Lipid nanoparticle loaded with antitumoral agent and functionnalized to target immosuppressive cells

Abstract

The present invention relates to lipid nanoparticle loaded with antitumoral agent and functionalized to target immunosuppressive cells. Inventors developpe valrubicin-loaded immunoliposomes (Val-ILs). A small amount of valrubicin incorporated into Val-ILs induces leukemia cell death in vivo, suggesting that Val-ILs could be used to treat acute leukemia cells. Inventors also demonstrated that Val-ILs could reduce the risk of contamination of CD34+ hematopoietic stem cells by acute leukemia cells during autologous peripheral blood stem cell transplantation. They also highlighted the potential of Val-ILs to target immunosuppressive cell populations in the spleen. The most efficient Val-ILs were found to be those loaded with CD11b,CD223, CD64, TIM1, CD200R3, CD204, CD49b, VEGFR2 and SIGLECF antibodies. This study provides the effectiveness and ease of preparation of Val-ILs as a novel nanoparticle technology. In the context of cancers, Val-ILs have the potential to be used as a precise and effective therapy based on targeted vesicle-mediated cell death.

Description

[0001] LIPID NANOPARTICLE LOADED WITH ANTITUMORAL AGENT AND FUNCTIONNALIZED TO TARGET IMMOSUPPRESSIVE CELLS

[0002] FIELD OF THE INVENTION:

[0003] The present invention relates to a composition comprising a lipid nanoparticle loaded with antitumoral agent and functionalized to target immunosuppressive cells. The present invention also relates to the composition of the invention for use in the treatment of cancer disease (solid cancer and malignant hematological disease).

[0004] BACKGROUND OF THE INVENTION:

[0005] Advancements in the treatments available for acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and lymphoma have led to increased complete remission rates, with 5-year survival rates exceeding 80% for ALL, 50% for AML and 70% for lymphoma (1- 10). Still, there remains a need to develop new treatment strategies aimed at reducing the intensity of therapies and enhancing patient prognosis following a relapse. Unilamellar vesicles (UVs) are vesicular structures characterized by a lipid bilayer; their unique structure enables them to transport a wide range of hydrophilic and hydrophobic compounds. In particular, liposomes have been extensively employed as pharmaceutical carriers for various drugs, especially in cancer treatment (11-17). Since their discovery over 50 years ago (18), liposomes have evolved into a promising tool in the fields of medicine, biology, and chemistry. This is a result of their biocompatibility and their capacity to encapsulate and deliver a diverse array of drugs for therapeutic purposes (11-17).

[0006] Drug-loaded liposomes can be directed to tumors using passive targeting, a method that has been employed in the treatment of hematological cancers. For example, "passive liposomes" used to treat hematological malignancies include liposomal daunorubicin, which has been developed for the treatment of relapsed and refractory AML (19-21). In addition, vincristine sulfate liposome injection has been developed to enhance the pharmacokinetics and pharmacodynamics of vincristine (22). This formulation has been utilized in clinical settings to treat patients with relapsed and refractory lymphoma (23), as well as young patients with refractory solid tumors or leukemia (22, 24). These advancements in liposomal drug delivery have shown promise in improving the treatment outcomes for patients with various hematological malignancies.

[0007] "Active liposomes" constitute another approach, involving the attachment of specific ligands to the surface of liposomes to bind to particular antigens on target cells (11-17). To actively target specific cells, a variety of ligands, including antibodies, proteins, carbohydrates, and even aptamers, have been attached to the surface of liposomes (11-17). Within the realm of immunotherapy, immunoliposomes (ILs) have emerged as a novel strategy that has been extensively investigated in preclinical solid cancer models, showing promising results (11-17). Further experiments with mouse models have demonstrated that ILs encapsulating the anthracycline doxorubicin can target cancer cells and enhance the immunotherapeutic effect by reshaping the immunosuppressive tumor microenvironment (25-28). In the field of hematology, therapeutic ILs targeting CD20 (29, 30) and CD19 (31-34) have been developed to treat B-cell lymphoma both in vitro and in vivo using xenograft models. ILs-aCD19, containing imatinib, have been shown to efficiently kill Philadelphia chromosome-positive ALL cells in vitro (35). The delivery of PEGylated liposomal doxorubicin using bispecific antibodies has shown improved efficacy in models of high-risk childhood leukemia (36). Additionally, antibody fragment-decorated liposomal conjugates have successfully targeted Philadelphia-like AML (37). Nanoparticle-mediated targeting of the fusion gene RUNX1 : :ETO has also been reported in AML (38). These are all exciting developments in the application of liposomal drug delivery for treating various hematological malignancies.

[0008] The majority of established ILs have relied on encapsulating hydrophilic drugs within their internal aqueous compartments (29-35, 39, 40). However, in this particular study, our objective was to create ILs loaded with valrubicin, a hydrophobic analogue of doxorubicin. Valrubicin is recognized for its use in intravesical chemotherapy for carcinoma in situ of the bladder (41-45). Given its lipophilic nature, valrubicin was found to integrate effectively into the liposomes. We thus investigated the efficacy of these liposomes with valrubicin incorporated into the lipids, which we called VaLILs, using in vitro and in vivo leukemia and lymphoma models. Our primary goal was to explore this novel nanoparticle technology designed to induce specific vesicle-mediated cell death for the treatment of hematological cancers. This approach offers a promising avenue for the targeted treatment of these malignancies.

[0009] SUMMARY OF THE INVENTION:

[0010] A first object of the invention relates to a composition comprising a lipid nanoparticle loaded with an antitumoral agent and said lipid nanoparticle being associated with an antigen binding agent to target immunosuppressive cells. A second object of the invention also relates to composition of the invention for use in the prevention or treatment of a patient affected with a cancer disease (solid cancer and or malignant haematological disease).

[0011] In a particular embodiment the cancer is a drug resistant cancer or tumor relapse.

[0012] DETAILED DESCRIPTION OF THE INVENTION:

[0013] Here the inventors created valrubicin-loaded immunoliposomes (Val-ILs) using the antitumor prodrug valrubicin, a hydrophobic analogue of daunorubicin. Being lipophilic, valrubicin readily incorporated Val-ILs that were loaded with specific antibodies. Val-ILs injected intravenously rapidly reached the bone marrow and spleen, indicating their potential to effectively target cancer cells in these areas. Following the transplantation of human pediatric B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), or acute myeloid leukemia (AML) in immunodeficient NSG mice, inventors generated patient- derived xenograft (PDX) models, which were treated with Val-ILs loaded with antibodies to target CD19, CD7 or CD33. Only a small amount of valrubicin incorporated into Val-ILs was needed to induce leukemia cell death in vivo, suggesting that this approach could be used to efficiently treat acute leukemia cells. Inventors also demonstrated that Val-ILs could reduce the risk of contamination of CD34+hematopoietic stem cells by acute leukemia cells during autologous peripheral blood stem cell transplantation, which is a significant advantage for clinical applications. Using EL4 lymphoma cells on immunocompetent C57BL / 6 mice, they also highlighted the potential of Val-ILs to target immunosuppressive cell populations in the spleen, which could be valuable in impairing cancer cell expansion, particularly in lymphoma cases. The most efficient Val-ILs were found to be those loaded with CDl lb or CD223 antibodies, which respectively target the myeloid-derived suppressor cells (MDSC) or the lymphocyte-activation gene 3 (LAG-3 or CD223) on T4 lymphocytes. This study provides a promising preclinical demonstration of the effectiveness and ease of preparation of Val-ILs as a novel nanoparticle technology. In the context of hematological cancers, Val-ILs have the potential to be used as a precise and effective therapy based on targeted vesicle-mediated cell death. Lipid nanoparticle according to the invention;

[0014] A first aspect of the invention consists to a composition comprising: a lipid nanoparticle loaded with lipidic antitumoral agent and said lipid nanoparticle being associated with an antigen binding agent targeting immunosuppressive cell.

[0015] The term “lipid nanoparticles”, as used in the present invention, relates to a nanometersize lipid particle of vesicular type comprising an oily internal phase (or an oily core) and an envelope (or a shell) comprising a polyglycol ester of a hydroxy fatty acid.

[0016] In some embodiments, the lipid nanoparticles according to the invention lipid nanoparticles include (cationic) liposomes and polymers. Lipid nanoparticles useful in the practice of the present invention has very well known in the art.

[0017] According to the invention, lipid nanoparticles include but are not limited to liposomes such as cationic liposomes, solid-lipid nanoparticles (SLNs or LNPs) such as [(4- hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate)-based nanoparticles; niosomes; polymers such as cationic polymers; polymers-based nanoparticles such polyethylenimine(PEI)-based nanoparticles; lipopeptides-based nanoparticles such as lipid 1,2- dilinoleyloxy-3 -dimethylaminopropane (DLin-DMA)-based nanoparticles, dilinoleylmethyl-4- dimethylaminobutyrate (DLin-MC3-DMA)-based nanoparticles, ALC-0315-based nanoparticles, ALC-0159-based nanoparticles SM-102-based nanonparticles and ; and chitosans as described in Toualbi L, et al. International Journal of Molecular Sciences, Maier.M et al. Molecular Therapy (2013), Shriane D et al. Biol Pharm Bull (2018). lipid nanoparticles according to the invention include also the lipid nanoparticles described in patent WO2017049245, WO2018081480 and WO2021016430.

[0018] Examples of lipid nanoparticles include but are not limited to any of the lipid nanoparticles described in Nadia Fattahia N. et al “Emerging insights on drug delivery by fatty acid mediated synthesis of lipophilic prodrugs as novel nanomedicines”Joumal of Controlled Release 326, 2020, Pages 556-598: all of which are herein incorporated by reference.

