Immunomodulatory complex and uses thereof for therapy

Description

Domaine technique

[0001] The invention relates to the field of modulating immunotherapies. The invention relates to a molecular complex consisting of at least one ligand of a sulfated sugar of the glycosaminoglycan family and at least one ligand of another surface molecule of antigen-presenting cells or NK or NKT cells linked together, for use as an immunostimulating drug, in particular in immunotherapy of cancer and infectious diseases. Technique antérieure

[0002] The majority of therapeutic approaches are based on drugs capable of acting directly on the target responsible for the pathology or on cells in a non-physiological state. However, pathological processes can be promoted by dysfunctions of the immune system. Immunosuppression mechanisms are notably observed during tumor progression (Galon J. and D. Bruni, Nat. Rev. Drug Discov., 2019 18(3):197-218) or during infections by an infectious pathogen (Wherry EJ and M. Kurachi, Nature Rev. Immunol., 2015, 15(8):486-499). These observations have led to the development of immunotherapeutic drugs capable of restoring the adequate functioning of the immune defenses, in particular by modulating the activity of T lymphocytes or NK or NKT cells, in order to allow the immune system to control pathological processes more effectively.These modulating immunotherapies aim to restore or inhibit the functionality of a large part of a lymphocyte repertoire, in particular the repertoire of CD4+ T, CD8+ T or regulatory T lymphocytes. They differ in this from vaccine immunotherapies which aim to induce a limited number of lymphocytes corresponding to the cells specific to the Ag(s) included in the vaccine.

[0003] To develop immunotherapeutic drugs, it is first necessary to identify targets that play a crucial role in controlling the immune response. In the 1990s, it was shown that molecules expressed on the surface of T lymphocytes, called PD-1 and CTLA-4 respectively, can induce inhibitory signals that negatively regulate the activity of these cells and can thus regulate certain immune defense mechanisms (Ishida, Y., Agata, Y., Shibahara, K., & Honjo, T. (1992). EMBO J., 11(11), 3887-3895.; Freeman, et al. (2000). J Exp Med, 192(7), 1027-1034.; Krummel MF, Allison JP, J. Exp. Med., 1995 Aug 1;182(2):459-65). These two molecules have been called immune checkpoints, inhibitory immune checkpoint molecules, or inhibitory ICPs.

[0004] The molecular mechanisms responsible for the activity of these inhibitory ICPs 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 called PD-L1 and B7, respectively. The PD-1 / PD-L1 or CTLA-4 / B7 interaction then mediates T-cell inhibition signals and thereby triggers various immunosuppression mechanisms. It has further been shown that antibodies (Ab) specific to PD-1, PD-L1 or CTLA-4 can block the PD-1 / PD-L1 or CTLA-4 / B7 interaction, thus allowing the lifting of inhibition and the reactivation of T lymphocytes (Hodi, FS et al., PNAS. (2003), 100(8), 4712-4717; Iwai, Y. et al., Int. Immunol., (2005). 17(2), 133-144).Some of these immunomodulatory Abs have been shown to be capable of limiting the growth of various cancers (melanoma, lung, etc.) and significantly increasing the life expectancy of patients. They are now commonly used as anti-tumor immunotherapeutic drugs in humans (Adachi K. and K. Tamada, Cancer Sci., 2015;106(8):945-50; Riley RS et al., Nat. Rev. Drug Discov. 2019 18(3):175-196). They are also being considered for other therapeutic areas, particularly for the treatment of infectious diseases (Rao M. et al., Int. J. Infect. Dis., 2017;56:221-228). These first inhibitory anti-ICP antibodies, however, have the disadvantage of only working in 10 to 30% of patients with cancer (Pitt JM et al., Immunity. 2016 Jun 21;44(6):1255-69).Many research groups therefore aim to discover new immunomodulatory compounds that are more effective and / or can be used in combination with the immunotherapies described above. As several new inhibitory ICPs have been identified, work is focused on the selection of compounds capable of binding these ICPs or their ICP-ligand and thus neutralizing the ICP / ICP-ligand binding in order to reactivate T lymphocytes. The discovery of activating ICPs expressed on the surface of the T cell has led to focusing other work on the selection of agonist ligands for these ICPs (Mahoney KM. Et al., Nat. Rev. Drug Discov., 2019, 14:561-584; De Sousa Linhares A., Front. Immunol., 2018, 31;9:1909.; Granier C. et al, ESMO Open, 2017 Jul 3;2(2):e000213).

[0005] To immunomodulate the immune response as effectively as possible, we seek to select therapeutic molecules capable of binding ICP or IC P-ligand selectively expressed on the surface of immune effector cells, such as T lymphocytes and NK or NKT cells, or Ag-presenting cells (APCs). Indeed, this selectivity of expression makes it possible to limit the dissemination of the molecule towards non-immune cells. This results in an increase in therapeutic efficacy and a reduction in the risk of side effects.

[0006] The glycocalyx is notably composed of proteoglycans, which are glycoproteins comprising one or more unbranched glycosaminoglycan (GAG) chains. Among these, the heparan sulfate proteoglycan (HSPG) family corresponds to proteins associated with sulfated GAGs: heparan sulfates (HS). HSPGs, which play a central role in many biological processes (cell proliferation, cell adhesion, inflammation, coagulation, cellular penetration of pathogenic microorganisms, particularly viruses and parasites), are found on the surface of most mammalian cells and in extracellular matrices (Dreyfuss et al., Annuals of the Brazilian Academy of Sciences, 2009, 81, 409-429). This ubiquitous expression therefore leads to the consideration that HSPGs, and their HS domains, do not represent relevant immunomodulatory targets.Furthermore, the ability of HS ligands to induce immune cell activation has not been identified to date. Résumé de l'invention

[0007] In their work, the inventors first discovered that a ligand of a sulfated sugar from the GAG ​​family, heparan sulfates (HS), is incapable of activating dendritic cells. in vitro. They then observed that a molecular complex containing this ligand and a ligand of a surface molecule of antigen-presenting cells (APCg) potentiates the immunomodulatory effect of the heparan sulfate (HS) ligand. Indeed, this molecular complex induces APCg even more strongly as shown by the increase in the secretion of the cytokines IL-6 and IL-12, and allows the subsequent activation of T lymphocytes. The inventors showed that the association between the two ligands can be carried out in the form of a covalent or non-covalent complex, in particular in the form of a fusion protein. The inventors further observed that the immunomodulatory properties of this complex make it possible to better control pathological processes and, in particular, to slow down the progression of a tumor.

[0008] These results are surprising because, although described as being able to play a role as a receptor or coreceptor, it had never been shown that HSPGs can potentiate the activation mediated by a receptor ligand expressed specifically on the surface of APCs. Finally, the molecular complex, which targets ubiquitously expressed HS, could disseminate to the extracellular matrix or to cells of no interest for the pathology. It should therefore theoretically not be able to modulate the activity of APCs and a large repertoire of T lymphocytes with sufficient efficiency for an immunotherapeutic effect to occur.

[0009] The inventors also discovered that the results obtained with APCs were transposable to other cells of the innate immune response, NK and NKT cells. Thus, the inventors showed that a ligand of a surface molecule of NK or NKT cells incapable of inducing these cells on its own becomes capable of increasing the number of activated cells when it is associated, in a molecular complex, with a ligand of a sulfated sugar of the GAG ​​family.

[0010] Accordingly, the present invention relates to a molecular complex for use as an immunomodulatory, preferably immunostimulatory, medicament, said complex consisting of at least one ligand of a sulfated sugar of the glycosaminoglycan family (first ligand or L1) and at least one ligand of another surface molecule of APC or NK or NKT cell (second ligand or L2) linked together, and said complex being devoid of an antigen specific to the disease to be treated.

[0011] According to preferred embodiments of the invention, the first ligand is a heparan sulfate binding peptide selected from the group consisting of: a peptide from the HIV Tat protein comprising at least the basic region Tat 49-57 (SEQ ID NO: 3) such as the peptides Tat 49-57 (SEQ ID NO: 3), Tat37-57 (SEQ ID NO: 8) and Tat22-57C(22-37)S (SEQ ID NO 9); a polyarginine peptide R7 to R11; and a peptide from the R domain of diphtheria toxin comprising the R domain of diphtheria toxin (SEQ ID NO: 5) or at least the fragment DT453-467 (SEQ ID NO 7) of said domain which comprises the heparan sulfate binding region.

[0012] According to preferred embodiments of the invention, the second ligand targets a surface molecule of the APCs selected from the group consisting of: C-type lectin receptors, membrane immunoglobulins, receptors for the constant region of immunoglobulins, and immune checkpoint (ICP) molecules.

[0013] According to preferred embodiments of the invention, the second ligand targets a surface molecule of NK or NKT cells selected from the group consisting of: NKG2D, NKp30, NKp44, NKp46, NKp80, Ly49H receptors, KIR receptors, NKG2A, and ICPs PD-1, CTLA-4, TIM-3, TIGIT, LAG-3.

[0014] Preferably, the second ligand is selected from the group consisting of: (i) antibodies directed against said surface molecules of APCs or NK or NKT cells and fragments thereof containing at least the paratope; (ii) immunoglobulins, preferably IgG, and fragments thereof comprising at least the Fc region; and (iii) proteins and protein fragments which bind the Fc and / or Fab region of the antibodies, in particular protein A of S. aureus, its fragment BB (SEQ ID NO: 1) and its derivative ZZ (SEQ ID NO: 2).

[0015] According to preferred embodiments of the invention, said complex is in the form of oligomers or a mixture of monomers and oligomers.

[0016] According to preferred embodiments of the invention, said complex consists of a fusion protein between the first and the second ligand. A more particularly preferred type of complex according to the invention consists of a fusion protein comprising a first ligand chosen 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 the DT453-467 fragment (SEQ ID NO 7), and a second ligand chosen from: the BB fragment of protein A (SEQ ID NO: 1) or its ZZ derivative (SEQ ID NO: 2).

[0017] According to other preferred embodiments of the invention, the second ligand is an antibody or an antibody fragment and the first ligand forms a fusion protein with an immunoglobulin binding member, preferably protein A of S. aureus, its BB fragment (SEQ ID NO: 1) or its ZZ derivative (SEQ ID NO: 2). Preferably, said 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 the DT453-467 fragment (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 a more particularly preferred type of complex according to the invention, the immunoglobulin binding element is the BB fragment of protein A (SEQ ID NO: 1), and said fusion protein being complexed with the second ligand which consists of a whole immunoglobulin.In another type of complex more particularly preferred according to the invention, said fusion protein is complexed with the second ligand which is chosen from an anti-RFcgamma 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-PD-L1, anti-OX40 antibody or a fragment of the preceding antibodies comprising at least the paratope.

[0018] According to preferred embodiments of the invention, the complex is for use as an immunostimulatory medicament, 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.

[0019] The present invention also relates to a composition for use as an immunomodulatory, preferably immunostimulatory, medicament, comprising at least one molecular complex according to the invention and at least one pharmaceutically acceptable vehicle, a carrier substance and / or an adjuvant. Preferably, the adjuvant is a CpG oligodeoxynucleotide, polyinosinic-polycytidylic acid or a mixture of CpG oligodeoxynucleotide(s) and polyinosinic-polycytidylic acid, and / or the carrier substance is a nanoparticle.

[0020] According to preferred embodiments of the invention, said composition comprises at least one other therapeutic agent, preferably at least one immune checkpoint inhibitor, preferably an anti-PD-1, an anti-PD-L1 or an anti-CTLA-4.

[0021] According to preferred embodiments of the invention, the composition is for use in immunotherapy of cancer or infectious diseases. Exposé de l'invention

[0022] The present invention relates to a molecular complex for use as an immunostimulating drug in immunotherapy of cancer or infectious diseases, said complex consisting of at least one ligand of a sulfated sugar of the glycosaminoglycan family expressed on the surface of antigen-presenting cells or NK or NKT cells (first ligand) and at least one ligand of another surface molecule of APC or NK or NKT cells (second ligand) linked together, and said complex being devoid of an antigen specific to the disease to be treated.

[0023] Such a complex is capable of inducing immune system cells in a way that is favorable to triggering the immune response. Thus, when splenocytes are incubated with these complexes, the activation of dendritic cells is observed, which become capable of secreting IL-6 and IL-12 cytokines. In addition, DC induction can be obtained for second ligands that are incapable of triggering such an effect when unassociated, such as the isolated form of the APC ligand called ZZ. The association of the HS ligand can also potentiate the effect of a ligand capable, in isolated form, of activating APCs, such as a non-specific human polyclonal Ab. The association of the HS ligand with an NK or NKT cell ligand can also activate these cell types, whereas it lacks this capacity in isolated form.Finally, an immunomodulatory complex, preferably an immunostimulant, according to the invention can induce a therapeutic effect, in particular an antitumor effect, greater than that obtained with an anti-ICP (anti-PD1) Ac, in particular when the composition comprises an adjuvant. Définitions

[0024] The term "innate immune cells" refers to dendritic cells, NK and NKT cells, granulocytes (mast cells, neutrophils, eosinophils and basophils) and phagocytes (monocytes, macrophages, neutrophils, etc.).

[0025] The term "antigen presenting cell" means Ag presenting cell, APC, APCg or APC for Antigen Presenting Cell, a cell expressing one or more molecules of the major histocompatibility complex (MHC) class I and class II (HLA class I and class II molecules in humans) and capable of presenting Ags to CD4+ and CD8+ T lymphocytes specific for this Ag. Examples of Ag-presenting cells that may be mentioned include dendritic cells (CD or DC for Dendritic Cell ), monocytes, macrophages, B lymphocytes, lymphoblastoid lines, and genetically modified human or animal cell lines expressing MHC class I and class II molecules, including HLA I and HLA II molecules.

[0026] An NK cell is a granular lymphocyte expressing CD56 and CD16 molecules and having cytotoxic activity that does not require prior exposure to Ag.

[0027] An NKT cell is a granular lymphocyte expressing NK cell markers, including CD56 and CD16 molecules, and T cell markers, including CD3. This cell has cytotoxic activity that does not require prior exposure to Ag.

[0028] A "CPAg surface molecule" means a molecule expressed on the surface of Ag-presenting cells.

[0029] The term "NK or NKT cell surface molecule" means a molecule expressed on the surface of NK or NKT cells.

[0030] The term "APC-specific surface molecule" refers to a molecule expressed primarily on Ag-presenting cells, i.e., expressed on a very limited number of cells other than APCs. It is therefore a molecule with high expression specificity for APCs.

[0031] The term "NK or NKT cell-specific surface molecule" means a molecule expressed primarily on NK or NKT cells, i.e., expressed on a very limited number of cells other than NK or NKT cells. It is therefore a molecule with a high expression specificity for NK or NKT cells.

[0032] Glycosaminoglycan (GAG) is a linear polysaccharide composed of a repeating disaccharide always containing a hexosamine (glucosamine (GlcN) or galactosamine (GaIN)) and another sugar (glucuronic acid (GlcA), iduronic acid (IdoA), galactose (Gal)). Glucosamine is either N-sulfated (GlcNS) or N-acetylated (GlcNac). Galactosamine is always N-acetylated (GalNac). Sulfated glycosaminoglycans include simple polymers of GIcA such as chondroitin sulfate and copolymers comprising both GIcA and / or IdoA and / or Gal residues, such as heparin, heparan sulfate, dermatan sulfate, and keratan sulfate. GAG chains can be covalently linked to proteins (proteoglycans) that are expressed on the surface of mammalian cells and / or secreted into the extracellular medium.The first ligand according to the invention binds to a sulfated sugar of the glycosaminoglycan family which is expressed on the surface of mammalian cells, including among others APCs and NK or NKT cells.

