctional reagents such as glutaraldehyde or dithiobis(succinimidyl propionate). Preferably, coupling is performed using heterobifunctional reagents, particularly m-maleimidobenzoyl-N-hydroxysuccinimide (SMCC) or sulfo-SMCC, each of which contains a maleimide group capable of reacting with free thiols. In this case, SMCC is first covalently attached to the amine functional groups present on the ligand.Simultaneously, another heterobifunctional reagent (such as N-succinimidyl-S-acetylthioacetate, which contains a thioester group that can be cleaved with hydroxylamine, or succinimidyl-pyridyl-dithiopropionate, which contains a disulfide bridge that can be reduced under mild conditions) is attached to the amine functional group of the second partner, one of the ligands. Subsequently, the second partner is treated with hydroxylamine or a reducing agent to allow the release of the thiol. The thiolated compound is then incubated with a compound incorporating a maleimide, and coupling is achieved by reaction of the thiol group on the maleimide group. This type of covalent bond is described, inter alia, by Leonetti et al., J. Exp. Med., 1999, 189, 1217-1228. It is also possible to release the thiol group already present on one of the compounds and couple it to another compound that has been previously modified using SMCC. This method, which is often used to couple Abs to ligands, is described inter alia in Ishikawa et al., J. Immunoassay, 1983, 4, 209-327.

[0092] The non-covalent complex is prepared by contacting the second ligand (L2) with the first ligand (L1) under conditions that allow the two partners to interact. This interaction may involve a linker, particularly a protein or peptide with high and specific affinity for one of the partners in the complex (L1 or L2). In particular, the affinity of the linker for this partner in the complex is sufficient so that it does not immediately dissociate 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, the immunoglobulin-binding element is covalently bound to L1 to form a non-covalent complex with L2.

[0093] The polynucleotides according to the present invention can be obtained by 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 by hybridization with homologous probes, or by total or partial chemical synthesis. Recombinant vectors can be constructed and introduced into host cells by conventional recombinant DNA and genetic engineering methods known per se.

[0094] Unless otherwise indicated, the practice of the present invention employs conventional methods of immunology, cell culture, cell biology, molecular biology, and recombinant DNA, which are known to those skilled in the art. [Brief explanation of the drawings]

[0095] Other features, details and advantages of the invention will become apparent on reading the following detailed description with reference to exemplary embodiments of the invention and on examining the accompanying drawings, in which: FIG.

[0096] [Figure 1] Figure 1 shows the molecular weight and homogeneity of molecular complexes produced in E. coli or HEK cells. Proteins ZZ-DTRBDHEK, ZZ-DTRBDcoli, BB-DTRBDHEK, BB-DTRBDcoli, and ZZ-Tat22-57c(22-37)S were analyzed by electrophoresis under denaturing conditions. Proteins were deposited in the presence of molecular weight markers. After electrophoresis, proteins were stained with Coomassie blue.

[0097] [Figure 2]Figure 2 shows the binding of various molecular complexes to heparin. Serial dilutions of the proteins ZZfree, ZZ-DTRBDHEK, ZZ-DTRBDcoli, BB-DTRBDHEK, BB-DTRBDcoli, and ZZ-Tat22-57c(22-37)S were incubated in microtiter plate wells pre-adsorbed with rabbit IgG at pH 7.2. After 4 hours, the plates were washed, and biotinylated heparin was added. After a 30-minute incubation, heparin binding to the plate was detected using streptavidin conjugated to peroxidase and its enzyme substrate (ABTS). Heparin binding was considered significant when the optical density signal was at least 50% of the signal measured when ZZ-Tat22-57c(22-37)S was incubated at 100 nM at pH 7.2 on the microtiter plate.

[0098] [Figure 3] Figure 3 shows the cell types bound by the fusion protein ZZ-DTRBDHEK in a population of mouse splenocytes. Splenocytes were incubated with or without a fixed amount of ZZ-DTRBDHEK (100 nM). A series of fluorescent Abs specific for CD11b+ pDCs, cDCs, and CD8+ cDCs (A); CD4+ T lymphocytes, CD8+ T lymphocytes, B lymphocytes, and monocytes (B) were then added. After 30 minutes at 4°C, cells were washed and analyzed by flow cytometry. The percentage of cells bound by ZZ-DTRBDHEK in the subpopulations of interest is shown.

[0099] [Figure 4] Figure 4 shows that ZZ-DTRBDHEK induces the secretion of IL-6 and IL-12 by splenocytes. A fixed amount of ZZ, DTRBD, or ZZ-DTRBDHEK (1 μM) was incubated with mouse splenocytes. After 24 hours, the supernatant was removed, and the presence of IL-6 and IL-12 was assessed by enzyme immunoassay.

[0100] [Figure 5]Figure 5 shows that ZZ-Tat22-57c(22-37)S induces the secretion of IL-6 and IL-12 by splenocytes. A fixed amount of ZZ, TatCY49-57, ZZ + TatCY49-57, or ZZ-Tat22-57c(22-37)S (1 μM) was incubated with mouse splenocytes. After 24 hours, the supernatants were collected, and the presence of IL-6 and IL-12 was assessed by enzyme immunoassay.

[0101] [Figure 6] Figure 6 shows that the proteins ZZ-DTRBDHEK and BB-DTRBDHEK induce greater secretion of IL-6 and IL-12 by splenocytes than ZZ-DTRBDcoli and BB-DTRBDcoli. A fixed amount of ZZ-DTRBDHEK, ZZ-DTRBDcoli, BB-DTRBDHEK, and BB-DTRBDcoli (1 μM) was incubated with mouse splenocytes. After 24 hours, the supernatants were collected, and the presence of IL-6 and IL-12 was assessed by enzyme immunoassay.

[0102] [Figure 7] Figure 7 shows that ZZ-DTRBDHEK induces the secretion of IL-6 and IL-12 by isolated mouse dendritic cells. Dendritic cells were isolated by magnetic sorting. Dendritic cells were incubated with or without a fixed amount of ZZ-DTRBDHEK (0.6 μM). After 24 hours, supernatants were collected and the presence of IL-6 and IL-12 was assessed by enzyme immunoassay.

[0103] [Figure 8AB]Figure 8A-B shows that the proteins ZZ-DTRBDHEK and ZZ-Tat22-57c(22-37)S can induce the proliferation of dendritic cells in mice. Three groups of mice were injected with PBS buffer at 3-day intervals in the presence or absence of a fixed amount of ZZ-DTRBDHEK (5 nmol per mouse) or ZZ-Tat22-57c(22-37)S (10 nmol per mouse). 24 h after the last injection, the animals were euthanized, and the spleens were removed. Splenocytes labeled with a cocktail of fluorescent Abs allowed the identification of cDC-CD8+ (CD11ChighB220-CD8+CD11b-) (A), cDC-CD11b+ (CD11ChighB220-CD8-CD11b+) (B), and pDC (CD11c+CD317+CD11b+).

[0104] [Figure 8C] Figure 8C shows cells analyzed by flow cytometry. The number of positive cells is shown as a percentage of the total number of viable splenocytes. Statistical analysis was performed using the Kruskall-Wallis test (*p<0.05).

[0105] [Figure 9] Figure 9 shows that ZZ-DTRBDHEK induces the secretion of IL-6 and IL-12 by in vitro isolated human DCs. Isolated DCs derived from healthy human donors were incubated in the presence of a fixed amount of ZZ-DTRBDHEK (1 μM). After 24 hours, supernatants were collected and the presence of IL-6 and IL-12 was assessed by enzyme immunoassay.

[0106] [Figure 10]Figure 10 shows that mice injected with colorectal cancer cells subsequently injected with ZZ-DTRBDHEK alone or with an adjuvant mixture exhibit delayed tumor growth. A: Sixteen C57BL / 6 mice were subcutaneously injected with 0.5 M MC38 cells. Three days later, eight mice were injected with the adjuvant mixture CpG-B1018 / PolyI:C (30 μg of each adjuvant), and eight mice were not (control). A second injection was administered three days later. B: Eighteen C57BL / 6 mice were subcutaneously injected with 0.5 M MC38 cells. Three days later, six mice were injected with ZZ-DTRBDHEK in PBS buffer, six mice were injected with ZZ-DTRBDHEK (2 nmol per mouse) in PBS buffer containing the adjuvant mixture CpG-B1018 / PolyI:C (30 μg of each adjuvant), and six mice were not (control). Two more injections were performed at 3-day intervals. Tumor growth was monitored by measuring the tumors using calipers. Each injection is depicted by an arrow.