[0019] In some embodiments, the composition according to the invention, wherein the lipid nanoparticles is selected from the list consisting of liposome, emulsion, micelle, polymeric nanoparticules, self-assembly of nanoparticle, dendrimer, nanostructure lipid carrier, solid lipid nanoparticle.

[0020] In particular embodiments, the composition according to the invention, wherein the lipid nanoparticles is a liposome. The term “liposome”, as used in the present invention, relates to a small artificial vesicle, spherical in shape, having at least one lipid bilayer. Due to their hydrophobicity and / or hydrophilicity, biocompatibility, particle size and many other properties (Akbarzadeh, A.; et al (2013). " Nanoscale Research Letters. 8 (1): 102.) liposomes can be used as drug delivery vehicles for administration of pharmaceutical drugs and nutrients, such as lipid nanoparticles in mRNA vaccines, and DNA vaccines. Liposomes can be prepared by disrupting biological membranes (such as by sonication).

[0021] Liposomes are most often composed of phospholipids, (Mashaghi S., et al. Lipid Nanotechnology. Int J Mol Sci. 2013 Feb; 14(2): 4242-4282) especially phosphatidylcholine, and cholesterol (Akbarzadeh, A.; et al (2013). " Nanoscale Research Letters. 8 (1): 102.) but may also include other lipids, such as those found in egg and phosphatidylethanolamine, as long as they are compatible with lipid bilayer structure (Cevc, G (1993). Journal of Controlled Release. 160 (2): 135-146). A liposome design may employ surface ligands for targeting to desired cells or tissues (Torchilin, V (2006). " Advanced Drug Delivery Reviews. 58 (14): 1532-55.).

[0022] Indeed, in a particular embodiment, in order to link “antigen binding agent” at least one lipid used to form liposome is a lipid with a reactive moiety. For instance; DSPE-PEG-NHS, MW 2,000 is a phospholipid PEG polymer can be used to bind antibodies in targeted drug delivery system. The NHS-ester is reactive with amine at pH 6.5 to 7.5 to form a stable amide bond with antibodies. Other reactive lipides with a reactive moiety might also be DSPE-PEG- DBCO, DSPE-PEG-Maleimide (for Fab), DSPE-PEG-NHS, DSPE-PEG-Carboxylic Acid, DSPE-PEG-Amine and DSPE-PEG-DBCO. There are also Alkyne PEG, Amino PEG, Aminooxy PEG, APN PEG, BCN-PEG, Benzyl-PEG, Biotin PEG, Bis-PEG-acid, Bis-PEG- NHS, Boc-PEG, Branched PEG, Bromo PEG, DBCO PEG, Diketone Linkers, DNP-PEG, Fluorine PEG, Fmoc PEG, Hydroxy PEG, Iodo PEG, Lipid PEG, Maleimide Linkers, MeNH- PEG, Non-PEG linker, NOTA reagent, PEG Acid, PEG Aldehyde, PEG Azide, PEG Hydrazide, PEG NHS ester, PEG PFP ester, PEG Phosphonate, PEG PNP carbonate, PEG Silane, PEG Sulfonic acid, PEG Tosylate, PEG-X-PEG, Poly PEG, Propargyl PEG, PROTAC Linker, SPDP PEG, Sugar PEG, TCO-PEG, Tetrazine-PEG, Thiol PEG.

[0023] By the term “immunosuppressive cells” refers to any innate immune cells involves in the modulation of immune response and also involves in the mechanisms underlying tumor immune resistance. Example of immunosuppressive cells is selected from the list consisting of myeloid-derived suppressive cells, tumor-associated macrophages, tumor-associated neutrophils, regulatory T cells (Tregs), and tumor-associated dendritic cells. These immunosuppressive cells are critical factors correlated with immune resistance. In addition, cytokines and factors secreted by tumor cells or these immunosuppressive cells also mediate the tumor progression and immune escape of cancers. Thus, targeting these immunosuppressive cells and the related signals is the promising therapy to improve the efficacy of immunotherapies and reverse the immune resistance.

[0024] By the term “antigen binding agent targeting immunosuppressive cells” refers to any compound liable to specifically bind to immunosuppressive cells as defined above or an agent (such cytokine) activating immunosuppressive cells. An antigen binding agent can thus comprise or can consist of one or several binding moieties.

[0025] In a particular embodiment in order to be linked or conjugated to lipid nanoparticles the antigen binding agent comprise free amine or thiol groups.

[0026] In particular, the antigen binding agent comprising free amine or thiol groups is chosen from a protein, a peptide, a single chain antibody, a monoclonal antibody. Preferably, the antigen binding agent comprising free amine or thiol groups is a monoclonal antibody.

[0027] In an embodiment, the conjugate comprises a lipid nanoparticle of the invention or a lipide nanoparticle of the invention and a molecule comprising at least a free amine or a thiol group. In particular, the molecule comprising at least a free amine or a thiol group a protein may be a peptide, a single chain antibody, a polyclonal antibody, an antibody fragment (Fab, F(ab')2 fragment ...) or a monoclonal antibody. More particularly, the molecule comprising at least a free amine or a thiol group a protein may be a nanobody, an antibody fragment (Fab, F(ab')2 fragment ...) or a monoclonal antibody. Even more particularly, the molecule comprising at least a free amine or a thiol group a protein may be an antibody fragment (Fab, F(ab')2 fragment . ..) or a monoclonal antibody. Preferably, the molecule comprising at least a free amine or a thiol group a protein may be a monoclonal antibody.

[0028] In an embodiment, the molecule comprising at least a free amine or a thiol group may be a molecule modified with a linker comprising at least a free amine or a thiol group. In other words, the molecule comprising at least a free amine or a thiol group is a molecule wherein the free amine or thiol group is held by a linker moiety.

[0029] In the context of the invention, the term “linker” or “linker moiety” refers to a connector for linking a molecule to a nanoparticle of the invention or a nanoparticle obtained by the process of the invention (described below and in Experimental section).

[0030] In the context of the invention, the term “conjugate” refers to a molecule composed of two or more molecules which are linked together. The conjugates of the invention are typically composed of a lipid nanoparticle according to the invention linked to a protein, a peptide, a single chain antibody, a polyclonal antibody an antibody fragment (Fab, F(ab')2 fragment . . .) or a monoclonal antibody (mAb).

[0031] Particularly, the antibody (mAb or related antibody fragment (Fab, F(ab')2) may be chosen from antibody directed against CD 11b which target the myeloid-derived suppressor cells (MDSC) or antibody directed against CD223 (the lymphocyte-activation gene 3 (LAG-3 or CD223 ) which target T4 lymphocytes (lymphocytes T CDD4 “auxiliaries”, or T « helper ») or antibody directed against CD25 (alpha subunit of interleukin-2 receptor) or CD 127 which target Treg cells (lymphocytes Treg »), antibody directed against IL17alpha , Interleukin which recruit myeloid derived suppressor cells (MDSCs), antibody directed against (CD14 / CD16 (human counterpart of mouse Ly6C,) which target inflammatory monocyte / macrophage M2) antibody targeting CD101 expressed on Neutrophile cells and plays a role as inhibitor of T- cells proliferation and inhibits expression of IL2RA and IL2 production, antibody targeting CD115 / M-CSF-l expressed by dendritic cells in inflammatory condition, and involved in macrophage colony-stimulating factor, antibody targeting CD 11c, expressed by monocytes, macrophages and Natural killer cells, inducing phagocytosis and cell adhesion, Antibody targeting CD123 / IL-3R expressed by monocytes and B lymphocytes, with a role in differentiation and maturation, antibody targeting CD141 / Thrombomodulin, and activate cofactors of coagulation mechanism, antibody targeting CD124 (Siglec F), CD170 / Siglec-F TAN, CD 182 / CXCR2, expressed by Neutrophile (N2) and inhibits the activation of several cell types including monocytes, macrophages and neutrophils, antibody targeting CD200R3, expressed by myeloid cells to limits inflammation by inhibiting the expression of proinflammation. antibody targeting CD204 expressed on macrophage M2, antibody targeting CD206 / MRC1 expressed by macrophages, immature dendritic cells, and mediates the endocytosis of glycoproteins by macrophages, Class I MHC mediated antigen processing and presentation, antibody targeting CD24 expressed on B cell, involved to promote AG-dependent proliferation of B-cells, and prevents their terminal differentiation into antibody-forming cells. In association with SIGLEC 10 may be involved in the selective suppression of the immune response, antibody targeting i expressed on many leukocytes and myeloid cells, antibody targeting JAML expressed on myeloid cells and involved in leukocytes activation, Class I MHC mediated antigen processing and presentation, antibody targeting CD4 expressed by T4 lymphocytes, antibody targeting CD49b expressed by natural killer cells, antibody targeting CD64 expressed by macrophage Ml and dendritic cells, and involved in anti-inflammatory cytokines production, antibody targeting FceRI expressed by B lymphocytes and macrophage to initiates presentation to T cells, antibody targeting TIM1 expressed on activated lymphocytes and having a role as a co-stimulatory receptor in T-cell.

[0032] The fragment antigen-binding region (Fab region) of all these cited antibodies might also be incorporated on lipid nanoparticle.

[0033] More particularly, the monoclonal antibody may be chosen from CDl lb or CD223 antibodies which selectively target the myeloid-derived suppressor cells (MDSC) or the lymphocyte-activation gene 3 (LAG-3 or CD223) on T4 lymphocytes.

[0034] In one embodiment, the monoclonal antibody may be chosen from CDl lb, CD223, CD25 CD127 or IL17 alpha antibodies.

