Immunomodulatory complex and uses thereof for therapy

Abstract

The invention relates to a molecular complex consisting of at least one ligand of a sulphated sugar of the glycosaminoglycan family linked to at least one ligand of a surface molecule of antigen-presenting cells or NK or NKT cells, for use as an immunomodulatory drug, in particular in the immunotherapy of cancer and infectious diseases.

Description

Immunomodulator Complex and its Applications for Therapy technical field

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

[0002] Most therapeutic approaches are based on drugs capable of acting directly on the target responsible for the pathology or on cells exhibiting a non-physiological state. However, pathological processes can be promoted by dysfunctions of the immune system. Immunosuppressive 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 consideration of developing immunotherapeutic drugs capable of restoring the proper functioning of immune defenses, particularly by modulating the activity of T lymphocytes or NK / NKT cells, in order to allow the immune system to more effectively control pathological processes.These modulating immunotherapies aim to restore or inhibit the functionality of a large part of a lymphocyte repertoire, particularly the repertoire of CD4+ T cells, CD8+ T cells, or regulatory T cells. They differ in this respect from vaccine immunotherapies, which aim to induce a limited number of lymphocytes corresponding to the specific cells of the antigen(s) included in the vaccine.

[0003] To develop immunotherapeutic drugs, it is first necessary to identify targets playing a crucial role in controlling the immune response. During the 1990s, it was shown that molecules expressed on the surface of T lymphocytes, respectively called PD-1 and CTLA-4, can induce inhibitory signals that negatively regulate the activity of these cells and can thus regulate certain immune defense mechanisms (Sshida, Y., Agata, Y., Shibahara, K., & Honjo, T. (1992). EMBO J., 77(11), 3887-3895.; Freeman, et al. (2000). J Exp Med, 792(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 ICP inhibitors.

[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 inhibitory signals to T lymphocytes and thus triggers various immunosuppressive mechanisms. It has also 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), 700(8), 4712-4717; Iwai, Y. et al., Int. Immunol., (2005). 77(2), 133-144).Some of these immunomodulatory antibodies have proven capable of limiting the growth of various cancers (melanoma, lung cancer, etc.) and significantly increasing patients' life expectancy. They are now commonly used as anti-tumor immunotherapeutic drugs in humans (Adachi K. and K. Tamada, Cancer Soi., 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 anti-ICP inhibitory antibodies, however, have the drawback of only working in 10 to 30% of cancer patients (Pitt JM et al., Immunity. 2016 Jun 21;44(6):1255-69). Many research groups are therefore aiming to... to discover new immunomodulatory compounds that are more effective and / or usable in combination with the immunotherapies described previously. As several new inhibitory ICPs have been identified, work is focused on selecting 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, in turn, led to a focus on selecting 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, the aim is to select therapeutic molecules capable of binding ICPs or IC P-ligands selectively expressed on the surface of effector immune cells, such as T lymphocytes and NK or NKT cells, or antigen-presenting cells (APCs). This selectivity of expression limits the spread of the molecule to non-immune cells, resulting in increased therapeutic efficacy and a reduced 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 consists of proteins associated with sulfated GAGs: heparan sulfates (HS). HSPGs, which play a central role in numerous biological processes (cell proliferation, cell adhesion, inflammation, coagulation, and cellular penetration by pathogenic microorganisms, particularly viruses and parasites), are found on the surface of most mammalian cells and in the extracellular matrix (Dreyfuss et al., Annuals of the Brazilian Academy of Sciences, 2009, 81, 409-429). This ubiquitous expression leads to the conclusion that HSPGs, and their HS domains, do not represent relevant immunomodulatory targets. Furthermore, the The ability of HS ligands to induce immune cell activation has not yet been identified. Summary of the invention

[0007] In their work, the inventors first discovered that a ligand of a sulfated sugar from the GAG ​​family, heparan sulfates (HS), is unable to activate dendritic cells in vitro. They then observed that a molecular complex containing this ligand and a ligand of an antigen-presenting cell (APC) surface molecule potentiates the immunomodulatory effect of the heparan sulfate (HS) ligand. Indeed, this molecular complex induces APCs even more strongly, as shown by the increased secretion of the cytokines IL-6 and IL-12, and allows for the subsequent activation of T lymphocytes. The inventors showed that the association between the two ligands can be achieved in the form of a covalent or non-covalent complex, including a fusion protein.The inventors also observed that the immunomodulatory properties of this complex allow for better control of pathological processes and, in particular, for slowing the progression of a tumor.

[0008] These results are surprising because, although described as potentially acting as a receptor or co-receptor, HSPGs had never before been shown to potentiate activation mediated by a receptor ligand specifically expressed on the surface of APCs. Furthermore, the molecular complex, which targets ubiquitously expressed HSs, could disseminate to the extracellular matrix or to cells irrelevant to the pathology. Therefore, theoretically, it should not be capable of modulating the activity of APCs and a broad repertoire of T lymphocytes with sufficient efficacy to produce an immunotherapeutic effect.

[0009] The inventors also discovered that the results obtained with APCs were applicable to other cells of the innate immune response, namely 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 associated, in a molecular complex, with a ligand of a sulfated sugar from the GAG ​​family.

[0010] Accordingly, the present invention relates to a molecular complex for use as an immunomodulatory drug, preferably an immunostimulatory drug, said complex consisting of at least one ligand of a sulfated sugar from the glycosaminoglycan family (first ligand or L1) and at least one ligand of another surface molecule of APCs or NK or NKT cells (second ligand or L2) linked together, and said complex being devoid of a specific antigen of 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 Tat 49-57 region (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 R7 to R11 peptide; 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 DT453-467 fragment (SEQ ID NO: 7) of said domain which includes 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: the receptors NKG2D, NKp30, NKp44, NKp46, NKp80, Ly49H, the receptors KIR, NKG2A, and the 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 their fragments containing at least the paratope; (ii) immunoglobulins, preferably IgG, and their fragments comprising at least the Fc region; and (iii) proteins and protein fragments that bind the Fc and / or Fab region of antibodies, including S. aureus protein A, its BB fragment (SEQ ID NO: 1) and its ZZ derivative (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 second ligands. A particularly preferred type of complex according to the invention consists of a fusion protein comprising a first ligand selected from: Tat49-57 (SEQ ID NO: 3), Tat37-57 (SEQ ID NO: 8), Tat22-57C(22-37)S (SEQ ID NO: 9), the R domain of diphtheria toxin (SEQ ID NO: 5) or 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).

[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 element, preferably S. aureus protein A, 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 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 is complexed with the second ligand, which consists of an entire immunoglobulin. In another particularly preferred type of complex according to the invention, said fusion protein is complexed with the second ligand, which is selected from an anti-FcγRF 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 drug, 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 cytokines IL-6 and / or IL-12.

[0019] The present invention also relates to a composition for use as an immunomodulatory, preferably immunostimulatory, drug, comprising at least one molecular complex according to the invention and at least one pharmaceutically acceptable vehicle, carrier substance, and / or 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. Description of the invention

[0022] The present invention relates to a molecular complex for use as an immunomodulatory drug, preferably an immunostimulatory drug, said complex consisting of at least one ligand of a sulfated sugar from the glycosaminoglycan family (first ligand) and at least one ligand of another surface molecule of APCs or NK or NKT cells (second ligand) linked together, and said complex being devoid of a specific antigen of the disease to be treated.

[0023] Such a complex is capable of inducing immune system cells in a way that favors triggering an immune response. Thus, when splenocytes are incubated with these complexes, activation of dendritic cells is observed, which then become capable of secreting the cytokines IL-6 and IL-6. 12. Furthermore, DC induction can be achieved with secondary ligands that are incapable of triggering such an effect when unassociated, such as the isolated form of the APC ligand known as ZZ. Association of the HS ligand can also potentiate the effect of a ligand capable, in its isolated form, of activating APCs, such as a non-specific human polyclonal antibody. Association of the HS ligand with an NK or NKT cell ligand can also activate these cell types, whereas it lacks this capacity in its isolated form. Finally, an immunomodulatory complex, preferably immunostimulatory, according to the invention can induce a therapeutic effect, particularly an antitumor effect, superior to that obtained with an anti-ICP (anti-PD1) antibody, especially when the composition includes an adjuvant. Definitions

[0024] The term “immune cells of the innate response” refers to dendritic cells, NK and NKT cells, granulocytes (mast cells, neutrophils, eosinophils and basophils) and phagocytes (monocytes, macrophages, neutrophils and).

[0025] The term "antigen-presenting cell" (APC) refers to a cell expressing one or more major histocompatibility complex (MHC) class I and class II molecules (HLA class I and class II molecules in humans) and capable of presenting antigens to CD4+ and CD8+ T lymphocytes specific for that antigen. Examples of antigen-presenting cells include dendritic cells (DCs), monocytes, macrophages, B lymphocytes, lymphoblastoid cell lines, and genetically modified human or animal cell lines expressing MHC class I and class II molecules, particularly HLA-I and HLA-II.

[0026] An NK cell is defined as a granulocyte lymphocyte expressing CD56 and CD16 molecules and possessing cytotoxic activity that does not require prior exposure to the Ag.

[0027] An NKT cell is defined as a granulocytic lymphocyte expressing NK cell markers, notably the CD56 and CD16 molecules, and T lymphocyte markers, notably the CD3 molecule. This cell possesses cytotoxic activity that does not require prior exposure to the Ag.

[0028] The term "AgC surface molecule" refers to a molecule expressed on the surface of Ag-presenting cells.

[0029] The term "NK or NKT cell surface molecule" refers to 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 antigen-presenting cells, meaning it is expressed on a very limited number of cells other than APCs. It is therefore a molecule with high expression specificity for APCs.

[0031] An "NK or NKT cell-specific surface molecule" is defined as a molecule expressed primarily on NK or NKT cells, meaning it is expressed on a very limited number of cells other than NK or NKT cells. It is therefore a molecule with high expression specificity for NK or NKT cells.

[0032] Glycosaminoglycans (GAGs) are linear polysaccharides composed of repeating disaccharide units, each containing a hexosamine (glucosamine (GIcN) or galactosamine (GaIN)) and another sugar (glucuronic acid (GIcA), iduronic acid (IdoA), or galactose (Gai)). The glucosamine is either N-sulfated (GIcNS) or N-acetylated (GIcNac). The galactosamine is always N-acetylated (GalNac). Sulfated glycosaminoglycans include simple polymers of GIcA, such as chondroitin sulfate, and copolymers comprising residues of both GIcA and / or IdoA and / or Gai, 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 environment.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] The term "Ab" refers to an immunoglobulin (IgG, IgM, IgA, IgD, IgE). The term Ab designates a specific Ab, that is, an Ab directed against a particular molecule x (anti-molecule x Ab), notably a surface molecule of antigen-presenting cells (anti-surface molecule of antigen-presenting cells). The term immunoglobulin designates a non-specific Ab.

[0034] The term "individual" refers to a human or animal individual, preferably a human.

[0035] The term "ligand" of a molecule means any agent capable of binding that molecule with a sufficiently high affinity to form a stable complex, in vitro and in vivo.

[0036] The term “APC ligand” refers to a ligand of a surface molecule of Ag-presenting cells.

[0037] The term "NK or NKT cell ligand" refers to a ligand of a surface molecule of NK or NKT cells. The term "heparan sulfate ligand" refers to an agent that binds heparin with an optical density signal at least equal to 50% of the signal measured when ZZ-Tat22-57C(22-37)s is incubated at 100nM pH7.2 in the ELISA test of Example 1.

[0038] An "antigen" is defined as any substance that can be specifically recognized by the immune system, and in particular by antibodies and immune system cells (B lymphocytes, CD4+ T lymphocytes, CD8+ T lymphocytes), and capable of inducing a specific immune response.

[0039] A "disease-specific antigen" is defined as an antigen that induces an immune response specifically directed against the disease being treated. This disease-specific immune response includes the production of antibodies and / or the induction of a cytotoxic T cell response (activation of CD8+ T lymphocytes) or a helper T cell response (activation of CD4+ T lymphocytes) directed against an antigen of a pathogen or tumor cell responsible for the disease being treated.