[0107] [Figure 11]Figure 11 shows that mice injected with colorectal cancer cells subsequently injected with ZZ-DTRBDHEK, ZZ-Tat22-57C(22-37)S, along with an adjuvant mixture, exhibited delayed tumor growth. Thirty-two C57BL / 6 mice were subcutaneously injected with 0.5 M MC38 cells. Three days later, groups of eight mice were injected with ZZ-DTRBDHEK (0.96 nmol per mouse) in 50 μl of PBS containing the adjuvant mixture CpG-B1018 / PolyI:C (30 μg of each adjuvant). Another group was injected with anti-PD-1 Ab (50 μl, 1.3 nmol per mouse), a third group with ZZ-Tat22-57C(22-37)S (0.96 nmol per mouse) in 50 μl of PBS containing the adjuvant mixture CpG-B1018 / PolyI:C (30 μg of each adjuvant), and a final group of eight mice was not injected (control). Two additional injections were given, 3 days apart. Tumor growth was monitored by measuring tumors using calipers. Each injection is depicted by an arrow.

[0108] FIG. 11A shows the kinetics of tumor growth. FIG. 11B shows the kinetics of tumor growth. FIG. 11C shows the survival of treated animals.

[0109] [Figure 12]Figure 12 shows that the formation of molecular complexes ZZ-Tat22-57C22-37)S, anti-DEC205 / ZZ-Tat22-57C22-37)S, and IgG / ZZ-Tat22-57C22-37)S promotes the increase in the percentage of activated monocytes and CD4+ T lymphocytes in vitro. Fixed concentrations (0.1 μM) of anti-DEC205 / ZZ-Tat22-57C22-37)S, IgG / ZZ-Tat22-57C22-37)S, anti-DEC205 / ZZ, IgG / ZZ, and anti-DEC205 Abs were incubated with human PBMCs. Fixed concentrations (1 μM) of ZZ-Tat22-57C22-37)S or ZZ were incubated with human PBMCs. After 24 h, cells were harvested and labeled with fluorescent Abs. The proportion of activated monocytes (CD3-CD14+) in the population of live PBMCs was assessed using the marker CD69.

[0110] FIG. 12A shows that the proportion of activated CD4+ T lymphocytes in the viable CD4+ T lymphocyte population (CD3+CD4+) was assessed by measuring the expression of the CD69 molecule. In Figure 12B, cells were analyzed by flow cytometry.

[0111] [Figure 13] Figure 13 shows that molecular complexes (anti-CD74 / ZZ-Tat22-57C22-37)S, anti-CD209 / ZZ-Tat22-57C22-37)S, and anti-CD275 / ZZ-Tat22-57C22-37)S) against receptors expressed by dendritic cells induce IL-6 secretion by PBMCs. Human PBMCs were incubated with or without free Ab, ZZ, or ZZ-Tat22-57C(22-37)S or molecular complexes targeting the molecules CD74 (A), CD209 (B), and CD275 (C), respectively. After 24 hours, supernatants were removed, and the presence of IL-6 was assessed by enzyme immunoassay.

[0112] [Figure 14]Figure 14 shows that the molecular complex (anti-CD335 / ZZ-Tat22-57C(22-37)S) against the CD335 receptor expressed by NK and NKT cells induces an increase in the percentage of activated NK and NKT cells. PBMCs were incubated with free Ab, ZZ, or ZZ-Tat22-57C(22-37)S, or with or without the molecular complex anti-CD335 / ZZ-Tat22-57C(22-37)S, anti-CD335 / ZZ. After 18 hours, cells were analyzed by cytometry to assess the expression of the costimulatory molecule CD69 on the surface of NK cells (A) and NKT cells (B).

[0113] [Figure 15] Figure 15 shows that molecular complexes directed against two receptors expressed by NK cells and NKT cells, respectively, induce an increase in the percentage of activated NKT cells. PBMCs were incubated with or without free Ab, ZZ, or ZZ-Tat22-57C(22-37)S, or the molecular complexes anti-CD56 / ZZ-Tat22-57C(22-37)S, anti-CD56 / ZZ, anti-CD336 / ZZ-Tat22-57C(22-37)S, or anti-CD336 / ZZ. After 18 hours, cells were analyzed by cytometry to assess the expression of the costimulatory molecule CD69 on the surface of NKT cells.

[0114] [Figure 16] Figure 16 shows that molecular complexes (anti-CD56 / ZZ-Tat22-57C22-37)S, anti-CD335 / ZZ-Tat22-57C22-37)S, and anti-CD336 / ZZ-Tat22-57C22-37)S) directed against receptors expressed by NK cells induce IL-6 secretion by human PBMCs. PBMCs were incubated with free Abs, ZZ or ZZ-Tat22-57C(22-37)S, and with or without the molecules CD56 (A), CD335 (B), and CD336 (C). After 24 hours, supernatants were removed, and the presence of IL-6 was assessed by enzyme immunoassay.

[0115] [Figure 17] Figure 17 shows that molecular complexes containing anti-ICP antibodies (anti-CTLA-4 / ZZ-Tat22-57C22-37)S, anti-PD-L1 / ZZ-Tat22-57C22-37)S, and anti-OX40 / ZZ-Tat22-57C22-37)S) induce IL-6 secretion by human PBMCs. PBMCs were incubated with or without the free forms of Ab, ZZ, or ZZ-Tat22-57C(22-37)S, and with or without molecular complexes targeting the molecules CTLA-4 (A), PD-L1 (B), and OX40 (C), respectively. After 24 hours, supernatants were removed, and the presence of IL-6 was assessed by enzyme immunoassay. [Example]

[0116] Example 1: Expression, purification, biochemical characterization, and heparan sulfate binding capacity study of fusion proteins in E. coli or HEK cells. material and method <Formation of various molecular complexes>

[0117] ZZ-DTRBD coli The expression of the fusion protein ZZ-DTRBD in E. coli, called ZZ-DTRBD, was described in a publication by Lobeck et al. (Infection and Immunity, 1998, 66, 418-423). coli (SEQ ID NO: 12) is encoded by a polynucleotide having the sequence of SEQ ID NO: 11. Fusion protein ZZ-Tat in E. coli 22-57C(22-37)S Expression of fusion ZZOVAT 22-57S The fusion protein ZZ-Tat was produced according to a protocol similar to that described in the publication by Knittel et al. (Vaccine, 2016, 34(27):3093-3101). 22-57C(22-37)Scoli (SEQ ID NO: 20) is encoded by a polynucleotide having the sequence of SEQ ID NO: 19. For expression in eukaryotic cells, HEK cells (2.5 x 10 in 250 ml of 293F Freestyle medium) were cultured in 250 ml of 293F Freestyle medium. 6cells / ml) in the presence of PEI (0.5 mg / ml) and ZZ-DTRBD HEK The cells were transfected with the pCDNA3.4 plasmid encoding the fusion protein ZZ-DTRBD (400 μg DNA preparation obtained from a transfection maxiprep). HEK The polynucleotide contained a polynucleotide having the sequence of SEQ ID NO: 13 encoding (SEQ ID NO: 14). The cells were then incubated at 37°C with stirring for 24 hours. 250 ml of Ex-Cell medium was then added. After incubation at 37°C with stirring for 4 days, the culture supernatant was collected, filtered under sterile conditions, and a protease inhibitor cocktail was added.

[0118] After expression of the three proteins, the supernatants were each diluted 1 / 2 with 0.1% PBS-Tween and then passed through an IgG Sepharose column (IgG Sepharose 6 Fast flow #17-0969-02, Amersham) to purify the molecular proteins by immunoaffinity. The acidity of the fusion proteins eluted from the column was neutralized with 1 M Tris-HCl buffer, pH 8. The fusion protein, ZZ-DTRBD, derived from expression in E. coli. coli and ZZ-Tat 22-57C(22-37)S was subjected to a second purification cycle using a Mono S5 / 50 cation exchange column (GE Healthcare). coli For the purification of ZZ-Tat, the column was equilibrated with 0.05 M phosphate-citrate buffer, pH 5.5. 22-57C(22-37)S For the purification of the fusion protein ZZ-DTRBD, the column was equilibrated with 0.05 M phosphate-citrate buffer, pH = 4. coli and ZZ-Tat 22-57C(22-37)S The protein was eluted with a linear gradient of 0 to 1 M NaCl. The protein was finally concentrated with PBS and stored at -20°C until use. HEK and BB-DTRBD coli ZZ-DTRBD HEK and ZZ-DTRBD coliThe fusion protein BB-DTRBD was produced according to the same protocol used in 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.

[0119] <Molecular complex ZZ-DTRBD by gel electrophoresis HEK , ZZ-DTRBD coli , BB-DTRBD HEK and BB-DTRBD coli Analysis of molecular weight and uniformity of Protein ZZ-DTRBD HEK , ZZ-DTRBD coli , BB-DTRBD HEK and BB-DTRBD coli The proteins, as well as molecular weight markers, were deposited on 4-12% SDS-PAGE gels under denaturing conditions and then subjected to electrophoretic migration. After migration, the presence of protein bands was revealed using Coomassie blue staining.