[0035] As used herein the term “CD223” refers to “Lymphocyte-activation gene 3”, also known as LAG-3, is a protein which in humans is encoded by the LAG3 gene (Gene ID: 3902). LAG3, also designated CD223 (cluster of differentiation 223) is a cell surface molecule with diverse biological effects on T cell function but overall has an immune inhibitory effect. It is an immune checkpoint receptor and as such is the target of various drug development programs by pharmaceutical companies seeking to develop new treatments for cancer and autoimmune disorders. In soluble form it is also being developed as a cancer drug in its own right (Syn, Nicholas L; et al (2017). The Lancet Oncology. 18 (12): e731-e741). Conservation of their respective cytoplasmic tail motifs, CxC / H in the case of CD4 and an ITIM-like motif in the case of LAG-3, supports that competition between CD4 and LAG-3 for binding of kinase LCK is a conserved core part of the jawed vertebrate immune system.

[0036] Example of antigen binding agent of CD223 that could be used according to the invention is anti CD223 monoclonal antibody described in literature (Andrews LP et al Immunological reviews Volume 276, Issuel, March 2017 Pages 80-96.; Poirier N et al Clinical and Experimental Immunology, Volume 164, Issue 2, May 2011, Pages 265-274; Bookman SH et al International Journal of Cancer (2018) Volumel43, Issuel2 Pages 3201-3208.) or in patent US2016108121, WO2016028672; WO2017198741, WO2017015560.

[0037] For example, the antigen binding agent of CD223 of the invention is the anti CD223 monoclonal antibody available from Termofisher (CD223 (LAG-3) Monoclonal Antibody (clone 3DS223H), PE, eBioscience) or from Miltenyi Biotec (CD223 (LAG-3) Monoclonal Antibody (3DS223H), PE, eBioscience Clone REA351), or from Biolegend (PE anti-human CD223 (LAG-3) Antibody (clone 11C3C65).

[0038] As used herein, the term "CD1 lb" (cluster of differentiation molecule 1 IB (CD1 IB) is one protein subunit that forms heterodimeric integrin alpha-M beta-2 (aMp2) molecule, also known as Integrin alpha M (ITGAM) or macrophage-1 antigen (Mac-1) or complement receptor 3 (CR3) (or as CR3A, (Solovjov DA, et al (2005). The Journal of Biological Chemistry. 280 (2): 1336-1345). The second chain of aMp2 is the common integrin P2 subunit known as CD18, and integrin aMp2 thus belongs to the P2 subfamily (or leukocyte) integrins. aMp2 is expressed on the surface of many leukocytes involved in the innate immune system, including monocytes, granulocytes, macrophages, and natural killer cells (Solovjov DA, et al (2005)) and subsets of T and B cells (Khan SQ, et al (2018). Frontiers in Medicine. 5: 52). It mediates inflammation by regulating leukocyte adhesion and migration and has been implicated in several immune processes such as phagocytosis, cell-mediated cytotoxicity, chemotaxis and cellular activation (Solovjov DA, et al (2005)).

[0039] CDl lb is also expressed at immunosuppressive myeloid derived suppressor cells (CD1 lb Gr-1 Ly6C MDSCs)

[0040] Example of antigen binding agent of CDl lb that could be used according to the invention is anti CDl lb monoclonal antibody described in literature (Violette SM et al J Immunol (1995) 155 (6): 3092-3101.; Duan M et al Mucosal Immunology Volume 9, Issue 2, 2016, Pages 550-563; Furie MB et al Blood (1991) 78 (8): 2089-2097.) or in patent WO2016197974, US5219997; WO9211870.

[0041] For example, the antigen binding agent of CDl lb of the invention is the anti- CDl lb monoclonal antibody available from Abeam (Anti-CDl lb antibody [MI / 70] clone ab8878) from Invitrogen (CDl lb Recombinant Rabbit Monoclonal Antibody (JU93-81)) from Proteintech (CD1 lb Monoclonal Antibody (CloneNo. ICRF44).

[0042] As used herein, the term "CD25" refers to the alpha subunit of interleukin-2 receptor, a single-chain glycoprotein with a molecular weight of 55 kD present on a Treg cell surface.

[0043] Example of antigen binding agent of CD25 that could be used according to the invention is anti CD25 monoclonal antibody described in literature (Amlot PL et al Transplantation 60(7):p 748-756, October 15, 1995; Huss DI et al Immunology. 2016 Jul; 148(3): 276-286) or in patent WO2022106663 WO2023028451; WO2023134766

[0044] As used herein, the term "CD127" refers to the interleukin-7 receptor, present on a Treg cell surface. The IL-7 receptor alpha chain is described in the literature (e.g., Goodwin et al. (1990) Cell 60:941-951).

[0045] Example of antigen binding agent of CD 127 that could be used according to the invention is anti CD127 monoclonal antibody described in literature (Le Mai H et al Amercican Journal of Transplantation Volume20, Issuel January 2020, Pages 101-111; Poirier N et al J Immunol (2023) 210 (6): 753-763.) or in patent WO2013056984; WO2015189302 WO2011094259CN11592595. As used herein, the term "IL17alpha" (or “Interleukin- 17A” (UniProt Q16552) or “CTLA8” in rodents) refers to a proinflammatory cytokine produced by activated T cells. This cytokine regulates the activities of NF-kappaB and mitogen-activated protein kinases. This cytokine can stimulate the expression of IL6 and cyclooxygenase-2 (PTGS2 / COX-2), as well as enhance the production of nitric oxide (NO). In tumorigenesis, IL-17A has been shown to recruit myeloid derived suppressor cells (MDSCs) to dampen anti-tumor immunity (He D, et al (2010). Journal of Immunology. 184 (5): 2281-2288). IL-17A can also enhance tumor growth in vivo through the induction of IL-6, which in turn activates oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) and upregulates pro-survival and pro-angiogenic genes in tumors (Wang L, et al (2009), The Journal of Experimental Medicine. 206 (7): 1457-1464).

[0046] Example of antigen binding agent of IL 17a that could be used according to the invention is anti IL17a monoclonal antibody described in literature (Hueber W, et al Gut 2012;61 : 1693- 1700.; Lui L et al. Journal of Inflammation Research Volume 9, 2016 - page 39-50; Kaul M et al Journal of the European Academy of Dermatology and Venereology Volume 35, Issue 5May 2021Pagesl031-1233; ) or in patent W02009136286; W02014161570 WO2017186631.

[0047] As used herein, the term "CD 14" (or cluster of differentiation 14) refers to a human protein expressed mostly by macrophages as part of the innate immune system (Simmons DL, et al (1989). Blood. 73 (1): 284-289). CD14 helps to detect bacteria in the body by binding lipopolysaccharide (LPS), a pathogen-associated molecular pattern (PAMP). CD14 exists in two forms, one anchored to the membrane by a glycosylphosphatidylinositol (GPI) tail (mCD14), the other a soluble form (sCD14).

[0048] The non-classical monocyte (as Macrophage M2) with inflammatory phenotype is associated with shows low level expression of CD 14 and additional co-expression of the CD 16 receptor (CD14+CD16++ monocyte) (Ziegler-Heitbrock, Loems (March 2007). Journal of Leukocyte Biology. 81 (3): 584-592)

[0049] As used herein, the term "CD 16" also known as FcyRIII, refers to a cluster of differentiation molecule found on the surface of natural killer cells, neutrophils, monocytes, macrophages, and certain T cells (Georg P, et al. (2021). Cell. 185 (3): 493-512. e25.). CD16 has been identified as Fc receptors FcyRIIIa (CD16a) and FcyRIIIb (CD16b), which participate in signal transduction. The most well-researched membrane receptor implicated in triggering lysis by NK cells, CD 16 is a molecule of the immunoglobulin superfamily (IgSF) involved in antibody-dependent cellular cytotoxicity (ADCC) (Mandelboim O et al (1999). PNAS. 96 (10): 5640-4. It can be used to isolate populations of specific immune cells through fluorescent- activated cell sorting (FACS) or magnetic-activated cell sorting, using antibodies directed towards CD 16.

[0050] Example of antigen binding agent of CD 14 / CD 16 that could be used according to the invention is anti CD14 / CD16 monoclonal antibodies described in literature (Korkosz M. et al. Blood (2012) 119 (22): 5329-5330.; Fingerle G, et al Blood (1993) 82 (10): 3170-3176.) or in patent US5877299; CN117310168, CN116559440.

[0051] As used herein the term “CD49b” or “integrin alpha-2” refers to a cluster of differentiation 49b. CD49b is expressed by NK cells, NK-T cells, monocytes and platelets (Gagliani N et al., 2013, Nat Med, DOI : 10.1038 / nm.3179). The Entrez reference number of the human gene coding for CD49b is 3673 and the Uniprot reference number of CD49b human protein is P17301.

[0052] Example of antigen binding agent of CD49b that could be used according to the invention is anti CD49b monoclonal antibody described in literature (Morton L.F et al., 1994, Biochem J, DOI : 10.1042 / bj2990791).