[0040] An "immunomodulator" is defined as an agent capable of controlling the immune response and, in particular, of regulating, positively or negatively, the The relative response of different populations or subpopulations of immune cells, such as T and B lymphocytes, antigen-presenting cells (APCs), and NK or NKT cells, is a key characteristic of immunomodulators. The term immunomodulator encompasses both immunosuppressants and immunostimulants. An immunostimulant is defined as an agent capable of positively regulating, or activating, the relative response of different populations or subpopulations of immune cells, such as T and B lymphocytes, APCs, and NK or NKT cells. The effect of an immunomodulator, preferably an immunostimulant, is exerted on a broad range of cells. It is independent of the presence of a specific antigen of the disease being treated, unlike a vaccine, which requires the presence of a disease-specific antigen and induces a specific immune response to that antigen.While the use of an immunogen or a vaccine is limited to the disease including the specific antigen of that disease, the immunomodulatory complex, preferably immunostimulatory according to the invention which is independent of the presence of a specific antigen of the pathology to be treated, is usable in immunotherapy of many diseases such as cancer and infectious diseases.

[0041] A peptide is defined as a sequence of natural or synthetic amino acids, possibly modified. The term peptide is used regardless of the length of the amino acid sequence.

[0042] 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, designated L1, which targets a sulfated GAG, and the second ligand, designated 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 some preferred embodiments of the invention, the molecular complex consists of an L1 ligand linked to an L2 ligand.

[0043] The molecular complex according to the invention, preferably an immunostimulatory complex, is devoid of a disease-specific antigen, such as a vaccine antigen specific to the pathology being treated. When one of the ligands of the complex according to the invention comprises a disease-specific antigen disease to be treated, then the treatment of said disease with said complex is excluded from the invention.

[0044] APC, NK cell, or NKT cell ligands are molecules or complexes of molecules, natural, recombinant, or synthetic, of a 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 a suitable expression system, particularly for the production of therapeutic proteins. For example, recombinant proteins, polypeptides, or peptides can be produced in E. coli or in HEK or CHO cells.

[0045] The first and second ligands are associated or linked to each other by any suitable means. They may be covalently or non-covalently linked, either directly or via a linker, thus forming a molecular complex. The molecular complex consists of two or more ligands, and possibly appropriate linker(s).

[0046] The covalent bonding or association of ligands is generated in particular by a covalent chemical coupling (formation of a covalent conjugate) or by the construction of a fusion protein (genetic fusion).

[0047] The immunomodulatory complex, preferably an immunostimulatory complex, is in monomeric, oligomeric, or mixed form (a mixture of monomers and oligomers). According to certain preferred embodiments of the invention, said complex is in oligomeric or mixed form.

[0048] 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 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 sequences in amino acids 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 to its receptor expressed on the surface of CPAgs, NK or NKT cells.

[0049] Non-covalent binding is generated, in particular, by adsorption onto a nanoparticle. It can also be achieved using a molecule (linking element) with a high and specific affinity for L1 or L2. This linking element is covalently bound to one of the ligands to non-covalently bind the other ligand. When one of the ligands is an antibody (Ab), the linking element is typically a protein or protein fragment that binds the Fc and / or Fab region of immunoglobulins, as described in Application FR 2759296. Such immunoglobulin-binding elements include, in particular, S. aureus protein A, its BB fragment (SEQ ID NO: 1), and its derivative ZZ (SEQ ID NO: 2). The first two proteins bind both the Fc and Fab regions of immunoglobulins, while ZZ binds only the Fc region.For example, when one of the ligands is an antibody 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 coupled to L1 and L2 respectively; 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 that it does not immediately dissociate from this complex in vivo.

[0050] 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 element, preferably S. aureus protein A, its BB fragment (SEQ ID NO: 1), or its ZZ derivative (SEQ ID NO: 2). Preferably, the second ligand is an antibody or an antibody fragment, and the first ligand forms a fusion protein with a immunoglobulin binding element, preferably S. aureus protein A, its BB fragment (SEQ ID NO: 1) or its ZZ derivative (SEQ ID NO: 2).

[0051] 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 specifically 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 (cDCs) and plasmacytoid dendritic cells (pDCs); NK cells; and NKT cells. The analysis of expansion is notably performed by flow cytometry using appropriate markers for the different cell populations to be analyzed.The analysis of immune cell activation can be performed by detecting activation markers (CD69) and / or maturation markers (CD86) using flow cytometry, or by measuring cytokines secreted into the extracellular environment, particularly IL-6 and / or IL-12, using conventional assays 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 a specific antigen of the pathology being treated.

[0052] The molecular complex according to the invention, preferably an immunostimulatory complex, binds to at least one sulfated sugar from the glycosaminoglycan family expressed on the surface of APCs, NK cells, or NKT cells (sulfated GAG) and another surface molecule of APCs, NK cells, or NKT cells, in particular a surface molecule specific to APCs, NK cells, or NKT cells. The sulfated GAG targeted by the first ligand is preferably heparan sulfate, chondroitin sulfate, dermatan sulfate, or keratan sulfate, most preferably heparan sulfate. The sulfated GAG ligand (first ligand) can originate from a mammalian cell or a pathogenic microorganism, including a virus (Adenovirus, Cytomegalovirus, HIV, Virus Sindbis), a bacterium (Mycobacterium bovis, Bordetella pertussis), a parasite (Leishmania sp.), or a toxin; it is specifically a molecular complex, a molecule, or one of its fragments. binds heparin, and / or heparan sulfates. Such ligands are described in particular in "Heparan Binding Proteins", H. Edward Conrad, Academy Press, San Diego and London and Dreyfuss et al., Annuals of the Brazilian Academy of Sciences, 2009, 81, 409-429 (see especially Table II). Examples of these ligands include, but are not limited to: endogenous GAG ligands (Thrombin, Urokinase, Vitronectin, fibroblast growth factor 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 and core regions of Tat (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, APand Griffin, DE, J. Virol., 1998, 2, 7349-7356) and the R domain of diphtheria toxin (DTR or DTRBD; fragment 382 to 535 of DT (SEQ ID NO: 5)); Lobeck et al., Infection and Immunity, 1998, 66, 418-423). We can also mention cell penetrating peptides (CPPs) that bind heparin, heparan sulfates and / or chondroitin sulfates such as peptides very rich in basic residues, especially 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 peptides derived from the homeodomain of the Antennapedia protein (penetratin; fragment 43-58 (SEQ ID NO: 6)).

[0053] Alternatively, the first ligand is a natural or recombinant antibody directed against a sulfated GAG, preferably heparin, heparan sulfates, or chondroitin sulfates, or a fragment of such an antibody containing at least the paratope (antigen-binding domain), such as a Fab, Fab', F(ab')2, Fv, or single-chain Fv fragment (scFv for single-chain Fv), Fabc, or a Fab fragment comprising a portion of the Fc domain. Such antibodies are described, in particular, 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 antibody or antibody fragment is human or humanized.

[0054] 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 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 R7 to R11 peptide; 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 includes the heparan sulfate-binding region.Preferably, 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 Tat 49-57 region (SEQ ID NO: 3), in particular the Tat22-57C(22-37)S peptide (SEQ ID NO: 9) and a peptide comprising the R domain of diphtheria toxin (SEQ ID NO: 5).

[0055] 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 as a fusion protein with the second ligand or an immunoglobulin-binding element, so as to form oligomers.

[0056] The other molecule expressed on the surface of 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—that is, 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—that is, a surface molecule specific to APCs and in particular to dendritic cells. Among these surface molecules expressed primarily on APCs and in particular on dendritic cells, we can mention, notably: MHC class II molecules, in particular the α, β, and γ chains. invariant chain (li, li fragment or CD74) of MHC II molecules; surface immunoglobulins or membrane immunoglobulins; integrins such as CD11c and MAC1; Transferrin receptors; C-type lectin receptors such as, notably, the 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 immunoglobulin constant region (FcR or RFc), notably FcyR such as FcyRI (CD64), FcyRII (CD32), and FcyRIII (CD16); the TNF receptor superfamily such as CD40; and complement receptors. Among the surface molecules expressed primarily on APCs, we can mention immune checkpoint (ICP) molecules and their ligands (ICP-ligands) expressed on APCs, such as, but not limited to, PD-L1. PDL2, CD155, CD80, CD86, CD40, OX40L, ICOSL (CD275), CD70 (Wykes MN, Nat. Rev. Immunol., 2018, 18:91-104).

[0057] The other molecule expressed on the surface of 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—that is, 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—that is, a surface molecule specific to NK or NKT cells. Examples of these surface molecules expressed primarily on NK or NKT cells include: 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.

[0058] According to advantageous embodiments of the molecular complex, preferably an immunostimulatory complex, of the invention, the second ligand targets 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, preferably selected from the group consisting of: type C lectin receptors, membrane immunoglobulins, receptors for the constant region of immunoglobulins.

[0059] According to other advantageous embodiments of the molecular complex, preferably immunostimulatory, of the invention, the second ligand targets a surface molecule of APC selected from the group consisting of: type C lectin receptors, membrane immunoglobulins, receptors for the constant region of immunoglobulins (FcR), and immune checkpoint (ICP) molecules and their ligands (ICP ligands).

[0060] According to advantageous embodiments of the invention, the second ligand targets a surface molecule expressed essentially on NK or NKT cells, that is to say 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 0X40; preferably NKp44 (CD336), NKp46 (CD335), NCAM (CD56), CTLA-4 and 0X40.

[0061] The second ligand is selected from among saccharides that bind type C lectin receptors; immunoglobulins and their fragments comprising the constant region that bind Fc receptors; proteins or protein fragments that bind the Fc and / or Fab region of membrane immunoglobulins, as described in Application FR 2759296, including S. aureus protein A, its BB fragment (SEQ ID NO: 1) and its ZZ derivative (SEQ ID NO: 2), preferably rZZ (SEQ ID NO: 2). Alternatively, the second ligand is an antibody directed against these surface molecules of APCs, NK cells, or NKT cells, or a fragment of this antibody containing at least the paratope (antigen-binding domain), such as the Fab, Fab', F(ab')2, Fv, or single-chain Fv (scFv), Fabc, and Fab fragments comprising a portion of the Fc domain.

[0062] The antibody or antibody fragment is specifically directed against a specific surface molecule of immune cells involved in the innate or adaptive response, including APCs, NK cells, and NKT cells, such as, but not limited to, PD-L1, PD-L2, CD155, CD80, CD86, CD40, OX40L, ICOSL (CD275), CD70; PD-L1, PD-L2, CD155, CD80, CD86, CD40, OX40L, ICOSL (CD275), CD70, PD-1, CT LA- 4, TIM-3, TIGIT, LAG-3; preferably PDL1 and ICOSL (CD275) or PDL1, ICOSL (CD275), CTLA-4 and 0X40.

[0063] The antibody or antibody fragment may also be directed against a specific surface molecule of APCs, particularly dendritic cells, such as an anti-cγRF (I, II, and / or III) or anti-C-type lectin receptor antibody, especially anti-DEC-205 or anti-DC-SIGN (CD209); or anti-DEC-205, anti-DC-SIGN (CD209), or anti-li invariant chain (CD74). The antibody may be an agonist or an antagonist of said surface molecule; for example, the antibody may be 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 are commercially available.

[0064] 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 their fragments containing at least the paratope such as the fragments Fab, Fab', F(ab')2, Fv, scFv, Fabc and Fab with at least a portion of the Fc domain (ii) immunoglobulins, preferably IgG, and their fragments 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 BB fragment (SEQ ID NO: 1) and its ZZ derivative (SEQ ID NO: 2).

[0065] Antibodies directed against said surface molecules of antigen-presenting cells are preferably chosen from anti-Fcgamma (I, II and / or III), anti-DC-SIGN (CD209), anti-DEC-205, anti-CD206, anti-CD40; anti-Fcgamma (I, II and / or III), anti-DC-SIGN (CD209), anti-DEC-205, anti-CD206, anti-CD40, anti-invariant chain li (CD74), and anti-ICOSL (CD275); preferably anti-DC-SIGN (CD209) and anti-DEC-205 or anti-DC-SIGN (CD209), anti-DEC-205, anti-invariant chain li (CD74), and anti-ICOSL (CD275) antibodies.

[0066] 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.

[0067] Additional ligands of the molecular complex, preferably an immunostimulatory complex, are advantageously chosen from sulfated GAG ligands and APC surface molecule ligands as defined above.