[0033] On means by "Ac", an immunoglobulin (IgG, IgM, IgA, IgD, IgE). The term Ac designates a specific Ac, that is to say an Ac directed against a particular molecule x (anti-molecule x Ac) in particular a surface molecule of APC (anti-APC surface molecule of APC). The term immunoglobulin designates a non-specific Ac.

[0034] “Individual” means a human or animal individual, preferably human.

[0035] The term "ligand" of a molecule means any agent capable of binding this molecule with an affinity high enough to form a stable complex, in vitro And in vivo.

[0036] The term "CPAg ligand" means a ligand of an Ag-presenting cell surface molecule.

[0037] "NK or NKT cell ligand" means a ligand for a surface molecule of NK or NKT cells. "Heparan sulfate ligand" means an agent that binds heparin with an optical density signal at least equal to 50% of the signal measured when ZZ-Tat 22-57C(22-37)S is incubated at 100nM pH7.2 in the ELISA of Example 1.

[0038] The term "antigen" means any substance that can be specifically recognized by the immune system and in particular by antibodies and cells of the immune system (B lymphocytes, CD4+ T lymphocytes, CD8+ T lymphocytes) and capable of inducing a specific immune response. The term "antigen specific to the disease to be treated" means an antigen that induces an immune response directed specifically against the disease to be treated. Said immune response specific to the disease to be treated includes the production of antibodies, and / or the induction of a cytotoxic T response (activation of CD8+ T lymphocytes) or a helper T response (activation of CD4+ T lymphocytes) directed against an antigen of a pathogen or a tumor cell responsible for said disease to be treated.

[0039] The term "immunomodulator" means an agent capable of controlling the immune response and in particular of regulating, in a positive or negative manner, the relative response 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 immunostimulant. The term "immunostimulant" means an agent capable of regulating in a positive manner, i.e. activating, the relative response of different populations or subpopulations of immune cells such as T and B lymphocytes, APCs, NK or NKT cells. The effect of the immunomodulator, preferably immunostimulant, is exerted on a broad cellular repertoire.It is independent of the presence of an antigen specific to the pathology to be treated, unlike a vaccine which requires the presence of an antigen specific to the disease to be treated and induces an immune response specific to said antigen. While the use of an immunogen or a vaccine is limited to the disease comprising the antigen specific to this disease, the immunomodulatory complex, preferably immunostimulatory according to the invention which is independent of the presence of an antigen specific to the pathology to be treated can be used in immunotherapy for many diseases such as cancer and infectious diseases.

[0040] A peptide is a sequence of natural or synthetic amino acids, possibly modified. The term peptide is used regardless of the size of the amino acid sequence.

[0041] The molecular complex according to the invention consists of at least two ligands of APC or NK or NKT cell surface molecules linked together: the first ligand, called L1, which targets a sulfated GAG and the second ligand, called L2, which targets another APC surface molecule. The molecular complex according to the invention may comprise one or more L1 ligands and one or more L2 ligands linked together. According to certain preferred embodiments of the invention, the molecular complex consists of an L1 ligand linked to an L2 ligand.

[0042] The molecular complex according to the invention, preferably an immunostimulating complex, is devoid of antigen specific to the disease to be treated, such as a vaccine antigen specific to the pathology to be treated. When one of the ligands of the complex according to the invention comprises an antigen specific to a disease to be treated, then the treatment of said disease with said complex is excluded from the invention.

[0043] The ligands of APCs, NK or NKT cells are molecules or complexes of molecules, natural, recombinant or synthetic, of protein (protein, peptide, polypeptide), lipid, carbohydrate, nucleic acid or mixed (glycolipid, glycoprotein, lipoprotein) nature. According to certain preferred embodiments of the invention, the first and second ligands are proteins, polypeptides or peptides, hereinafter referred to as "peptides", preferably recombinant or synthetic. The recombinant proteins, polypeptides or peptides are advantageously produced in prokaryotic or eukaryotic cells, in an appropriate expression system, in particular for the production of therapeutic proteins. For example, the recombinant proteins, polypeptides or peptides can be produced in E. coli or in HEK or CHO cells.

[0044] The first and second ligands are associated or linked to each other by any suitable means. They may be linked covalently or non-covalently, either directly or through a linker, and thus form a molecular complex. The molecular complex or complex consists of two or more ligands, and optionally suitable linker(s).

[0045] The covalent bond or association of ligands is notably generated by covalent chemical coupling (formation of a covalent conjugate) or by the construction of a fusion protein (genetic fusion).

[0046] The immunomodulatory complex, preferably an immunostimulatory complex, is in monomeric, oligomeric or mixed form (mixture of monomers and oligomers). According to certain preferred embodiments of the invention, said complex is in oligomeric or mixed form.

[0047] According to certain preferred embodiments of the invention, the immunomodulatory molecular complex, preferably an immunostimulatory complex, consists of a fusion protein between the first (L1) and the second (L2) ligand(s). The amino acid sequences of L1 and L2 are fused in the appropriate order, either directly or via a suitable spacer peptide. Depending on the respective sizes of the amino acid sequences of L1 and L2, they are either fused at their ends (N-terminus of one of the sequences fused to the C-terminus of the other sequence) or one of the sequences is inserted into the other sequence at an appropriate site that does not have a deleterious effect on the binding of the ligand to its receptor expressed on the surface of APCs, NK or NKT cells.

[0048] The non-covalent bond is generated in particular by adsorption on a nanoparticle. It can also be obtained by using a molecule (binding element), having a high and specific affinity for L1 or L2. This binding element is covalently bound to one of the ligands to non-covalently associate the other ligand. When one of the ligands is an antibody (Ab), the binding element is in particular a protein or a protein fragment which binds the Fc and / or Fab region of immunoglobulins, as described in Application FR 2759296. Such immunoglobulin binding elements include in particular protein A of S. aureus, its fragment BB (SEQ ID NO: 1) and its derivative ZZ (SEQ ID NO: 2), the first two proteins binding the Fc and Fab regions of immunoglobulins while ZZ binds only the Fc region. For example, when one of the ligands is an Ab or an antibody fragment, the immunoglobulin binding element is covalently linked (covalent chemical coupling or fusion protein) to the other ligand. The binding element can also bind to partners respectively coupled to L1 and L2, for example, the binding element is Streptavidin which binds to biotinylated L1 and L2 ligands (L1-biot / Streptavidin / biot-L2). The affinity of the binding element for its partner, in the L1-L2 complex, is sufficient so that it does not immediately dissociate from this complex in vivo.

[0049] According to certain preferred embodiments of the immunomodulatory complex of the invention, preferably an immunostimulatory complex, one of the ligands is an antibody or an antibody fragment and the other ligand forms a fusion protein with an immunoglobulin binding member, preferably protein A of S. aureus, its BB fragment (SEQ ID NO: 1) or its ZZ derivative (SEQ ID NO: 2). Preferably, the second ligand is an antibody or an antibody fragment and the first ligand forms a fusion protein with an immunoglobulin binding element, preferably protein A of S. aureus, its fragment BB (SEQ ID NO: 1) or its derivative ZZ (SEQ ID NO: 2).

[0050] The immunomodulatory, preferably immunostimulatory, properties of the complex according to the 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 in particular evaluated by analyzing the expansion and / or activation of different populations or subpopulations of immune cells such as CD4+, CD8+ and B T lymphocytes; monocytes, dendritic cells including conventional dendritic cells (cDC) and plasmacytoid dendritic cells (pDC), NK cells, NKT cells. The analysis of the expansion is in particular carried out by flow cytometry using appropriate markers for the different cell populations to be analyzed.The analysis of the activation of immune cells can be carried out by the detection of activation markers (CD69), and / or maturation (CD86) in flow cytometry or by assaying cytokines secreted in the extracellular medium, in particular IL-6 and / or IL-12, by conventional tests such as ELISA. As mentioned above, the immunomodulatory, preferably immunostimulatory, effect of the molecular complex according to the invention is independent of the presence of an antigen specific to the pathology to be treated.

[0051] The molecular complex according to the invention, preferably an immunostimulatory complex, binds to at least one sulfated sugar of the glycosaminoglycan family expressed on the surface of APCs, NK or NKT (sulfated GAG) and another surface molecule of APCs, NK or NKT, in particular a surface molecule specific to APCs, NK or NKT. The sulfated GAG which is targeted by the first ligand is preferably a heparan sulfate, a chondroitin sulfate, a dermatan sulfate, or a keratan sulfate, preferably a heparan sulfate. The sulfated GAG ligand (first ligand) may be derived from a mammalian cell or a pathogenic microorganism, in particular a virus (Adenovirus, Cytomegalovirus, HIV, Sindbis Virus), a bacterium ( Mycobacterium bovis, Bordetella pertussis ), a parasite ( Leishmania sp. ) or a toxin, it is in particular a molecular complex, a molecule or one of its fragments which binds heparin, and / or heparan sulfates. Such ligands are notably described in "Heparan Binding Proteins", H. Edward Conrad, Academic Press, San Diego and London and Dreyfuss et al., Annuals of the Brazilian Academy of Sciences, 2009, 81, 409-429 (see in particular Table II). Examples of these ligands include, but are not limited to: endogenous ligands of GAG (Thrombin, Urokinase, Vitronectin, fibroblast growth factor and others), the HIV Tat protein (SEQ ID NO: 10) and its fragments, in particular fragments comprising only the basic region of Tat (Tat49-57 (SEQ ID NO: 3)) or the basic region and the central region of Tat (core; Tat38-48 (SEQ ID NO: 4)); dodecahedra derived from the Adenovirus penton (Vivès 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-), the Sindbis virus envelope glycoprotein (Byrnes, AP and Griffin, DE, J. Virol., 1998, 2, 7349-7356) and the diphtheria toxin R domain (DTR or DTRBD; fragment 382 to 535 of DT (SEQ ID NO: 5)); Lobeck et al., Infection and Immunity, 1998, 66, 418-423). Cell penetration peptides can also be mentioned (. CPP pour Cell Penetrating Peptide ) which bind heparin, heparan sulfates and / or chondroitin sulfates such as peptides very rich in basic residues, in particular in arginines which include the peptides derived from the basic region of the HIV Tat protein (Tat49-57), mentioned above, and the polyarginine peptides (R7 to R11), as well as basic / amphiphilic peptides such as the peptides derived from the homeodomain of the Antennapedia protein (penetratin; fragment 43-58 (SEQ ID NO: 6)).

[0052] Alternatively, the first ligand is a natural or recombinant Ac directed against a sulfated GAG, preferably heparin, heparan sulfates or chondroitin sulfates or a fragment of this Ac containing at least the paratope (Ag binding domain) such as a Fab, Fab', F(ab') 2 , Fv or single chain Fv fragment (scFv for single-chain Fv ), Fabc, and Fab fragment comprising a portion of the Fc domain. Such Ac are notably described in Thompson et al., J. Biol. Chem., 2009, 284, 35621-35631 and van Kuppevelt et al., J. Biol. Chem., 1998, 273, 12960-12966. Preferably, said Ac or fragment of Ac is human or humanized.

[0053] According to advantageous embodiments of the molecular complex, preferably immunostimulatory, of the invention, the first ligand 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) such as the peptides Tat 49-57 (SEQ ID NO: 3), Tat37-57 (SEQ ID NO: 8) and Tat22-57C(22-37)S (SEQ ID NO 9); a polyarginine peptide R7 to R11; and a peptide derived from the R domain of diphtheria toxin comprising the R domain of diphtheria toxin (SEQ ID NO: 5) or at least the fragment DT453-467 (SEQ ID NO 7) of said domain which comprises the heparan sulfate binding region.Preferably, the first ligand 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 Tat 49-57 (SEQ ID NO: 3), in particular the peptide Tat22-57C(22-37)S (SEQ ID NO: 9) and a peptide comprising the R domain of diphtheria toxin (SEQ ID NO: 5).

[0054] In advantageous embodiments of the molecular complex, preferably immunostimulatory, of the invention, when the complex comprises the R domain of diphtheria toxin (DTR: SEQ ID NO: 5), then the domain is produced in mammalian cells, in particular in the form of a fusion protein with the second ligand or an immunoglobulin binding element, so as to form oligomers.

[0055] The other molecule expressed on the surface of the APCs that is targeted by the second ligand is either a ubiquitous surface molecule other than a sulfated GAG; a surface molecule expressed primarily on immune cells of the innate or adaptive response including APCs, i.e., a surface molecule specific to immune cells of the innate or adaptive response including APCs; or a surface molecule expressed primarily on APCs, and in particular on dendritic cells, i.e., a surface molecule specific to APCs and in particular to dendritic cells.Among these surface molecules expressed essentially on APCs and in particular on dendritic cells, we can cite in particular: MHC class II molecules, in particular the α, β chains and the γ chain or invariant chain (li, fragment li or CD74) of MHC II molecules; surface immunoglobulins or membrane immunoglobulins; integrins such as in particular CD11c and MAC1; transferrin receptors, C-type lectin receptors 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), including FcγR such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16); the TNF receptor superfamily such as CD40; and complement receptors.Among the surface molecules expressed mainly on APCs, we can cite immune checkpoint molecules (ICPs) and their ligands (ICP-ligands) expressed on APCs, such as, but not limited to, PDL1, PDL2, CD155, CD80, CD86, CD40, OX40L, ICOSL (CD275), CD70 (Wykes MN, Nat. Rev. Immunol., 2018, 18:91-104).

[0056] The other molecule expressed on the surface of the NK or NKT cells that is targeted by the second ligand is either a ubiquitous surface molecule other than a sulfated GAG; a surface molecule expressed primarily on immune cells of the innate or adaptive response including NK or NKT cells, i.e., a surface molecule specific to immune cells of the innate or adaptive response including NK or NKT cells; or a surface molecule expressed primarily on NK or NKT cells, i.e., a surface molecule specific to NK or NKT cells. Among these surface molecules expressed primarily on NK or NKT cells, we can cite in particular: NKG2D, NKp30, NKp44, NKp46, NKp80, CD56, CD16, the KIR receptors, NKG2A, and the ICPs PD-1, CTLA-4, TIM-3, TIGIT, LAG-3 and OX40.

[0057] According to advantageous embodiments of the molecular complex, preferably immunostimulatory, of the invention, the second ligand targets a surface molecule expressed essentially on the APCs, and in particular on the dendritic cells, that is to say a surface molecule specific to the APCs and in particular to the dendritic cells, preferably selected from the group consisting of: type C lectin receptors, membrane immunoglobulins, receptors for the constant region of immunoglobulins.

[0058] According to other advantageous embodiments of the molecular complex, preferably immunostimulatory, of the invention, the second ligand targets a CPAg surface molecule selected from the group consisting of: C-type lectin receptors, membrane immunoglobulins, receptors for the constant region of immunoglobulins (RFc or FcR), and immune checkpoint molecules (ICP) and their ligands (ICP ligands).