[0053] As used herein the term “TIM1” or “HAVcr-1 refers to a transmembrane protein expressed on activated lymphocytes and appears to have a role as a co-stimulatory receptor in T cells. Hepatitis A virus cellular receptor 1 (HAVcr-1) also known as T-cell immunoglobulin and mucin domain 1 (TIM-1) is a protein that in humans is encoded by the HAVCR1. TIM-1 is preferentially expressed on T4 lymphocytes Th2 cells and has been identified as a stimulatory molecule for T-cell activation (FeigelstockD et al., 1998, J Virol, DOI : 10.1128 / jvi.72.8.6621- 6628.1998 ; McIntire J.J et al., 2001, Nat Immunol, DOI : 10.1038 / ni739). TIM-1 is overexpressed in various cancers, where it promotes tumor cell proliferation, survival, and migration through signaling pathways such as MEK / ERK and PI3K / AKT. This overexpression directly contributes to tumor progression by enhancing key processes like invasion and epithelial-mesenchymal transition (EMT) (Cao. J et al., 2024, Frontiers in cell and developmental biology, DOI : 10.3389 / fcell.2024.1307806). In addition to its role in tumor cells, TIM-1 is also expressed on B cells, where it plays a crucial role in modulating the tumor microenvironment. Specifically, TIM-1 inhibits Thl immune responses and promotes Th2 cytokine production, which indirectly contributes to immune evasion and tumor growth by suppressing anti-tumor immunity (Cao. J et al., 2024, Frontiers in cell and developmental biology, DOI : 10.3389 / fcell.2024.1307806). TIM-1 functions as a critical checkpoint for B cell activation. It restricts B cell-mediated antigen presentation and co-stimulation, thereby dampening the immune response. Blocking TIM-1 in B cells improves their responsiveness to type I interferon (IFN-I), leading to enhanced B cell activation, increased antigen presentation, and improved co-stimulatory signals. This results in elevated production of inflammatory cytokines and boosts effector T cell responses, which are key to mounting an effective antitumor response (Tian X et al., 2023, Signal Transduction and targeted therapy, DOI : 10.1038 / s41392-023-01643-w). By inhibiting TIM-1, not only is the suppressive tumor microenvironment altered, but CD8+ and CD4+ T cell responses are also significantly enhanced, leading to reduced tumor growth (Bod L et al., 2023, Nature, DOI : 10.1038 / s41586- 023-06231-0). The Entrez reference number of the human gene coding for TIM1 is 26762 and the Uniprot reference number of TIM1 human protein is Q96D42.

[0054] Example of antigen binding agent of TIM1 that could be used according to the invention is anti TIM1 monoclonal antibody described in literature (Qiang L et al., 2022, Pharmazie, DOI : 10.1691 / ph.2022.1670 ; Hye Sung K et al., 2010, J Immunol, DOI : 10.4049 / jimmunol.0901991).

[0055] The term of “antitumoral agent” is meant an anticancer drug that blocks cell growth by interfering with DNA, the genetic material in cells. Also called anticancer antibiotic and antineoplastic antibiotic or drug. Antitumoral agent is generally associated with chemotherapy treatment.

[0056] By a “chemotherapy treatment” is meant a drug that has proved its efficacy for the treatment of cancer, namely a drug having a marketing approval or a drug undergoing clinical or preclinical trial for the treatment of cancer.

[0057] Non-limiting examples of chemotherapeutic compounds include, for example, conventional chemotherapeutic and anti-angiogenic agents.

[0058] Exemplary chemotherapeutic compounds include alkylating agents, cytotoxic antibiotics such as topoisomerase I inhibitors, topoisomerase II inhibitors, plant derivatives, RNA / DNA antimetabolites, and antimitotic agents. Preferred examples may include, for example, cisplatin (CDDP), carboplatin, cytarabine (Ara-c), azacitidine (5-Azacitidine), decitabine (5 -aza-2'-deoxy cytidine), procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, chlofarabine, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, mitoxantrone, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, taxol, gemcitabine, navelbine, transplatinum, 5- fluorouracil, vincristin, vinblastin and methotrexate, or any analog or derivative variant of the foregoing.

[0059] In preferred embodiment, chemotherapeutic compound are hydrophobic compounds (lipophilic prodrugs) where chemotherapeutic compounds are linked to Fatty acid (FA) in order to improve half-life, absorption, specificity, rapid degradation, and resistance development. Saturated FAs (SFAs) or Unsatured FAs (UFAs) can be used to be conjugated to anticancer drug.

[0060] Examples of chemotherapeutic hydrophobic compounds include but are not limited to any of the chemotherapeutic hydrophobic compounds described in Nadia Fattahia N. et al “Emerging insights on drug delivery by fatty acid mediated synthesis of lipophilic prodrugs as novel nanomedicines, Journal of Controlled Release 326, 2020, Pages 556-598: all of which are herein incorporated by reference.

[0061] In a particular embodiment anti-tumor (chemotherapeutic) hydrophobic compounds is selected from the list consisting of : daunorubicin-UFAs such as Valrubicin (the lipophilic analogue of daunorubicin); Doxorubicin (DOX)-UFAs such as DOX-DHA, DOX-LNA, DOX- PA; Taxane-UFAs such as Paclitaxel (PTX)-UFAs, PTX-DHA (Taxoprexin) PTX-CLA (conjugate linoleic acid), SB-T-1213 and SB-T-1214, camptothecin (CPT) and its synthetic analogs, such as 9-nitro-CPT, 10-hydroxy-CPT (HCPT), irinotecan and topotecan; Propofol (diisopropyl phenol; PP)UFAs; Illudin-UFAs and derivative Illudin M DHA (IM- DHA); Cytarabine (ara-C)-UFAs and most potent derivative, CP-4055 (elacytarabine); Gemcitabine (GEM)-UFAs and derivative, CP-4125; GEM-CLA conjugate, GEM-DHA CP-4126; Azacytidine (AZA)-UFAs such as CP-4200 (EA ester of AZA); Chlorambucil (CHL)-UFAs such as CHL- DHA, CHL-ACA and CHL-OA; Mitomycin C((MMC)-UFAs such as MMC- DHA MMC-LNA, MMC-EPA; MMC-GLA, MMC-ACA; Daunomycin (DMC)-UFAs such as DMC-DHA and DMC-ACA, 2-Deoxy-5-fluorouridine ((FUDR)-UFAs such as FUDR-OA and FUDR-DHA; Tegafur (TF)-UFAs (FT oral prodrug of 5-FU) such as FT-OA, FT-LA, FT- ACA; Combretastatin (CA-4a-UFAs ((3,4,5,4'-tetramethoxy-3'-hydroxy-cis-stilbene; CA-4) such as CA-4-ACA, CA-LA, CA-DHA, CA-LNA; mesalamine (5-ASA) -UFAs such as 5- ASA-SFA and 5-ASA-PUFA (CLX-103).

[0062] In a particular embodiment anti-tumor hydrophobic compounds is Valrubicin.

[0063] Another object of the invention is a suspension of lipid nanoparticles according to the invention. Such a suspension of lipid nanoparticles contains a solvent which can be selected from an aqueous solution, for example water, or saline solution, or glycerol, or mannitol in which the particles of the invention described above are suspended.

[0064] In other words, the suspension of particles according to the invention comprises lipid nanoparticles according to the invention suspended in a solvent selected from an aqueous solution, for example water, a saline solution, a glycerol solution and a mannitol solution. In particular, the suspension of particles according to the invention comprises particles according to the invention suspended in an aqueous solution or a saline solution. More particularly, the suspension of particles according to the invention comprises particles according to the invention suspended in an aqueous solution, preferably water, more preferably distilled water.

[0065] In a particular embodiment the lipidic nanoparticle of the invention has a hydrodynamic diameter comprised between 100 and 1000 nm, preferably between 100 and 500 nm, more preferably between 100 and 300 nm, still more preferably between 100 and 200 nm, said nanoparticle comprising lipid nanoparticles, preferably liposome, loaded with an antitumoral agent and associated with an antigen binding agent to target immunosuppressive cells.

[0066] As used herein, the expression “comprised between . .. and ...” should be understood to include the boundaries of the recited range.

[0067] In the context of the invention, the term “hydrodynamic diameter” refers to the diameter of a hypothetical hard sphere that diffuses with the same speed as the particle being measured. It reflects the size of the particle when in solution and includes coatings or surface modifications made to the particle in question.

[0068] The hydrodynamic diameter of the particles of the invention may be determined according to any method known by the person skilled in the art. In particular, the hydrodynamic diameter of the particles of the invention may be determined by dynamic light scattering (DLS), with for example a NanoZS® apparatus (Malvern Instruments, Worcestershire, UK) equipped with a 633 nm laser at a fixed scattering angle of 173°, with the temperature of the cell being kept constant at 25°C. The nanoparticles are for this measure put in suspension in water at a concentration of 20 pg to 200 pg of iron per mL of water. Other known methods are particle tracking analysis (PTA) or its variant nanoparticle tracking analysis (PTA).

[0069] For example, the hydrodynamic diameter of the particle of the invention may be comprised between 100 and 1000 nm, between 100 and 900 nm, between 100 and 800 nm, between 100 and 700 nm, between 100 and 600 nm, between 100 and 500 nm, between 100 and 400 nm, between 100 and 300 nm, between 100 and 200 nm, between 150 and 1000 nm, between 150 and 900 nm, between 150 and 800 nm, between 150 and 700 nm, between 150 and 600 nm, between 150 and 500 nm, between 150 and 400 nm, between 150 and 300 nm, between 150 and 200 nm between 200 and 1000 nm, between 200 and 900 nm, between 200 and 800 nm, between 200 and 700 nm, between 250 and 1000 nm, between 250 and 900 nm, between 250 and 800 nm, between 250 and 700 nm, between 300 and 1000 nm, between 300 and 900 nm, between 300 and 800 nm, between 300 and 700 nm, between 400 and 1000 nm, between 400 and 900 nm, between 400 and 800 nm, between 400 and 700 nm, between 500 and 1000 nm, between 500 and 900 nm, between 500 and 800 nm or between 500 and 700 nm.

[0070] For example, the hydrodynamic diameter of the particle of the invention may also between 100 and 200 nm, be comprised between 100 and 200 nm, between 100 and 150 nm, between 100 and 140 nm, between 100 and 130 nm, between 100 and 110 nm.