[0068] 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 that binds the Fc and / or Fab region of immunoglobulins such as the BB fragment of Staphylococcus aureus protein A and its derivative ZZ (second ligand), preferably in the form of a fusion protein comprising the first and second ligands. A first type of complex particularly preferred 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 chosen from the BB fragment of protein A (SEQ ID NO: 1) or its derivative ZZ (SEQ ID NO: 2); preferably its derivative ZZ (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, notably chosen from SEQ ID NO: 21 to 23. More preferably, the complex comprises one of the sequences SEQ ID NO: 12, 14, 16, 18, and 20. When the complex includes the R domain of diphtheria toxin (DTR: SEQ ID NO: 5), then the domain is... advantageously produced in mammalian cells, particularly as a fusion protein with the second ligand, so as to form oligomers.

[0069] 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 that binds the Fc and / or Fab region of antibodies, preferably only the Fab region, such as Staphylococcus aureus protein A and its BB fragment, preferably in the form of a fusion protein comprising the first ligand and the immunoglobulin-binding element, said first ligand covalently associated with the immunoglobulin-binding element being complexed to (ii) an immunoglobulin, preferably an IgG, or a fragment comprising at least the Fc region (second ligand). Preferably, an entire immunoglobulin, most preferably an entire IgG.The first ligand and the immunoglobulin-binding element 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 with an entire 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, the complex comprises one of the sequences SEQ ID NO: 16 or 18. When the complex includes the R domain of diphtheria toxin (DTR: SEQ ID NO: 5), then the domain is advantageously produced in mammalian cells, in particular as a fusion protein with the immunoglobulin-binding element, so as to form oligomers.

[0070] 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 Ac selected from the group consisting of an anti-RFcgamma (I, II, and / or III) Ac, an anti-DEC-205 Ac, an anti-DC-SIGN (CD209) Ac, an anti-invariant chain li (CD74) Ac, an anti-ICOSL (CD275) Ac, an anti-NKp46 (CD335) Ac, an anti-NKp44 (CD336) Ac, an anti-NCAM (CD56) Ac, an anti-CTLA-4 Ac, an anti-PDL1 Ac, an anti-OX40 Ac, and a fragment of the preceding Ac 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 to the Fc and / or Fab region of immunoglobulins, such as the BB fragment of Staphylococcus aureus protein A and its derivative ZZ, preferably in the form of a fusion protein of the first ligand with the immunoglobulin-binding element. The first ligand and the immunoglobulin-binding element 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 derivative ZZ (SEQ ID NO: 2). This fusion protein is complexed with an anti-fGFR (I, II, and / or III), anti-DEC-205, anti-DC-SIGN, anti-CD74, anti-CD275, anti-CD335, anti-CD336, anti-CD56, anti-CTLA-4, or anti-PDL1 antibody. , anti-0X40 or to a fragment of the preceding antibodies comprising 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 is C-terminal to the fusion protein; the immunoglobulin-binding element (BB or ZZ) and the first ligand are separated by a suitable spacer peptide, notably 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, particularly as 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.

[0071] 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 cytokines IL-6 and / or IL-12.

[0072] The invention further relates to an immunomodulatory composition, preferably immunostimulatory, comprising at least one immunomodulatory complex, preferably immunostimulatory according to the invention, and at least one pharmaceutically acceptable vehicle, carrier substance and / or adjuvant.

[0073] Pharmaceutically acceptable vehicles are those that are conventionally used.

[0074] Adjuvants are the humoral and / or cellular immune adjuvants classically used in immunotherapy. 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(l)-poly(C) or poly lC) 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.

[0075] The carrier substances are those conventionally used. These include unilamellar or multilamellar liposomes, ISCOMS, virosomes (virus-like particles), saponin micelles, solid microspheres of a saccharidic (poly(lactide-co-glycolide)) or gold 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.

[0076] 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(s) optionally being 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.

[0077] According to particular embodiments of the invention, the composition comprises a polynucleotide or a mixture of polynucleotides encoding APC, NK, or NKT ligands that are proteins, polypeptides, or peptides. The polynucleotide consists of a recombinant, synthetic, or semi-synthetic nucleic acid that is expressible in host cells 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, the composition comprises a polynucleotide including a sequence encoding a fusion protein comprising at least the coding sequences of the first and second ligands, fused in phase in an appropriate manner, and optionally a sequence encoding a linker element as defined above.Alternatively, the composition comprises a mixture of polynucleotides including at least one first polynucleotide comprising a sequence encoding the first ligand and a second polynucleotide comprising a sequence encoding the second ligand, said first or second nucleotide also comprising a sequence encoding a linker element as defined above. Said polynucleotide(s) are preferably inserted into one or more expression vectors comprising appropriate transcription and / or translation regulatory sequences (promoter, transcription activator, etc.). transcription terminator, polyadenylation signal) for the expression of the first and second ligands, and possibly other ligands in vivo in individuals receiving the composition. Many vectors suitable for therapeutic use are known in themselves. These include viral vectors (adenoviruses, retroviruses, lentiviruses, AAVs) and non-viral vectors (naked DNA), notably a plasmid, into which the sequence of interest has been pre-inserted. Alternatively, the polynucleotide(s) in question are mRNAs, preferably modified. The use of mRNA in therapy is well known to those skilled in the art (see, for example, Drew Weissman, Expert Reviews of Vaccines, October 2014, 1-17, doi: 10.1586 / 14760584.2015.973859).

[0078] 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 from an individual to be treated, in particular dendritic cells that are modified ex vivo before being re-administered to the individual (Ex vivo Cell Therapy).

[0079] The immunomodulatory composition may further comprise at least one other therapeutic agent, in particular an anticancer agent, an anti-infective agent, another immunomodulatory agent, or a vaccine antigen specific to the disease to be treated, said vaccine antigen advantageously being 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. 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.

[0080] The immunomodulatory composition, preferably immunostimulatory, comprises an effective dose of complex(s), polynucleotide(s), vector(s), or cell(s) sufficient to induce an immune response capable of producing a therapeutic effect on the disease being treated, i.e., reducing the symptoms of that disease. This includes, in particular, mitigating the consequences of action by a pathogen (infectious or non-infectious) or reducing tumor growth in an individual treated with this composition. This dose is determined and adjusted according to factors such as the subject's age, sex, and weight. The immunomodulatory composition, preferably immunostimulatory, according to the invention is presented in a pharmaceutical form suitable for the chosen method of 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 disease being treated. Administration may be intratumoral, subcutaneous, intramuscular, intravenous, intradermal, intraperitoneal, oral, sublingual, rectal, vaginal, intranasal, by inhalation, or by transdermal application. The composition is available in a pharmaceutical form adapted to the chosen administration method.

[0081] Isolated polynucleotides or polynucleotides inserted into a plasmid vector are introduced into the individual to be treated, either using physical methods such as electroporation, or by combining them with any substance(s) that allow passage through the plasma membrane, such as carriers like nanotransporters, liposomes, lipids, or cationic polymers. Furthermore, these methods can be advantageously combined, for example, by using electroporation in conjunction with liposomes.

[0082] The immunomodulatory composition, preferably immunostimulatory, according to the present invention is used in immunotherapy, particularly for antitumor or anti-infective purposes. The immunomodulatory composition, preferably immunostimulatory, according to the present invention is used preventively or curatively, that is, to prevent a disease in individuals or to treat individuals suffering from a disease. According to preferred embodiments of the invention, said composition is used curatively, that is, to treat individuals suffering from a disease. It can be used in combination with other treatments, therapeutic or surgical, particularly 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.

[0083] 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) cancers, skin, ovarian, uterine, brain, liver, and kidney cancers.

[0084] According to other preferred embodiments of the invention, said composition is used in the treatment of infectious diseases, particularly those in 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, including infections with HIV, HBV, HCV, Mycobacterium tuberculosis, or Plasmodium falciparum. When said complex comprises Tat or a fragment of Tat 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 diseases infectious diseases other than diphtheria (infection by the diphtheria bacillus, Corynebacterium diphteriae).

[0085] The immunomodulatory composition, preferably immunostimulatory, according to the present invention is used either in conventional therapy, or in cell therapy, or even by a combination of the two approaches.

[0086] Cell therapy involves the preparation of antigen-presenting cells, including dendritic cells, or NK or NKT cells, using a standard protocol that includes isolating peripheral blood mononuclear cells (PBMCs) from the patient to be treated and culturing the dendritic, NK, or NKT cells in the presence of molecular complex(s), polynucleotide(s), or vector(s) as defined above. In a second step, the antigen-presenting, NK, or NKT cells loaded with the aforementioned molecular complex(s) or modified with the aforementioned polynucleotide(s) or vector(s) are re-infused into the patient.

[0087] The present invention also relates to a method of immunotherapy, in particular anti-tumor or anti-infective, characterized in that it comprises the administration of an immunomodulatory composition, preferably immunostimulatory as defined above, to an individual, by any appropriate means as defined above.

[0088] The present invention also relates to the use of an immunomodulatory composition, preferably immunostimulatory as defined above, for the preparation of a drug intended for immunotherapy, preferably anti-tumor or anti-infective.

[0089] The APC, NK or NKT cell ligand complexes according to the invention are prepared by conventional techniques known to those skilled in the art, namely:

[0090] - Ligands for the surface molecules of APCs, NK cells, or NKT cells can be produced by chemical synthesis or by expression of recombinant DNA in a suitable eukaryotic or prokaryotic cell system. Peptides and proteins can be synthesized in solid phase, according to the technique Fmoc, originally described by Merrifield et al. (J. Am. Chem. Soc., 1964, 85, 2149-), and purified by reversed-phase high-performance liquid chromatography. Polypeptides and proteins can be produced from corresponding cDNAs, cloned into a suitable eukaryotic or prokaryotic expression vector. Polypeptides or proteins produced in cells modified by the recombinant vector are purified by any suitable means, including affinity chromatography. Antibodies directed against surface molecules of APCs, NK cells, 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) are available from BECTON-DICKINSON, while the anti-CD56 antibody is available from Biolegend (#304622) and the anti-CD335 (AM31284AF-N) and anti-CD336 (#AM50346PU-N) antibodies are available from Origene.Alternatively, monoclonal antibodies can be produced by conventional techniques known to those skilled in the art. For example, monoclonal antibodies are produced from hybridomas obtained by fusing B lymphocytes from an animal immunized with the surface molecule of antigen-presenting cells (APCs) with myeloma cells, according to the technique of Kohler and Milstein (Nature, 1975, 256, 495-497); the hybridomas are cultured in vitro, notably in fermenters, or produced in vivo, in the form of ascites; alternatively, said monoclonal antibodies are produced by genetic engineering as described in US patent 4,816,567. Humanized antibodies are produced by general methods such as those described in International Application WO 98 / 45332.Antibody fragments are produced from cloned VH and VL regions, from mRNAs of hybridomas or splenic lymphocytes from an immunized mammal; for example, Fv, scFv, or Fab fragments are expressed on the surface of filamentous phages according to the Winter and Milstein technique (Nature, 1991, 349, 293-299). After several selection steps, antigen-specific antibody fragments are isolated and expressed in a suitable expression system using conventional cloning and recombinant DNA expression techniques. The antibodies, or their fragments as defined above, are purified using standard techniques known to the field, such as affinity chromatography.

[0091] The covalent association of the first ligand (L1) to the second ligand (L2) of a surface molecule of APCs or NK or NKT cells can be achieved by constructing a fusion protein in which the nucleotide sequences encoding L1 and L2 are fused in phase, in the appropriate order, either directly or via a nucleotide sequence encoding a suitable spacer peptide. Depending on the respective amino acid lengths of L1 and L2, they are either fused at their ends (the N-terminus of one sequence fused to the C-terminus of the other sequence) or one of the sequences is inserted into the other sequence at a suitable site that does not adversely affect 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 covalently coupled by any appropriate means.Coupling is achieved via reactive groups initially present or previously introduced onto the ligand(s). Coupling can notably be carried out at amino acid residues whose side chain includes a reactive function. Among these amino acids, examples include polar amino acids containing a -OH function [serine (S), threonine (T) or tyrosine (Y)], -SH [cysteine ​​(C)], -NH2 [lysine (K) or arginine (R)], -COOH [aspartic acid (D) or glutamic acid (E)], and polar amino acids with a side chain functionalized by the addition of a reactive function, notably a chloro- or bromo-acetyl group reactive with thiol groups or a hydrazine group reactive with aldehydes. The ligand is coupled by any suitable means; These methods, 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 achieved using heterobifunctional reagents, notably m-maleimidobenzoyl-N-hydroxysuccinimide (SMCC) or sulfo-SMCC, each of which contains a maleimide group capable of reacting with free thiols. In this case, the SMCC is first covalently linked to an amine function present on the ligand. Simultaneously, another heterobifunctional reagent (such as N-succinimidyl S-acetylthioacetate, which contains a hydroxylamine-cleavable thioester group, or succinimidyl-pyridyl-) is used. Dithiopropionate (which contains a disulfide bridge reducible under mild conditions) is associated with an amine group of the second partner, which is one of the ligands. The second partner is then treated with hydroxylamine or a reducing agent to release the thiol. The thiol compound is then incubated with the compound incorporating the maleimide, and coupling is achieved by reaction of the thiol group with the maleimide group. This type of covalent coupling is described in Leonetti 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 antibodies to ligands, is notably described in Ishikawa et al., J. Immunoassay, 1983, 4, 209-327.