[0120] <Binding of molecular complexes to heparin> To assess the binding of the molecular complex to heparan sulfate, heparin, a sulfated sugar representative of the heparan sulfate family, was used.

[0121] The interaction was evaluated using an enzyme immunoassay. For this purpose, a series of microtiter plates was pre-adsorbed with rabbit IgG (1 μg / 100 μl / well in 0.1 M phosphate buffer, pH 7.2) and then saturated with a buffer solution containing 0.3% bovine serum albumin (200 μl / well in 0.1 M phosphate buffer, pH 7.2). Another series of microtiter plates was saturated with a buffer solution containing 0.3% bovine serum albumin (300 μl / well in 0.1 M phosphate buffer, pH 7.2). Subsequently, two series of plates were washed and the protein ZZ-DTRBD was added. HEK , ZZ-DTRBD coli , BB-DTRBD HEK and BB-DTRBDcoli and ZZ-Tat 22-57C(22-37)S Serial dilutions of ZZ-Tat (0.1% bovine serum albumin in 0.1 M phosphate buffer, pH 7.4) and free ZZ were added to the wells. After 4 hours of incubation at ambient temperature, the plates were washed, and 100 μl of heparin-biotin (1 μM) was added per well. After 1 hour at ambient temperature, the plates were washed, and 100 μl of streptavidin conjugated to peroxidase (1 / 2000 dilution) was added. After 30 minutes of incubation, the plates were washed, and the substrate (ABTS) was added. Staining was measured at 414 nm after 30 minutes of incubation. To exclude nonspecific binding to albumin, the optical signal measured on the plate adsorbed with bovine serum albumin alone was subtracted from the signal measured on the plate adsorbed with IgG. The optical density signal was determined by subtracting the optical density signal measured on the plate adsorbed with IgG from the ZZ-Tat. 22-57C(22-37)S Heparin binding is considered significant if it is 50% or greater than the signal measured when incubated at 100 nM, pH 7.2 on a microtiter plate.

[0122] <Result> We previously constructed a fusion protein called ZZ-DTRBD, which incorporates both the double ZZ domain from Staphylococcus aureus protein A and the DTRBD domain from diphtheria toxin (Lobeck et al., Infection and Immunity, 1998, 66, 418-423). Like protein A, ZZ can bind to the Fc region of immunoglobulins. DTRBD binds to the diphtheria toxin receptor and contains a heparan sulfate-binding site located in region 453-467 (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 various cell types bearing surface immunoglobulins and proteoglycan heparan sulfate via interaction with heparan sulfate.

[0123] Similarly, we constructed a fusion protein called BB-DTRBD by replacing the coding sequence for ZZ with a coding sequence called BB, which, like ZZ, corresponds to a dual domain derived from protein A of Staphylococcus aureus, but has the specific feature of also binding to the Fc and Fab regions of immunoglobulins (Jansson 1998 FEMS Immunol. and Med. Microbiol., Leonetti et al. 1999 J. Exp. Med. 189, 1217-28).

[0124] The present inventors have developed ZZ-Tat 22-57C(22-37)S They also constructed a fusion protein called , which contains a double ZZ domain derived from Staphylococcus aureus protein A, which contains a heparan sulfate-binding site, and Tat, derived from the HIV transcriptional transactivator. 22-57C(22-37)S (WO2011 / 092675 and Knittel et al., Vaccine, 2016, 34(27):3093-3101). These properties make ZZ-Tat 22-57C(22-37)S Through its interaction with HS, it can target a variety of cell types bearing surface immunoglobulins and the proteoglycan heparan sulfate.

[0125] We investigated the effects of ZZ-DTRBD, BB-DTRBD and ZZ-Tat 22-57C(22-37)S The complex expressed in E. coli was 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-loaded gel. coli , BB-DTRBD coli , and ZZ-Tat 22-57C(22-37)SThe complexes were subjected to ion exchange chromatography to remove contaminating LPS. Finally, the specific properties of these complexes were evaluated by gel electrophoresis. As can be seen in Figure 1, ZZ-DTRBD coli and BB-DTRBD coli ZZ-DTRBD migrates as a predominant band with a molecular weight of approximately 35 kDa, close to the theoretical weight of the molecule (32230), indicating that this band corresponds to the monomer. Minor bands corresponding to degradation products are also present in the complex of these two molecules. HEK and BB-DTRBD HEK ZZ-DTRBD migrated in several bands distributed substantially between 35 and 150 kDa, indicating that expression in HEK cells resulted in a heterogeneous mixture consisting of monomeric and oligomeric forms of the ZZ-Tat molecular complex. 22-57C(22-37)S migrates as a predominant band, reflecting the homogeneity of the purified protein.

[0126] The molecular weight of approximately 25 kDa is slightly larger than that calculated for the theoretical weight of the molecule (MW = 19145). However, Tat and its derivatives tend to migrate anomalously (e.g., Kittiworakarn et al.), and the fusion protein ZZ-Tat expressed in E. coli and purified 22-57C(22-37)S This strongly suggests that the cellulose is essentially composed of monomers.

[0127] Next, we investigated the suitability of free ZZ and the five molecular complexes for binding to heparin, a sulfated polysaccharide representative of the heparan sulfate family. In enzyme immunoassays, no significant optical signal from free ZZ could be observed (Figure 2). In contrast, ZZ-DTRBD coli , BB-DTRBD HEK , BB-DTRBD coli , ZZ-DTRBD HEK , and ZZ-Tat 22-57C(22-37)S For the DTRBD domain or Tat, the increase in optical density based on the incubation dose is measured. 22-57C(22-37)SThe presence of the domain indicates that the molecular complex can bind to heparin. Furthermore, the ZZ-DTRBD HEK and the BB-DTRBD HEK fusion contains oligomers (see Figure 1), and this data indicates that the presence of oligomers does not inhibit the interaction.

[0128] <Example 2: The fusion protein ZZ-DTRBD HEK can bind to various types of cells in the mouse immune system.> Materials and Methods <Binding of ZZ-DTRBD to immune cells Resuspend C57BI / 6 mouse spleen cells in PBS buffer, 0.5% BSA, 2 mM EDTA at 10×10 6 cells / ml. Place 100 μl of the cell suspension into a 96-well round-bottom plate. Add 100 μl of buffer, 0.5% BSA, 2 mM EDTA per well in the absence or presence of ZZ-DTRBD HEK (100 nM). Incubate the mixture at 4°C for 30 minutes, then wash twice with PBS 0.5% BSA 2 mM EDTA. Add 2 μg of rabbit IgG per well to the cells, incubate at 4°C for 20 minutes, then wash twice with q.s. PBS 0.5% BSA 2 mM EDTA 200 μl. Resuspend the cells in 50 μl of labeling buffer (PBS 0.5% BSA 2 mM EDTA) containing a different mixture of Abs from BioLegend. Incubate the mixture at 4°C in the dark for 20 minutes, then wash the spleen cells twice with q.s. PBS 0.5% BSA 2 mM EDTA 200 μl. Next, fix the cells at ambient temperature for 30 minutes. For this, add 100 μl of �4% PFA buffer, then 100 μl of PBS 0.5% BSA 2 mM EDTA before detection with a BD FACSAria™ cytometer.

[0129] Dendritic cell mixture 1 (replication): B220-FITC (#103206, 1 / 200), CD11c-PE-Cy7 (#117318, 1 / 200), CD317-APC (#127016, 1 / 100), CD 8a-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)

[0130] Mix 2 for monocytes and T and B lymphocytes (duplicate): 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)

[0131] <Result> To assess the ability of the molecular complex to bind to cells of the immune system, ZZ-DTRBD HEK The fusion protein was incubated in the presence of mouse splenocytes. Thus, we observed that the fusion protein preferentially bound to cDC-CD8+ cells, which are specialized APCs (Figure 3A). It also preferentially interacted with monocytes, which also function as APCs (Figure 3B). Finally, it bound to lymphocytes to a lesser extent. Therefore, this data indicates that the fusion protein preferentially targets APCs.

[0132] Example 3: Fusion protein ZZ-DTRBD HEK and ZZ-Tat 22-57C(22-37)S induces in vitro secretion of IL-6 and IL-12 by cells of the immune system material and method C57BL / 6 mouse splenocytes were cultured at 2 × 10 in RPMI medium containing 10% FCS and 1% penicillin / streptomycin. 6 Resuspend 100 μl of cell suspension in 100 μl of the molecular complex (ZZ, DTRBD, ZZ-DTRBD) HEK , or ZZ-Tat 22-57C(22-37)S) Distribute the supernatants into a 96-well plate in the presence or absence of IL-6 and finally incubate at 1 µM. After 24 h of incubation, collect the supernatants for ELISA assays of cytokines IL-6 and IL-12, performed according to the manufacturer's instructions (R&D #DY406-05 and #DY419).