[0059] According to advantageous embodiments of the invention, the second ligand targets a surface molecule expressed essentially on NK or NKT cells, i.e. a surface molecule specific to NK or NKT cells, preferably selected from the group consisting of: the receptors NKG2D, NKp30, NKp44, NKp46, NKp80, CD56, CD16, the receptors KIR, NKG2A, and the ICPs PD-1, CTLA-4, TIM-3, TIGIT, LAG-3 and OX40; preferably NKp44 (CD336), NKp46 (CD335), NCAM (CD56), CTLA-4 and OX40.

[0060] The second ligand is in particular chosen from saccharides which bind C-type lectin receptors; immunoglobulins and fragments thereof comprising the constant region which bind FcRs; proteins or fragments of proteins which bind the Fc and / or Fab region of membrane immunoglobulins, as described in Application FR 2759296, in particular protein A of S. aureus, its fragment BB (SEQ ID NO: 1) and its derivative ZZ (SEQ ID NO: 2), preferably r ZZ (SEQ ID NO: 2). Alternatively, the second ligand is an antibody directed against these surface molecules of APC, NK or NKT cell or a fragment of this antibody containing at least the paratope (Ag binding domain), such as the fragments Fab, Fab', F(ab') 2 , Fv or single chain Fv (scFv for single-chain Fv), Fabc, and Fab fragment comprising a portion of the Fc domain.

[0061] The antibody or antibody fragment is in particular directed against a specific surface molecule of immune cells of the innate or adaptive response including APCs, NK and 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.

[0062] .The antibody or antibody fragment may also be directed against a surface molecule specific to APCs and in particular dendritic cells such as an anti-RFcgamma (I, II and / or III) or anti-C-type lectin receptor antibody, in particular anti-DEC-205 or anti-DC-SIGN (CD209); anti-DEC-205, anti-DC-SIGN (CD209) or anti-invariant chain 11 (CD74). The antibody may be an agonist or an antagonist of said surface molecule; for example, the antibody is an agonist of an activating surface molecule or an antagonist of an inhibitory surface molecule. Preferably, said antibody or antibody fragment is human or humanized. Such antibodies are well known to those skilled in the art and commercially available.

[0063] According to advantageous embodiments of the molecular complex, preferably immunostimulatory, of the invention, the second ligand is selected from the group consisting of: (i) antibodies directed against said surface molecules of antigen-presenting cells, NK or NKT cells and fragments thereof containing at least the paratope such as Fab, Fab', F(ab') 2 , Fv, scFv, Fabc and Fab fragments with at least a portion of the Fc domain (ii) immunoglobulins, preferably IgG, and fragments thereof comprising at least the Fc region, and (iii) proteins and protein fragments which bind the Fc and / or Fab region of the antibodies, in particular protein A of S. aureus, its fragment BB (SEQ ID NO: 1) and its derivative ZZ (SEQ ID NO: 2).

[0064] The antibodies directed against said surface molecules of antigen-presenting cells are preferably chosen from anti-RFcgamma (I, II and / or III), anti-DC-SIGN (CD209), anti-DEC-205, anti-CD206, anti-CD40 antibodies; anti-RFcgamma (I, II and / or III), anti-DC-SIGN (CD209), anti-DEC-205, anti-CD206, anti-CD40, anti-invariant chain 11 (CD74), and anti-ICOSL (CD275) antibodies; preferably anti-DC-SIGN (CD209) and anti-DEC-205 antibodies or anti-DC-SIGN (CD209), anti-DEC-205, anti-invariant chain 11 (CD74), and anti-ICOSL (CD275) antibodies.

[0065] The antibodies directed against said NK or NKT cell surface molecules are preferably chosen 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.

[0066] The additional ligands of the molecular complex, preferably an immunostimulatory complex, are advantageously chosen from sulfated GAG ligands and CPAg surface molecule ligands as defined above.

[0067] A first type of preferred immunostimulatory complex according to the invention consists of a sulfated GAG ligand peptide as defined above (first ligand) associated with a protein or protein fragment which binds the Fc and / or Fab region of immunoglobulins such as the BB fragment of protein A of Staphylococcus aureus and its ZZ derivative (second ligand), preferably in the form of a fusion protein comprising the first and second ligands. A particularly preferred first type of complex 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 (DTR: SEQ ID NO: 5) or the DT453-467 fragment (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, either directly or via a suitable spacer peptide.In this first particularly preferred type of complex, the second ligand is preferably N-terminal and the first ligand C-terminal of the fusion protein; the first and second ligands are separated by a suitable spacer peptide, in particular chosen from SEQ ID NO: 21 to 23. More preferably, said complex comprises one of the sequences SEQ ID NO: 12, 14, 16, 18 and 20. When the complex comprises the R domain of diphtheria toxin (DTR: SEQ ID NO: 5), then the domain is advantageously produced in mammalian cells, in particular in the form of a fusion protein with the second ligand, so as to form oligomers.

[0068] A second type of preferred immunostimulatory complex according to the invention consists of: (i) a sulfated GAG ligand peptide as defined above (first ligand) covalently linked to an immunoglobulin binding element as defined above, in particular a protein or protein fragment binding the Fc and / or Fab region of Ac, preferably only the Fab region, such as protein A of Staphylococcus aureus and its BB fragment, preferably in the form of a fusion protein comprising the first ligand and the immunoglobulin binding member, said first ligand covalently associated with the immunoglobulin binding member being complexed with (ii) an immunoglobulin, preferably an IgG, or a fragment comprising at least the Fc region (second ligand). Preferably, a whole immunoglobulin, more preferably a whole IgG. The first ligand and the immunoglobulin binding member are fused, either directly or via a suitable spacer peptide.A second particularly preferred type of complex consists of a fusion protein comprising a first ligand selected from Tat49-57 (SEQ ID NO: 3), Tat 37-57 (SEQ ID NO: 8), Tat22-57C(22-37)S (SEQ ID NO 9), the R domain of diphtheria toxin (DTR: SEQ ID NO: 5) or the DT453-467 fragment (SEQ ID NO 7) and an immunoglobulin binding element comprising the BB fragment of protein A (SEQ ID NO: 1), said fusion protein being complexed to a whole immunoglobulin. In this second particularly preferred type of complex, the immunoglobulin binding element (BB) is preferably N-terminal and the first ligand C-terminal of the fusion protein; the immunoglobulin binding element (BB) and the first ligand are separated by a suitable spacer peptide, in particular chosen from SEQ ID NO: 21 to 23. More preferably, said complex comprises one of the sequences SEQ ID NO: 16 or 18.When the complex comprises the R domain of diphtheria toxin (DTR: SEQ ID NO: 5), then the domain is advantageously produced in mammalian cells, in particular in the form of a fusion protein with the immunoglobulin binding element, so as to form oligomers.

[0069] A third type of preferred immunostimulatory complex according to the invention consists of: a sulfated GAG ligand peptide as defined above (first ligand) associated with an Ab selected from the group consisting of an anti-RFcgamma (I, II, and / or III) Ab, an anti-DEC-205 Ab, an anti-DC-SIGN (CD209) Ab, an anti-invariant chain 11 (CD74) Ab, an anti-ICOSL (CD275) Ab, an anti-NKp46 (CD335) Ab, an anti-NKp44 (CD336) Ab, an anti-NCAM (CD56) Ab, an anti-CTLA-4 Ab, an anti-PDL1 Ab, an anti-OX40 Ab, and a fragment of the preceding Abs comprising at least the paratope. Preferably, the sulfated GAG ligand peptide as defined above (first ligand) is covalently linked to an immunoglobulin binding element as defined above, in particular a protein or protein fragment that binds the Fc and / or Fab region of immunoglobulins such as the BB fragment of protein A of Staphylococcus aureus and its ZZ derivative, preferably in the form of a fusion protein of the first ligand with the immunoglobulin binding member. The first ligand and the immunoglobulin binding member are fused, either directly or via a suitable spacer peptide.A third particularly preferred type of complex 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 (DTR: SEQ ID NO: 5), or the DT453-467 fragment (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), said fusion protein being complexed with an anti-RFcgamma (I, II, and / or III), anti-DEC-205, anti-DC-SIGN, anti-CD74, anti-CD275, anti-CD335, anti-CD336, anti-CD56, anti-CTLA-4 antibody, anti-PDL1, anti-OX40 or a fragment of the preceding antibodies including at least the paratope.In this third particularly preferred type of complex, the immunoglobulin binding element (BB or ZZ) is preferably N-terminal and the first ligand C-terminal of the fusion protein; the immunoglobulin binding element (BB or ZZ) and the first ligand are separated by a suitable spacer peptide, in particular chosen from SEQ ID NO: 21 to 23. More preferably, said complex comprises one of the sequences SEQ ID NO: 12, 14, 16, 18 or 20. When the complex comprises the R domain of diphtheria toxin (DTR: SEQ ID NO: 5), then the domain is advantageously produced in mammalian cells, in particular in the form of a fusion protein with the immunoglobulin binding element, so as to form oligomers. More preferably, said complex 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.

[0070] According to preferred embodiments of the invention, said complex is used as an immunostimulant, preferably to activate antigen-presenting cells, in particular dendritic cells or monocytes, to activate NK or NKT cells, and / or to activate the secretion of the cytokines IL-6 and / or IL-12.

[0071] The invention further relates to an immunomodulatory, preferably immunostimulatory, composition comprising at least one immunomodulatory, preferably immunostimulatory, complex according to the invention, and at least one pharmaceutically acceptable vehicle, a carrier substance and / or an adjuvant.

[0072] Pharmaceutically acceptable vehicles are those conventionally used.

[0073] Adjuvants are the adjuvants of humoral and / or cellular immunity classically used in immunotherapy. The adjuvants are advantageously chosen from the group consisting of: oily emulsions, mineral substances, bacterial extracts, saponin, aluminum hydroxide, monophosphoryl lipid A, squalene and TLR ligands, in particular oligodeoxynucleotides comprising at least one CpG sequence (CpG oligodeoxynucleotide) which are TLR9 ligands, or polyinosinic-polycytidylic acid (poly(I)-poly(C) or poly IC) which is a TLR3 ligand. According to preferred embodiments of the invention, said composition comprises at least one adjuvant, preferably a CpG oligodeoxynucleotide, polyinosinic-polycytidylic acid or a mixture of CpG oligodeoxynucleotide(s) and polyinosinic-polycytidylic acid.

[0074] Les carrier substances are those classically used. These include unilamellar or multilamellar liposomes, ISCOMS, virosomes ( virus-like particles ), saponin micelles, solid microspheres of saccharide (poly(lactide-co-glycolide)) or gold-containing nature, and nanoparticles. According to preferred embodiments of the invention, said composition comprises at least one carrier substance, for example a nanoparticle or a mixture of nanoparticles.

[0075] The immunomodulatory, preferably immunostimulatory, composition according to the invention comprises a complex comprising 2 or more ligands or a mixture of different complexes, said complex(es) being optionally linked together by covalent or non-covalent bonds and / or incorporated inside or on the surface of a particle such as a liposome, a virosome or a nanoparticle.

[0076] According to particular embodiments of the invention, the composition comprises a polynucleotide or a mixture of polynucleotides encoding CPAg, NK or NKT ligands which are proteins, polypeptides or peptides. The polynucleotide consists of a recombinant, synthetic or semi-synthetic nucleic acid which is expressible in cells of the host to which the composition is administered. The nucleic acid may be DNA, RNA, in particular mRNA, a mixed nucleic acid (DNA / RNA) and may be modified. For example, said composition comprises a polynucleotide comprising a sequence encoding a fusion protein comprising at least the coding sequences of the first and second ligand, suitably fused in frame, and optionally a sequence encoding a binding element as defined above.Alternatively, the composition comprises a mixture of polynucleotides comprising at least a first polynucleotide comprising a sequence coding for the first ligand and a second polynucleotide comprising a sequence coding for the second ligand, said first or second nucleotide also comprising a sequence coding for a binding element as defined above. Said polynucleotide(s) are preferably inserted into one or more expression vectors comprising appropriate transcriptional and / or translational regulatory sequences (promoter, transcriptional activator, transcriptional terminator, polyadenylation signal) for the expression of the first ligand and the second ligand, and optionally the other ligands. in vivo in individuals to whom the composition is administered. Many vectors that can be used in therapy are known per se. Among others, viral vectors (adenovirus, retrovirus, lentivirus, AAV) and non-viral vectors (naked DNA), in particular a plasmid, into which the sequence of interest has been previously inserted, can be used. Alternatively, said polynucleotide(s) are mRNAs, preferably modified. The use of mRNA in therapy is well known to those skilled in the art (review, for example, Drew Weissman, Expert Reviews of Vaccines, October 2014, 1-17, doi: 10.1586 / 14760584.2015.973859).

[0077] According to particular embodiments of the invention, the composition comprises cells modified by the composition according to the invention. For example, the cells are modified by a polynucleotide, a mixture of polynucleotides or a vector as defined above or loaded with a ligand complex as defined above. The cell is in particular a natural antigen-presenting cell such as a dendritic cell or an artificial antigen-presenting cell, such as exosomes derived from dendritic cells or vesicles derived from cells expressing the ligands of the molecular complex according to the invention. For example, the cells are antigen-presenting cells of an individual to be treated, in particular dendritic cells which are modified ex vivo before being re-administered to the individual (Cell Therapy ex vivo ).

[0078] The immunomodulatory composition may further comprise at least one other therapeutic agent, in particular an anticancer, anti-infectious agent, another immunomodulatory agent or a vaccine antigen specific to the disease to be treated, said vaccine antigen being advantageously associated with a carrier substance or included in a suitable vector. According to preferred embodiments of the invention, said composition further comprises at least one immune checkpoint inhibitor (Marin-Acevedo J. et al., 2018, Hematol. Oncol., 11:39) such as, but not limited to, an anti-PD-1, an anti-PDL-1 or an anti-CTLA4, in particular an antibody, preferably monoclonal, directed against the PD-1, PDL-1 or CTLA4 molecule, preferably the human hPD-1, hPDL-1 or hCTLA4 molecule. The composition according to the invention advantageously comprises an anti-PD-1, in particular an anti-PD-1 monoclonal antibody, preferably anti-hPD-1.According to other preferred embodiments of the invention, said composition further comprises at least one vaccine antigen specific to the disease to be treated, preferably associated with a carrier substance or included in a suitable vector.

[0079] The immunomodulatory, preferably immunostimulatory, composition comprises an effective dose of complex(es), polynucleotide(s), vector(s), cell(s) sufficient to induce an immune response capable of producing a therapeutic effect on the disease to be treated, i.e. of reducing the symptoms of this disease. This includes in particular the attenuation of the consequences of the action by a pathogen (infectious or non-infectious) or the reduction of the growth of a tumor, in an individual treated with this composition. This dose is determined and adjusted according to factors such as the age, sex and weight of the subject. The immunomodulatory, preferably immunostimulatory, composition according to the invention is presented in a galenic form adapted to the chosen administration.The composition is generally administered according to standard immunotherapy protocols at doses and for a duration sufficient to induce an effective immune response against the pathology to be treated. Administration may be intratumoral, subcutaneous, intramuscular, intravenous, intradermal, intraperitoneal, oral, sublingual, rectal, vaginal, intranasal, by inhalation or by transdermal application. The composition is presented in a galenic form suitable for a chosen administration.