[0071] Therapeutic methods and uses:

[0072] The present invention provides methods and compositions (such as pharmaceutical compositions) for preventing or treating cancer diseases (solid cancer or a malignant haematological disease). The present invention also provides methods and compositions for inhibiting or preventing cancer disease.

[0073] In the context of the invention, the term "treatment or prevention" means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. In particular, the treatment of the disorder may consist in reducing the number of malignant cells. Most preferably, such treatment leads to the complete depletion of the malignant cells.

[0074] Preferably, the individual to be treated is a human or non-human mammal (such as a rodent (mouse, rat), a feline, a canine, or a primate) affected or likely to be affected with cancer. Preferably, the individual is a human.

[0075] According to a second aspect, the present invention relates to a composition of the invention for use in the prevention or the treatment of a patient affected with cancer disease.

[0076] The terms "cancer" and "tumor" refer to or describe the pathological condition in mammals that is typically characterized by unregulated cell growth. In particular, the cancer may be associated with a solid tumor or malignant haematological disease (from hematopoietic cell). Examples of cancers that are associated with solid tumor formation include breast cancer, uterine / cervical cancer, lymphoma oesophageal cancer, pancreatic cancer, colon cancer, colorectal cancer, kidney cancer, ovarian cancer, prostate cancer, head and neck cancer, nonsmall cell lung cancer stomach cancer, tumor of mesenchymal origin (i.e; fibrosarcoma and rhabdomyoscarcoma), tumor of the central and peripheral nervous system (i.e; including astrocytoma, neuroblastoma, glioma, glioblatoma), thyroid cancer.

[0077] Preferably the solid tumor is selected from the group consisting of lymphoma, breast cancer, lung cancer, colorectal cancer. The term "malignant haematological disease" refers to or describe the pathological condition in mammals that is typically characterized by unregulated haematological cell growth. More precisely, malignant haematological disease according to the invention is due to an unregulated growth of undifferentiated hematopoietic bone marrow cells (hematopoietic stem cell).

[0078] As intended herein the expression “hematopoietic stem cell (HSC)” refers to adult multipotent stem cells that give rise to all the blood cell types including for example myeloid lineages (monocytes and macrophages, neutrophils, basophils, eosinophils), erythrocytes, megakaryocytes / platelets, and lymphoid lineages (T-cells, B-cells, NK-cells).

[0079] The expression “hematopoietic stem cell malignancy” or “hematopoietic malignancy” according to the invention comprises acute myeloid leukemia (AML), acute lymphoblastic leukemia, chronic myeloid, lymphoid leukemia, lymphoma and myelodysplastic syndrome (as defined in 2001 WHO classification).

[0080] Preferably, the hematopoietic malignancy according to the invention is selected from the group consisting of B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), or acute myeloid leukemia (AML).

[0081] In particular embodiment malignant haematological disease is acute myeloid leukemia (AML).

[0082] The present invention further contemplates a method of preventing or treating malignant haematological disease in a subject comprising administering to the subject a therapeutically effective amount of a composition of the invention.

[0083] The present invention further also provides a method of preventing or treating drug resistant cancer or tumor relapse in a subject suffering from cancer disease comprising administering to the subject a therapeutically effective amount of a composition of the invention.

[0084] "Drug resistance" as used in expressions such as “drug resistant cancer” or “drug resistant cells” or “drug resistant disease” means a circumstance where a disease (e g., cancer disease) does not respond to a therapeutic agent. Drug resistance can be intrinsic, which means that the disease has never been responsive to the therapeutic agent, or acquired, which means that the disease cases responding to the agent or agents to which the disease had previously been responsive. For cancers, such therapeutic agent may be a chemotherapeutic drug such as colchicine, vinblastine, doxorubicin, vinca alkaloids, etoposide, taxanes, or other small molecules used in cancer chemotherapy (Cytarabine, Mitoxantrone, Decitabine, Azacitidine and Daunorubicin in AML therapy) Drug resistance may be associated with cancer and other conditions, such as bacterial, viral, protozoal, and fungal diseases.

[0085] By “tumor relapse” or “cancer recurrence” is meant the return of cancer after treatment and after a period of time during which the cancer cannot be detected: in another term it means reappearance of cancer after a disease-free period.

[0086] In one aspect, the present invention provides a method of inhibiting tumor growth in a subject comprising administering a therapeutically effective amount of a composition of the invention.

[0087] By a "therapeutically effective amount" of a composition of the invention as above described is meant a sufficient amount of the antagonist to prevent or treat a cancer disease (solid cancer and malignant haematological disease). It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0.001 mg / kg to 7 mg / kg of body weight per day. Pharmaceutical compositions of the invention:

[0088] The composition of the invention as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

[0089] Accordingly, the present invention relates to a pharmaceutical composition comprising a composition of the invention according to the invention and a pharmaceutically acceptable carrier.

[0090] The present invention also relates to a pharmaceutical composition for use in the prevention or treatment of cancer disease comprising a composition of the invention according to the invention and a pharmaceutically acceptable carrier.

[0091] The present invention further relates to a pharmaceutical composition for preventing or treatment of drug resistant cancer or tumor relapse in a subject suffering from cancer disease comprising a composition according to the invention and a pharmaceutically acceptable carrier.

[0092] "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0093] In therapeutic applications, compositions are administered to a patient already suffering from a disease, as described, in an amount sufficient to cure or at least partially stop the symptoms of the disease and its complications. An appropriate dosage of the pharmaceutical composition is readily determined according to any one of several well-established protocols. For example, animal studies (for example on mice or rats) are commonly used to determine the maximal tolerable dose of the bioactive agent per kilogram of weight. In general, at least one of the animal species tested is mammalian. The results from the animal studies can be extrapolated to determine doses for use in other species, such as humans for example. What constitutes an effective dose also depends on the nature and severity of the disease or condition, and on the general state of the patient's health.

[0094] In therapeutic treatments, the antagonist contained in the pharmaceutical composition can be administered in several dosages or as a single dose until a desired response has been achieved. The treatment is typically monitored and repeated dosages can be administered as necessary. The daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0.001 mg / kg to 10 mg / kg of body weight per day. It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability, and length of action of that compound, the age, the body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.

[0095] In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

[0096] The appropriate unit forms of administration include forms for oral administration, such as tablets, gelatine capsules, powders, granules and solutions or suspensions to be taken orally, forms for sublingual and buccal administration, aerosols, implants, forms for subcutaneous, intramuscular, intravenous, intranasal or intraocular administration and forms for rectal administration.

[0097] In the pharmaceutical compositions of the present invention, the active principle is generally formulated as dosage units containing from 0.5 to 1000 mg, preferably from 1 to 500 mg, more preferably from 2 to 200 mg of said active principle per dosage unit for daily administrations.

[0098] When preparing a solid composition in the form of tablets, a wetting agent such as sodium laurylsulfate can be added to the active principle optionally micronized, which is then mixed with a pharmaceutical vehicle such as silica, gelatine, starch, lactose, magnesium stearate, talc, gum arabic or the like. The tablets can be coated with sucrose, with various polymers or other appropriate substances or else they can be treated so as to have a prolonged or delayed activity and so as to release a predetermined amount of active principle continuously.

[0099] A preparation in the form of gelatin capsules is obtained by mixing the active principle with a diluent such as a glycol or a glycerol ester and pouring the mixture obtained into soft or hard gelatine capsules.

[0100] A preparation in the form of a syrup or elixir can contain the active principle together with a sweetener, which is preferably calorie-free, methyl-paraben and propylparaben as an antiseptic, a flavoring and an appropriate color.

[0101] The water-dispersible powders or granules can contain the active principle mixed with dispersants or wetting agents, or suspending agents such as polyvinyl-pyrrolidone, and also with sweeteners or taste correctors.

[0102] Rectal administration is effected using suppositories prepared with binders which melt at the rectal temperature, for example cacao butter or polyethylene glycols.

[0103] Parenteral, intranasal or intraocular administration is effected using aqueous suspensions, isotonic saline solutions or sterile and injectable solutions which contain pharmacologically compatible dispersants and / or wetting agents, for example propylene glycol, butylene glycol, or polyethylene glycol.

[0104] Thus a cosolvent, for example an alcohol such as ethanol or a glycol such as polyethylene glycol or propylene glycol, and a hydrophilic surfactant such as Tween. RTM. 80, can be used to prepare an aqueous solution injectable by intravenous route. The active principle can be solubilized by a triglyceride or a glycerol ester to prepare an oily solution injectable by intramuscular route.

[0105] Transdermal administration is effected using multilaminated patches or reservoirs into which the active principle is in the form of an alcoholic solution.

[0106] Administration by inhalation is effected using an aerosol containing for example sorbitan trioleate or oleic acid together with trichlorofluoromethane, di chlorotetrafluoroethane or any other biologically compatible propellant gas.

[0107] The active principle can also be formulated as microcapsules or microspheres, optionally with one or more carriers or additives.

[0108] Among the prolonged-release forms which are useful in the case of chronic treatments, implants can be used. These can be prepared in the form of an oily suspension or in the form of a suspension of microspheres in an isotonic medium. The active principle can also be presented in the form of a complex with a cyclodextrin, for example .alpha.-, .beta - or .gamma. -cyclodextrin, 2-hydroxypropyl-.beta. -cyclodextrin or methyl - . b eta. -cy cl odextrin .

[0109] Combination therapies of the invention:

[0110] In other embodiments, the Composition of the invention may be administered to a subject with an appropriate additional therapeutic agent useful in prevention or treatment of the condition from which the patient suffers or is susceptible to; examples of such agents include a chemotherapeutic agent, an immunomodulatory agent, a hormonal agent, an immunotherapeutic agent (like antibody anti CD33, ....), an immune checkpoint inhibitor, etc.