[0133] <Result> The inventors wondered whether the molecular complex could induce activation of cells of the immune system. Since cell activation can lead to cytokine secretion, the inventors decided to evaluate the presence of two cytokines in vitro in the supernatants resulting from incubation of mouse splenocytes with the molecular complex and different control proteins. The first cytokine is IL-6, which represents a pro-inflammatory cytokine important for the initiation of immune responses. The second cytokine is IL-12, which is essential for the induction of cellular immune responses. The inventors investigated the effects of ZZ-DTRBD on the immune system. HEK They found increased amounts of these two cytokines in the supernatant resulting from incubation with ZZ-DTRBD, indicating that this molecular complex induces splenocyte activation (Figure 4). In contrast, they found no increase in the amount of cytokines in the supernatant resulting from incubation with free ZZ or free DTRBD, indicating that the dual domain that binds to the Ig and heparan sulfate binding domains alone is unable to activate immune cells. Therefore, these results support the conclusion that ZZ-DTRBD HEK We show that is capable of inducing activation of cells of the immune system, and that this effect absolutely requires binding of the dual Ig-binding domain and the DTRBD HS-binding domain.

[0134] The present inventors have developed ZZ-Tat 22-57C(22-37)SThey proceeded according to principles similar to those described in the previous paragraph to assess whether Tat can activate cells of the immune system and whether the Tat domains involved in ZZ association and binding to heparan sulfate are absolutely necessary for the stimulatory effect. CY49-57 They used a peptide called ZZ, which contains the basic Tat domain and represents the interaction of Tat and its derivatives with heparan sulfate. They then transformed mouse splenocytes into ZZ, Tat, and CY49-57 , ZZ+TatCY 49-57 , and ZZ-Tat 22-57C(22-37)S They then assessed the presence of IL-6 and IL-12 in the supernatant. 22-57C(22-37)S We found these two cytokines in the supernatant obtained from incubation with ZZ, Tat CY49-57 , ZZ+Tat CY49-57 These results were therefore consistent with those of ZZ-Tat. 22-57C(22-37)S can induce activation of cells of the immune system, and this property is also observed in the free forms of ZZ and Tat CY49-57 These results show that the dual Ig-binding domain is not shared by Tat and that the binding of the basic Tat region involved in HS binding is absolutely necessary for efficacy.

[0135] Example 4: A molecular complex i) maintains the ability to induce the secretion of IL-6 and IL-12 by cells of the immune system when its ZZ domain is replaced by BB, and ii) has an increased stimulatory capacity when it consists of an oligomeric form. material and method C57BL / 6 mouse splenocytes were cultured at 2 × 10 in RPMI medium containing 10% FCS and 1% penicillin / streptomycin. 6 100 μl of the cell suspension was resuspended at 100 cells / ml. 100 μl of the cell suspension was incubated with different molecular complexes (ZZ-DTRBD) at a final concentration of 1 μM. HEK , BB-DTRBD HEK , ZZ-DTRBD coli and BB-DTRBD coliDistribute the supernatant into a 96-well plate in the presence or absence of IL-6 and IL-12. After 24 h of incubation, collect the supernatant for ELISA assays of cytokines IL-6 and IL-12, performed according to the manufacturer's instructions (R&D #DY406-05 and #DY419).

[0136] <Result> The work described in Example 3 was carried out using two molecular complexes containing a ZZ double domain. This double domain, which binds to the Fc region of Abs, can therefore target Abs located on the surface of APCs. The inventors then wondered whether a molecular complex capable of targeting both the Fc and Fab regions of Ig could also activate cells of the immune system. To this end, they focused on the BB double domain derived from protein A of Staphylococcus aureus, which has the particular feature of being able to bind not only to the Fc region of Abs in the same way as Z, but also to the Fb region, unlike ZZ (Jansson B. et al., Fems Immunol. Med. Microbiol. 1998, 20:69-78). They used the complex BB-DTRBD described in Example 1. HEK and BB-DTRBD coli Prepare ZZ-DTRBD HEK and ZZ-DTRBD coli and compared them for their ability to induce secretion of the cytokines IL-6 and IL-12 in vitro when incubated with mouse splenocytes.

[0137] Because we noted that ZZ-DTRBD and BB-DTRBD were in oligomeric form after expression in HEK cells, while being monomeric after expression in E. coli (see Figure 1 ), this study also allowed us to assess whether the oligomerization state affected their ability to induce the immune system.

[0138] The inventors have developed ZZ-DTRBD HEK The supernatant resulting from incubation with BB-DTRBD HEKWe observed that the ZZ-DTRBD cells contained higher amounts of cytokines than those resulting from incubation with ZZ-DTRBD (Figure 6). coli The supernatant obtained from incubation with BB-DTRBD coli The amount of cytokines contained was greater than that obtained from incubation with BB. These results indicate that the BB-containing molecular complex maintains its ability to induce cytokine secretion in vitro, but to a lesser extent than the ZZ-containing molecular complex. Thus, this data indicates that the activating effect may be mediated by a molecular complex containing domains that can bind to different sites on Abs.

[0139] Comparison of the cytokine content in the supernatant based on the cell type used to produce the molecular complex (i.e., HEK vs. E. coli) allowed us to demonstrate differences in the stimulatory effect depending on the type of production. Indeed, the HEK cell-derived complex (ZZ-DTRBD) HEK , BB-DTRBD HEK The supernatant from incubation with the complex (ZZ-DTRBD) produced by expression in E. coli was coli OyoBB-DTRBD coli IL-6 and IL-12 are present in greater amounts than those obtained from incubation with IL-6 (Figure 6). Because the proteins resulting from expression in HEK cells are primarily oligomers (see Figure 1), we speculated that the immune system would be more effectively induced if the molecular complex had a high degree of oligomerization.

[0140] Example 5: Isolated dendritic cells were cultured using ZZ-DTRBD HEK secrete IL-6 and IL-12 when incubated in the presence of material and method After euthanasia of C57BL / 6J mice, spleens were collected in RPMI medium 10% FCS, 1% penicillin / streptomycin. The spleens were perfused with 3 ml of 2 mg / ml collagenase D in HBSS 0.5% BSA and then incubated at 37°C for 30 minutes. Splenocytes were collected, and magnetic sorting of dendritic cells (DCs) was performed according to the manufacturer's instructions (Miltenyi Biotec #130-100-875). DCs were centrifuged at 390 x g for 5 minutes at 4°C, and then 2 x 10 DCs were collected in RPMI medium 10% FCS, 1% penicillin / streptomycin. 6 Resuspend 100 μl of cell suspension (200,000 cells) in a 96-flat-bottom well plate and add 100 μl of RPMI medium 10% FCS 1% penicillin / streptomycin to ZZ-DTRBD HEK Cells are incubated at 37°C for 24 hours and supernatants are collected for ELISA assays of cytokines IL-6 and IL-12 according to the manufacturer's instructions (R&D #DY406-05 and #DY419).

[0141] <Result> The establishment of immune defense mechanisms relies on cooperation between different cellular partners. Among them, dendritic cells (DCs) represent a central APC, as they contribute to the activation of other cell types through direct interactions or through the cytokines they secrete. To evaluate whether ZZ-DTRBD induces cytokine secretion by DCs, DCs were purified from mouse splenocytes C57BI / 6. These DCs were then incubated with or without ZZ-DTRBD for 24 hours, after which the supernatants were removed to assay for the presence of IL-6 and IL-12 (Figure 7). ZZ-DTRBD HEK The amount of IL-6 present in the supernatant obtained from incubation with ZZ-DTRBD is approximately five times the amount found in the supernatant obtained from incubation without the molecular complex. Large amounts of IL-12 (approximately 8700 pg / ml) were also detected in the supernatant obtained from incubation with ZZ-DTRBD. HEKZZ-DTRBD was detected in the supernatants obtained from incubation with the complex, but was barely detectable in those obtained from incubation without the complex. Thus, these data indicate that ZZ-DTRBD may induce the secretion of IL-6 and IL-12 by mouse dendritic cells, potentially contributing to the establishment of immune defense mechanisms.