[0080] The polynucleotides isolated or inserted into a plasmid vector are introduced into the individual to be treated, either using physical methods such as electroporation, or by associating them with any substance(s) allowing the passage of the plasma membrane, such as carriers like nanocarriers, liposomes, lipids or cationic polymers. In addition, these methods can advantageously be combined, for example by using electroporation associated with liposomes.

[0081] The immunomodulatory, preferably immunostimulatory, composition according to the present invention is used in immunotherapy, in particular antitumor or anti-infectious. The immunomodulatory, preferably immunostimulatory, composition according to the present invention is used for preventive or curative purposes, i.e. to prevent a pathology in individuals or to treat individuals suffering from a pathology. According to preferred embodiments of the invention, said composition is used for curative purposes, i.e. to treat individuals suffering from a pathology. It can be used in combination with other treatments, therapeutic or surgical, in particular in combination with other therapeutic agents as defined above, the composition according to the invention and the other therapeutic agents being able to be administered simultaneously, separately or sequentially.

[0082] According to preferred embodiments of the invention, said composition is used in the treatment of cancer. Cancers are any type of cancer that can benefit from immunotherapy such as, but not limited to: breast, colon, prostate, esophageal, stomach, lung, ENT (ear, nose and throat), skin, ovarian, uterine, brain, liver, kidney cancers.

[0083] According to other preferred embodiments of the invention, said composition is used in the treatment of infectious diseases, in particular those for which the infectious agents persist in the body. Infectious diseases are any type of infectious disease that can benefit from immunotherapy (Wykes MN et al., Nat. Rev. Immunol., 2018, 18:91-104) such as, but not limited to: viral, bacterial, fungal or parasitic infections, in particular infections by HIV, HBV, HCV, mycobaterium tuberculosis or plasmodium falciparum. When said complex comprises Tat or a Tat fragment as defined above, then said composition is used in the treatment of infectious diseases other than HIV infection (AIDS).When said complex comprises DTR or a fragment of DTR as defined above, then said composition is used in the treatment of infectious diseases other than diphtheria (infection by the diphtheria bacillus, . Corynebacterium diphteriae).

[0084] The immunomodulatory, preferably immunostimulatory, composition according to the present invention is used either in conventional therapy or in cell therapy, or even by a combination of the two approaches.

[0085] Cell therapy comprises the preparation of antigen-presenting cells, in particular dendritic cells, or NK or NKT cells, by a conventional protocol comprising the isolation of peripheral blood mononuclear cells (PBMC) from a patient to be treated and the culturing of the dendritic, NK or NKT cells, in the presence of molecular complex(es), polynucleotide(s), vector(s) as defined above. In a second step, the antigen-presenting cells, NK or NKT loaded with said molecular complex(es) or modified by said polynucleotide(s) or vector(s) are reinjected into the patient.

[0086] The present invention also relates to an immunotherapy method, in particular anti-tumor or anti-infectious, characterized in that it comprises the administration of an immunomodulatory composition, preferably an immunostimulant as defined above, to an individual, by any appropriate means as defined above.

[0087] The present invention also relates to the use of an immunomodulatory composition, preferably immunostimulant as defined above for the preparation of a medicament intended for immunotherapy, preferably anti-tumor or anti-infectious.

[0088] The complexes of ligands of APCs, NK or NKT cells according to the invention are prepared by conventional techniques known to those skilled in the art, namely: Ligands for the surface molecules of APCs, NK or NKT cells can be produced by chemical synthesis or by expression of recombinant DNA in an appropriate cellular system, eukaryotic or prokaryotic. Peptides and proteins can be synthesized in solid phase, according to the Fmoc technique, originally 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 cDNAs, cloned into an appropriate eukaryotic or prokaryotic expression vector, the polypeptides or proteins produced in cells modified by the recombinant vector are purified by any suitable means, in particular by affinity chromatography. Abs directed against surface molecules of APCs, NK or NKT cells are well known and commercially available.For example, by way of non-limiting example, anti-CD205 (#555831); anti-CD206 (#555952); anti-CD209 (#551186); anti-HLA-DR (#555556) Abs are available from BECTON-DICKINSON, while anti-CD56 Ab is available from Biolegend (#304622) and anti-CD335 (AM31284AF-N) and anti-CD336 (#AM50346PU-N) Abs are available from Origene. Alternatively, monoclonal Abs can be produced by conventional techniques known to those skilled in the art. For example, monoclonal Abs are produced from hybridomas obtained by fusion of B lymphocytes from an animal immunized with the surface molecule of APCs with myelomas, according to the technique of Köhler and Milstein (Nature, 1975, 256, 495-497); the hybridomas are cultured. in vitro, in particular in fermenters or produced in vivo, in the form of ascites; alternatively, said monoclonal Ac are produced by genetic engineering as described in US patent US 4,816,567. Humanized Ac are produced by general methods such as those described in International Application WO 98 / 45332. The Ac fragments are produced from the cloned VH and VL regions, from the mRNAs of hybridomas or splenic lymphocytes of an immunized mammal; for example, the Fv, scFv or Fab fragments are expressed on the surface of filamentous phages according to the technique of Winter and Milstein (Nature, 1991, 349, 293-299); after several selection steps, the Ag-specific Ac fragments are isolated and expressed in an appropriate expression system, by conventional recombinant DNA cloning and expression techniques.The Ac or their fragments as defined above are purified by conventional techniques known to those skilled in the art, such as affinity chromatography. The covalent association of the first ligand (L1) with the second ligand (L2) of a surface molecule of the APCs or NK or NKT cells can be achieved by the construction of a fusion protein in which the nucleotide sequences coding for L1 and L2 are fused in phase, in the appropriate order, either directly or via a nucleotide sequence coding for an appropriate spacer peptide.Depending on the respective sizes of the amino acid sequences of L1 and L2, they are either fused at their ends (N-terminal end of one of the sequences fused to the C-terminal end of the other sequence) or one of the sequences is inserted into the other sequence at an appropriate site that does not have a deleterious effect on the binding of the ligand(s) to its receptor expressed on the surface of APCs or NK or NKT cells. Alternatively, the ligand(s) may be coupled covalently, by any appropriate means. The coupling is carried out via reactive groups initially present or previously introduced on the ligand(s). The coupling may in particular be carried out at the level of amino acid residues whose side chain comprises a reactive function.Among these amino acids, mention may be made of polar amino acids comprising a function: -OH [serine (S), threonine (T) or tyrosine (Y)], -SH [cysteine ​​(C)], -NH 2 [lysine (K) or arginine (R)], -COOH [aspartic acid (D) or glutamic acid (E)], and polar amino acids with a side chain functionalized by the addition of a reactive function, in particular a chloro- or bromo-acetyl reactive with thiol groups or a hydrazine group reactive with aldehydes. The ligand is coupled by any suitable means; these means which are known to those skilled in the art include in particular coupling using homobifunctional reagents such as glutaraldehyde or dithiobis-(succinimidyl propionate). Preferably, the coupling is carried out using heterobifunctional reagents, in particular m-maleimidobenzoyl-N-hydroxysuccinimide (SMCC) or sulfo-SMCC which each contain a maleimide group capable of reacting with free thiols.In this case, the SMCC is previously covalently bound to an amine function present on the ligand. At the same time, another heterobifunctional reagent (such as N-succinimidyl S-acetylthioacetate, which contains a thioester group cleavable with hydroxylamine, or succinimidyl-pyridyl-dithiopropionate, which contains a disulfide bridge reducible under mild conditions), is associated with an amine function of the second partner, which is one of the ligands. The second partner is then treated with hydroxylamine or a reducing agent to allow the release of the thiol. The thiolated compound is then incubated with the compound having incorporated the maleimide, and the coupling is obtained by reaction of the thiol group with the maleimide group. This type of covalent coupling is notably described in Léonetti et al., J. Exp. Med., 1999, 189, 1217-1228.It is also possible to release a thiol group already present on one of the compounds and then couple it to another compound that has been previously modified using SMCC. This method, which is often used to couple Ac to ligands, is notably described in Ishikawa et al., J. Immunoassay, 1983, 4, 209-327. Non-covalent complexes are prepared by bringing the second ligand (L2) into contact with the first ligand (L1) under conditions allowing the two partners to interact. This interaction may involve a binding element, in particular a protein or a peptide, which has a high and specific affinity for one of the partners of the complex (L1 or L2). In particular, the affinity of the binding element for this partner, in the complex, is sufficient so that it does not immediately dissociate from this complex. in vivo. When one of the ligands is an immunoglobulin, the binding element is an immunoglobulin binding element as described in Application FR 2759296. For example, an immunoglobulin binding element is covalently bound to L1, so as to form a non-covalent complex with L2. The polynucleotides according to the invention are obtained by conventional methods, known per se, following standard protocols known to those skilled in the art. For example, they can be obtained by amplification of a nucleic sequence by PCR or RT-PCR, by screening genomic DNA libraries by hybridization with a homologous probe, or by total or partial chemical synthesis. The recombinant vectors are constructed and introduced into host cells by conventional recombinant DNA and genetic engineering methods, which are known per se.

[0089] The implementation of the invention uses, unless otherwise indicated, conventional methods of immunology, cell culture, cell biology, molecular biology and recombinant DNA which are known to those skilled in the art. Brève description des dessins