[0111] The administration of the composition of the invention and the other therapeutic agent, (e.g., a chemotherapeutic agent) can be carried out simultaneously, e.g., as a single composition or as two or more distinct compositions using the same or different administration routes. Alternatively, or additionally, the administration can be done sequentially, in any order. Alternatively, or additionally, the steps can be performed as a combination of both sequentially and simultaneously, in any order. In certain embodiments, intervals ranging from minutes to days, to weeks to months, can be present between the administrations of the two or more compositions. For example, the additional therapeutic agent may be administered first, followed by the composition of the invention. However, simultaneous administration or administration of the Composition of the invention first is also contemplated.

[0112] Accordingly, in one aspect, the present invention relates to a pharmaceutical composition comprising a composition according to the invention and an additional therapeutic agent.

[0113] In another aspect, the present invention relates to a kit-of-part composition comprising a composition of the invention according to the invention and an additional therapeutic agent.

[0114] Also provided, is a pharmaceutical composition for use in the treatment or prevention of drug resistant cancer or tumor relapse in a subject suffering from cancer disease comprising a Composition of the invention according to the invention and an additional therapeutic agent.

[0115] Combination composition

[0116] Combination of two lipid nanoparticles

[0117] It has been shown that when two or more different treatments are combined, the treatments may work synergistically and allow reduction of dosage of each of the treatments, - l' l - thereby reducing the detrimental side effects exerted by each compound at higher dosages. In other instances, malignancies that are refractory to a treatment may respond to a combination therapy of two or more different treatments. Combination means also co administration of a treatment which target an immune population of cells together with a treatment which target cancer cells.

[0118] Accordingly, a third object of the invention relates to a composition comprising:

[0119] 1) a lipid nanoparticle loaded with lipidic antitumoral agent and said lipid nanoparticle being associated with an antigen binding agent targeting immunosuppressive cell and

[0120] 2) a lipid nanoparticle loaded with lipidic antitumoral agent and said lipid nanoparticle being associated with an antigen binding agent targeting tumor cells

[0121] The present invention also provides a method for treating a cancer disease in subject in need thereof comprising the combination of a lipid nanoparticle loaded with lipidic antitumoral agent and said lipid nanoparticle being associated with a lipid nanoparticle loaded with lipidic antitumoral agent and said lipid nanoparticle being associated with an antigen binding agent targeting tumor cells.

[0122] In particular, the antigen binding agent targeting tumor cells comprise free amine or thiol groups is chosen from a protein, a peptide, a single chain antibody, a monoclonal antibody. Preferably, the antigen binding agent comprising free amine or thiol groups is a monoclonal antibody.

[0123] In an embodiment, the conjugate comprises a lipid nanoparticle of the invention or a lipide nanoparticle of the invention and a molecule comprising at least a free amine or a thiol group. In particular, the molecule comprising at least a free amine or a thiol group a protein may be a peptide, a single chain antibody, a polyclonal antibody, an antibody fragment (Fab, F(ab')2 fragment ...) or a monoclonal antibody. More particularly, the molecule comprising at least a free amine or a thiol group a protein may be a nanobody, an antibody fragment (Fab, F(ab')2 fragment ...) or a monoclonal antibody. Even more particularly, the molecule comprising at least a free amine or a thiol group a protein may be an antibody fragment (Fab, F(ab')2 fragment . ..) or a monoclonal antibody. Preferably, the molecule comprising at least a free amine or a thiol group a protein may be a monoclonal antibody.

[0124] In an embodiment, the molecule comprising at least a free amine or a thiol group may be a molecule modified with a linker comprising at least a free amine or a thiol group. In other words, the molecule comprising at least a free amine or a thiol group is a molecule wherein the free amine or thiol group is held by a linker moiety.

[0125] In the context of the invention, the term “linker” or “linker moiety” refers to a connector for linking a molecule to a nanoparticle of the invention or a nanoparticle obtained by the process of the invention (described in Experimental section).

[0126] In the context of the invention, the term “conjugate” refers to a molecule composed of two or more molecules which are linked together. The conjugates of the invention are typically composed of a lipid nanoparticle according to the invention linked to a protein, a peptide, a single chain antibody, a polyclonal antibody an antibody fragment (Fab, F(ab')2 fragment . . .) or a monoclonal antibody.

[0127] The antigen binding agent targeting tumor is well known known by the men in the art.

[0128] Particularly, in some embodiments, the tumor-antigen targeting antibody is specific for a human tumor-antigen.

[0129] In some embodiments, the tumor-antigen targeting antibody is specific for a tumorantigen selected from the group consisting of human epithelial cell adhesion molecule (hEpCAM), Isocitrate dehydrogenase [NADP] cytoplasmic (IDH1), Aldehyde Dehydrogenase 1 Family Member Al (ALDH1), CD274, CD45, cyclin DI (BCL1), Dickkopf-Related Protein 1 (DKK-1), Enhancer Of Zeste Homolog 2 (EZH2), Heat Shock Protein Family H (Hspl lO) Member 1(HSPH1), Kallikrein Related Peptidase 4 (KLK4), Kinesin Family Member 20A (KIF20A), Papillomavirus Regulatory Factor 1 (PBF), Vascular Endothelial Growth Factor A (VEGF), VEGF2 receptor (VEGFR2), B Cell Maturation Antigen (BCMA), ICOS, CD19, CD20, CD24, CD27 CD28, CD33, CD37, CD38, CD157, CD40, CD44, CD47, CD86, CD122, CD123, CD137, CD160, Human 5 '-nucleotidase (NT5E), Recombinant Human Lysosome- associated membrane glycoprotein 1 (LAMP1), Tumor necrosis factor receptor superfamily member 4 (TNFRSF4), Tumor necrosis factor receptor superfamily member 18 (TNFRSF18) CTL-recognized antigen on melanoma (CAMEL), Differentiation antigen melanoma 6 (DAM- 6), Differentiation antigen melanoma 10 (DAM- 10), G antigen 1 (GAGE-1), G antigen 2 (GAGE-2), G antigen 3 (GAGE-3), G antigen 4 (GAGE-4), G antigen 5 (GAGE-5), G antigen 6 (GAGE-6), G antigen 7 (GAGE-7), G antigen 8 (GAGE-8), Interleukin 13 receptor alpha2 chain (IL-13Ra2), Melanoma antigen Al (MAGE-A1), Melanoma antigen A2 (MAGE-A2), Melanoma antigen A3 (MAGE- A3), Melanoma antigen A4 (MAGE-A4), Melanoma antigen A6 (MAGE-A6), Melanoma antigen A9 (MAGE-A9), Melanoma antigen A10 (MAGE-A10), Melanoma antigen A12 (MAGE-A12), Melanoma antigen Cl (MAGE-CI), Melanoma antigen C2 (MAGE-C2), NA cDNA clone of patient M88 (NA88-A), New York esophageous 1 (NY- ESO-1), Synovial sarcoma, X breakpoint 2 (SSX-2), Synovial sarcoma, X breakpoint 4 (SSX4), Taxol resistant associated protein 3 (TRAG-3), carcinoembryonic antigen (CEA), Epithelial cell adhesion molecule (Ep-CAM), Basal cell adhesion molecule (BCAM), orphan G protein- coupled receptor, class C group 5 member D (GPRC5D), Fc Receptor-Like 5 (FCRL5), DLL3 (Delta Like Canonical Notch Ligand 3) Melanoma-Associated ME20 Antigen (GP100), mammaglobin-A, Melanoma antigen recognized by T cells- 1 / melanoma antigen A (Melan- A / MART-1), Melanocortin 1 receptor (MC1R), Ocular albinism type 1 protein (OA1), Prostate-specific antigen (PSA), Tyrosinase-related protein 1 (TRP-1), Tyrosinase-related protein 2 (TRP-2), tyrosinase, adipophilin, alpha-fetoprotein (AFP), interferon- inducible protein absent in melanoma 2 (AIM-2), acute lymphoblastic leukemia (ALL), 707 alanine proline (707-AP), acute promyelocytic leukemia (APL), adenocarcinoma antigen recognized by T cells 4 (ART-4), B antigen (BAGE), Ephrin type-A receptor 2 (EphA2), Ephrin type-A receptor 3 (EphA3), Fibroblast growth factor 5 (FGF5), Glycoprotein 250 (G250), AlphaMannoside Beta-1,6-N-Acetylglucosaminyltransferase V (GnTV), hER2, human signet-ring tumor 2(HST-2), human telomerase reverse transcriptase (hTERT), M-CSF, mucin-1 (MUC1), mucin-2 (MUC2), mucin- 16 (MUC16), mucin- 17 (MUC17), Preferentially expressed antigen of melanoma (PRAME), Prostate-specific membrane antigen (PSMA), protein 15 (p 15), protein 53 (p53), renal antigen (RAGE), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), squamous antigen rejecting tumor 1 (SART-1), squamous antigen rejecting tumor 2 (SART-2), squamous antigen rejecting tumor 3 (SART-3), SRY-Box Transcription Factor 10 (SOX10), Wilms Tumor 1 (WT1), 707 alanine proline (707-AP), a-actinin-4, P-catenin, Casein Kinase 1 Alpha 1 (CSNK1A1), Cyclin Dependent Kinase Inhibitor 2A (CDKN2A), Caseinolytic Mitochondrial Matrix Peptidase Proteolytic Subunit (CLPP), Colorectal Turn or- Associated Antigen- 1 (COA- 1), Elongation factor 2 (ELF2), Melanoma Ag recognized by T cells-2 (MART2), Melanoma ubiquitous mutated 1 (MUM1), Melanoma ubiquitous mutated 2 (MUM2), Melanoma ubiquitous mutated 3 (MUM3), myosin, OS9 Endoplasmic Reticulum Lectin (OS-9), Transforming Protein P21 (K-ras), Neuroblastoma RAS Viral Oncogene Homolog (N-ras), O- Linked N-acetylglucosamine transferase gene (OGT), TGFaRII, L antigen (LAGE-1), annexin II, cell division cycle 27 (CDC27), Neo-Poly(A) Polymerase (neo-PAP), Receptor-type proteintyrosine phosphatase kappa (PTPRK), TGF RII, Adaptor Related Protein Complex 2 Subunit Sigma 1 (AP2S1), BBXHigh Mobility Group Box Domain Containing (ARTCI), B-Raf ProtoOncogene, Serine / Threonine Kinase (B-RAF), caspase-5 (CASP-5), caspase-8 (CASP-8), elongation factor 2, FLT3, fibronectin 1 (FN1), Fibronectin Type III Domain Containing 3B (FNDC3B), Growth Arrest Specific 7 (GAS7), Glycoprotein Nmb (GPNMB), HAUS Augmin Like Complex Subunit 3 (HAUS3), HLA-A11, HLA-A2, Hydroxy steroid Dehydrogenase Like 1 (HSDL1), Heat shock protein 70-2 (HSP70-2), Matrilin-1 (MATN), Malic Enzyme 1 (MEI), Protein Phosphatase 1 Regulatory Subunit 3B (PPP1R3B), Peroxiredoxin 5 (PRDX5), Ubiquitin Protein Ligase E3 Component N-Recognin 4 (RBAF600), Sirtuin 2 (SIRT2), triosephosphate isomerase, Cancer / Testis Antigen 37 (CT37 / FMR1NB), cylcin-Al, Cancer / Testis Antigen 83 (KK-LC-1), Cancer / Testis Antigen KM-HN-1 (KM-HN-1), LDL Receptor Related Protein Associated Protein 1 (LRPAP1), lymphocyte Antigen 6 Family Member K (LY6K), sarcoma antigen (SAGE), Ankyrin Repeat Domain 30A (NY-BR-1), prostatic Acid Phosphatase (PAP), prostate stem-cell antigen (PSCA), A-kinase anchor protein 4 (AKAP-4) Bcl-2-Like Protein (BCLX), WD Repeat Domain 46 (BING-4), calcitonin (CALCA), Programmed Cell Death 1 Ligand 1 (PDL1), Leukocyte Common Antigen (LCA), Cleavage And Polyadenylation Specific Factor 1 (CPSF), Dickkopf-Related Protein 1, cyclin DI, cyclin-dependent kinase 4 (CDK4), nectin-2, nectin-4, Enhancer Of Zeste Homolog 2 (EZH2), P53 -Binding Protein Mdm2 (MDM2), Matrix Metallopeptidase 2 (MMP2), Matrix Metallopeptidase 7 (MMP-7), glypican-3, hepsin, Hepatocyte Cell Adhesion Molecule (HEPACAM), Heat Shock Protein Family H Member 1 (HSPH1), Indoleamine 2,3- Dioxygenase 1 (IDO1), HLA-G, Mitochondrial Ribosomal Protein S4 (IMP3), carboxylesterase 2 (CES2), kallikrein 4, Mitotic Kinesin-Like Protein 2 (KIF20A), lengsin, midkine, Paired Box Protein Pax-5 (PAX5), Cancer / Testis Antigen 92 (PLAC1), Regulator Of G-Protein Signaling 5 (RGS5), Ras Homolog Family Member C (RHOC), Ring Finger Protein 43 (RNF43), Doublecortin Domain Containing 2 (RU2), secernin 1, survivin, telomerase, Six Transmembrane Epithelial Antigen Of The Prostate 1 (STEAP1), and Trophoblast Glycoprotein (TPBG).