[0142] Example 6: Proteins ZZ-DTRBD and ZZ-Tat 22-57C(22-37)S can induce dendritic cells in mice material and method Three groups of eight C57BL / 6 mice were treated with ZZ-DTRBD HEK (5 nmol per mouse) or ZZ-Tat 22-57C(22-37)S The animals were injected three times with 100 μl of PBS at 3-day intervals, with or without IgG (10 nmol per mouse). 24 hours after the final injection, the animals were euthanized, and the spleens were harvested to collect splenocytes. The cells were resuspended in 50 μl of labeling buffer (PBS 0.5% BSA 2 mM EDTA) containing different Ab mixtures. The cells were then incubated for 20 minutes at 4°C in the dark and then washed twice with PBS 0.5% BSA 2 mM EDTA. The cells were then fixed in 100 μl of 4% PFA buffer for 20 minutes at 4°C, then washed in PBS 0.5% BSA 2 mM EDTA. Finally, they were resuspended in 20 μl of PBS 0.5% BSA 2 mM EDTA and analyzed using a BD FACSAria™ flow cytometer. The cells were analyzed by flow cytometry. cDC-CD8+ expressed CD11c high B220-CD8+CD11b-, and cDC-CD11b+ are identified as CD11c high identified as B220-CD8-CD11b+, and pDCs as CD11c int Identified as CD317+CD11b+.

[0143] The mixture of Abs used for the phenotypic analysis of dendritic cells is as follows: 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), Live Dead Violet (ThermoFischer #L34964, 1 / 1000)

[0144] <Results> Since the molecular complex preferentially binds to APCs in vitro and induces cytokine secretion, the inventors wondered whether such a complex could induce the expansion of APCs in mice. To evaluate this, the inventors injected a PBS solution into three groups of 8 C57BI / 6 mice in the presence or absence of ZZ-DTRBD HEK or ZZ-Tat 22-57C(22-37)S Next, the animals were euthanized and the spleen was sampled to evaluate the frequency of the various subpopulations of dendritic cells in the splenocytes. As seen in Figure 8, the three mouse DC populations (i.e., DC-CD8+, DC-CD11b+, and pDC) are present in the spleens of the group of mice injected with only PBS. However, in the groups of animals injected with ZZ-DTRBD HEK or ZZ-Tat 22-57C(22-37)S the frequency of these three cell types is significantly increased. Thus, this data indicates that the molecular complex can induce the proliferation of dendritic cells in vivo. Since these cells are central to the initiation of the immune response, these results strongly suggest that the complex can promote the induction of the immune response mechanism.

[0145] <Example 7: ZZ-DTRBD HEK induces the secretion of IL-6 and IL-12 by human dendritic cells in vitro> Materials and Methods <Isolation and incubation of human dendritic cells for the study of IL-6 and IL-12 secretion> The leukocyte-platelet layer bag is diluted 1 / 2 with AIM V medium and then incubated overnight at ambient temperature with agitation. Next, 15 ml of Histopaque medium is added to four Leusep tubes, to which 4 x 25 ml of diluted blood is added. The tubes are centrifuged at 1000 x g for 15 minutes at ambient temperature. The peripheral blood mononuclear cell (PBMC) ring is collected and washed with PBS containing 2 mM EDTA without calcium or magnesium. The PBMC are centrifuged at 150 x g for 10 minutes at ambient temperature, followed by the addition of red blood cell lysis buffer (8.3 mg / ml NH4Cl, 0.84 mg / ml NaHCO3, 0.1 mM EDTA) and incubation at 4°C for 10 minutes. 40 ml of PBS is added, and the cells are then centrifuged at ambient temperature and 150 x g for 10 minutes. DCs are sorted according to the manufacturer's instructions (Miltenyi Biotec #130-091-379). DCs were centrifuged at 390 × g for 5 min at 4°C and then cultured at 2 × 10 in RPMI medium 10% FCS, 1% penicillin / streptomycin. 6 Place 100 μl of sorted DCs (200,000 cells) in a 96-well plate and resuspend in 100 μl of RPMI medium 10% FCS 1% penicillin / streptomycin at 2000 cells / ml. HEK Add 1 μM IgG in the absence or presence of IgG (final concentration 1 μM). Incubate the cells at 37 °C for 24 h and collect the supernatant for ELISA assay of cytokines IL-6 and IL-12 according to the manufacturer's instructions (R&D #DY206-05 and #DY1270-05).

[0146] <Result> ZZ-DTRBD HEK To assess whether ZZ-DTRBD can induce the secretion of IL-6 and IL-12 by DCs from healthy human donors in vitro, the presence of these two cytokines was assessed in the cell culture supernatants after 24 hours of incubation. As can be seen in Figure 9, IL-6 was secreted by ZZ-DTRBD. HEKIL-12 is found in approximately 10-fold greater amounts in supernatants obtained from incubation in the presence of ZZ-DTRBD than in supernatants obtained from incubation in the absence of the fusion protein. HEK ZZ-DTRBD was present in large amounts (2400 pg / ml) in the supernatant obtained from incubation of DCs in the presence of the complex, and in very small amounts (8 pg / ml) in the supernatant obtained from incubation without the molecular complex. Thus, all these data indicate that ZZ-DTRBD induces increased secretion of IL-6 and IL-12 by human DCs. Therefore, ZZ-DTRBD may contribute to the establishment of human immune defense mechanisms related to the secretion of these cytokines.

[0147] Example 8: ZZ-DTRBD and ZZ-Tat 22-57C(22-37)S slows the progression of colorectal tumor growth material and method <Study on the effect of the adjuvant mixture CpG / PolyI:C on tumor growth of mouse colorectal cancer cell lines> Two groups of eight C57BL / 6 mice were subcutaneously injected in the paws with 0.5 μM MC38 cells. Mice were then either uninjected (control) or injected with the adjuvant mixture CpG-B1018 / PolyI:C (30 μg of each adjuvant) 3 and 6 days later. Tumor growth was monitored by measuring tumors using calipers. Mice were euthanized when the discontinuation criteria were met.

[0148] < Molecular complex ZZ-DTRBD injected in the absence or presence of adjuvant HEK Study of the effect of on tumor growth in mouse colorectal cancer cell lines Three groups of six C57BL / 6 mice were injected subcutaneously into the paws with 0.5 M MC38 cells. Three, six, and nine days later, mice were either not injected (control) or injected with ZZ-DTRBD in the absence or presence of CpG-B1018+PolyI:C. HEK(2 nmol per mouse) was injected (30 μg of each adjuvant per mouse). Mice were individually monitored (tumor measurements using calipers) for 15 days before euthanasia.

[0149] Anti-PD-1 Ab and molecular complex ZZ-DTRBD inhibit tumor growth in mouse colorectal cancer cell lines HEK and ZZ-Tat 22-57c(22-37)s Comparison of the effects of Four groups of eight C57BL / 6 mice were injected subcutaneously in the paws with 0.5 M MC38 cells. Three, six, and nine days later, mice were either uninjected (control) or injected with anti-PD-1 antibody (Euromedex #BE0146-100MG, clone RMP1-14), ZZ-DTRBD, in the presence of CpG-B1018+PolyI:C (30 μg of each adjuvant per mouse). HEK or ZZ-Tat 22-57C(22-37)s (0.96 nmol per mouse). Mice are monitored individually over time (tumor measurements using calipers). Once the discontinuation criteria are met, they are euthanized.

[0150] <Result> To assess whether the molecular complex can affect tumor growth, a study was conducted in a syngeneic mouse cancer model. This model is based on a colon tumor cell line called MC38 and C57BI / 6 mice. Tumor induction was induced by injecting 500,000 MC38 cells per mouse.

[0151] First, the effect of the adjuvant mixture CpG1018 / PolyI:C on tumor growth was evaluated. As can be seen in Figure 10A, tumor growth is substantially unaffected in the group of mice injected with the CpG1018 / PolyI:C mixture compared to the untreated (control) group. Therefore, this data indicates that the adjuvant mixture alone is unable to slow the growth of this colorectal tumor.

[0152] Second, ZZ-DTRBD HEKZZ-DTRBD by injection alone or in the presence of the adjuvant mixture CpG1018 / polyI:C HEK The effect of ZZ-DTRBD without adjuvant on tumor growth was evaluated. As shown in Figure 10B, compared to the untreated (control) group, HEK In the group injected with ZZ-DTRBD, tumor growth slowed on days 8 and 10. HEK and even larger in the group treated with the CpG1018 / PolyI:C adjuvant mixture. In fact, in this group, tumor growth slows down throughout the entire period of animal observation (days 6 to 15). Therefore, all of this data supports the conclusion that ZZ-DTRBD HEK These results demonstrate that MC38 cell line proliferation is affected by MC38 mAb, and that this effect is amplified when mixed with the adjuvant CpG1018 / PolyI:C.