[0090] Other characteristics, details and advantages of the invention will appear on reading the detailed description below which refers to examples of implementation of the present invention, and on analysis of the appended drawings, in which: Fig. 1 [ Fig. 1 ] shows the molecular mass and degree of homogeneity of molecular complexes produced in E. coli or in HEK cells. The proteins ZZ-DTRBD HEK, ZZ-DTRBD coli, BB-DTRBD HEK, BB-DTRBD coli and ZZ-Tat 22-57C(22-37)S were analyzed by electrophoresis under denaturing conditions. The proteins were deposited in the presence of molecular weight markers. After electrophoretic migration the proteins were stained with Coomassie blue. Fig. 2 [ Fig. 2 ] shows the binding of different molecular complexes to heparin. Dilution series of free ZZ, ZZ-DTRBD HEK, ZZ-DTRBD coli, BB-DTRBD HEK, ZZ-DTRBD coli and ZZ-Tat 22-57C(22-37)S proteins were respectively incubated at pH 7.2 in microtiter plate wells previously adsorbed with rabbit IgG. After 4 hours the plates were washed and biotinylated heparin was added. After 30 minutes of incubation the binding of heparin to the plates was detected using streptavidin coupled to peroxidase and a substrate of this enzyme (ABTS). Heparin binding is considered significant when the optical density signal is at least 50% of the signal measured when ZZ-Tat 22-57C(22-37)S is incubated at 100nM pH7.2 on microtiter plates. Fig. 3 [ Fig. 3 ] shows the cell types bound by the ZZ-DTRBD HEK fusion protein in a mouse splenocyte population. Splenocytes were incubated in the presence or absence of a fixed amount of ZZ-DTRBD HEK (100nM). A series of fluorescent Abs specific for pDCs, CD11b+ cDCs, and CD8+ cDCs (A) ; CD4+ T lymphocytes, CD8+ T lymphocytes, B lymphocytes, monocytes (B) was then added. After 30 minutes at 4 °C, cells were washed and analyzed by flow cytometry. The percentage of cells bound by ZZ-DTRBD HEK in the subpopulation considered is shown. Fig. 4 [ Fig. 4 ] shows that ZZ-DTRBD HEK induces the secretion of IL-6 and IL-12 by splenocytes. A fixed amount of ZZ, DTRBD or ZZ-DTRBD HEK (1 µM) was incubated with mouse splenocytes. After 24 h the supernatants were collected and the presence of IL-6 and IL-12 was assessed by enzyme immunoassay. Fig. 5 [ Fig. 5 ] shows that ZZ-Tat 22-57C(22-37)S induces the secretion of IL-6 and IL-12 by splenocytes. A fixed amount of ZZ, Tat CY49-57, ZZ + Tat CY49-57 or ZZ-Tat 22-57C(22-37)S (1µM) was incubated with mouse splenocytes. After 24h the supernatants were collected and the presence of IL-6 and IL-12 was assessed by enzyme immunoassay. Fig. 6 [ Fig. 6 ] shows that ZZ-DTRBD HEK, BB-DTRBD HEK proteins induce the secretion of greater amounts of IL-6 and IL-12 by splenocytes than ZZ-DTRBD coli and BB-DTRBD coli. A fixed amount of ZZ-DTRBD HEK, ZZ-DTRBD coli, BB-DTRBD HEK and BB-DTRBD coli (1µM) was incubated with mouse splenocytes. After 24h the supernatants were collected and the presence of IL-6 and IL-12 was assessed by enzyme immunoassay. Fig. 7 [ Fig. 7 ] shows that ZZ-DTRBD HEK induces the secretion of IL-6 and IL-12 by isolated murine dendritic cells. Dendritic cells were isolated by magnetic sorting. The dendritic cells were then incubated in the presence or absence of a fixed amount of ZZ-DTRBD HEK (0.6µM). After 24h, the supernatants were collected and the presence of IL-6 and IL-12 was assessed by enzyme immunoassay. Fig. 8 [ Fig. 8A-B ] shows that ZZ-DTRBD HEK and ZZ-Tat 22-57C(22-37)S proteins are capable of inducing the expansion of murine dendritic cells in mice. Three groups of mice were injected three times at three-day intervals with PBS buffer in the absence or presence of a fixed amount of ZZ-DTRBD HEK (5 nmol per mouse) or ZZ-Tat 22-57C(22-37)S (10 nmol per mouse). 24 hours after the last injection, the animals were euthanized, the spleens were removed, and the splenocytes were labeled using a cocktail of fluorescent Abs to distinguish CD8+ cDCs (CD11c high< B220 -< CD8 +< CD11b -< ) (A), cDC-CD11b+ (CD11chighB220 -< CD8 -< CD11b +< ) (B) and pDCs (CD11c int< CD317 +< CD11b +< ). [ Fig. 8C ] shows cells analyzed by flow cytometry. The number of positive cells was represented as a percentage of the total number of live splenocytes. Statistical analyses were performed by a Kruskall-Wallis test (* p < 0.05). Fig. 9 [ Fig. 9 ] shows that ZZ-DTRBD HEK induces in vitro the secretion of IL-6 and IL-12 by isolated human DCs. Isolated DCs from healthy human donors were incubated in the presence of a fixed amount of ZZ-DTRBD HEK (1µM). After 24 hours, the supernatants were harvested to assess the presence of IL-6 and IL-12 cytokines by enzyme immunoassay. Fig. 10 [ Fig. 10 ] shows that mice injected with colorectal cancer cells exhibit slowed tumor growth when subsequently injected with ZZ-DTRBD HEK alone or in combination with an adjuvant mixture. A: Sixteen C57BL / 6 mice were injected with 0.5M MC38 cells subcutaneously. Three days later, eight mice were injected with the CpG-B 1018 / Poly I:C adjuvant mixture (30 µg for each adjuvant) and eight were not injected (Controls). A second injection was performed three days later. B : Eighteen C57BL / 6 mice were injected with 0.5M MC38 cells subcutaneously. Three days later, six mice were injected with ZZ-DTRBD HEK in PBS buffer, six others were injected with ZZ-DTRBD HEK (2 nmol per mouse) in PBS buffer containing the CpG-B 1018 / Poly I:C adjuvant mixture (30 µg for each adjuvant), and six were not injected (Controls). Two additional injections were performed three days apart. Tumor growth was monitored by measuring tumors with a caliper. Each injection is represented by an arrow. Fig. 11 [ Fig. 11 ] shows that mice injected with colorectal cancer cells exhibit slowed tumor growth when subsequently injected with ZZ-DTRBD HEK, ZZ-Tat 22-57C(22-37)S in combination with an adjuvant mixture. Thirty-two C57BL / 6 mice were injected with 0.5M MC38 cells subcutaneously. Three days later, a group of eight mice was injected with ZZ-DTRBD HEK (0.96 nmol per mouse) in 50 µl of PBS containing the CpG-B 1018 / Polyl:C adjuvant mixture (30 µg for each adjuvant), another group with an anti-PD-1 Ab (50 µl, 1.3 nmol per mouse), a third group with ZZ-Tat 22-57C(22-37)S (0.96 nmol per mouse) in 50 µl of PBS containing the CpG-B 1018 / Polyl:C adjuvant mixture (30 µg for each adjuvant), and a final group of eight mice was uninjected (Controls). Two additional injections were performed three days apart. Tumor growth was monitored by measuring tumors with a caliper.Each injection is represented by an arrow. [. Fig. 11A ] Tumor growth kinetics. [ Fig. 11B ] Tumor growth kinetics. [ Fig. 11C ] Survival of treated animals. Fig. 12 [ Fig. 12 ] shows that the formation of molecular complexes ZZ-Tat 22-57C22-37)S, anti-DEC205 / ZZ-Tat 22-57C22-37)S and IgG / ZZ-Tat 22-57C22-37)S induce in vitro the increase in the proportion of activated monocytes and CD4+ T lymphocytes. A fixed concentration (0.1µM) of anti-DEC205 / ZZ-Tat 22-57C22-37)S , IgG / ZZ-Tat 22-57C22-37)S , anti-DEC205 / ZZ, IgG / ZZ, anti-DEC205 Ab was incubated with human PBMCs. A fixed concentration (1µM) of ZZ-Tat 22-57C22-37)S , or ZZ was incubated with human PBMCs. After 24h the cells were harvested and labeled with fluorescent Ab. The proportion of activated monocytes (CD3 -< CD14 +< ) in the live PBMC population was assessed using the CD69 marker. [ Fig. 12A ]. The proportion of activated CD4+ T lymphocytes in the population of living CD4+ T lymphocytes (CD3 +< CD4 +< ) was assessed by measuring the expression of the CD69 molecule. [ Fig. 12B ]. Cells were analyzed by flow cytometry. Fig. 13 [ Fig. 13 ] shows that molecular complexes directed against receptors expressed by dendritic cells (anti-CD74 / ZZ-Tat 22-57C22-37)S, anti-CD209 / ZZ-Tat 22-57C22-37)S, and anti-CD275 / ZZ-Tat 22-57C22-37)S) induce the secretion of IL-6 by PBMCs. Human PBMCs were incubated in the presence or absence of free forms of Ab, ZZ or ZZ-Tat 22-57C(22-37)S as well as molecular complexes targeting the molecules CD74 (A), CD209 (B) and CD275 (C), respectively. After 24 hours, the supernatants were collected and the presence of IL-6 was assessed by enzyme immunoassay. Fig. 14 [ Fig. 14 ] shows that a molecular complex (anti-CD335 / ZZ-Tat 22-57C22-37)S ) directed against the CD335 receptor expressed by NK and NKT cells induces an increase in the proportion of activated NK and NKT cells. PBMCs were incubated in the presence or absence of free forms of Ab, ZZ or ZZ-Tat 22-57C(22-37)S as well as anti-CD335 / ZZ-Tat 22-57C22-37)S , anti-CD335 / ZZ molecular complexes. After 18 hours the cells were analyzed by cytometry to evaluate the expression of the co-stimulatory molecule CD69 on the surface of NK cells (A) and NKT (B). Fig. 15 [ Fig. 15 ] shows that molecular complexes directed against two receptors expressed by NK and NKT cells respectively induce an increase in the proportion of activated NKT cells. PBMCs were incubated in the presence or absence of free forms of Ab, ZZ or ZZ-Tat 22-57C(22-37)S as well as anti-CD56 / ZZ-Tat 22-57C(22-37)S, anti-CD56 / ZZ, anti-CD336 / ZZ-Tat 22-57C(22-37)S, or anti-CD336 / ZZ molecular complexes. After 18 hours, the cells were analyzed by cytometry to evaluate the expression of the co-stimulatory molecule CD69 on the surface of NKT cells. Fig. 16 [ Fig. 16 ] shows that molecular complexes directed against receptors expressed by NK cells (anti-CD56 / ZZ-Tat 22-57C22-37)S , anti-CD335 / ZZ-Tat 22-57C22-37)S , and anti-CD336 / ZZ-Tat 22-57C22-37)S ) induce the secretion of IL-6 by human PBMCs. PBMCs were incubated in the presence or absence of free forms of Ab, ZZ or ZZ-Tat 22-57C(22-37)S as well as molecular complexes targeting CD56 molecules respectively (A), CD335 (B) and CD336 (C). After 24 hours, the supernatants were collected and the presence of IL-6 was assessed by enzyme immunoassay. Fig. 17 [Fig. 18] shows that molecular complexes containing anti-ICP Abs (anti-CTLA-4 / ZZ-Tat 22-57C22-37)S, anti-PD-L / ZZ-Tat 22-57C22-37)S , and anti-OX40 / ZZ-Tat 22-57C22-37)S ) induce IL-6 secretion by human PBMCs. PBMCs were incubated in the presence or absence of free forms of Abs, ZZ or ZZ-Tat 22-57C(22-37)S as well as molecular complexes targeting CTLA-4 molecules respectively (A) , PD-L1 (B) and OX40 (C) After 24 hours, the supernatants were collected and the presence of IL-6 was assessed by enzyme immunoassay. Exemples Exemple 1 : Expression of fusion proteins in E. coli or in HEK cells, purification, biochemical characterization, and study of their capacity to bind heparan sulfates Matériels et Méthodes Production des différents complexes moléculaires

[0091] Expression of the ZZ-DTRBD fusion protein in E. coli, called ZZ-DTRBD coli, was previously described in the publication of Lobeck et al. (Infection and Immunity, 1998, 66, 418-423). The ZZ-DTRBD coli fusion protein (SEQ ID NO: 12) is encoded by a polynucleotide of sequence SEQ ID NO: 11. Expression of the ZZ-Tat 22-57C(22-37)S fusion protein in E. coli was performed following a protocol similar to that described for the ZZOVATat 22-57S fusion in the publication by Knittel et al. (Vaccine, 2016, 34(27):3093-3101). The ZZ-Tat 22-57C(22-37)Scoli fusion protein (SEQ ID NO: 20) is encoded by a polynucleotide of sequence SEQ ID NO: 19. For expression in eukaryotic cells, HEK cells (2.5 × 10 6 cells / ml in 250 ml 293F freestyle medium) were transfected with a pCDNA3.4 plasmid encoding ZZ-DTRBD HEK (400 µg of DNA preparation from maxiprep per transfection) in the presence of PEI (0.5 mg / ml). This plasmid includes the polynucleotide of sequence SEQ ID NO: 13 which codes for the ZZ-DTRBD HEK fusion protein (SEQ ID NO: 14). The cells were then incubated for 24 hours at 37°C with shaking. 250 ml of Ex Cell medium was then added. After 4 days of incubation at 37°C with shaking, the culture supernatants were collected, sterile filtered and a protease inhibitor cocktail was added.

[0092] After expression of the three proteins, the supernatants were diluted ½ in PBS-Tween 0.1% and then passed through an IgG sepharose column (IgG sepharose 6Fast flow #17-0969-02, Amersham) to purify the molecular complexes by immunoaffinity. The acidity of the fusion proteins eluted from the column was neutralized in 1M Tris-HCl buffer, pH 8. The fusion proteins resulting from expression in E. coli,ZZ-DTRBD coli and ZZ-Tat 22-57C(22-37)S , underwent a second round of purification using a mono S 5 / 50 cation exchange column (GE Healthcare). The column was equilibrated with 0.05M phosphate citrate buffer pH=5.5 for the purification of ZZ-DTRBD coli . The column was equilibrated with 0.05M phosphate citrate buffer pH=4 for the purification of ZZ-Tat 22-57C(22-37)S . The fusion proteins ZZ-DTRBD coli and ZZ-Tat 22-57C(22-37)S were then eluted with a linear gradient from 0 to 1M NaCl. The proteins were finally concentrated in PBS and stored at -20°C until use. The production of the BB-DTRBD HEK and BB-DTRBD coli molecular complexes was carried out following the same protocol as that used for ZZ-DTRBD HEK and ZZ-DTRBD coli. The BB-DTRBD HEK fusion protein (SEQ ID NO: 18) is encoded by the polynucleotide of sequence SEQ ID NO: 17.The BB-DTRBD coli fusion protein (SEQ ID NO: 16) is encoded by the polynucleotide of sequence SEQ ID NO: 15. Analysis of molecular mass and degree of homogeneity of molecular complexes ZZ-DTRBD HEK, ZZ-DTRBD coli, BB-DTRBD HEK and BB-DTRBD coli by electrophoresis gel

[0093] The proteins ZZ-DTRBD HEK, ZZ-DTRBD coli, BB-DTRBD HEK and BB-DTRBD coli as well as molecular weight markers were loaded onto a 4-12% SDS-PAGE gel under denaturing conditions and then subjected to electrophoretic migration. Following migration, the presence of protein bands was revealed by Coomasie blue staining. Binding of molecular complexes to heparin

[0094] To assess the binding of molecular complexes to heparan sulfates, heparin, which is a sulfated sugar representative of the heparan sulfate family, was used.

[0095] The interaction was assessed by an immunoenzymatic technique. For this, a series of microtiter plates was previously adsorbed with rabbit IgG (1 µg / 100 µl / well in 0.1M phosphate buffer pH7.2) and then saturated with a buffer solution containing 0.3% bovine serum albumin (200 µl / well in 0.1M phosphate buffer pH7.2). Another series of microtiter plates was saturated with a buffer solution containing 0.3% bovine serum albumin (300 µl / well in 0.1M phosphate buffer pH7.2). Both sets of plates were then washed and dilution series (in 0.1M phosphate buffer pH7.4 containing 0.1% bovine serum albumin) of ZZ-DTRBD HEK, ZZ-DTRBD coli, BB-DTRBD HEK and BB-DTRBD coli and ZZ-Tat 22-57C(22-37)S and free ZZ were deposited in the wells. After 4 hours of incubation at room temperature the plates were washed and 100µl of heparin-biotin (1µM) was added per well.After 1 hour at room temperature, the plates were washed and 100µl of peroxidase-coupled streptavidin (dilution 1 / 2000) was added. After 30 minutes of incubation, the plates were washed and a substrate (ABTS) was added. The staining was measured at 414 nm after 30 minutes of incubation. To eliminate non-specific binding to albumin, the optical signal measured on plates adsorbed only with bovine serum albumin was deduced from the signal measured on plates adsorbed with IgG. Heparin binding is considered significant when the optical density signal is greater than or equal to 50% of the signal measured when ZZ-Tat 22-57C(22-37)S is incubated at 100nM pH 7.2 on microtiter plates. Results

[0096] The inventors previously constructed a fusion protein, called ZZ-DTRBD, incorporating, on the one hand, a double ZZ domain derived from protein A of Staphylococcus aureusand, on the other hand, the DTRBD domain derived from diphtheria toxin (Lobeck et al., Infection and Immunity, 1998, 66, 418-423). ZZ can bind to the Fc region of immunoglobulins in a similar manner to protein A. DTRBD binds the diphtheria toxin receptor and also has a heparan sulfate binding site located in the 453-467 region (Knittel et al. J. Immunol., 2015, 194(8):3601-11; Knittel et al. Vaccine, 2016, 34(27):3093-3101). These binding characteristics allow ZZ-DTRBD to target different cell types bearing surface immunoglobulins and heparan sulfate proteoglycans via interaction with heparan sulfates.

[0097] The inventors similarly constructed a fusion protein, called BB-DTRBD, by replacing the coding sequence ZZ with a coding sequence called BB. The BB protein corresponds to a double domain which, like ZZ, is derived from protein A of Staphylococcus aureusbut has the particularity of binding the Fc region as well as the Fab region of immunoglobulins (Jansson 1998 FEMS Immunol. and Med. Microbiol., Léonetti et al. 1999, J. Exp. Med., 189, 1217-28).

[0098] The inventors also constructed a fusion protein, called ZZ-Tat 22-57C(22-37)S. It contains a double ZZ domain derived from the protein A of Staphylococcus aureus and a Tat 22-57C(22-37)S domain derived from the HIV transcriptional transactivator (WO 2011 / 092675, and Knittel et al. Vaccine, 2016, 34(27):3093-3101) that possesses a heparan sulfate binding site. These binding characteristics allow ZZ-Tat 22-57C(22-37)S to target different cell types bearing surface immunoglobulins and heparan sulfate proteoglycans via interaction with HS.

[0099] The inventors expressed ZZ-DTRBD, BB-DTRBD and ZZ-Tat 22-57C(22-37)S recombinantly using different expression systems. The complexes expressed in E. coli are called ZZ-DTRBD coli , BB-DTRBD coli and ZZ-Tat 22-57C(22-37)S and those expressed in HEK cells are called ZZ-DTRBD HEK and BB-DTRBD HEK . After expression, the complexes were purified using a column containing an IgG-carrying gel. Then the ZZ-DTRBD coli , BB-DTRBD coli and ZZ-Tat 22-57C(22-37)S proteins were subjected to ion exchange chromatography to remove contaminating LPS. Finally, some characteristics of these complexes were evaluated by gel electrophoresis. As can be seen in the Fig. 1 ,ZZ-DTRBD coli and BB-DTRBD coli migrate in a predominant band with a molecular mass of approximately 35kDa which is close to the theoretical mass of the molecule (32230) which indicates that this band corresponds to a monomer. Minor bands corresponding to degradation products are also present in these two molecular complexes. ZZ-DTRBD HEK and BB-DTRBD HEK migrate in several bands mainly distributed between 35 and 150k Da which indicates that expression in HEK cells leads to a heterogeneous mixture consisting of monomeric and oligomeric forms of ZZ-DTRBD. The molecular complex ZZ-Tat 22-57C(22-37)S migrates in a majority band which reflects the homogeneity of the purified protein. The molecular mass of approximately 25 kDa is slightly higher than that calculated for the theoretical mass of the molecule (MW = 19145). However, Tat and its derivatives tend to migrate abnormally (Kittiworakarn et al.etc) which strongly suggests that the ZZ-Tat 22-57C(22-37)S fusion protein expressed in . E. coli and purified is essentially made up of a monomer.