[0092] Non-covalent complexes are prepared by contacting the second ligand (L2) with the first ligand (L1) under conditions that allow the two partners to interact. This interaction may involve a linker, such as a protein or peptide, that has a high and specific affinity for one of the complex partners (L1 or L2). In particular, the affinity of the linker for this partner in the complex is sufficient to prevent its immediate dissociation from the complex in vivo. When one of the ligands is an immunoglobulin, the linker is an immunoglobulin-binding element as described in Application FR 2759296. For example, an immunoglobulin-binding element is covalently linked to L1 to form a non-covalent complex with L2.

[0093] 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 acid sequence by PCR or RT-PCR, by screening of 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.

[0094] 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. Brief description of the drawings

[0095] Other features, details, and advantages of the invention will become apparent upon reading the detailed description below, which refers to examples of implementation of the present invention, and upon analysis of the accompanying drawings, in which: Fig. 1

[0096] Figure 1 shows the molecular mass and degree of homogeneity of molecular complexes produced in E. coli or HEK cells. The proteins ZZ-DTRBDHEK, ZZ-DTRBDcoii, BB-DTRBDHEK, BB-DTRBDcoii, and ZZ-Tat22-57C(22-37)s were analyzed by electrophoresis under denaturing conditions. The proteins were loaded in the presence of molecular weight markers. After electrophoretic migration, the proteins were stained with Coomassie blue. Fig. 2

[0097] [Fig. 2] shows the binding of different molecular complexes to heparin. Series of dilutions of the proteins ZZ free, ZZ-DTRBDHEK, ZZ-DTRBD ¥N, BB-DTRBDHEK, ZZ-DTRBDCON, and ZZ-Tat22-57C(22-37)s were 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, heparin binding to the plates was detected using streptavidin-peroxidase coupled to a substrate of this enzyme (ABTS). Heparin binding was considered significant when the optical density signal was at least 50% of the signal measured when ZZ-Tat22-57C(22-37)s was incubated at 100 nM pH 7.2 on the microtiter plates. Fig. 3

[0098] [Fig. 3] shows the cell types linked by the ZZ-DTRBDHEK fusion protein in a mouse splenocyte population. Splenocytes were Cells were incubated in the presence or absence of a fixed amount of ZZ-DTRBDHEK (100 nM). A series of fluorescent antibodies specific for pDCs, CD11 b+ cDCs, and CD8+ cDCs (A); CD4+ T lymphocytes, CD8+ T lymphocytes, B lymphocytes, and monocytes (B) was then added. After 30 minutes at 4 °C, the cells were washed and analyzed by flow cytometry. The percentage of cells bound by ZZ-DTRBDHEK in the considered subpopulation is shown. Fig. 4 [Fig. 4] shows that ZZ-DTRBDHEK induces the secretion of IL-6 and IL-12 by splenocytes. A fixed amount of ZZ, DTRBD, or ZZ-DTRBDHEK (1 mM) was incubated with mouse splenocytes. After 24 hours, the supernatants were collected, and the presence of IL-6 and IL-12 was assessed by enzyme immunoassay. Fig. 5 [Fig. 5] shows that ZZ-Tat22-57C(22-37)s induces the secretion of IL-6 and IL-12 by splenocytes. A fixed amount of ZZ, TatcY49-57, ZZ + TatcY49-57, or ZZ-Tat22-57C(22-37)s (1 mM) 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. 6

[0099] Figure 6 shows that the proteins ZZ-DTRBDHEK and BB-DTRBDHEK induce the secretion of greater amounts of IL-6 and IL-12 by splenocytes than ZZ-DTRBDHEK and BB-DTRBDHEK. A fixed amount of ZZ-DTRBDHEK, ZZ-DTRBDHEK, BB-DTRBDHEK, and BB-DTRBDcoii (1 mM) was incubated with mouse splenocytes. After 24 hours, the supernatants were collected, and the presence of IL-6 and IL-12 was assessed by enzyme immunoassay. Fig. 7

[0100] [Fig. 7] shows that ZZ-DTRBDHEK 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 of or in the absence of a fixed amount of ZZ-DTRBDHEK, (0.qmM). After 24h, the supernatants were collected and the presence of IL-6 and IL-12 was evaluated by enzyme immunoassay. Fig. 8

[0101] [Fig. 8A-B] shows that the proteins ZZ-DTRBDHEK and ZZ-Tat22-57C(22-37)s 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 either ZZ-DTRBDHEK (5 nmoles per mouse) or ZZ-Tat22-57C(22-37)s (10 nmoles per mouse). 24 h after the last injection, the animals were euthanized, the spleens were harvested, and the splenocytes were labeled using a cocktail of fluorescent antibodies to distinguish CD8+ cDCs (CD11c high B220 CD8 + CD11 b ) (A), cDC-CD11 b+ (CD11 chighB220 CD8 CD11 b + ) (B) and the pDCs (CD11 c int CD317 + CD11 b + ) .

[0102] [Fig. 8C] shows the 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 using a Kruskal-Wallis test ( * p<0.05). Fig. 9

[0103] Figure 9 shows that ZZ-DTRBDHEK induces the secretion of IL-6 and IL-12 in vitro by isolated human dendritic cells (DCs). DCs isolated from healthy human donors were incubated in the presence of a fixed amount of ZZ-DTRBDHEK (1 mM). After 24 hours, the supernatants were collected to assess the presence of the cytokines IL-6 and IL-12 by enzyme-linked immunosorbent assay (ELISA). Fig. 10

[0104] [Fig. 10] shows that mice injected with colorectal cancer cells exhibit slowed tumor growth when subsequently injected with ZZ-DTRBDHEK alone or in combination with an adjuvant mixture. A: Sixteen C57BL / 6 mice were injected subcutaneously with 0.5M MC38 cells. Three days later, eight mice were injected with the adjuvant mixture CpG-B 1018 / Poly I:C (30 pg of each adjuvant), and eight were Eighteen C57BL / 6 mice were injected subcutaneously with 0.5M MC38 cells. Three days later, six mice were injected with ZZ-DTRBDHEK in PBS buffer, six others were injected with ZZ-DTRBDHEK (2 nmol per mouse) in PBS buffer containing the adjuvant mixture CpG-B 1018 / Poly I:C (30 pg of each adjuvant), and six were not injected (Controls). Two additional injections were administered three days apart. Tumor growth was monitored by measuring tumors with calipers. Each injection is represented by an arrow. Fig. 11

[0105] [Fig. 11] shows that mice injected with colorectal cancer cells exhibit slowed tumor growth when subsequently injected with ZZ-DTRBDHEK, ZZ-Tat22-57C(22-37)s in combination with an adjuvant mixture. Thirty-two C57BL / 6 mice were injected subcutaneously with 0.5M MC38 cells. Three days later, one group of eight mice was injected with ZZ-DTRBDHEK (0.96 nmol per mouse) in 50 mL of PBS containing the adjuvant mixture CpG-B 1018 / Polyl:C (30 pg of each adjuvant), another group with an anti-PD-1 antibody (50 mL, 1.3 nmol per mouse), a third group with ZZ-Tat22-57C(22-37)s (0.96 nmol per mouse) in 50 mL of PBS containing the adjuvant mixture CpG-B 1018 / Polyl:C (30 pg of each adjuvant), and a final group of eight mice was not injected (Controls). Two additional injections were administered three days apart. Tumor growth was monitored by measuring tumors with calipers.Each injection is represented by an arrow.

[0106] [Fig. 11 A] Tumor growth kinetics.

[0107] [Fig. 11 B] Tumor growth kinetics.

[0108] [Fig. 11 C] Survival of treated animals. Fig. 12

[0109] [Fig. 12] shows that the formation of the molecular complexes ZZ-Tat22-57C22-37)s, anti-DEC205 / ZZ-Tat22-57C22-37)s and lgG / ZZ-Tat22-57C22-37)s induce in vitro an increase in the proportion of activated monocytes and CD4+ T lymphocytes. A fixed concentration (0.1 mM) of anti-DEC205 / ZZ-Tat22-57C22-37, IgG / ZZ-Tat22-57C22-37, anti-DEC205 / ZZ, IgG / ZZ, and anti-DEC205 antibodies was incubated with human DMSCs. A fixed concentration (1 mM) of ZZ-Tat22-57C22-37, or ZZ, was incubated with human DMSCs. After 24 hours, the cells were harvested and labeled with fluorescent antibodies. The proportion of monocytes (CD3 CD14) +) activated in the live CMSP population was assessed using the CD69 marker.

[0110] [Fig. 12A] The proportion of activated CD4+ T lymphocytes in the CD4+ T lymphocyte population (CD3 + CD4 + ) living was assessed by measuring the expression of the CD69 molecule. [Fig. 12B] The cells were analyzed by flow cytometry. Fig. 13

[0111] [Fig. 13] shows that molecular complexes directed against receptors expressed by dendritic cells (anti-CD74 / ZZ-Tat22-57C22-37), anti-CD209 / ZZ-Tat22-57C22-37, and anti-CD275 / ZZ-Tat22-57C22-37 induce IL-6 secretion by CMSPs. Human CMSPs were incubated in the presence or absence of free antibodies, ZZ or ZZ-Tat22-57C(22-37), and molecular complexes targeting 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

[0112] [Fig. 14] shows that a molecular complex (anti-CD335 / ZZ-Tat22-57C22-37) directed against the CD335 receptor expressed by NK and NKT cells induces an increase in the proportion of activated NK and NKT cells. Microsatellite complexing sequences (MSCSs) were incubated in the presence or absence of free antibody, ZZ, or ZZ-Tat22-57C22-37, as well as the anti-CD335 / ZZ-Tat22-57C22-37 and anti-CD335 / ZZ molecular complexes. After 18 hours, the cells were analyzed by flow cytometry to assess the expression of the co-stimulatory molecule CD69 on the surface of NK (A) and NKT (B) cells. Fig. 15

[0113] [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. CMSPs were incubated in the presence or absence of free forms of antibodies, ZZ, or ZZ-Tat22-57C(22-37)s, as well as anti-CD56 / ZZ-Tat22-57C(22-37)s, anti-CD56 / ZZ, anti-CD336 / ZZ-Tat22-57C(22-37)s, or anti-CD336 / ZZ molecular complexes. After 18 hours, the cells were analyzed by flow cytometry to assess the expression of the co-stimulatory molecule CD69 on the surface of NKT cells. Fig. 16

[0114] [Fig. 16] shows that molecular complexes directed against receptors expressed by NK cells (anti-CD56 / ZZ-Tat22-57C22-37), anti-CD335 / ZZ-Tat22-57C22-37, and anti-CD336 / ZZ-Tat22-57C22-37 induce IL-6 secretion by human CMSPs. CMSPs were incubated in the presence or absence of free antibody, ZZ, or ZZ-Tat22-57C(22-37), as well as molecular complexes targeting CD56 (A), CD335 (B), and CD336 (C), respectively. After 24 hours, the supernatants were collected, and the presence of IL-6 was assessed by enzyme immunoassay. Fig. 17