[0153] In another series of experiments, the effects of the following treatments on the growth of MC38 tumors in C57BI / 6 mice were compared: ZZ-DTRBD HEK / CpG1018 / PolyI:C, ZZ-Tat 22-57c(22-37)s / CpG1018 / PolyI:C, anti-PD-1 Ab. As can be seen in Figures 11A and 11B, treatment with anti-PD-1 Ab did not significantly affect tumor growth compared to the control group. In contrast, ZZ-DTRBD HEK / CPG1018 / PolyI:C and ZZ-Tat 22-57C(22-37)S Mice injected with IFN-γ / CpG1018 / PolyI:C, respectively, showed reduced tumor growth, which translates into increased survival rates in these two groups of animals (Figure 11C).

[0154] Example 9: CD4+ activated monocytes and lymphocytes can be induced by molecular complexes that target the surface molecule DEC205 and the Fc gamma receptor, respectively. material and method To prepare molecular complexes targeting the DEC205 molecule, we used anti-DEC205 Ab (BioLegend; clone NLDC-145, ref. BLE138202). ZZ-Tat was added at a fixed concentration (0.2 μM for each molecule) in RPMI medium without FCS. 22-57C22-37)S The antibodies were incubated for 24 hours in the absence or presence of anti-DEC205 / ZZ-Tat due to the ability of the ZZ region to bind to the Fc region of the anti-DEC205 Ab. 22-57c22-37)S Using the same protocol, we formed a complex between the ZZ molecule and anti-DEC205 Ab, termed anti-DEC205 / ZZ.

[0155] To prepare molecular complexes targeting the FC gamma receptor, a nonspecific human polyclonal Ab was used. This Ab was synthesized by inducing ZZ-Tat following a protocol identical to that used for the anti-DEC205 Ab. 22-57C(22-37)S or ZZ. Therefore, IgG / ZZ-Tat 22-57C(22-37)S and were able to form a non-covalent molecular complex called the IgG / ZZ complex.

[0156] Human PBMCs prepared as described in Example 7 were then incubated in the presence or absence of the following compounds: anti-DEC205 / ZZ-Tat 22-57C(22-37)S , IgG / ZZ-Tat 22-57C(22-37)S , anti-DEC205 / ZZ, IgG / ZZ, Z-DTRBD HEK , and anti-DEC205 Ab and free IgG (final concentration of each compound: 0.1 μM). In these experiments, PBMCs were treated with ZZ-Tat 22-57C(22-37)SThe cells were incubated with 1 μM of PBMCs and free ZZ protein. After 24 hours, the cells were harvested and labeled with fluorescent antibodies that allow differentiation of 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 analyzed by flow cytometry.

[0157] <Result> The previous example demonstrates that molecular complexes targeting Abs can effectively induce specific immune response mechanisms and slow tumor growth. Abs located on the surface of APCs are the molecules targeted by these complexes. However, APCs express numerous other molecules that may also represent targets for the molecular complexes of the present invention. Therefore, the inventors suspected that molecular complexes targeting receptors other than HS and immunoglobulins on the surface of APCs might also activate these cells. To assess this point, they decided to evaluate two types of receptors. The first is the protein DEC205, a lectin selectively expressed in humans by specific populations of monocytes and dendritic cells (Kato M. et al., 2006, Int. Immunol., 18:857-869). The second is the Fc gamma receptor, most forms of which are expressed by specific populations of monocytes and dendritic cells.

[0158] Next, the inventors prepared two molecular complexes that could target each of these receptors. To target the DEC205 protein, they used a monoclonal antibody specific for this receptor and combined it with the fusion protein ZZ-Tat. 22-57C(22-37)S They conjugated this molecular conjugate to anti-DEC205 / ZZ-Tat 22-57C(22-37)S To target Fc receptors, they used a nonspecific human polyclonal IgG Ab that can interact with these receptors via the Fc domain. They combined this IgG with ZZ-Tat22-57C(22-37)S Between IgG / ZZ-Tat 22-57C22-37)S Therefore, these two molecular complexes are called ZZ-Tat 22-57C(22-37)S Tat 22-57C(22-37)S The Tat domain has the ability to bind to APC receptors and heparan sulfate. In these molecular complexes, the two proteins are non-covalently linked via their respective Fc and ZZ domains. Therefore, since the ZZ domain has already interacted with Ab, it can no longer target immunoglobulins on the surface of APCs. We investigated the ability of Tat to bind to heparan sulfate in order to use it as a control for subsequent activation experiments. 22-57C(22-37)S Two conjugates were also prepared that did not contain the region, designated anti-DEC205 / ZZ and IgG / ZZ, respectively.

[0159] Since the DEC205 molecule and Fc receptors are expressed by monocytes, the inventors next wondered whether a molecular complex could activate this subpopulation of APCs. To this end, they used a fixed concentration (0.1 μM) of each of the following: anti-DEC205 / ZZ-Tat 22-57C(22-37)S , IgG / ZZ-Tat 22-57C(22-37)S PBMCs were incubated in the absence or presence of anti-DEC205 / ZZ IgG / ZZ, anti-DEC205 IgG, and ZZ-Tat at the same concentrations found to be activating in Examples 3, 4, and 5. 22-57C(22-37)S To evaluate the effect of ZZ-Tat 22-57C(22-37)S and free ZZ protein at a concentration of 1 μM. After 24 hours, they assessed the percentage of activated monocytes and CD4+ T lymphocytes. As seen in Figure 12A, ZZ did not alter the percentage of activated monocytes in PBMCs, whereas ZZ-Tat 22-57C(22-37)S The percentage of activated cells was significantly increased by anti-DEC205 Ab, either free or contained in the anti-DEC205 / ZZ complex, indicating that this molecular complex is capable of activating human APCs. The percentage of activated cells was decreased by anti-DEC205 Ab either free or contained in the anti-DEC205 / ZZ complex. In contrast, anti-DEC205 / ZZ-Tat 22-57C(22-37)SThe percentage of activated monocytes increases in the presence of the complex. A similar behavior is observed when considering CD4+ T lymphocytes (Figure 12B). Thus, this data indicates that Abs that are not capable of targeting and activating APCs may be able to do so if they are included in a molecular complex that also allows them to target HS.

[0160] Analysis of the activation state of monocytes after incubation with free human IgG shows that this Ab allows increasing the percentage of activated monocytes. IgG / ZZ is unable to significantly increase the percentage of activated cells. In contrast, incubation of PBMCs with the molecular complex IgG / ZZ-Tat 22-57C(22-37)S When incubated with HCl, the number of activated monocytes more than doubles. A similar behavior is observed when considering CD4+ T lymphocytes (Figure 12B).

[0161] Thus, the data indicate that Abs targeting Fc gamma receptors on the surface of APCs can activate them, and that the activating effect is increased when the Abs are included in a molecular complex that can also target HS.

[0162] Interestingly, these results show a joint increase in the proportion of activated monocytes and T lymphocytes, indicating that the molecular complex induces several cellular actors that play a central role in immune defense mechanisms.

[0163] Example 10: Dendritic cell-targeting molecular complexes induce IL-6 secretion by cells of the immune system in vitro. material and method To prepare molecular complexes targeting DCs, we used three antibodies specific for molecules expressed by DCs. The first Ab (BD Reference 555538) is designated anti-CD74 Ab and targets the CD74 molecule. The second Ab (BD Reference 551186) is designated anti-CD209 Ab and targets the CD209 molecule. The third Ab (BD Reference 552501) is designated anti-CD275 Ab and targets the CD275 molecule.

[0164] Each of these three Abs (30 nM for each molecule) was injected into ZZ-Tat 22-57C(22-37)S The antibodies were incubated in RPMI medium with 5% human Ab serum at 4°C for 24 hours in the absence or presence of anti-CD74 / ZZ-Tat or ZZ (5 nM for each molecule). The ability of the ZZ region to bind to the Fc region of these Abs was assessed by the following methods: anti-CD74 / ZZ-Tat, ... 22-57C(22-37)S , anti-CD209 / ZZ-Tat 22-57C(22-37)S , anti-CD275 / ZZ-Tat 22-57C(22-37)S , allowing them to form different non-covalent molecular complexes, termed anti-CD74 / ZZ, anti-CD209 / ZZ, and anti-CD275 / ZZ.

[0165] Human PBMCs were cultured at 5x10 in RPMI medium with 5% human AB serum. 6 100 μl of the cell suspension was resuspended at 200 cells / ml. 100 μl of the cell suspension was added to the free Ab, ZZ, or ZZ-Tat 22-57C(22-37)S and the cells were dispensed into a 96-well plate in the presence or absence of molecular complexes. After 24 hours of incubation, the supernatants were collected for an ELISA assay of the cytokine IL-6, which was performed according to the manufacturer's instructions (R&D#DY406-05).