[0100] Next, the inventors investigated the ability of free ZZ and the five molecular complexes to bind heparin, which is a sulfated polysaccharide representative of the heparan sulfate family. The enzyme immunoassay did not observe a significant optical signal for free ZZ ( Fig. 2 ). In contrast, an increase in optical density as a function of the incubated dose is measured for ZZ-DTRBD coli, BB-DTRBD HEK, BB-DTRBD coli, ZZ-DTRBD HEK and ZZ-Tat 22-57C(22-37)S. These data therefore indicate that the presence of the DTRBD domain or the Tat 22-57C(22-37)S domain allows the binding of molecular complexes to heparin. Moreover, as the ZZ-DTRBD HEK and BB-DTRBD HEK fusions contain oligomers (cf Figure 1 )These data indicate that the presence of oligomers does not disrupt the interaction. Example 2: The ZZ-DTRBD HEK fusion protein is capable of binding different types of cells of the murine immune system Materials and methods ZZ-DTRBD binding to immune system cells

[0101] C57BI / 6 mouse splenocytes are resuspended at 10.10 6< cells / mL in PBS 0.5% BSA 2mM EDTA buffer. 100 µL of the cell suspension are placed in a 96-well round-bottom plate. 100 µL of 0.5% BSA 2 mM EDTA buffer are added per well in the absence or presence of ZZ-DTRBD HEK (100 nM). The mixtures are incubated for 30 minutes at 4°C then washed twice in PBS 0.5% BSA 2 mM EDTA. 2 µg per well of rabbit IgG are added to the cells which are incubated for 20 minutes at 4°C then washed twice qsp 200 µL in PBS 0.5% BSA 2 mM EDTA. The cells are resuspended in 50 µL of labeling buffer (PBS 0.5% BSA 2 mM EDTA) containing different mixtures of Ac from BioLegend. The mixtures are incubated for 20 minutes at 4°C away from light and then the splenocytes are washed twice qsp 200 µL in PBS 0.5% BSA 2 mM EDTA. The cells are then fixed for 30 minutes at room temperature.For this, 100 µL of 4% PFA buffer then 100 µL of PBS 0.5% BSA 2 mM EDTA were added before acquisition on a BD FACSAria ™ cytometer.

[0102] Dendritic Cell Mix 1 (duplicates): B220-FITC (#103206, 1 / 200), CD11c-PE-Cy7 (#117318, 1 / 200), CD317-APC (#127016, 1 / 100), CD8a-PerCP-Cy5.5 (#100734, 1 / 200), CD11b-APC-Cy7 (#101226, 1 / 800), Donkey anti-rabbit-BV421 (#406410, 1 / 100), Live Dead Aqua (ThermoFischer #L34966, 1 / 1000).

[0103] Mix 2 for monocytes and T, B lymphocytes (duplicates): 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). Results

[0104] To assess the ability of a molecular complex to bind immune system cells, ZZ-DTRBD HEK was incubated in the presence of mouse splenocytes. It was observed that this fusion protein preferentially binds to cDC-CD8+ cells, which are specialized APCs ( Fig. 3A ). It also preferentially interacts with monocytes which can also play the role of APCs ( Fig. 3B ). Finally, it binds to lymphocytes to a lesser extent. These data therefore indicate that the fusion protein preferentially targets APCs. Example 3: The ZZ-DTRBD HEK and ZZ-Tat 22-57C(22-37)S fusion proteins induce in vitro the secretion of IL-6 and IL-12 by cells of the immune system. Materials and Methods

[0105] C57BL / 6 mouse splenocytes are resuspended at a rate of 2.10 6< cells / mL in RPMI 10% FCS 1% Penicillin / Streptomycin medium. 100 µL of the cell suspension are distributed in 96-well plates in the presence or absence of molecular complexes (ZZ, DTRBD, ZZ-DTRBD HEK, or ZZ-Tat 22-57C(22-37)S) incubated at 1 µM final. After 24 hours of incubation, the supernatants are harvested for IL-6 and IL-12 cytokine assay by ELISA performed according to the manufacturer's instructions (R&D #DY406-05 and #DY419). Results

[0106] The inventors wondered whether molecular complexes could induce the activation of immune system cells. Since cell activation can lead to the secretion of cytokines, the inventors decided to evaluate in vitrothe presence of two of them in supernatants from the incubation of mouse splenocytes with molecular complexes and different control proteins. The first cytokine is IL-6 because it is representative of the inflammatory cytokines important for the initiation of the immune response. The second is IL-12 because it proves crucial for the induction of cellular immune responses. They found these two cytokines in increased quantities in the supernatants from the incubation with ZZ-DTRBD HEK which indicates that this molecular complex induces the activation of splenocytes ( Fig. 4 ).In contrast, they did not find an increase in the amount of cytokines in the supernatants resulting from incubation with free ZZ or free DTRBD, which indicates that the double Ig-binding domain and the heparan sulfate-binding domain alone cannot activate immune cells. These results therefore demonstrate that ZZ-DTRBD HEK is capable of inducing the activation of immune system cells and indicate that the association of the double Ig-binding domain and the HS-binding DTRBD domain is absolutely required for the effect.

[0107] The inventors proceeded according to a principle similar to that described in the previous paragraph to evaluate whether ZZ-Tat 22-57C(22-37)S is capable of activating cells of the immune system and whether the association of ZZ and the domain of Tat involved in binding to heparan sulfates is also absolutely required for the stimulatory effect. For this, they notably used a peptide, named Tat CY49-57 , which contains the basic region of Tat and is therefore representative of the interaction of Tat and its derivatives with heparan sulfates. They incubated mouse splenocytes with ZZ, Tat CY49-57 , ZZ + Tat CY49-57 , and ZZ-Tat 22-57C(22-37)S , respectively. They then evaluated the presence of IL-6 and IL-12 in the supernatants. They found these two cytokines in the supernatants from incubation with ZZ-Tat 22-57C(22-37)S but not in the supernatants from incubation with ZZ, Tat CY49-57, ZZ + Tat CY49-57 ( Fig. 5 ).These results therefore demonstrate that ZZ-Tat 22-57C(22-37)S is capable of inducing the activation of immune system cells and that this characteristic is not shared by the free forms ZZ and Tat CY49-57, which indicates that the association of the double Ig-binding domain and the basic region of Tat responsible for HS binding is absolutely required for the effect. Example 4: A molecular complex i) remains capable of triggering the secretion of IL-6 and IL-12 by cells of the immune system when its ZZ domain is substituted by BB, ii) exhibits increased stimulatory capacities when it is made up of oligomeric forms. Materials and Methods

[0108] C57BL / 6 mouse splenocytes are resuspended at a rate of 2.10 6< cells / mL in RPMI 10% FCS 1% Penicillin / Streptomycin medium. 100 µL of the cell suspension are distributed in 96-well plates in the presence or absence of different molecular complexes (ZZ-DTRBD HEK, BB-DTRBD HEK, ZZ-DTRBD coli and BB-DTRBD coli) incubated at 1 µM final. After 24 hours of incubation, the supernatants are harvested for IL-6 and IL-12 cytokine assay by ELISA performed according to the manufacturer's instructions (R&D #DY406-05 and #DY419). Results

[0109] The work described in Example 3 was carried out with two molecular complexes containing the double ZZ domain. This double domain, which binds the Fc of the Abs, can thus target the Abs located on the surface of the APCs. The inventors then asked themselves whether molecular complexes that can jointly target the Fc region and the Fab region of the Igs can also be capable of activating cells of the immune system. To do this, they relied on the double BB domain derived from the protein A of Staphylococcus aureus, which has the particularity of being able to bind the Fc region of Ac in the same way as Z but also the Fab region of Ac, unlike ZZ (Jansson B. et al., Fems Immunol. Med. Microbiol. 1998, 20:69-78). They prepared the BB-DTRBD HEK and BB-DTRBD coli complexes which are described in Example 1 and compared them to ZZ-DTRBD HEK and ZZ-DTRBD coli for the ability to induce the secretion of cytokines IL-6 and IL-12 in vitro when incubated with murine splenocytes.

[0110] As the inventors realized that ZZ-DTRBD and BB-DTRBD are in oligomeric form following their expression in HEK cells (cf. Fig. 1 ) while they are monomeric after expression in E. coli This work also made it possible to assess whether the state of oligomerization can affect the ability to induce the immune system.

[0111] The inventors realized that the supernatants from incubation with ZZ-DTRBD HEK contain higher amounts of cytokines than those from incubation with BB-DTRBD HEK. ( fig. 6 ).Similarly, supernatants from incubation with ZZ-DTRBD coli contain higher amounts of cytokines than those from incubation with BB-DTRBD coli. These results demonstrate that a molecular complex containing BB remains capable of inducing cytokine secretion. in vitro, but in lesser proportions than a molecular complex containing ZZ. These data therefore indicate that the activation effect can be mediated by molecular complexes containing domains capable of binding different sites on the Ac.

[0112] Comparison of cytokine levels in supernatants depending on the cell type used (i.e. HEK versus E. coli) for the production of molecular complexes has made it possible to highlight the difference in stimulating efficiency depending on the type of production. Indeed, in the supernatants resulting from incubation with complexes from HEK cells (ZZ-DTRBD HEK , BB-DTRBD HEK ) IL-6 and IL-12 are present in greater quantities than in those resulting from incubation with complexes resulting from expression in E. coli (ZZ-DTRBD coli and BB-DTRBD coli) ( Fig. 6 ) . As proteins resulting from expression in HEK cells are mainly oligomeric (cf. Fig. 1 ), The inventors deduced that the immune system is more effectively induced when the molecular complexes have a high oligomerization rate. Example 5 : Isolated dendritic cells secrete IL-6 and IL-12 when incubated in the presence of ZZ-DTRBD HEK Materials and methods

[0113] A C57BL / 6J mouse was euthanized and its spleen was collected in RPMI 10% FCS 1% Penicillin / Streptomycin medium. The spleen was perfused with 3 mL of collagenase D at 2 mg / mL in HBSS 0.5% BSA and then incubated for 30 minutes at 37°C. Splenocytes were collected and magnetic sorting of dendritic cells (DC) was performed according to the manufacturer's instructions (Miltenyi Biotec #130-100-875). DC were centrifuged for 5 minutes at 4°C at 390g and then resuspended at 2.10 6< cells / mL in RPMI 10% FCS 1% Penicillin / Streptomycin medium. 100 µL of the cell suspension (200,000 cells) are placed in a 96-well flat-bottom plate and 100 µL of RPMI 10% FCS 1% Penicillin / Streptomycin medium is added in the absence or presence of ZZ-DTRBD HEK (0.6 µM final). The cells are incubated for 24 hours at 37°C, then the supernatants are harvested for IL-6 and IL-12 cytokine assay by ELISA according to the manufacturer's instructions (R&D #DY406-05 and #DY419). Results

[0114] The establishment of immune defense mechanisms depends on the collaboration between different cellular partners. Among these, dendritic cells (DCs) represent APCs that play a central role. Indeed, they contribute to the activation of other cell types via direct interactions or through cytokines they secrete. To assess whether ZZ-DTRBD induces cytokine secretion by DCs, DCs were purified from splenocytes of C57BI / 6 mice. Then, these DCs were incubated for 24 hours in the presence or absence of ZZ-DTRBD and the supernatants were collected to assay the presence of IL-6 and IL-12. ( Fig. 7 ).The amount of IL-6 present in supernatants from incubation with ZZ-DTRBD HEK is approximately 5 times higher than that found in supernatants from incubation without molecular complex. A significant amount of IL-12 (approximately 8700 pg / ml) is also detected in supernatants from incubation with ZZ-DTRBD HEK but almost none in those from incubation without molecular complex. These data therefore indicate that ZZ-DTRBD induces the secretion of IL-6 and IL-12 by murine dendritic cells and could thus contribute to the establishment of immune defense mechanisms. Example 6 : ZZ-DTRBD and ZZ-Tat 22-57C(22-37)S proteins are capable of inducing dendritic cells in mice. Materials and Methods

[0115] Three groups of eight C57BL / 6 mice were injected three times at three-day intervals with 100 µl of PBS in the absence or presence of ZZ-DTRBD HEK (5 nmol per mouse) or ZZ-Tat 22-57C(22-37)S (10 nmol per mouse), respectively. 24 hours after the last injection, the animals were euthanized, and the spleens were harvested to recover 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 protected from light and washed twice in PBS 0.5% BSA 2 mM EDTA. The cells were then fixed for 20 minutes at 4°C in 100 µL of 4% PFA buffer and then washed in PBS 0.5% BSA 2 mM EDTA. They were finally resuspended in 200 µL of PBS 0.5% BSA 2 mM EDTA and then analyzed using a BD FACSAria ™ flow cytometer. The cells were analyzed by flow cytometry.CD8+ cDCs are identified as CD11c high< B220 -< CD8 +< CD11b -< , CD11b+ cDCs are identified as CD11c high< B220 -< CD8 -< CD11b +< and pDCs are identified as CD11c int< CD317 +< CD11b +< .

[0116] The Ab mixtures used for phenotypic analysis of dendritic cells were: 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). Results

[0117] As molecular complexes preferentially bind to APCs in vitroand induce cytokine secretion, the inventors wondered whether such complexes could induce the expansion of APCs in mice. To evaluate this aspect, the inventors injected three groups of 8 C57BI / 6 mice with a PBS solution in the presence or absence of ZZ-DTRBD HEK or ZZ-Tat 22-57C(22-37)S. They then euthanized the animals and removed their spleens to evaluate the frequency of different dendritic cell subpopulations in splenocytes. As can be seen in the figure 8 , the three murine DC populations (i.e., DC-CD8+, DC-CD11b+, and pDC) are present in the spleens of the group of mice injected only with PBS. The frequency of these three cell types is, however, significantly increased in the groups of animals injected with ZZ-DTRBD HEK or ZZ-Tat 22-57C(22-37)S. These data therefore indicate that molecular complexes are capable of inducing the expansion of dendritic cells in vivo.As these cells are central in the initiation of the immune response, these results strongly suggest that the complexes can thus promote the induction of immune response mechanisms. Example 7 : ZZ-DTRBD HEK induces IL-6 and IL-12 secretion by human dendritic cells in vitro Materials and Methods Isolation of human dendritic cells and incubation for the study of IL-6 and IL-12 secretion.