[0115] [Fig. 18] shows that molecular complexes containing anti- ICPs (anti-CTLA-4 / ZZ-Tat22-57C22-37)s, anti-PD-L1 / ZZ-Tat22-57C22-37)s, and anti- OX40 / ZZ-Tat22-57C22-37)s induce IL-6 secretion by human CMSPs. CMSPs were incubated in the presence or absence of free forms of the antibodies, ZZ or ZZ-Tat22-57C(22-37)s, and molecular complexes targeting CTLA-4 (A), PD-L1 (B), and OX40 (C), respectively. After 24 hours, the supernatants were collected, and the presence of IL-6 was assessed by enzyme immunoassay. Examples Example 1: Expression of fusion proteins in E. coli or HEK cells, purification, biochemical characterization, and study of their ability to bind heparan sulfates. Materials and Methods Production of the different molecular complexes

[0116] The expression of the ZZ-DTRBD fusion protein in E. coli, referred to as ZZ-DTRBDcoii, was previously described in the publication by Lobeck et al. (Infection and Immunity, 1998, 66, 418-423). The ZZ-DTRBDcoii fusion protein (SEQ ID NO: 12) is encoded by a polynucleotide with sequence SEQ ID NO: 11. The expression of the ZZ-Tat22-57C(22-37)s fusion protein in E. coli was achieved using a protocol similar to that described for the ZZOVATat22-57s fusion in the publication by Knittel et al. (Vaccine, 2016, 34(27):3093-3101). The ZZ-Tat22-57C(22-37)Scoii fusion protein (SEQ ID NO: 20) is encoded by a polynucleotide with sequence SEQ ID NO: 19. For expression in eukaryotic cells, HEK cells (2.5 x 10⁻¹⁰ 6Cells (800 cells / ml in 250 ml of 293F freestyle medium) were transfected with a pCDNA3.4 plasmid encoding ZZ-DTRBDHEK (400 pg of DNA prepared from MaxiPrep by transfection) in the presence of PEI (0.5 mg / ml). This plasmid includes the polynucleotide sequence SEQ ID NO: 13, which encodes the ZZ-DTRBDHEK fusion protein (SEQ ID NO: 14). The cells were then incubated for 24 h 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, sterilely filtered, and a protease inhibitor cocktail was added.

[0117] After expression of the three proteins, the supernatants were each diluted 1:2 in 0.1% PBS-Tween 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 1 M Tris-HCl buffer, pH 8. The fusion proteins derived from the expression in E. coli, ZZ-DTRBD ¥iiZZ-DTRBD22-57C(22-37)s and ZZ-Tat22-57C(22-37)s underwent a second purification cycle using a mono S 5 / 50 cation exchange column (GE Healthcare). The column was equilibrated with 0.05M phosphate citrate buffer at pH 5.5 for the purification of ZZ-DTRBDcoii. The column was equilibrated with 0.05M phosphate citrate buffer at pH 4 for the purification of ZZ-Tat22-57C(22-37)s. The fusion proteins ZZ-DTRBDcoii and ZZ-Tat22-57C(22-37)s were then eluted with a linear gradient from 0 to 1 M NaCl. The proteins were finally concentrated in PBS and stored at -20°C until use. The production of the BB-DTRBDHEK and BB-DTRBD¥ii molecular complexes was carried out following the same protocol as that used for ZZ-DTRBDHEK and ZZ-DTRBD¥ii. The BB-DTRBDHEK fusion protein (SEQ ID NO: 18) is encoded by the polynucleotide of sequence SEQ ID NO: 17. The BB-DTRBD fusion protein ¥ N (SEQ ID NO: 16) is coded by the polynucleotide of sequence SEQ ID NO: 15. Analysis of the molecular mass and degree of homogeneity of the ZZ-DTRBDHFK molecular complexes. ZZ-DTRBD ¥ii BB-DTRBDHEK and BB-DTRBD COii by electrophoresis gel

[0118] ZZ-DTRBDHEK, ZZ-DTRBD proteins ¥ii, BB-DTRBDHEK and BB-DTRBDcoii, along with 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 Coomassie blue staining. Binding of molecular complexes to heparin

[0119] To evaluate the binding of molecular complexes to heparan sulfates, heparin, which is a sulfated sugar representative of the heparan sulfate family, was used.

[0120] The interaction was assessed using an enzyme-linked immunosorbent assay (ELISA). For this purpose, a series of microtiter plates were first adsorbed with rabbit IgG (1 pg / 1 00 mIU / well in 0.1 M phosphate buffer, pH 7.2) and then saturated with a buffer solution containing 0.3% bovine serum albumin (200 µl / well in 0.1 M phosphate buffer, pH 7.2). Another series of microtiter plates... The plates were saturated with a buffer solution containing 0.3% bovine serum albumin (300 µl / well in 0.1 M phosphate buffer, pH 7.2). Both sets of plates were then washed, and sets of dilutions (in 0.1 M phosphate buffer, pH 7.4, containing 0.1% bovine serum albumin) of the proteins ZZ-DTRBDHEK, ZZ-DTRBDCON, BB-DTRBDHEK, BB-DTRBDcoii, ZZ-Tat22-57C(22-37)s, and free ZZ were loaded into the wells. After 4 hours of incubation at room temperature, the plates were washed, and 100 mL of heparin-biotin (1 mM) was added per well. After 1 hour at room temperature, the plates were washed, and 100 mL of streptavidin-peroxidase (1 / 2000 dilution) was added. After 30 minutes of incubation, the plates were washed and a substrate (ABTS) was added. The color intensity was measured at 414 nm after 30 minutes of incubation.To eliminate non-specific binding to albumin, the optical signal measured on plates adsorbed solely with bovine serum albumin was subtracted from the signal measured on plates adsorbed with IgG. Heparin binding was considered significant when the optical density signal was greater than or equal to 50% of the signal measured when ZZ-Tat22-57C(22-37)s was incubated at 100 nM pH 7.2 on microtiter plates. Results

[0121] The inventors previously constructed a fusion protein, called ZZ-DTRBD, incorporating, on the one hand, a double ZZ domain derived from Staphylococcus aureus protein A and, 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 manner similar to protein A. DTRBD, for its part, binds to 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 carrying surface immunoglobulins and heparan sulfate proteoglycans via interaction with heparan sulfates.

[0122] The inventors similarly constructed a fusion protein, called BB-DTRBD, by replacing the ZZ-coding sequence with a sequence coding called BB. The BB protein corresponds to a double domain which, like ZZ, is derived from the Staphylococcus aureus A protein but 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).

[0123] The inventors also constructed a fusion protein, called ZZ-Tat22-57C(22-37)s. It contains a double ZZ domain derived from Staphylococcus aureus protein A and a Tat22-57C(22-37)s domain derived from the HIV transcriptional transactivator (WO 2011 / 092675, and Knittel et al. Vaccine, 2016, 34(27):3093-3101) which has a heparan sulfate binding site. These binding characteristics allow ZZ-Tat22-57C(22-37)s to target different cell types expressing surface immunoglobulins and heparan sulfate proteoglycans via interaction with HS.

[0124] The inventors expressed ZZ-DTRBD, BB-DTRBD, and ZZ-T at22-57C(22-37)s recombinantly using different expression systems. The complexes expressed in E. coli are named ZZ-DTRBD¥ii, BB-DTRBDcoii, and ZZ-Tat22-57C(22-37)s, and those expressed in HEK cells are named ZZ-DTRBDHEK and BB-DTRBDHEK. After expression, the complexes were purified using a column containing an IgG-carrying gel. The ZZ-DTRBDcoii, BB-DTRBD¥N, and ZZ-Tat22-57C(22-37)s proteins were then subjected to ion-exchange chromatography to remove contaminating LPS. Finally, certain characteristics of these complexes were evaluated by gel electrophoresis. As can be seen in Fig. 1, ZZ-DTRBDcoii and BB-DTRBD ¥iiThey migrate in a predominant band with a molecular mass of approximately 35 kDa, which is close to the theoretical mass of the molecule (32230), indicating that this band corresponds to a monomer. Minor bands corresponding to degradation products are also present in these two molecular complexes. ZZ-DTRBDHEK and BB-DTRBDHEK, on ​​the other hand, migrate in several bands mainly distributed between 35 and 150 kDa, indicating that expression in HEK cells leads to a heterogeneous mixture consisting of monomeric and oligomeric forms of ZZ-DTRBD. The molecular complex ZZ-Tat22-57C(22-37)s migrates in a single band The majority reflects the homogeneity of the purified protein. The molecular mass of approximately 25 kDa is slightly higher than the calculated theoretical mass of the molecule (MW=19145). However, Tat and its derivatives tend to migrate abnormally (Kittiworakarn et al., etc.), strongly suggesting that the purified ZZ-Tat22-57C(22-37)s fusion protein expressed in E. coli is essentially composed of a monomer.

[0125] Next, the inventors investigated the ability of free ZZ and the five molecular complexes to bind heparin, a sulfated polysaccharide representative of the heparan sulfate family. The enzyme immunoassay did not reveal a significant optical signal for free ZZ (Fig. 2). In contrast, an increase in optical density with increasing incubated dose was observed for ZZ-DTRBDcoii, BB-DTRBDHEK, BB-DTRBD¥ii, ZZ-DTRBDHEK, and ZZ-Tat22-57C(22-37)s. These data therefore indicate that the presence of the DTRBD domain or the Tat22-57C(22-37)s domain enables the binding of the molecular complexes to heparin. Furthermore, since the ZZ-DTRBDHEK and BB-DTRBDHEK fusions contain oligomers (see Figure 1), these data indicate that the presence of oligomers does not disrupt the interaction. Example 2: The ZZ-DTRBDHEK fusion protein is capable of binding to different types of cells in the murine immune system. Materials and methods Binding of ZZ-DTRBD to immune system cells

[0126] C57BI / 6 mouse splenocytes are resuspended at 10.10 6 cells / mL in 0.5% BSA 2 mM EDTA buffer. 100 pL of the cell suspension are placed in a 96-well round-bottom plate. 100 pL of 0.5% BSA 2 mM EDTA buffer is added per well, with or without ZZ-DTRBDHEK (100 nM). The mixtures are incubated for 30 minutes at 4°C and then washed twice in 0.5% BSA 2 mM EDTA buffer. 2 pg of rabbit IgG per well is added to the cells, which are incubated for 20 minutes at 4°C and then washed twice to a total of 200 pL in 0.5% BSA 2 mM EDTA buffer. The cells are resuspended in 50 pL of labeling buffer (PBS 0.5% BSA 2 mM EDTA) containing different mixtures of antibodies from BioLegend. The mixtures were incubated for 20 minutes at 4°C in the dark, then the splenocytes were washed twice to 200 pL in 0.5% PBS, 2 mM BSA, and EDTA. The cells were then fixed for 30 minutes at room temperature. For this purpose, 100 pL of 4% PFA buffer followed by 100 pL of 0.5% PBS, 2 mM EDTA, were added before acquisition on a BD FACSAria™ flow cytometer.

[0127] Mixture 1 for dendritic cells (duplicates): B220-FITC (#103206, 1 / 200), CD11 c-PE-Cy7 (#117318, 1 / 200), CD317-APC (#127016, 1 / 100), CD8a-PerCP-Cy5.5 (#100734, 1 / 200), CD11 b-APC-Cy7 (#101226, 1 / 800), Donkey anti-rabbit-BV421 (#406410, 1 / 100), Live Dead Aqua (ThermoFischer#L34966, 1 / 1000).

[0128] Mixture 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) (#406410, 1 / 100), Live Dead Aqua (ThermoFischer #L34966, 1 / 1000). Results

[0129] To assess the ability of a molecular complex to bind immune system cells, ZZ-DTRBDHEK was incubated in the presence of mouse splenocytes. It was observed that this fusion protein binds preferentially to cDC-CD8+ cells, which are specialized APCs (Fig. 3A). It also interacts preferentially with monocytes, which can also act as 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 fusion proteins ZZ-DTRBDHEK and ZZ-Tat22-57C(22-37)s induce in vitro the secretion of IL-6 and IL-12 by immune system cells. Materials and Methods

[0130] C57BL / 6 mouse splenocytes are resuspended at a rate of 2.10 6cells / mL in RPMI medium containing 10% FBS and 1% penicillin / streptomycin. 100 pL of the cell suspension are distributed into 96-well plates with or without molecular complexes (ZZ, DTRBD, ZZ-DTRBDHEK, or ZZ-Tat22-57C(22-37)s) and incubated at a final concentration of 1 mM. After 24 hours of incubation, the supernatants are collected for IL-6 and IL-12 cytokine assays by ELISA, performed according to the manufacturer's instructions (R&D #DY406-05 and #DY419). Results

[0131] The inventors wondered whether molecular complexes could induce the activation of immune system cells. Since cell activation can lead to cytokine secretion, the inventors decided to evaluate in vitro the presence of two cytokines in supernatants obtained from the incubation of mouse splenocytes with molecular complexes and various control proteins. The first cytokine was IL-6, as it is representative of inflammatory cytokines important for initiating the immune response. The second was IL-12, as it is crucial for inducing cellular immune responses. They found both cytokines in increased quantities in supernatants obtained from incubation with ZZ-DTRBDHEK, indicating that this molecular complex induces splenocyte activation (Fig. 4).However, they did not find an increase in cytokine levels in the supernatants obtained from incubation with free ZZ or free DTRBD, indicating that the dual Ig-binding domain and the heparan sulfate-binding domain cannot, on their own, activate immune cells. These results therefore demonstrate that ZZ-DTRBDHEK is capable of inducing the activation of immune system cells and indicate that the combination of the dual Ig-binding domain and the FIS-binding DTRBD domain is absolutely necessary for this effect.