[0166] <Result> The inventors wondered whether a molecular complex targeting three molecules expressed by DCs could induce activation of cells of the immune system. Because cell activation can lead to the secretion of cytokines, the inventors decided to evaluate the presence of IL-6 in vitro. They detected this cytokine as a compound called ZZ-Tat 22-57C(22-37)SThey detected IL-6 in the supernatants obtained from incubation with anti-CD74 / ZZ-Tat but not with ZZ. They found none in the supernatants obtained from incubation of PBMCs with free anti-CD74 Ab (Fig. 13A), indicating that in the free form, this Ab is unable to induce activation. We observed that incubation of PBMCs with anti-CD74 / ZZ complexes increased IL-6 secretion, indicating that the ZZ bidomain allows activation mediated by this Ab (Fig. 13A). However, secretion of this cytokine was not observed in the supernatants obtained from incubation of PBMCs with anti-CD74 / ZZ-Tat. 22-57C(22-37)S We show that the complex is even more potent at 100 kJ / s, and that the addition of a Tat domain that allows HSPG binding can increase the cell activation mediated by this complex.

[0167] We observed that free anti-CD209 and anti-CD275 Abs could induce IL-6 secretion when incubated with PBMCs (FIGS. 13B and 13C).

[0168] When these two Abs were complexed with ZZ (anti-CD209 / ZZ and anti-CD275 / ZZ), respectively, secretion of this cytokine was also observed. However, this secretion was not observed when these two Abs were complexed with ZZ-Tat. 22-57C(22-37)S This indicates that the activation potential of the molecular targeting complex may be enhanced if it is able to bind to HSPG.

[0169] Example 11: Molecular complexes directed against the CD335 receptor expressed by NK and NKT cells induce an increase in the proportion of activated NK and NKT cells in vitro. material and method To prepare a molecular complex targeting the CD335 molecule on the surface of NK and NKT cells, we used an antibody called anti-CD335 Ab (Origene AM31284AF-N). 22-57C(22-37)SThe antibodies were incubated in RPMI medium with 5% human Ab serum at 37°C for 5 hours in the absence or presence of either anti-CD335 / ZZ-Tat or ZZ (5 nM for each molecule). The ability of the ZZ region to bind to the Fc region of these Abs resulted in the formation of anti-CD335 / ZZ-Tat Abs, respectively. 22-57C(22-37)S This allowed the formation of a two-molecule complex called anti-CD335 / ZZ.

[0170] Human PBMCs were cultured at 5x10 in RPMI medium with 5% human AB serum. 6 100 μl of the cell suspension was added to the 100 μl of the anti-CD335 Ab, ZZ, or ZZ-Tat 22-57C(22-37)S PBMCs were plated in a 96-well plate in the presence or absence of the molecular complex. After 18 hours, cells were harvested and labeled with fluorescent antibodies that allow differentiation of NK cells (CD56+, BioLegend Reference 362510, diluted 1 / 100), NKT cells (CD56+, BioLegend Reference 362510; CD3+ cells; Miltenyi ref. 130-113-136, diluted 1 / 200), and CD69 molecules (Biolegend Reference 310932, diluted 1 / 100). After a 30-minute incubation, PBMCs were fixed with a solution containing 4% paraformaldehyde and analyzed by flow cytometry.

[0171] <Result> The inventors wondered whether a molecular complex targeting the CD335 molecule expressed on the surface of NK and NKT cells might induce activation of these two cell types. To assess this, they used anti-CD335 Abs either in their isolated form or in molecular complexes. They incubated PBMCs in the absence or presence of different mixtures. Because NK and NKT cells play an important role in immune defense mechanisms, after 18 hours of incubation, they assessed the percentage of activated NK and NKT cells by monitoring the expression of the costimulatory molecule CD69.

[0172] As can be seen in Figure 14, ZZ, isolated anti-CD335 Ab, and anti-CD335 / ZZ complexes do not increase the percentage of CD69-expressing NK (A) or NKT (B) cells in PBMCs. 22-57C(22-37)S In fact, this molecular complex does not increase the percentage of NKT cells expressing CD69, but it does increase the percentage of NK cells expressing this marker, and ZZ-Tat 22-57C(22-37)S The binding of ZZ to HS ligands in the complex enables enhanced activation of NK cells. 22-57C(22-37)S The anti-CD335 Ab affects the activation of both cell subpopulations. In fact, it increases the proportion of NK and NKT cells expressing CD69. Thus, all this data indicates that the anti-CD335 Ab is not capable of activating these two cell types, but may be able to induce them when included in a molecular complex that also allows targeting HS.

[0173] Example 12: Molecular complexes targeting CD56 (A) and CD336 (B), molecules expressed by NK and NKT cells, respectively, induce an increase in the percentage of activated NKT cells in vitro. material and method To prepare molecular complexes targeting the CD56 and CD336 molecules on the surface of NK and NKT cells, we used Ab anti-CD5 (Biolegend reference 304622), which targets the CD56 molecule, and Ab anti-CD336 (Origene AM50346PU-N), which targets the CD336 molecule.

[0174] These Abs inhibited ZZ-Tat at 30 nM. 22-57C(22-37)S The antibodies were incubated in RPMI medium with 5% human Ab serum at 37°C for 5 hours in the absence or presence of either anti-CD56 or ZZ (5 nM for each molecule). The ability of the ZZ region to bind to the Fc region of these Abs resulted in the formation of anti-CD56 / ZZ-Tat Abs, respectively. 22-57C(22-37)S , anti-CD56 / ZZ, anti-CD336 / ZZ-Tat 22-57C(22-37)S, and anti-CD336 / ZZ.

[0175] Human PBMCs were cultured at 5x10 in RPMI medium with 5% human AB serum. 6 Resuspend 100 μl of cell suspension in 100 μl of anti-CD56 Ab, anti-CD336 Ab, anti-CD336 Ab, ZZ or ZZ-Tat 22-57C(22-37)S and dispensed into 96-well plates in the presence or absence of isolated forms of noncovalent molecular complexes.

[0176] After 18 hours, cells were harvested and labeled using fluorescent Abs that allow the identification of NKT cells (CD56+CD3+) and CD69 molecules as described in Example 11. After a 30-minute incubation, PBMCs were fixed using a solution containing 4% paraformaldehyde and analyzed by flow cytometry.

[0177] <Result> The inventors wondered whether a molecular complex targeting the expressed molecules CD56 and CD336 might induce NKT cell activation. To assess this, they used anti-CD56 and anti-CD336 Abs, either individually or in a molecular complex. They incubated PBMCs in the absence or presence of the different mixtures. After 18 hours of incubation, the percentage of activated NKT cells was assessed by monitoring the expression of the costimulatory molecule CD69.

[0178] As can be seen in Figure 15, ZZ, ZZ-Tat 22-57C(22-37)S Anti-CD56 Ab, isolated anti-CD336 Ab, and anti-CD336 / ZZ complex do not increase the percentage of CD69-expressing NKT cells in PBMCs. 22-57C(22-37)S (Figure 15A), anti-CD336 / ZZ-Tat 22-57C(22-37)S In (Figure 15B) a different behavior is observed.

[0179] Indeed, when these two molecular complexes were incubated with PBMCs, an increase in the percentage of NKT cells expressing CD69 was observed. Thus, all these data indicate that isolated anti-CD56 and anti-CD336 Abs do not have the ability to activate NKT cells, but may be able to do so when included in a molecular complex that also targets HS.

[0180] Example 13: Molecular complexes targeting NK and NKT cells induce IL-6 secretion by cells of the immune system in vitro.

[0181] material and method To prepare molecular complexes targeting NK and NKT cells, the three antibodies described in Examples 10 and 11 were used. Each of these three Abs (30 nM for each molecule) was injected into ZZ-Tat 22-57C(22-37)S The antibodies were incubated in RPMI medium with 5% human Ab serum for 24 hours in the absence or presence of either anti-CD56 or ZZ (5 nM for each molecule). The ability of the ZZ region to bind to the Fc region of these Abs resulted in the formation of anti-CD56 / ZZ-Tat Abs, respectively. 22-57C(22-37)S , anti-CD335 / ZZ-Tat 22-57C(22-37)S , anti-CD336 / ZZ-Tat 22-57C(22-37)S , which allowed the formation of various non-covalent molecular complexes called anti-CD56 / ZZ, anti-CD335 / ZZ, and anti-CD336 / ZZ.

[0182] Human PBMCs were cultured at 5x10 in RPMI medium with 5% human AB serum. 6 100 μl of the cell suspension was resuspended at 200 cells / ml. 100 μl of the cell suspension was added to the free Ab, ZZ, or ZZ-Tat 22-57C(22-37)S The cells were then aliquoted into a 96-well plate in the presence or absence of the molecular complex. After 24 h of incubation, the supernatant was collected for an ELISA assay of the cytokine IL-6, which was performed according to the manufacturer's instructions (R&D #DY406-05).