[0118] A bag of buffy coat is diluted ½ in AIMV medium and then incubated overnight at room temperature with shaking. 15 mL of histopaque medium is then added to 4 leucosep tubes to which 4 x 25 mL of diluted blood are added. The tubes are centrifuged for 15 minutes at room temperature at 1000 g without brake. The peripheral blood mononuclear cell rings (PBMCs) are recovered and washed with PBS 2 mM EDTA without calcium or magnesium. The PBMCs are centrifuged at 150 g for 10 minutes at room temperature and then a red blood cell lysis buffer (8.3 mg / mL NH 4 Cl, 0.84 mg / mL NaHCOs, 0.1 mM EDTA) is added and incubated for 10 minutes at 4°C. 40 mL of PBS are added and the cells are centrifuged for 10 minutes at room temperature at 150 g. DC sorting is then carried out according to the manufacturer's instructions (Miltenyi Biotec #130-091-379). The DC are centrifuged for 5 minutes at 4°C at 390 g and then resuspended at 2.10 6< cells / mL in RPMI 10% FCS 1% Penicillin / Streptomycin medium. 100 µl of sorted DC (200,000 cells) are placed in a 96-well plate and 100 µL of RPMI 10% FCS 1% Penicillin / Streptomycin medium in the absence or presence of ZZ-DTRBD HEK (1 µM final). The cells are incubated for 24 hours at 37°C then the supernatants are harvested for IL-6 and IL-12 cytokine assay by ELISA according to the manufacturer's instructions (R&D #DY206-05 and #DY1270-05). Results

[0119] To assess whether ZZ-DTRBD HEK can induce in vitro the secretion of IL-6 and IL-12 by DCs from healthy human donors, after 24 hours of incubation the presence of these two cytokines was evaluated in the culture supernatants of these cells. As can be seen in the Fig. 9 ,IL-6 is found in approximately 10 times greater quantities in supernatants from incubation in the presence of ZZ-DTRBD HEK compared to supernatants from incubation in the absence of the fusion protein. IL-12 is present in large quantities (2400 pg / ml) in supernatants from incubation of DCs in the presence of ZZ-DTRBD HEK and in very small quantities (8 pg / ml) in those from incubation in the absence of the molecular complex. All of these data therefore indicate that ZZ-DTRBD induces an increase in the secretion of IL-6 and IL-12 by human DCs. ZZ-DTRBD could thus contribute to the establishment of immune defense mechanisms in humans that would be linked to the secretion of these cytokines. Example 8 : ZZ-DTRBD and ZZ-Tat 22-57C(22-37)S slow the progression of colorectal tumor growth. Materials and Methods Study of the effect of the CpG / Polyl:C adjuvant mixture on tumor growth of a murine colorectal cancer line

[0120] Two groups of eight C57BL / 6 mice were injected with 0.5M MC38 cells subcutaneously in the paw. The mice were then left uninjected (Controls) or injected with the CpG-B 1018 / Poly I:C adjuvant mixture (30 µg for each adjuvant) three and six days later. Tumor growth was monitored by measuring tumors with a caliper. Upon reaching the stopping criterion, they were euthanized. Study of the effect of the ZZ-DTRBD HEK molecular complex, injected in the absence or presence of adjuvant, on tumor growth of a murine colorectal cancer line

[0121] Three groups of six C57BL / 6 mice were injected with 0.5M MC38 cells subcutaneously in the paw. Three, six, and nine days later, the mice were either uninjected (Controls) or injected with ZZ-DTRBD HEK (2 nmol per mouse) in the absence or presence of CpG-B 1018 + Poly I:C (30 µg for each adjuvant per mouse). The mice were monitored individually for 15 days (tumor measurements with a caliper) before euthanasia. Comparison of the effect of an anti-PD-1 Ab and ZZ-DTRBD HEK molecular complexes And ZZ-Tat 22-57C(22-37)S on tumor growth of a murine colorectal cancer line

[0122] Four groups of eight C57BL / 6 mice were injected with 0.5M MC38 cells subcutaneously in the paw. Three, six and nine days later, the mice were either not injected (Controls) or injected with the anti-PD-1 antibody (Euromedex #BE0146-100MG, clone RMP1-14), ZZ-DTRBD HEK or ZZ-Tat 22-57C(22-37)S (0.96 nmol per mouse) in the presence of CpG-B 1018 + Poly I:C. (30 µg for each adjuvant per mouse). The mice were monitored individually (tumor measurements with a caliper) over time. Upon reaching the stopping criterion, they were euthanized. Results

[0123] To assess whether molecular complexes can impact tumor growth, studies were conducted in a syngeneic mouse cancer model. This model is based on a colon tumor line, called MC38, and C57BI / 6 mice. Cancer induction is caused by injecting 500,000 MC38 cells per mouse.

[0124] First, the impact of the CpG1018 / polyl:C adjuvant mixture on tumor growth was assessed. As can be seen in the Fig. 10A , Compared to the untreated group (controls), tumor growth is almost not affected in the group of mice injected with the CpG1018 / polyl:C mixture. These data therefore indicate that this adjuvant mixture cannot slow the growth of this colorectal tumor on its own.

[0125] In a second step, the impact of ZZ-DTRBD HEK injected alone or in the presence of the adjuvant mixture CpG1018 / polyl:C on tumor growth was evaluated. As can be seen in the Fig. 10B , Compared to the untreated group (controls), tumor growth is slowed on day 8 and day 10 in the group injected with ZZ-DTRBD HEK without adjuvant. The effect is even more significant in the group treated with ZZ-DTRBD HEK and the CpG1018 / polyl:C adjuvant mixture. Indeed, in this group, tumor growth is slowed over the entire period of observation of the animals (D6 to D15). All of these data therefore indicate that ZZ-DTRBD HEK has an impact on the growth of the MC38 line and that this effect is amplified when it is mixed with the CpG1018 / polyl:C adjuvant.

[0126] In another series of experiments, the effect on MC38 tumor growth in C57BI / 6 mice was compared for the following treatments: ZZ-DTRBD HEK / CpG1018 / polyl:C, ZZ-Tat 22-57C(22-37)S / CpG1018 / polyl:C, anti-PD-1 Ab. As can be seen in the Fig. 11A And 11B , Treatment with anti-PD-1 Ab does not significantly impact tumor growth compared to the control group. Mice injected with ZZ-DTRBD HEK / CpG1018 / polyl:C and ZZ-Tat 22-57C(22-37)S / CpG1018 / polyl:C, respectively, show a decrease in tumor growth. This slowdown in tumor growth leads to increased survival of animals in these two groups. ( Fig. 11C ). Example 9 : Activated monocytes and CD4+ lymphocytes can be induced by molecular complexes targeting the surface molecule DEC205 and Fc gamma receptors, respectively. . Materials and Methods

[0127] To prepare a molecular complex targeting the DEC205 molecule, an anti-DEC205 Ab was used (BioLegend; clone NLDC-145, ref BLE138202). It was incubated in the absence or presence of ZZ-Tat 22-57C22-37)S at a fixed concentration (0.2µM for each molecule), for 24 hours in RPMI medium without FCS. The ability of the ZZ region to bind the Fc region of the anti-DEC205 Ab allowed the formation of a non-covalent molecular complex, called anti-DEC205 / ZZ-Tat 22-57C22-37)S. The same protocol was used to form a complex between the ZZ molecule and the anti-DEC205 Ab, called anti-DEC205 / ZZ.

[0128] To prepare a molecular complex targeting FC gamma receptors, a non-specific human polyclonal Ab was used. This Ab was incubated with ZZ-Tat 22-57C22-37)S or ZZ following a protocol identical to that used for the anti-DEC205 Ab. It was thus possible to form a non-covalent molecular complex, called IgG / ZZ-Tat 22-57C22-37)S and an IgG / ZZ complex.

[0129] Human PBMCs prepared as described in Example 7 were then incubated in the absence or presence of the following compounds: anti-DEC205 / ZZ-Tat 22-57C22-37)S, IgG / ZZ-Tat 22-57C22-37)S, anti-DEC205 / ZZ, IgG / ZZ ZZ-DTRBD HEK, and free anti-DEC205 Ab and IgG (final concentration of 0.1 µM for each compound). In these experiments, PBMCs were also incubated with free ZZ-Tat 22-57C22-37)S and ZZ proteins at a concentration of 1 µM. After 24 hours, the cells were harvested and labeled with fluorescent Abs to identify monocytes (CD14, anti-CD14-BV605, Biolegend), CD4+ T lymphocytes (CD4, anti-CD4-PerCP-Cy5.5, Biolegend) and the CD69 molecule (anti-CD69-BV785, Biolegend). After 30 minutes of incubation, the PBMCs were fixed using a solution containing 4% paraformaldehyde and analyzed by flow cytometry. Results

[0130] The previous examples show that molecular complexes targeting Abs can effectively induce certain immune response mechanisms and effectively slow tumor growth. The Abs located on the surface of APCs are the molecules targeted by these complexes. However, APCs express a large number of other molecules that can also represent targets for molecular complexes according to the invention. The inventors therefore wondered whether molecular complexes targeting HS and receptors other than immunoglobulins located on the APC surface can also activate these cells. To evaluate this aspect, they chose to evaluate two types of receptors. The first is the DEC205 protein, which is a lectin selectively expressed in humans by monocytes and certain populations of 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 monocytes and certain populations of dendritic cells.

[0131] The inventors then prepared two molecular complexes that could respectively target these receptors. To target the DEC205 protein, they used a monoclonal Ab specific for this receptor, which they complexed with the fusion protein ZZ-Tat 22-57C22-37)S . They called this molecular complex anti-DEC205 / ZZ-Tat 22-57C22-37)S . To target the Fc receptors, they used a non-specific human polyclonal IgG Ab that can interact with these receptors via its Fc domain. They formed a molecular complex between this IgG and ZZ-Tat 22-57C22-37)S , called IgG / ZZ-Tat 22-57C22-37)S . These two aforementioned molecular complexes thus possess the capacity to bind CPAg receptors and heparan sulfates via the Tat 22-57C22-37)S domain of ZZ-Tat 22-57C22-37)S. In these molecular complexes, the two proteins are non-covalently associated via their respective Fc and ZZ domains.ZZ can therefore no longer target immunoglobulins located on the surface of APCs because it is already engaged in the interaction with the Abs. The inventors also prepared two complexes, respectively called anti-DEC205 / ZZ and IgG / ZZ, lacking the Tat 22-57C22-37)S heparan sulfate binding region in order to use them as controls in subsequent activation experiments.

[0132] Since the DEC205 molecule and Fc receptors are expressed by monocytes, the inventors then asked whether the molecular complexes can enable the activation of this APC subpopulation. For this, they incubated PBMCs in the absence or presence of a fixed concentration (0.1 µM) of anti-DEC205 / ZZ-Tat 22-57C22-37)S , IgG / ZZ-Tat 22-57C22-37)S , anti-DEC205 / ZZ, IgG / ZZ, anti-DEC205, IgG, respectively. They also incubated the free ZZ-Tat 22-57C22-37)S and ZZ proteins at a concentration of 1µM to evaluate the effect of ZZ-Tat 22-57C22-37)S at a concentration identical to that which proved activating in examples 3, 4 and 5. After 24h, they evaluated the proportion of activated monocytes and CD4+ T lymphocytes. As can be seen in the Fig. 12A ,ZZ does not modify the proportion of activated monocytes in PBMCs while it is significantly increased by ZZ-Tat 22-57C22-37)S which indicates that this molecular complex allows to activate human APCs. The proportion of activated cells is for its part reduced with the free anti-DEC205 Ab or included in the anti-DEC205 / ZZ complex. On the other hand, the proportion of activated monocytes is increased in the presence of the anti-DEC205 / ZZ-Tat 22-57C22-37)S complex. A similar behavior is observed when CD4+ T lymphocytes are considered ( Fig. 12B ). These data therefore indicate that an Ac targeting APCs and lacking the capacity to activate them can become capable of inducing them when it is included in a molecular complex allowing HS to also be targeted.

[0133] Analysis of the activation status of monocytes after incubation with free human IgG shows that this Ab increases the proportion of activated monocytes. The IgG / ZZ complex does not allow a significant increase in the proportion of activated cells. On the other hand, the number of activated monocytes is more than twice as high when PBMCs are incubated with the molecular complex IgG / ZZ-Tat 22-57C22-37)S . A similar behavior is observed when CD4+ T lymphocytes are considered ( Fig. 12B ). These data therefore indicate that an Ac targeting Fc gamma receptors on the surface of APCs is capable of activating them but that the activation effect is increased when the Ac is included in a molecular complex allowing it to also target HS.

[0134] Interestingly, these results showing a joint increase in the proportion of activated monocytes and T lymphocytes indicate that molecular complexes induce several cellular actors that play a central role in immune defense mechanisms. Example 10 : Molecular complexes targeting dendritic cells induce in vitro the secretion of IL-6 by cells of the immune system. Materials and Methods

[0135] To prepare molecular complexes targeting DCs, three antibodies specific to molecules expressed by DCs were used. The first Ab (BD reference 555538), called anti-CD74 Ab, targets the CD74 molecule. The second Ab (BD reference 551186), called anti-CD209 Ab, targets the CD209 molecule. The third Ab (BD reference 552501), called anti-CD275 Ab, targets the CD275 molecule.

[0136] Each of these three Abs (30nM for each molecule) was incubated in the absence or presence of ZZ-Tat 22-57C22-37)S or ZZ (5nM for each molecule), for 24 hours at 4°C in RPMI medium, 5% human serum AB. The ability of the ZZ region to bind the Fc region of these Abs allowed the formation of different non-covalent molecular complexes called anti-CD74 / ZZ-Tat 22-57C22-37)S, anti-CD209 / ZZ-Tat 22-57C22-37)S, anti-CD275 / ZZ-Tat 22-57C22-37)S, anti-CD74 / ZZ, anti-CD209 / ZZ, and anti-CD275 / ZZ, respectively.

[0137] Human PBMCs were resuspended at a rate of 5.10 6< cells / mL in RPMI 5% Human Serum AB medium. 100 µL of the cell suspension was distributed in a 96-well plate in the presence or absence of free ZZ or ZZ-Tat 22-57C(22-37)S Abs and molecular complexes. After 24 hours of incubation, the supernatants were harvested for IL-6 cytokine assay by ELISA performed according to the manufacturer's instructions (R&D #DY406-05). Results

[0138] The inventors wondered whether molecular complexes targeting three molecules expressed by DCs could induce the activation of immune system cells. Since cell activation can lead to the secretion of cytokines, the inventors decided to evaluate in vitro the presence of IL-6. They found this cytokine in the supernatants from incubation with ZZ-Tat 22-57C22-37)S but not in those from incubation with ZZ. They did not find it in the supernatants from incubation of SMCs with free anti-CD74 Ab ( Fig. 13A ) indicating that this Ac cannot induce activation when it is in free form. The inventors observed that IL-6 secretion is increased when the PBMCs are incubated with the anti-CD74 / ZZ complex, indicating that the double ZZ domain allows activation mediated by this Ac ( Fig. 13A). However, the secretion of this cytokine is even stronger with anti-CD74 / ZZ-Tat 22-57C22-37)S , indicating that the addition of the Tat region allowing binding of HSPGs allows to increase the cellular activation mediated by this complex.

[0139] The inventors noticed that free anti-CD209 and anti-CD275 Ac are capable of inducing the secretion of IL-6 when they are incubated with CMSPs ( Fig. 13B, and 13C ). When these two Abs are respectively complexed with ZZ (anti-CD209 / ZZ and anti-CD275 / ZZ) the secretion of this cytokine is also observed. This secretion is however increased when these two Abs are complexed with ZZ-Tat 22-57C22-37)S, which indicates that the activating power can be increased when the targeting molecular complexes are able to bind HSPGs. Example 11: A molecular complex directed against the CD335 receptor expressed by NK and NKT cells induces in vitro an increase in the proportion of activated NK and NKT cells. Materials and Methods

[0140] To prepare molecular complexes targeting the CD335 molecule on the surface of NK and NKT cells, an Ab (Origene AM31284AF-N), called anti-CD335 Ab, was used. This Ab was incubated at 30nM in the absence or presence of ZZ-Tat 22-57C22-37)S or ZZ (5nM for each molecule), for 5 hours at 37°C in RPMI medium, 5% AB human serum. The ability of the ZZ region to bind the Fc region of these Abs allowed the formation of two molecular complexes called anti-CD335 / ZZ-Tat 22-57C22-37)S, and anti-CD335 / ZZ, respectively.