[0130] As used herein the term “VEGFR2” refers to a tyrosine-protein kinase that acts as a cellsurface receptor for VEGFA, VEGFC and VEGFD. VEGFR2 activity on myeloid cells mediates immune suppression in the tumor microenvironment. VEGFR2 playing a critical role in tumor angiogenesis. VEGF-A can modulate immune cells (DC, MDSC, TAM) to induce the accumulation of regulatory T-cells. VEGRF2 is expressed by myeloid cells (DC, MDSC, TAM).

[0131] In some embodiments, the tumor-antigen targeting antibody is selected from the list consisting of CD7, CD19 and CD33 antibody.

[0132] Example of antigen binding agent of CD 19 that could be used according to the invention is anti CD19 monoclonal antibody described in literature (Hekman, Aet al. Cancer Immunol Immunother 32, 364-372 (1991); Naddafi F et al Int J Mol Cell Med. 2015 ; 4(3): 143-151; Breton CS et al Journal of Hematology & Oncology (2014) Volume 7, article number 33) or in patent WO2021178253, US2023287114; US2023272070, US2020062843.

[0133] For example, the antigen binding agent of CD19 of the invention is the anti CD19 monoclonal antibody available from Termofisher (CD 19 Monoclonal Antibody (HIB 19), Ebioscience) or from Sigma Aldrich (Anti-Human B cells (CD 19) Antibody, clone FMC63), or from Bio Techne (Human CD19 Antibody Clone MAB4867)

[0134] Example of antigen binding agent of CD7 that could be used according to the invention is anti CD7 monoclonal antibody described in literature (Arthur E. et al (1997) Leukemia & Lymphoma, 26:3-4, 287-298; Rabinowich H; et al. J Immunol (1994) 152 (2): 517-526.; Arnlot, P , Cammisuli, S. (1990). CD7 Monoclonal Antibodies. In: Borrebaeck, C.A.K., Larrick, J.W. (eds) Therapeutic Monoclonal Antibodies. Palgrave Macmillan, London) or in patent US6689362, WO9948534; DE4345200, CN116970082.

[0135] For example, the antigen binding agent of CD7 of the invention is the anti CD7 monoclonal antibody available from Termofisher (CD7 Monoclonal Antibody (eBio 124- ID 1 (124-1D1)), APC, eBioscience) or from Miltenyi (CD7 Antibody, anti-human, Bright FITC (Clone no. 130-105-844), or from Biorad (anti Human CD7 Antibody Clone LT7)

[0136] In some embodiments, the CD33 -targeting antibody is monoclonal antibody ZZ, as described in.

[0137] Example of antigen binding agent of CD33 that could be used according to the invention is anti CD33 monoclonal antibody (or ScFv) described in literature (Feldman E, et a. Leukemia volume 17, pages314-318 (2003); Caron C N et al. Cancer Res (1992) 52 (24): 6761-6767;) or in patent JP2023134445, WO2021119400; W02019006280, WO2017214333.

[0138] For example, the antigen binding agent of CD33 of the invention is the anti CD33 monoclonal antibody available from Termofisher (CD33 Monoclonal Antibody (WM-53 (WM53)), PE-Cyanine7, eBioscience) or from Boster Bio (Anti-Human CD33 Monoclonal Antibody PE-Cy7 / Catalog# FC01508-PE-Cy7), or from BioTechne (Human Siglec-3 / CD33 Antibody / Catalog #: MAB11373).

[0139] The fragment antigen-binding region (Fab region) of all these cited antibodies might also be incorporated on lipid nanoparticle.

[0140] More particularly, the monoclonal antibody may be chosen from CD 19 or CD33 antibodies which selectively target tumor antigen. Therefore, the present invention provides a combination of

[0141] 1) a lipid nanoparticle loaded with lipidic antitumoral agent and said lipid nanoparticle being associated with an antigen binding agent targeting immunosuppressive cell and

[0142] 2) a lipid nanoparticle loaded with lipidic antitumoral agent and said lipid nanoparticle being associated with an antigen binding agent targeting tumor cells for simultaneous or sequential use in the treatment of a cancer disease.

[0143] In the present invention, the cancer disease is selected from the group consisting a solid cancer or malignant haematological disease. In particular, the solid cancer is lymphoma breast cancer, lung cancer, colorectal cancer. In particular, the malignant haematological disease is selected from the group consisting of B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML) or lymphoma.

[0144] The invention also provides a composition comprising the combination for use in the treatment of drug resistant tumor or tumor relapse in a patient affected with a cancer disease.

[0145] Combination of the lipid nanoparticle of the invention with an immune checkpoint inhibitor

[0146] Another object of the invention relates to a composition comprising a combination of

[0147] 1) a lipid nanoparticle loaded with lipidic antitumoral agent and said lipid nanoparticle being associated with an antigen binding agent targeting immunosuppressive cell and

[0148] 2) an immune checkpoint inhibitor.

[0149] The present invention also provides a method for treating a cancer disease in subject in need thereof comprising the combination of a lipid nanoparticle loaded with lipidic antitumoral agent and said lipid nanoparticle being associated with an antigen binding agent targeting immunosuppressive cell with an immune checkpoint inhibitor.