[0183] <Result> We wondered whether a molecular complex targeting NK and NKT cells might induce activation of cells of the immune system. Because cell activation can lead to cytokine secretion, we decided to evaluate the presence of IL-6 in vitro.

[0184] They identified this cytokine as ZZ-Tat 22-57C(22-37)S They found IL-6 in the supernatants obtained from incubation with anti-CD56 and anti-CD335 Abs (Fig. 16A and 16B), indicating that these two proteins are unable to induce activation when in the free form. In contrast, they detected the cytokine in the supernatants obtained from incubation with anti-CD336 Ab (Fig. 16C), indicating that this Ab has stimulatory activity in its free form.

[0185] We performed immunofluorescence assays of PBMCs with anti-CD56 / ZZ-Tat antibodies for the three Abs. 22-57C(22-37)S (Figure 16A), anti-CD335 / ZZ-Tat 22-57C(22-37)S (Figure 16B) and anti-CD336 / ZZ-Tat 22-57C(22-37)S We observed that IL-6 secretion increased when the NK cells were incubated with the complex (Figure 16C). Therefore, this data indicates that the molecular complex targeting NK cells and HSPGs can effectively activate immune cells.

[0186] Example 14: Molecular complexes targeting immune checkpoints (ICPs) induce IL-6 secretion by cells of the immune system in vitro. material and method To prepare molecular complexes targeting ICPs, we used three antibodies specific to molecules thought to be ICPs. The first Ab (BioXCell reference BE0190) is designated anti-CTLA-4 Ab and targets the CTLA-4 molecule. The second Ab (BioXCell reference BE0285) is designated anti-PD-LIAb and targets the PD-L1 molecule. The third Ab (R&D reference MAB10542) is designated anti-OX40 Ab and targets the OX40 molecule.

[0187] Each of these three Abs (30 nM for each molecule) was administered to ZZ-Tat 22-57C(22-37)S The antibodies were incubated in RPMI medium with 5% human Ab serum in the absence or presence of anti-CTLA-4 / ZZ-Tat or ZZ (5 nM for each molecule) for 24 hours at 4°C. The ability of the ZZ region to bind the Fc region of these Abs was assessed by the following methods: anti-CTLA-4 / ZZ-Tat, ... 22-57C(22-37)S , anti-PD-L1 / ZZ-Tat 22-57C(22-37)S , anti-OX40 / ZZ-Tat 22-57C(22-37)S This allowed them to form different non-covalent molecular complexes, termed anti-CTLA-4 / ZZ, anti-PD-LI / ZZ, and anti-OX40 / ZZ.

[0188] Human PBMCs were cultured at 5x10 in RPMI medium with 5% human AB serum. 6 The cells were resuspended at 200 cells / ml. 100 μl of the cell suspension was added to free Ab, ZZ, or ZZ-Tat. 22-57C(22-37)S and distributed into 96-well plates in the presence or absence of molecular complexes. After 24 h of incubation, the supernatants were collected for ELISA assay of the cytokine IL-6, which was performed according to the manufacturer's instructions (R&D#DY406-05).

[0189] <Result> The inventors wondered whether molecular complexes targeting ICP could induce activation of cells of the immune system. Because cell activation can lead to cytokine secretion, the inventors decided to evaluate the presence of IL-6 in vitro. They found that none was detected in the supernatants obtained from incubation of PBMCs with three different free Abs or free ZZ, indicating that these compounds, in their free form, are unable to induce activation (Figures 17A, 17B, and 17C). In contrast, they found that this cytokine was not secreted by ZZ-Tat. 22-57C(22-37)S The supernatant obtained from incubation with Tat and the dual Ig-binding domains were identified. 22-57C(22-37)S The association of the HS binding domains allows activation of the cells in the system. However, the inventors did not differentiate PBMCs into either anti-CTLA-4 or ZZ-Tat antibodies. 22-57C(22-37)S (Figure 17A), anti-PD-LI / ZZ-Tat 22-57C(22-37)S (Figure 17B), and anti-OX40 / ZZ-Tat 22-57C(22-37)S We observed a further increase in IL-6 secretion when cells were incubated with the complex (Figure 17C). In contrast, IL-6 was absent in the supernatants obtained from incubation with anti-CTLA-4 / ZZ, anti-PD-L1 / ZZ, or anti-OX40 / ZZ, indicating that the double ZZ domain does not contribute to the activation mediated by these Abs. Therefore, all of this data indicates that molecular complexes targeting ICPs and HSPGs can induce immune cell activation.

Claims

1. A molecular complex for use as an immunostimulant in the immunotherapy of cancer or infectious diseases, the complex comprising a ligand (first ligand) of at least one sulfated sugar of the glycosaminoglycan family expressed on the surface of an antigen-presenting cell or a natural killer or NKT cell, and a ligand (second ligand) other than a sulfated sugar of the glycosaminoglycan family of a surface molecule of at least one antigen-presenting cell or a natural killer or NKT cell, the ligands bind to each other, and the complex does not contain an antigen specific to the disease of interest.

2. The first ligand is a peptide Tat49-57 (SEQ ID NO:3), Tat37-57 (SEQ ID NO:8) and Tat22-57C, which contain a heparan sulfate binding domain. （２２－３７）Ｓ 2. The molecular complex of claim 1, which is a heparan sulfate binding peptide selected from the group consisting of a peptide derived from the HIV Tat protein comprising at least the basic region Tat49-57 (SEQ ID NO:3) of (SEQ ID NO:9); a polyarginine peptide of R7 to R11; and a peptide comprising the R domain of diphtheria toxin (SEQ ID NO:5) or at least a fragment of said domain DT453-467 (SEQ ID NO:7).

3. 3. The molecular complex of claim 1 or 2, wherein the second ligand targets a surface molecule of an antigen-presenting cell selected from the group consisting of a C-type lectin receptor, a membrane-bound immunoglobulin, a receptor for the constant region of an immunoglobulin, and an immune checkpoint molecule and its ligand.

4. The molecular complex according to any one of claims 1 to 3, wherein the second ligand is selected from the group consisting of: (i) antibodies and fragments thereof comprising at least a paratope directed against a surface molecule of an antigen-presenting cell or a NK or NKT cell; (ii) immunoglobulins and fragments thereof comprising at least the Fc region; and (iii) proteins and protein fragments that bind to the Fc and / or Fab region of an antibody.

5. The molecular complex described in claim 4, wherein the second ligand is selected from the group consisting of Staphylococcus aureus protein A, its BB fragment (sequence number 1) and its ZZ derivative (sequence number 2).

6. A molecular complex according to any one of claims 1 to 5, in the form of an oligomer or a mixture of monomers and oligomers.

7. The molecular complex of any one of claims 1 to 6, which consists of a fusion protein between the first and second ligands.

8. 8. The molecular complex of claim 7, consisting of a fusion protein comprising a first ligand 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 a second ligand selected from the BB fragment of protein A (SEQ ID NO:1) or its ZZ derivative (SEQ ID NO:2).

9. 7. A molecular complex according to any one of claims 1 to 6, wherein the second ligand is an antibody or an antibody fragment and the first ligand forms a fusion protein with an immunoglobulin binding element which is protein A of Staphylococcus aureus, its BB fragment (SEQ ID NO: 1) or its ZZ derivative (SEQ ID NO: 2).

10. 10. The molecular complex of claim 9, wherein the fusion protein comprises a first ligand 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 an immunoglobulin binding element selected from the BB fragment of protein A (SEQ ID NO:1) or its ZZ derivative (SEQ ID NO:2).

11. 11. The molecular complex of claim 10, wherein 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 consisting of a whole immunoglobulin.

12. 9. The molecular complex of claim 8, wherein 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-CD56, anti-CD-335, anti-CD336, anti-CTLA-4, anti-PD-L1, anti-OX40 antibodies, or fragments of the above antibodies comprising at least the paratope.

13. A molecular complex described in any one of claims 1 to 12 for use as an immunostimulant for activating antigen-presenting cells.

14. The molecular complex of claim 13 for use as an immunostimulant for activating dendritic cells or monocytes, for activating NK or NKT cells, and / or for activating the secretion of the cytokines IL-6 and / or IL-12.

15. A composition for use in the immunotherapy of cancer or infectious diseases as an immunostimulant comprising at least one molecular complex according to any one of claims 1 to 14 and at least one pharma- ceutically acceptable carrier, carrier substance and / or adjuvant.

16. The composition of claim 15, wherein the adjuvant is a CpG oligodeoxynucleotide, polyinosinic-polycytidylic acid or a mixture of CpG oligodeoxynucleotide and polyinosinic:polycytidylic acid, and / or the carrier material is a nanoparticle.

17. 17. The composition of claim 15 or 16, comprising at least one other therapeutic agent.

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