[0141] Human PBMCs were resuspended at a rate of 5.10 6< cells / ml in RPMI medium 5% Human serum AB. 100 µL of the cell suspension was distributed in 96-well plates in the presence or absence of free anti-CD335, ZZ or ZZ-Tat 22-57C(22-37)S Ab as well as molecular complexes. After 18 hours, the cells were harvested, labeled with fluorescent Abs to identify NK cells (CD56+, biolegend reference 362510 1 / 100 dilution), NKT cells (CD56+ biolegend reference 362510; CD3+; Miltenyi ref 130-113-136 1 / 200 dilution), and the CD69 molecule (biolegend reference 310932, 1 / 100 dilution). After 30 minutes of incubation, the PBMCs were fixed using a solution containing 4% paraformaldehyde and then analyzed by flow cytometry. Results

[0142] The inventors wondered whether molecular complexes targeting the CD335 molecule expressed on the surface of NK and NKT cells could induce the activation of these two cell types. To assess this, they used anti-CD335 Ab in isolated form or included in a molecular complex. They incubated PBMCs in the absence or presence of the different mixtures. Since NK and NKT cells play a crucial role in immune defense mechanisms, after 18 hours of incubation, they assessed the proportion of activated NK and NKT cells by monitoring the expression of the co-stimulatory molecule CD69.

[0143] As can be seen on the Figure 14 , ZZ, isolated anti-CD335 Ab and anti-CD335 / ZZ complex do not increase the proportion of NK cells (A) or NKT (B)expressing CD69 in PBMCs. A different behavior is observed with free ZZ-Tat 22-57C22-37)S. Indeed, this molecular complex does not increase the proportion of NKT cells expressing CD69 but increases the percentage of NK cells expressing this marker, which indicates that the association of ZZ and the HS ligand in the ZZ-Tat 22-57C(22-37)S complex increases the activation of NK cells. The anti-CD335 / ZZ-Tat 22-57C(22-37)S molecular complex, for its part, has an impact on the activation of both cell subpopulations. Indeed, it increases the proportion of NK and NKT cells expressing CD69. All of these data therefore indicate that the anti-CD335 Ac lacks the ability to activate both cell types but can become capable of inducing them when it is included in a molecular complex that also targets HS. Example 12 : Molecular complexes directed respectively against the CD56 (A) and CD336 (B) molecules expressed by NK and NKT cells induce in vitro an increase in the proportion of activated NKT cells. Materials and Methods

[0144] To prepare molecular complexes targeting CD56 and CD336 molecules on the surface of NK and NKT cells, anti-CD56 Ab (Biolegend reference 304622) which targets the CD56 molecule and anti-CD336 Ab (Origene AM50346PU-N) which targets the CD336 molecule were used.

[0145] These Ac were incubated at 30nM in the absence or presence of ZZ-Tat 22-57C22-37)S or ZZ (5nM for each molecule), for 5 hours at 37°C in RPMI medium, 5% AB human serum. The ability of the ZZ region to bind the Fc region of these Ac allowed the formation of four molecular complexes called respectively anti-CD56 / ZZ-Tat 22-57C22-37)S, anti-CD56 / ZZ, anti-CD336 / ZZ-Tat 22-57C22-37)S, and anti-CD336 / ZZ.

[0146] Human PBMCs were resuspended at a rate of 5.10 6< cells / ml in RPMI medium 5% Human serum AB. 100 µL of the cell suspension was distributed in 96-well plates in the presence or absence of isolated forms of anti-CD56 Ab, anti-CD336 Ab, ZZ or ZZ-Tat 22-57C(22-37)S as well as non-covalent molecular complexes. After 18 hours, the cells were harvested, labeled using fluorescent Abs described in Example 11 which allow the identification of NKT cells (CD56+CD3+), and the CD69 molecule. After 30 minutes of incubation, the PBMCs were fixed using a solution containing 4% paraformaldehyde and then analyzed by flow cytometry. Results

[0147] The inventors wondered whether molecular complexes targeting the expressed CD56 and CD336 molecules could induce NKT cell activation. To assess this, they used anti-CD56 and anti-CD336 Abs in isolated form or included in a molecular complex. They incubated PBMCs in the absence or presence of the different mixtures. After 18 hours of incubation, they assessed the proportion of activated NKT cells by monitoring the expression of the co-stimulatory molecule CD69.

[0148] As can be seen on the Figure 15 , ZZ, ZZ-Tat 22-57C22-37)S , anti-CD56 Ab, isolated anti-CD336 Ab and the anti-CD336 / ZZ complex do not increase the proportion of NKT cells expressing CD69 in PBMCs. A different behavior is observed with the anti-CD56 / ZZ-Tat 22-57C22-37)S molecular complexes ( fig. 15A ), anti-CD336 / ZZ-Tat 22-57C22-37)S. ( Fig. 15B). Indeed, an increase in the proportion of NKT cells expressing CD69 is found when these two molecular complexes are incubated with PBMCs. All these data therefore indicate that isolated anti-CD56 Ab and anti-CD336 Ab lack the ability to activate NKT cells but can become capable of inducing them when they are included in a molecular complex also targeting HS. Example 13: Molecular complexes targeting NK and NKT cells induce in vitro the secretion of IL-6 by cells of the immune system. Materials and Methods

[0149] 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 (30nM for each molecule) was incubated in the absence or presence of ZZ-Tat 22-57C22-37)S or ZZ (5nM for each molecule), for 24 hours in RPMI medium, 5% Human serum AB. The ability of the ZZ region to bind the Fc region of these Abs made it possible to form different non-covalent molecular complexes called anti-CD56 / ZZ-Tat 22-57C22-37)S, anti-CD335 / ZZ-Tat 22-57C22-37)S, anti-CD336 / ZZ-Tat 22-57C22-37)S. anti-CD56 / ZZ, anti-CD335 / ZZ, and anti-CD336 / ZZ, respectively.

[0150] Human PBMCs are resuspended at a rate of 5.10 6< cells / mL in RPMI medium 5% Human serum AB. 100 µL of the cell suspension is distributed in 96-well plates in the presence or absence of free ZZ or ZZ-Tat 22-57C(22-37)S Abs as well as molecular complexes. After 24 hours of incubation, the supernatants are harvested for an IL-6 cytokine assay by ELISA performed according to the manufacturer's instructions (R&D #DY406-05). Results

[0151] The inventors wondered whether molecular complexes targeting NK and NKT cells could induce the activation of immune system cells. Since cell activation can lead to the secretion of cytokines, the inventors decided to evaluate in vitrothe presence of IL-6. They found this cytokine in the supernatants from incubation with ZZ-Tat 22-57C22-37)S but not in those from incubation with ZZ. They did not find IL-6 in the supernatants from incubation of SMCs with free anti-CD56 and anti-CD335 Abs ( Fig. 16A and 16B ), indicating that these two proteins cannot induce activation when in free form. However, they detected the cytokine in the supernatants resulting from incubation with the anti-CD336 antibody ( Fig. 16C ) which indicates that, in its free form, this Ac has a stimulating activity. The inventors observed, for the three Ac, an increased secretion of IL-6 when the CMSPs are respectively incubated with the anti-CD56 / ZZ-Tat complexes 22-57C22-37)S, ( Fig. 16A ), anti-CD335 / ZZ-Tat 22-57C22-37)S, ( Fig. 16B ) and anti-CD336 / ZZ-Tat 22-57C22-37)S( Fig. 16C ). These data therefore indicate that molecular complexes targeting NK cells and HSPGs can induce effective activation of immune cells. Example 14 : Molecular complexes targeting immune response checkpoints (ICPs) induce in vitro the secretion of IL-6 by cells of the immune system. Materials and Methods

[0152] To prepare molecular complexes targeting ICPs, three antibodies specific to molecules considered as ICPs were used. The first Ab (BioXCell reference BE0190), called anti-CTLA-4 Ab, targets the CTLA-4 molecule. The second Ab (BioXCell reference BE0285), called anti-PD-L1 Ab, targets the PD-L1 molecule. The third Ab (R&D reference MAB10542), called anti-OX40 Ab, targets the OX40 molecule.

[0153] Each of these three Abs (30nM for each molecule) was incubated in the absence or presence of ZZ-Tat 22-57C22-37)S or ZZ (5nM for each molecule), for 24 hours at 4°C in RPMI medium, 5% human serum AB. The ability of the ZZ region to bind the Fc region of these Abs allowed the formation of different non-covalent molecular complexes, called anti-CTLA-4 / ZZ-Tat 22-57C22-37)S, anti-PD-L1 / ZZ-Tat 22-57C22-37)S, anti-OX40 / ZZ-Tat 22-57C22-37)S, anti-CTLA-4 / ZZ, anti-PD-L1 / ZZ, and anti-OX40 / ZZ, respectively.

[0154] Human PBMCs were resuspended at 5.10 6< cells / mL in RPMI 5% Human Serum AB medium. 100 µL of the cell suspension was distributed in 96-well plates in the presence or absence of free Ab, ZZ or ZZ-Tat 22-57C(22-37)S as well as molecular complexes. After 24 hours of incubation, the supernatants were harvested for IL-6 cytokine assay by ELISA performed according to the manufacturer's instructions (R&D #DY406-05). Results

[0155] The inventors wondered whether molecular complexes targeting ICPs could induce the activation of immune system cells. Since cell activation can lead to the secretion of cytokines, the inventors decided to evaluate in vitro the presence of IL-6. They did not find any in the supernatants resulting from the incubation of CMSPS with the three different free Abs or with free ZZ, which indicates that these compounds cannot induce activation when in free form ( Fig. 17A, 17B, and 17C ).On the other hand, they found this cytokine in the supernatants resulting from the incubation with ZZ-Tat 22-57C22-37)S which demonstrates that the association of the double Ig binding domain and the HS binding Tat 22-57C22-37)S domain allows the activation of cells of the system. The inventors however observed that the secretion of IL-6 is further increased when the CMSPs are respectively incubated with the anti-CTLA-4 / ZZ-Tat 22-57C22-37)S complexes, ( Fig. 17A ), anti-PD-L1 / ZZ-Tat 22-57C22-37)S, ( Fig. 17B ), anti-OX40 / ZZ-Tat 22-57C22-37)S ( Fig. 17C ).In contrast, IL-6 was absent from supernatants resulting 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. Together, these data indicate that molecular complexes targeting ICPs and HSPGs can induce immune cell activation.

Claims

1. Molecular complex for use as an immunostimulant medicament in the immunotherapy of cancer or infectious diseases, said complex consisting of at least one ligand of a sulphated sugar of the glycosaminoglycan family expressed on the surface of antigen-presenting cells or NK or NKT cells (first ligand) and at least one ligand of a surface molecule of antigen-presenting cells or of NK or NKT cells other than a sulphated sugar of the glycosaminoglycan family (second ligand) linked together, and said complex being devoid of an antigen specific for the disease to be treated.

2. Molecular complex for use according to claim 1, wherein the first ligand is a heparan sulphate 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) 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); a polyarginine peptide R7 to R11; and a peptide comprising the diphtheria toxin R domain (SEQ ID NO: 5) or at least the fragment DT453-467 (SEQ ID NO:7) of said domain comprising the heparan sulphate binding region.

3. Molecular complex for use according to any one of the preceding claims, wherein the second ligand targets an antigen-presenting cell surface molecule selected from the group consisting of: type C lectin receptors, membrane immunoglobulins, receptors for the constant region of immunoglobulins, and immune checkpoint molecules and their ligands.

4. Molecular complex for use according to any one of the preceding claims, wherein the second ligand is selected from the group consisting of: (i) antibodies directed against said surface molecules of antigen-presenting cells or NK or NKT cells and fragments thereof containing 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 the Fc and / or Fab region of antibodies, in particular S. aureus protein A, its fragment BB (SEQ ID NO: 1) and its derivative ZZ (SEQ ID NO: 2).

5. Molecular complex for use according to any one of the preceding claims, in the form of oligomers or a mixture of monomers and oligomers.

6. Molecular complex for use according to any one of the preceding claims, consisting of a fusion protein between the first and second ligand.

7. Molecular complex for use according to claim 6, consisting of a fusion protein comprising a first ligand selected from: Tat49-57 (SEQ ID NO: 3), Tat37-57 (SEQ ID NO: 8) and Tat22-57C(22-37)S (SEQ ID NO:9); the diphtheria toxin R domain (SEQ ID NO: 5) or the fragment DT453-467 (SEQ ID NO:7) and a second ligand selected from: the fragment BB of protein A (SEQ ID NO: 1) or its derivative ZZ (SEQ ID NO: 2).

8. Molecular complex for use according to any one of claims 1 to 5, wherein the second ligand is an antibody or an antibody fragment and the first ligand forms a fusion protein with an immunoglobulin binding element, preferably S. aureus protein A, its fragment BB (SEQ ID NO: 1) or its derivative ZZ (SEQ ID NO: 2).

9. Molecular complex for use according to claim 8, wherein said fusion protein comprises a first ligand selected from Tat49-57 (SEQ ID NO: 3), Tat37-57 (SEQ ID NO: 8) and Tat22-57C(22-37)S (SEQ ID NO: 9); the diphtheria toxin R domain (SEQ ID NO: 5) or the fragment DT453-467 (SEQ ID NO:7) and an immunoglobulin-binding element selected from the fragment BB of protein A (SEQ ID NO: 1) or its derivative ZZ (SEQ ID NO: 2).

10. Molecular complex for use according to claim 9, wherein the immunoglobulin-binding element is the fragment BB of protein A (SEQ ID NO: 1), and said fusion protein being complexed with the second ligand, which consists of a whole immunoglobulin.

11. Molecular complex for use according to claim 8, wherein said fusion protein is complexed with the second ligand, which is selected from an anti-Fc gamma RI, RII and / or RIII, anti-DEC-205 , anti-DC-SIGN, anti-CD74, anti-CD275, anti-CD56, anti-CD335, anti-CD336, anti-CTLA-4, anti-PD-L1 or anti-OX40 antibody, or a fragment of the preceding antibodies comprising at least the paratope.

12. Molecular complex for use according to any one of the preceding claims as an immunostimulant medicament, preferably to activate antigen-presenting cells, in particular dendritic cells or monocytes, to activate NK or NKT cells, and / or to activate the secretion of IL-6 and / or IL-12 cytokines.

13. Composition for use as an immunostimulant medicament in the immunotherapy of cancer or infectious diseases, comprising at least one molecular complex as defined in any one of the preceding claims, and at least one pharmaceutically acceptable vehicle, a carrier substance and / or an adjuvant.

14. Composition for use according to claim 13, wherein the adjuvant is a CpG oligodeoxynucleotide, polyinosinic-polycytidylic acid or a mixture of CpG oligodeoxynucleotide(s), and polyinosinic-polycytidylic acid, and / or the carrier substance is a nanoparticle.

15. The composition for use according to claim 13 or 14, comprising at least another therapeutic agent, preferably at least one immune checkpoint inhibitor, preferably an anti-PD-1, an anti-PDL-1 or an anti-CTLA4.