[0132] The inventors proceeded according to a principle similar to that described in the preceding paragraph to evaluate whether ZZ-Tat22-57C(22-37)s is capable of activating immune system cells and whether the association of ZZ and the Tat domain involved in binding to heparan sulfates is also absolutely necessary for the stimulatory effect. To this end, they notably used a peptide, named TatcY49-57, 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, TatcY49-57, ZZ + TatcY49-57, and ZZ-Tat22-57C(22-37)s, respectively. They then assessed the presence of IL-6 and IL-12 in the supernatants. They found these two cytokines in the supernatants from incubation with ZZ-Tat22-57C(22-37)s but not in the supernatants from incubation with ZZ, TatcY49-57, ZZ + TatcY49-57 (Fig. 5).These results therefore demonstrate that ZZ-Tat22-57C(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 T atcY49-57, 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 immune system cells when its ZZ domain is substituted by BB, ii) exhibits increased stimulatory capabilities when it is made up of oligomeric forms. Materials and Methods

[0133] C57BL / 6 mouse splenocytes are resuspended at a rate of 2.10 6cells / mL in RPMI medium 10% SVF 1% Penicillin / Streptomycin. 100 pL of the cell suspension are distributed into 96-well plates in the presence or absence of different molecular complexes (ZZ-DTRBDHEK, BB-DTRBDHEK, ZZ-DTRBD). ¥ N and BB-DTRBDcoii) incubated to 1 mM final. After 24h of incubation, the supernatants are collected for IL-6 and IL-12 cytokine assay by ELISA performed according to the manufacturer's instructions (R&D #DY406-05 and #DY419). Results

[0134] The work described in Example 3 was carried out with two molecular complexes containing the ZZ double domain. This double domain, which binds the Fc region of antibodies, can thus target antibodies located on the surface of antigen-presenting cells (APCs). The inventors then wondered whether molecular complexes capable of jointly targeting the Fc and Fab regions of immunoglobulins could also activate immune system cells. To this end, they relied on the BB double domain derived from Staphylococcus aureus protein A, which has the unique characteristic of being able to bind the Fc region of antibodies in the same way as Z, but also the Fab region of antibodies, unlike ZZ (Jansson B. et al., Fems Immunol. Med. Microbiol. 1998, 20:69-78).They prepared the BB-DTRBDHEK and BB-DTRBDcoii complexes described in Example 1 and compared them to ZZ-DTRBDHEK and ZZ-DTRBDcoii for the ability to induce the secretion of IL-6 and IL-12 cytokines in vitro when incubated with murine splenocytes.

[0135] As the inventors noticed that ZZ-DTRBD and BB-DTRBD are in oligomeric form following their expression in HEK cells (see Fig. 1) whereas 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.

[0136] The inventors observed that supernatants obtained from incubation with ZZ-DTRBDHEK contain higher levels of cytokines than those obtained from incubation with BB-DTRBDHEK (Fig. 6). Similarly, supernatants obtained from incubation with ZZ-DTRBD¥ii contain higher levels of cytokines than those obtained from incubation with BB-DTRBD ¥ii These results demonstrate that a molecular complex containing BB remains capable of inducing cytokine secretion in vitro, but to a lesser extent 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 antibodies.

[0137] Comparison of cytokine levels in supernatants according to the cell type used (i.e., HEK versus E. coli) for complex production Molecular analysis has highlighted the difference in stimulatory efficacy depending on the type of production. Indeed, in the supernatants obtained from incubation with complexes derived from HEK cells (ZZ-DTRBDHEK, BB-DTRBDHEK), IL-6 and IL-12 are present in greater quantities than in those obtained from incubation with complexes derived from expression in E. coli (ZZ-DTRBD¥ii and BB-DTRBDcoii) (Fig. 6). Since the proteins derived from HEK cell expression are primarily oligomeric (see Fig. 1), the inventors deduced that the immune system is more effectively induced when the molecular complexes exhibit a high degree of oligomerization. Example 5: Isolated dendritic cells secrete IL-6 and IL-12 when incubated in the presence of ZZ-DTRBDHEK Materials and methods

[0138] A C57BL / 6J mouse was euthanized, and its spleen was collected in RPMI 10% FBS 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 (DCs) was performed according to the manufacturer's instructions (Miltenyi Biotec #130-100-875). The DCs were centrifuged for 5 minutes at 4°C at 390g and then resuspended at 2.10 6 cells / mL in RPMI 10% FBS 1% Penicillin / Streptomycin medium. 100 pL of the cell suspension (200,000 cells) are placed in a 96-well flat-bottom plate, and 100 pL of RPMI 10% FBS 1% Penicillin / Streptomycin medium is added, with or without ZZ-DTRBDHEK (0.6 pM final concentration). The cells are incubated for 24 hours at 37°C, and the supernatants are then collected for IL-6 and IL-12 cytokine assays by ELISA according to the manufacturer's instructions (R&D #DY406-05 and #DY419). Results

[0139] The establishment of immune defense mechanisms depends on the collaboration between different cellular partners. Among these, dendritic cells (DCs) play a central role as antigen-presenting cells (APCs). Indeed, they contribute to the activation of other cell types through direct or indirect interactions. via cytokines they secrete. To evaluate whether ZZ-DTRBD induces cytokine secretion by DCs, DCs were purified from C57BI / 6 mouse splenocytes. These DCs were then incubated for 24 h in the presence or absence of ZZ-DTRBD, and the supernatants were collected to measure the presence of IL-6 and IL-12 (Fig. 7). The amount of IL-6 in the supernatants from incubation with ZZ-DTRBDHEK was approximately 5 times greater than that found in supernatants from incubation without the molecular complex. A significant amount of IL-12 (approximately 8700 pg / ml) was also detected in the supernatants from incubation with ZZ-DTRBDHEK, but virtually none in those from incubation without the molecular complex. These data therefore indicate that ZZ-DTRBD causes 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: The proteins ZZ-DTRBD, and ZZ-Tat22-57C(22-37)s are capable of inducing dendritic cells in mice. Materials and Methods

[0140] Three groups of eight C57BL / 6 mice were injected three times at three-day intervals with 100 mIU of PBS in the absence or presence of ZZ-DTRBDHEK (5 nmoles per mouse) or ZZ-Tat22-57C(22-37)s (10 nmoles per mouse), respectively. Twenty-four hours after the last injection, the animals were euthanized, and the spleens were collected to recover the splenocytes. The cells were resuspended in 50 pL of labeling buffer (PBS 0.5% BSA 2 mM EDTA) containing different antibody mixtures. The cells were then incubated for 20 minutes at 4°C in the dark and subsequently washed twice in PBS 0.5% BSA 2 mM EDTA. The cells were then fixed for 20 minutes at 4°C in 100 pL of 4% PFA buffer and washed in 0.5% BSA PBS and 2 mM EDTA. They were then resuspended in 200 pL of 0.5% BSA PBS and 2 mM EDTA and analyzed using a BD FACSAria™ flow cytometer. The cells were analyzed by flow cytometry. CD8+ cDCs were identified as CD11 c highB220 CD8 + CD11 b, the cDC-CD11 b+ are identified CD11c high B220 CD8 CD11b + and the pDCs are identified as CD11c int CD317 + CD11 b + .

[0141] The antibody mixtures used for the phenotypic analysis of dendritic cells are as follows: B220-FITC (#103206, 1 / 200), CD11c-PE-Cy7 (#117318, 1 / 200), CD317-APC (#127016, 1 / 100), CD11b-APC-Cy7 (#101226, 1 / 800), CD8a-PerCP-Cy5.5 (#100734, 1 / 200), Live Dead Violet (ThermoFischer #L34964, 1 / 1000). Results

[0142] Because molecular complexes bind preferentially to APCs in vitro and induce cytokine secretion, the inventors wondered whether such complexes could induce APC expansion in mice. To evaluate this, the inventors injected three groups of eight C57BI / 6 mice with a PBS solution in the presence or absence of ZZ-DTRBDHEK or ZZ-Tat22-57C(22-37)s. They then euthanized the animals and harvested their spleens to assess the frequency of different dendritic cell subpopulations in splenocytes. As shown in Figure 8, all three murine DC populations (i.e., DC-CD8+, DC-CD11b+, and pDC) are present in the spleens of the group of mice injected with only PBS. The frequency of these three cell types is, however, significantly increased in groups of animals injected with ZZ-DTRBDHEK OR ZZ-Tat22-57C(22-37)s.These data therefore indicate that molecular complexes are capable of inducing the expansion of dendritic cells in vivo. Since these cells are central to 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-DTRBDHEK induces the secretion of IL-6 and IL-12 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.

[0143] A 1:1 dilution of leukocyte-platelet fluid is used to dilute a 1:2 ratio in AIMV medium and incubated overnight at room temperature with shaking. 15 mL of Histopaque medium is then added to four Leucosep tubes, to which 4 x 25 mL of diluted blood are added. The tubes are centrifuged at 1000 g for 15 minutes at room temperature without a brake. Peripheral blood mononuclear cell (PBMC) rings are collected and washed with 2 mM PBS and calcium- and magnesium-free EDTA. The PBMCs are centrifuged at 150 g for 10 minutes at room temperature, then a red blood cell lysis buffer (8.3 mg / mL NFUCI, 0.84 mg / mL NaHCO, 0.1 mM EDTA) is added and the tube is 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 performed according to the manufacturer's instructions (Miltenyi Biotec #130-091-379). The DCs are centrifuged for 5 minutes at 4°C at 390 g and then resuspended at 2.10 6cells / mL in RPMI 10% FBS 1% Penicillin / Streptomycin medium. 100 mL of sorted DCs (200,000 cells) are placed in a 96-well plate and 100 pL of RPMI 10% FBS 1% Penicillin / Streptomycin medium in the absence or presence of ZZ-DTRBDHEK (1 mM final concentration). The cells are incubated for 24 h at 37°C, and the supernatants are then collected for IL-6 and IL-12 cytokine assay by ELISA according to the manufacturer's instructions (R&D #DY206-05 and #DY1270-05). Results

[0144] To assess whether ZZ-DTRBDHEK can induce the in vitro secretion of IL-6 and IL-12 by DCs from healthy human donors, the presence of these two cytokines was evaluated in the culture supernatants of these cells after 24 hours of incubation. As can be seen in Fig. 9, IL-6 is found in significant quantities. The concentration of IL-12 is approximately 10 times higher in supernatants from incubations in the presence of ZZ-DTRBDHEK compared to supernatants from incubations in the absence of the fusion protein. IL-12, on the other hand, is present in large quantities (2400 pg / ml) in supernatants from DC incubations in the presence of ZZ-DTRBDHEK and in very small quantities (8 pg / ml) in those from incubations in the absence of the molecular complex. These data indicate that ZZ-DTRBD induces an increase in IL-6 and IL-12 secretion by human DCs. ZZ-DTRBD could therefore contribute to the establishment of immune defense mechanisms in humans that are linked to the secretion of these cytokines. Example 8: ZZ-DTRBD and ZZ-Tat22-57C(22-37)s slow the progression of colorectal tumor growth. Materials and Methods Study of the effect of the adjuvant CpG / Polyl:C mixture on tumor growth in a murine colorectal cancer cell line

[0145] Two groups of eight C57BL / 6 mice were injected subcutaneously into the paw with 0.5M MC38 cells. The mice were then either uninjected (Controls) or injected with the adjuvant mixture CpG-B 1018 / Poly I:C (30 pg of each adjuvant) three and six days later. Tumor growth was monitored by measuring tumors with calipers. Upon reaching the stopping criterion, the mice were euthanized. Study of the effect of the ZZ-DTRBDHEK molecular complex, injected in the absence or presence of adjuvant, on tumor growth in a murine colorectal cancer cell line

[0146] Three groups of six C57BL / 6 mice were injected subcutaneously in the paw with 0.5 M MC38 cells. Three, six, and nine days later, the mice were either uninjected (Controls) or injected with ZZ-DTRBDHEK (2 nmoles per mouse) in the absence or presence of CpG-B 1018 + Poly l:C (30 pg per mouse). adjuvant by mouse). The mice are monitored individually for 15 days (tumor measurements with calipers) before euthanasia. Comparison of the effect of an anti-PD-1 antibody and the molecular complexes ZZ-DTRBDHEK and ZZ-Tat22-57C(22-37)s on tumor growth in a murine colorectal cancer cell line

[0147] Four groups of eight C57BL / 6 mice were injected subcutaneously in the paw with 0.5M MC38 cells. Three, six, and nine days later, the mice were either uninjected (Controls) or injected with the anti-PD-1 antibody (Euromedex #BE0146-100MG, clone RMP1-14), ZZ-DTRBDHEK, or ZZ-Tat22-57C(22-37)s (0.96 nmol per mouse) in the presence of CpG-B 1018 + Poly l:C (30 pg of each adjuvant per mouse). The mice were monitored individually (tumor measurements with calipers) over time. Upon reaching the stopping criterion, they were euthanized. Results

[0148] 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 cell line, called MC38, and C57BI / 6 mice. Cancer induction is triggered by injecting 500,000 MC38 cells per mouse.

[0149] Initially, the impact of the adjuvant CpG1018 / polyl:C mixture on tumor growth was evaluated. As can be seen in Fig. 10A, compared to the untreated group (controls), tumor growth was virtually unaffected in the group of mice injected with the CpG1018 / polyl:C mixture. These data therefore indicate that this adjuvant mixture alone cannot slow the growth of this colorectal tumor.

[0150] In a second step, the impact of ZZ-DTRBDHEK injected alone or in the presence of the adjuvant mixture CpG1018 / polyl:C on tumor growth was evaluated. As can be seen in Fig. 10B, compared to the untreated group (controls), tumor growth was slowed on day 8 and on day 10 in the group injected with ZZ-DTRBDHEK without adjuvant. The effect was even more pronounced in the group treated with ZZ-DTRBDHEK and the CpG1018 / polyl:C adjuvant mixture. Indeed, in this group, tumor growth was slowed throughout the entire observation period (days 6 to 15). All of these data therefore indicate that ZZ-DTRBDHEK has an impact on the growth of the MC38 cell line and that this effect is amplified when mixed with the CpG1018 / polyl:C adjuvant.

[0151] In another series of experiments, the effect on MC38 tumor growth in C57BI / 6 mice was compared for the following treatments: ZZ-DTRBDHEK / CpG1018 / polyl:C, ZZ-Tat22-57C(22-37)s / CpG1018 / polyl:C, and anti-PD-1 antibodies. As can be seen in Figures 11A and 11B, treatment with anti-PD-1 antibodies did not significantly impact tumor growth compared to the control group. In contrast, mice injected with ZZ-DTRBDHEK / CpG1018 / polyl:C and ZZ-Tat22-57C(22-37)s / CpG1018 / polyl:C, respectively, exhibited a decrease in tumor growth. This slowing of tumor growth led to increased survival in both groups (Figure 11C). Example 9: Activated monocytes and CD4+ lymphocytes can be induced by molecular complexes targeting the DEC205 surface molecule and Fc receptors, respectively ga mma. Materials and Methods

[0152] To prepare a molecular complex targeting the DEC205 molecule, an anti-DEC205 antibody (BioLegend; clone NLDC-145, ref BLE138202) was used. It was incubated in the absence or presence of ZZ-Tat22-57C22-37)s at a fixed concentration (0.2 mM for each molecule) for 24 hours in RPMI medium without FBS. The ability of the ZZ region to bind the Fc region of the anti-DEC205 antibody allowed the formation of a non-covalent molecular complex, called anti-DEC205 / ZZ-Tat22-57C22-37)s. The same protocol was used to form a complex between the ZZ molecule and the anti-DEC205 antibody, called anti-DEC205 / ZZ.

[0153] To prepare a molecular complex targeting Fc receptors ga A non-specific human polyclonal antibody was used. This antibody was incubated with ZZ- Tat22-57C22-37)s or ZZ following a protocol identical to that used for the anti-DEC205 antibody. It was thus possible to form a non-covalent molecular complex, called lgG / ZZ-Tat22-57C22-37)s and an IgG / ZZ complex.

[0154] Human CMSPs prepared as described in Example 7 were then incubated in the absence or presence of the following compounds: anti-DEC205 / ZZ-Tat22-57C22-37, IgG / ZZ-Tat22-57C22-37, anti-DEC205 / ZZ, IgG / ZZ ZZ-DTRBDHEK, and free anti-DEC205 and IgG (final concentration of 0.1 mM for each compound). In these experiments, CMSPs were also incubated with free ZZ-Tat22-57C22-37 and ZZ proteins at a concentration of 1 mM. After 24 hours, the cells were harvested and labeled with fluorescent antibodies 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 microsatellite microscopy cells (MSMCs) were fixed with a 4% paraformaldehyde solution and then analyzed by flow cytometry. Results

[0155] The preceding examples show that molecular complexes targeting antibodies can effectively induce certain immune response mechanisms and effectively slow tumor growth. Antibodies located on the surface of antigen-presenting cells (APCs) are the molecules targeted by these complexes. However, APCs express a large number of other molecules that could also represent targets for molecular complexes according to the invention. The inventors therefore wondered whether molecular complexes targeting hemiglobulins (HS) and other receptors besides immunoglobulins located on the APC surface could also activate these cells. To evaluate this aspect, they chose to assess two types of receptors. The first is the DEC205 protein, which is a lectin selectively expressed in humans by monocytes and certain dendritic cell populations (Kato M. et al. 2006, Int. Immunol., 18:857-869). The second is the Fc receptor gamma, most forms of which are expressed by monocytes and certain populations of dendritic cells.

[0156] The inventors then prepared two molecular complexes capable of targeting these receptors. To target the DEC205 protein, they used a monoclonal antibody specific to this receptor, which they complexed with the fusion protein ZZ-Tat22-57C22-37s. They named this molecular complex anti-DEC205 / ZZ-Tat22-57C22-37s. To target the Fc receptors, they used a non-specific human polyclonal IgG antibody that can interact with these receptors via its Fc domain. They formed a molecular complex between this IgG and ZZ-Tat22-57C22-37s, called lgG / ZZ-Tat 22-57C22-37)s. These two aforementioned molecular complexes thus possess the ability to bind APC receptors and heparan sulfates via the Tat22-57C22-37)s domain of ZZ-Tat22-57C22-37)s. In these molecular complexes, the two proteins are associated non-covalently 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 antibodies. The inventors also prepared two complexes, respectively named anti-DEC205 / ZZ and IgG / ZZ, lacking the Tat22-57C22-37)s heparan sulfate-binding region, in order to use them as controls in subsequent activation experiments.

[0157] Since the DEC205 molecule and Fc receptors are expressed by monocytes, the inventors then wondered whether molecular complexes could enable the activation of this APC subpopulation. To this end, they incubated CMSPs in the absence or presence of a fixed concentration (0.1 mM) of anti-DEC205 / ZZ-Tat22-57C22-37)s, lgG / ZZ-Tat22-57C22-37)s, anti-DEC205 / ZZ, IgG / ZZ, anti-DEC205, IgG, respectively. They also incubated free ZZ-Tat22-57C22-37s and ZZ proteins at a concentration of 1 mM to assess the effect of ZZ-Tat22-57C22-37s at the same concentration found to be activating in Examples 3, 4, and 5. After 24 hours, they assessed the proportion of activated monocytes and CD4+ T lymphocytes. As shown in Fig. 12A, ZZ does not alter the proportion of activated monocytes in the CMSPs, whereas it is significantly increased by ZZ-Tat22-57C22-37s, indicating that this molecular complex can activate human APCs.The proportion of activated cells is decreased with free anti-DEC205 or included in the anti-DEC205 / ZZ complex. Conversely, the proportion of activated monocytes is increased in the presence of the anti-DEC205 / ZZ-Tat22-57C22-37 complex. A similar behavior is observed when CD4+ T lymphocytes are considered (Fig. 12B). These data therefore indicate that an antibody targeting APCs but lacking the ability to activate them can become capable of inducing their activation when included in a molecular complex that also targets HS.

[0158] Analysis of monocyte activation status after incubation with free human IgG shows that this antibody increases the proportion of activated monocytes. The IgG / ZZ complex does not significantly increase the proportion of activated cells. In contrast, the number of activated monocytes is more than twice as high when CMSPs are incubated with the IgG / ZZ-Tat22-57C22-37 molecular complex. A similar behavior is observed when CD4+ T lymphocytes are considered (Fig. 12B). These data therefore indicate that an antibody targeting Fc receptors ga mma on the surface of CPAgs is capable of activating them, but the activation effect is increased when the Ac is included in a molecular complex allowing it to also target HS.

[0159] Interestingly, these results showing a joint increase in the proportion of activated monocytes and T lymphocytes indicate that molecular complexes induce several cellular players 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 immune system cells. Materials and Methods

[0160] To prepare molecular complexes targeting DCs, three antibodies specific to molecules expressed by DCs were used. The first antibody (BD reference 555538), called anti-CD74, targets the CD74 molecule. The second antibody (BD reference 551186), called anti-CD209, targets the CD209 molecule. The third antibody (BD reference 552501), called anti-CD275, targets the CD275 molecule.

[0161] Each of these three antibodies (30nM for each molecule) was incubated in the absence or presence of ZZ-Tat22-57C22-37)s or ZZ (5nM for each molecule), for 24 hours at 4°C in RPMI medium, 5% AB human serum. The ability of the ZZ region to bind the Fc region of these antibodies allowed the formation of different non-covalent molecular complexes called anti-CD74 / ZZ-Tat22-57C22-37)s, anti-CD209 / ZZ-Tat22-57C22-37)s, anti-CD275 / ZZ-Tat22-57C22-37)s, anti-CD74 / ZZ, anti-CD209 / ZZ, and anti-CD275 / ZZ, respectively.

[0162] Human CMSPs are resuspended at a rate of 5.10 6 cells / mL in RPMI 5% AB human serum. 100 pL of the cell suspension was dispensed into 96-well plates in the presence or absence of antibodies, ZZ or ZZ-Tat22-57C(22-37)s free, as well as molecular complexes. After 24 h of incubation, the supernatants were collected for IL-6 cytokine assay by ELISA performed according to the manufacturer's instructions (R&D #DY406-05). Results

[0163] 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 cytokine secretion, the inventors decided to evaluate the presence of IL-6 in vitro. They found this cytokine in supernatants from incubation with ZZ-Tat22-57C22-37s but not in those from incubation with ZZ. They did not find it in supernatants from incubation of CMSPS with free anti-CD74 antibody (Fig. 13A), indicating that this antibody cannot induce activation in its free form. The inventors observed that IL-6 secretion is increased when CMSPs are incubated with the anti-CD74 / ZZ complex, indicating that the ZZ double domain enables activation mediated by this antibody (Fig. 13A).However, the secretion of this cytokine is even stronger with anti-CD74 / ZZ-Tat22-57C22-37), which indicates that the addition of the Tat region allowing HSPG binding increases cell activation mediated by this complex.

[0164] The inventors discovered that free anti-CD209 and anti-CD275 antibodies are capable of inducing IL-6 secretion when incubated with CMSPs (Fig. 13B and 13C). When these two antibodies are complexed with ZZ (anti-CD209 / ZZ and anti-CD275 / ZZ, respectively), secretion of this cytokine is also observed. However, this secretion is increased when these two antibodies are complexed with ZZ-Tat22-57C22-37), indicating that the activating power can be increased when the targeting molecular complexes are capable of binding 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

[0165] To prepare molecular complexes targeting the CD335 molecule on the surface of NK and NKT cells, an antibody (Origene AM31284AF-N), called anti-CD335, was used. This antibody was incubated at 30 nM in the absence or presence of ZZ-Tat22-57C22-37s or ZZ (5 nM for each molecule) for 5 hours at 37°C in RPMI medium, 5% AB human serum. The ability of the ZZ region to bind to the Fc region of these antibodies allowed the formation of two molecular complexes called anti-CD335 / ZZ-Tat22-57C22-37s and anti-CD335 / ZZ, respectively.

[0166] Human CMSPs were resuspended at a rate of 5.10 6cells / ml in RPMI 5% AB human serum. 100 pL of the cell suspension was distributed into 96-well plates in the presence or absence of free anti-CD335, ZZ, or ZZ-Tat22-57C(22-37)s antibodies, as well as molecular complexes. After 18 hours, the cells were harvested and labeled with fluorescent antibodies to identify NK cells (CD56+, BioLegend reference 362510, 1 / 100 dilution), NKT cells (CD56+, BioLegend reference 362510; CD3+; Miltenyi reference 130-113-136, 1 / 200 dilution), and the CD69 molecule (BioLegend reference 310932, 1 / 100 dilution). After 30 minutes of incubation, the CMSPs were fixed using a solution containing 4% paraformaldehyde and then analyzed by flow cytometry. Results

[0167] 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 was assessed. To evaluate this, they used the anti-CD335 antibody in isolated form or included in a molecular complex. They incubated MSPCs 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.

[0168] As can be seen in Figure 14, ZZ, the isolated anti-CD335 antibody, and the anti-CD335 / ZZ complex do not increase the proportion of NK (A) or NKT (B) cells expressing CD69 in CMSPs. A different behavior is observed with free ZZ-Tat22-57C(22-37)s. Indeed, this molecular complex does not increase the proportion of NKT cells expressing CD69 but, on the other hand, increases the percentage of NK cells expressing this marker, indicating that the association of ZZ and the HS ligand in the ZZ-Tat22-57C(22-37)s complex increases NK cell activation. The anti-CD335 / ZZ-Tat22-57C(22-37)s molecular complex, for its part, impacts the activation of both cell subpopulations. Indeed, it increases the proportion of NK and NKT cells expressing CD69.All of this data therefore indicates that the anti-CD335 antibody lacks the ability to activate both cell types but can become capable of inducing them when included in a molecular complex that also targets HS. Example 12: Molecular complexes directed against CD56 (A) and CD336 (B) molecules respectively, expressed by NK and NKT cells, induce in vitro an increase in the proportion of activated NKT cells. Materials and Methods

[0169] To prepare molecular complexes targeting CD56 and CD336 molecules on the surface of NK and NKT cells, the anti-CD56 antibody (Biolegend reference 304622) which targets the CD56 molecule and the anti-CD336 antibody (Origene AM50346PU-N) which targets the CD336 molecule were used.

[0170] These antibodies were incubated at 30 nM in the absence or presence of ZZ-Tat22-57C22-37 or ZZ (5 nM 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 enabled the formation of four molecular complexes called respectively anti-CD56 / ZZ-Tat22-57C22-37)s, anti-CD56 / ZZ, anti-CD336 / ZZ-Tat22-57C22-37)s, and anti-CD336 / ZZ.

[0171] Human CMSPs were resuspended at a rate of 5.10 6cells / ml in RPMI 5% AB human serum. 100 mIU of the cell suspension was distributed into 96-well plates in the presence or absence of isolated forms of anti-CD56, anti-CD336, ZZ, or ZZ-Tat22-57C(22-37)s antibodies, as well as non-covalent molecular complexes. After 18 hours, the cells were harvested and labeled with fluorescent antibodies described in Example 11, which allow for the identification of NKT cells (CD56+CD3+) and the CD69 molecule. After 30 minutes of incubation, the microsatellite microscopy (MSMP) was fixed with a 4% paraformaldehyde solution and then analyzed by flow cytometry. Results

[0172] The inventors investigated whether molecular complexes targeting expressed CD56 and CD336 molecules could induce NKT cell activation. To evaluate this, they used anti-CD56 and anti-CD336 antibodies in isolated form or within a molecular complex. They incubated microsatellite complexing proteins (MSCPs) 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.

[0173] As can be seen in Figure 15, the anti-CD56 antibody, the isolated anti-CD336 antibody, and the anti-CD336 / ZZ complex do not increase the proportion of CD69-expressing NKT cells in CMSPs. A different behavior is observed with the anti-CD56 / ZZ-Tat22-57C22-37 molecular complexes (Fig. 15A) and the anti-CD336 / ZZ-Tat22-57C22-37 molecular complexes (Fig. 15B). Indeed, an increase in the proportion of CD69-expressing NKT cells is found when these two molecular complexes are incubated with CMSPs. All of these data therefore indicate that isolated anti-CD56 and anti-CD336 antibodies lack the ability to activate NKT cells but can become capable of inducing them when 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 immune system cells. Materials and Methods

[0174] To prepare molecular complexes targeting NK and NKT cells, the three antibodies described in Examples 10 and 11 were used. Each of these three antibodies (30 nM for each molecule) was incubated in the absence or presence of ZZ-Tat22-57C22-37s or ZZ (5 nM for each molecule) for 24 hours in RPMI medium, 5% AB human serum. The ability of the ZZ region to bind to the Fc region of these antibodies allowed the formation of different non-covalent molecular complexes called anti-CD56 / ZZ-Tat22-57C22-37s, anti-CD335 / ZZ-Tat22-57C22-37s, anti- CD336 / ZZ-Tat22-57C22-37)s. anti-CD56 / ZZ, anti-CD335 / ZZ, and anti-CD336 / ZZ, respectively.

[0175] Human CMSPs are resuspended at a rate of 5.10 6cells / mL in RPMI 5% AB human serum. 100 pL of the cell suspension are distributed into 96-well plates in the presence or absence of free antibodies, ZZ or ZZ-Tat22-57C(22-37)s, and molecular complexes. After 24 hours of incubation, the supernatants are collected for IL-6 cytokine assay by ELISA performed according to the manufacturer's instructions (R&D #DY406-05). Results

[0176] 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 cytokine secretion, the inventors decided to evaluate the presence of IL-6 in vitro. They found this cytokine in supernatants from incubation with ZZ-Tat22-57C22-37 but not in those from incubation with ZZ. They did not find IL-6 in supernatants from incubation of CMSPS with free anti-CD56 and anti-CD335 antibodies (Fig. 16A and 16B), indicating that these two proteins cannot induce activation in their free form. However, they did detect the cytokine in supernatants from incubation with the anti-CD336 antibody (Fig. 16C). This indicates that, in its free form, this antibody possesses stimulatory activity. The inventors observed, for all three antibodies, increased IL-6 secretion when CMSPs were incubated with the anti-CD56 / ZZ-Tat22-57C22-37 complexes (Fig. 16A), anti-CD335 / ZZ-Tat22-57C22-37 complexes (Fig. 16B), and anti-CD336 / ZZ-Tat22-57C22-37 complexes (Fig. 16C), respectively. 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 immune system cells. Materials and Methods

[0177] To prepare molecular complexes targeting ICPs, three antibodies specific to molecules considered to be ICPs were used. The first antibody (BioXCell reference BE0190), called anti-CTLA-4, targets the CTLA-4 molecule. The second antibody (BioXCell reference BE0285), called anti-PD-L1, targets the PD-L1 molecule. The third antibody (R&D reference MAB10542), called anti-OX40, targets the OX40 molecule.

[0178] Each of these three antibodies (30nM for each molecule) was incubated in the absence or presence of ZZ-Tat22-57C22-37)s or ZZ (5nM for each molecule), for 24 hours at 4°C in RPMI medium, 5% AB human serum. The ability of the ZZ region to bind the Fc region of these antibodies allowed the formation of different non-covalent molecular complexes, called anti-CTLA-4 / ZZ-Tat22-57C22-37)s, anti-PD-L1 / ZZ-Tat22-57C22-37)s, anti-OX40 / ZZ-Tat22-57C22-37)s, anti-CTLA-4 / ZZ, anti-PD-L1 / ZZ, and anti-OX40 / ZZ, respectively.

[0179] Human CMSPs were resuspended at a rate of 5.10 6 cells / mL in RPMI 5% AB human serum. 100 pL of the cell suspension was distributed into 96-well plates in the presence or absence of free antibodies, ZZ or ZZ-Tat22-57C(22-37)s, and molecular complexes. After 24 hours of incubation, the supernatants were collected for IL-6 cytokine assay by ELISA performed according to the manufacturer's instructions (R&D #DY406-05). Results

[0180] The inventors wondered whether molecular complexes targeting ICPs could induce the activation of immune system cells. Since cell activation can lead to cytokine secretion, the inventors decided to evaluate the presence of IL-6 in vitro. They did not find it in the supernatants from the incubation of CMSPS with the three different free antibodies or with free ZZ, indicating that these compounds cannot induce activation in their free form (Figs. 17A, 17B, and 17C). In contrast, they found this cytokine in the supernatants from the incubation with ZZ-Tat22-57C22-37s, demonstrating that the association of the dual Ig-binding domain and the HS-binding Tat22-57C22-37s domain enables the activation of immune system cells. The inventors observed, however, that IL-6 secretion is further increased when CMSPs are respectively incubated with anti-CTLA-4 / ZZ-Tat22-57C22-37 complexes, (Fig.17A), anti-PD-L1 / ZZ-Tat22-57C22-37)s (Fig. 17B), anti-OX40 / ZZ-Tat22-57C22-37)s (Fig. 17C). In contrast, IL-6 is absent from the supernatants obtained from incubation with anti-CTLA-4 / ZZ, anti-PD-L1 / ZZ, or anti-OX40 / ZZ, indicating that the ZZ double domain does not contribute to activation mediated by these antibodies. Taken together, these data therefore indicate that molecular complexes targeting ICPs and HSPGs can induce immune cell activation.

Claims

Demands 1. Molecular complex for use as an immunomodulatory drug, preferably an immunostimulatory drug, said complex consisting of at least one ligand of a sulfated sugar from the glycosaminoglycan family (first ligand) and at least one ligand of a surface molecule of antigen-presenting cells or NK or NKT cells (second ligand) linked together, and said complex being devoid of a disease-specific antigen.

2. A molecular complex for use according to claim 1, wherein 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 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 R domain of diphtheria toxin (SEQ ID NO: 5) or at least the DT453-467 fragment (SEQ ID NO: 7) of said domain comprising the heparan sulfate 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: C-type lectin receptors, membrane immunoglobulins, receptors for the constant region of immunoglobulins, and immune checkpoint molecules and their ligands.

4. A 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 their fragments containing at least the paratope; (ii) immunoglobulins, preferably IgG, and their fragments comprising at least the Fc region; and (iii) proteins and protein fragments that bind the Fc and / or Fab region of antibodies, in particular the S. aureus protein A, its BB fragment (SEQ ID NO: 1) and its ZZ derivative (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), 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 selected from: the BB fragment 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 BB fragment (SEQ ID NO: 1) or its ZZ derivative (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), 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 derivative ZZ (SEQ ID NO: 2).

10. Molecular complex for use according to claim 9, wherein 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 an entire immunoglobulin.

11. Molecular complex for use according to claim 9, wherein said fusion protein is complexed with the second ligand which is selected from an anti-RFcgamma I, II and / or III, anti-DEC-205, anti-DC-SIGN, anti-CD74, anti-CD275, anti-CD56, anti-CD335, anti-CD336, anti-CTLA-4, anti-PD-L1, anti-OX40 antibody, or a fragment of the preceding antibodies comprising at least the paratope.

12. Molecular complex for use according to any of the preceding claims as an immunostimulatory drug, 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 cytokines IL-6 and / or IL-12.

13. Composition for use as an immunomodulatory drug, preferably an immunostimulant, comprising at least one molecular complex as defined in any one of the preceding claims, and at least one pharmaceutically acceptable vehicle, carrier substance and / or 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. Composition for use according to claim 13 or 14, comprising at least one other therapeutic agent, preferably at least one immune checkpoint inhibitor, preferably an anti-PD-1, an anti-PDL-1 or an anti-CTLA4.

16. Composition for use according to any one of claims 13 to 15, for use in immunotherapy of cancer or infectious diseases.

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