[0150] As used herein, the expression “immune checkpoint inhibitor" or "checkpoint blockade cancer immunotherapy agent” (both expressions will be used interchangeably) has its general meaning in the art and refers to any compound inhibiting the function of an immune inhibitory checkpoint protein. Inhibition includes reduction of function and full blockade. Preferred immune checkpoint inhibitors are antibodies that specifically recognize immune checkpoint proteins. A number of immune checkpoint inhibitors are known and in analogy of these known immune checkpoint protein inhibitors, alternative immune checkpoint inhibitors may be developed in the (near) future. The immune checkpoint inhibitors include peptides, antibodies, nucleic acid molecules and small molecules. In particular, the immune checkpoint inhibitor of the present invention is administered for enhancing the proliferation, migration, persistence and / or cytotoxic activity of CD8+ T cells in the subject and in particular the tumor-infiltrating of CD8+ T cells of the subject. As used herein “CD8+ T cells” has its general meaning in the art and refers to a subset of T cells that express CD8 on their surface. They are MHC class I- restricted, and function as cytotoxic T cells. “CD8+ T cells” are also called CD8+ T cells are called cytotoxic T lymphocytes (CTL), T-killer cell, cytolytic T cells, CD8+ T cells or killer T cells. CD8 antigens are members of the immunoglobulin supergene family and are associative recognition elements in major histocompatibility complex class I-restricted interactions. The ability of the immune checkpoint inhibitor to enhance T CD8 cell killing activity may be determined by any assay well known in the art. Typically said assay is an in vitro assay wherein CD8+ T cells are brought into contact with target cells (e.g. target cells that are recognized and / or lysed by CD8+ T cells). For example, the immune checkpoint inhibitor of the present invention can be selected for the ability to increase specific lysis by CD8+ T cells by more than about 20%, preferably with at least about 30%, at least about 40%, at least about 50%, or more of the specific lysis obtained at the same effector: target cell ratio with CD8+ T cells or CD8 T cell lines that are contacted by the immune checkpoint inhibitor of the present invention, Examples of protocols for classical cytotoxicity assays are conventional.

[0151] Typically, the checkpoint blockade cancer immunotherapy agent is an agent which blocks an immunosuppressive receptor expressed by activated T lymphocytes, such as cytotoxic T lymphocyte-associated protein 4 (CTLA4) and programmed cell death 1 (PDCD1, best known as PD-1), or by NK cells, like various members of the killer cell immunoglobulin- like receptor (KIR) family, or an agent which blocks the principal ligands of these receptors, such as PD-1 ligand CD274 (best known as PD-L1 or B7-H1).

[0152] Typically, the checkpoint blockade cancer immunotherapy agent is an antibody.

[0153] In some embodiments, the checkpoint blockade cancer immunotherapy agent is an antibody selected from the group consisting of anti-CTLA4 antibodies, anti-PDl antibodies, anti-PDLl antibodies, anti-PDL2 antibodies, anti-TIM-3 antibodies, anti-LAG3 antibodies, anti-IDOl antibodies, anti-TIGIT antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti- BTLA antibodies, and anti-B7H6 antibodies.

[0154] Examples of anti-CTLA-4 antibodies are described in US Patent Nos: 5,811,097; 5,811,097; 5,855,887; 6,051,227; 6,207,157; 6,682,736; 6,984,720; and 7,605,238. One anti- CDLA-4 antibody is tremelimumab, (ticilimumab, CP-675,206). In some embodiments, the anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-D010) a fully human monoclonal IgG antibody that binds to CTLA-4.

[0155] Examples of PD-1 and PD-L1 antibodies are described in US Patent Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149, and PCT Published Patent Application Nos: W003042402, WO2008156712, W02010089411, W02010036959, WO2011066342, WO2011159877, WO2011082400, and WO2011161699. In some embodiments, the PD-1 blockers include anti-PD-Ll antibodies. In certain other embodiments the PD-1 blockers include anti-PD-1 antibodies and similar binding proteins such as nivolumab (MDX 1106, BMS 936558, ONO 4538), a fully human IgG4 antibody that binds to and blocks the activation of PD-1 by its ligands PD-L1 and PD-L2; lambrolizumab (MK-3475 or SCH 900475), a humanized monoclonal IgG4 antibody against PD-1 ; CT-011 a humanized antibody that binds PD-1 ; AMP-224 is a fusion protein of B7-DC; an antibody Fc portion; BMS-936559 (MDX- 1105-01) for PD-L1 (B7-H1) blockade.

[0156] Other immune-checkpoint inhibitors include lymphocyte activation gene-3 (LAG-3) inhibitors, such as IMP321, a soluble Ig fusion protein (Brignone et al., 2007, J. Immunol. 179:4202-4211).

[0157] Other immune-checkpoint inhibitors include B7 inhibitors, such as B7-H3 and B7-H4 inhibitors. In particular, the anti-B7-H3 antibody MGA271 (Loo et al., 2012, Clin. Cancer Res. July 15 (18) 3834).

[0158] Also included are TIM3 (T-cell immunoglobulin domain and mucin domain 3) inhibitors (Fourcade et al., 2010, J. Exp. Med. 207:2175-86 and Sakuishi et al., 2010, J. Exp. Med. 207:2187-94). As used herein, the term “TIM-3” has its general meaning in the art and refers to T cell immunoglobulin and mucin domain-containing molecule 3. The natural ligand of TIM-3 is galectin 9 (Gal9). Accordingly, the term “TIM-3 inhibitor” as used herein refers to a compound, substance or composition that can inhibit the function of TIM-3. For example, the inhibitor can inhibit the expression or activity of TIM-3, modulate or block the TIM-3 signaling pathway and / or block the binding of TIM-3 to galectin-9. Antibodies having specificity for TIM-3 are well known in the art and typically those described in WO2011155607, W02013006490 and WO2010117057. In some embodiments, the immune checkpoint inhibitor is an Indoleamine 2, 3 -dioxygenase (IDO) inhibitor, preferably an IDO1 inhibitor. Examples of IDO inhibitors are described in WO 2014150677. Examples of IDO inhibitors include without limitation 1 -methyl -tryptophan (IMT), P- (3-benzofuranyl)-alanine, P-(3-benzo(b)thienyl)- alanine), 6-nitro-tryptophan, 6- fluoro-tryptophan, 4-methyl-tryptophan, 5 -methyl tryptophan, 6-methyl-tryptophan, 5 -methoxy -tryptophan, 5 -hydroxy-tryptophan, indole 3-carbinol, 3,3'- diindolylmethane, epigallocatechin gallate, 5-Br-4-Cl-indoxyl 1,3-diacetate, 9- vinylcarbazole, acemetacin, 5 -bromo-tryptophan, 5 -bromoindoxyl diacetate, 3- Amino-naphtoic acid, pyrrolidine dithiocarbamate, 4-phenylimidazole a brassinin derivative, a thiohydantoin derivative, a P-carboline derivative or a brassilexin derivative. Preferably the IDO inhibitor is selected from 1-methyl-tryptophan, -(3- benzofuranyl)-alanine, 6-nitro-L-tryptophan, 3- Amino-naphtoic acid and -[3- benzo(b)thienyl] -alanine or a derivative or prodrug thereof

[0159] In some embodiments, the immune checkpoint inhibitor is an anti-TIGIT (T cell immunoglobin and ITIM domain) antibody.

[0160] In a preferred embodiment, the checkpoint blockade cancer immunotherapy agent is a CTLA4 blocking antibody, such as Ipilimumab, or a PD-1 blocking antibody, such as Nivolumab or Pembrolizumab, or a combination thereof.

[0161] Therefore, the present invention provides a combination of :

[0162] 1) a lipid nanoparticle loaded with lipidic antitumoral agent and said lipid nanoparticle being associated with an antigen binding agent targeting immunosuppressive cell and

[0163] 2) an immune checkpoint inhibitor for simultaneous or sequential use in the treatment of a cancer disease.

[0164] In the present invention, the cancer disease is selected from the group consisting a solid cancer or malignant haematological disease. In particular, the solid cancer is lymphoma breast cancer, lung cancer, colorectal cancer. In particular, the malignant haematological disease is selected from the group consisting of B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML) or lymphoma.

[0165] The invention also provides a composition comprising the combination for use in the treatment of drug resistant tumor or tumor relapse in a patient affected with cancer disease.

[0166] The present invention also provides a lipid nanoparticle loaded with lipidic antitumoral agent and said lipid nanoparticle being associated with an antigen binding agent targeting immunosuppressive cell, for use in a method for enhancing sensitivity of a patient suffering from cancer diseases to an immune checkpoint inhibitor. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0167] Method of preparation

[0168] Another object of the invention is a process for preparing a suspension of liposomes of the invention, comprising the steps of: a) Mixing a phospholipid typically phosphatidylcholine, and cholesterol and a lipid with a reactive moiety with an organic solvent; b) Adding a lipophilic antitumoral agent and removing organic solvent by evaporation; c) incubating the mixture at a temperature between 30 to 100°C preferably 60 and 70°C, and vortex the mixture d) sonicated the vesicles for several cycles to obtain unilamellar vesicles (UVs) e) adding antigen bidding agent directed against immunosuppressive cells just before the purification process via dialysis f) Recovering a suspension of liposome loaded with antitumoral agent and associated antigen bidding agent directed against immunosuppressive cells,

[0169] Said liposome having a diameter ranging from 100 nm to 1 pm

[0170] The process of the invention consists in preparing a suspension of lipid nanoparticles, preferably liposome, in particular in water, more particularly in distilled water.

[0171] The process of the invention may be performed according to any suitable method known by the person skilled in the art, including, for example, co-precipitation, solvothermal synthesis, thermal decomposition, polyol process, sonochemical reaction and sol-gel reaction.

[0172] The step a) of the process of the invention is perfomed by mixing phospholipids (typically phosphatidylcholine, and cholesterol preferably with a respective molar ratio of 100; 37; 1.5; 0.2,)and a lipid with a reactive moiety (as described above), with an organic solvent.

[0173] The step a) of the process of the invention may be performed using any organic solvent known by the person skilled in the art, including, for example, Acetone, Ethanol, Isopropanol, Methanol, Ether, Ethyl acetate, Acetonitrile, Chloroform, Hexane, Benzene, Toluene, Acetic acid, Butanol, Aniline, Carbon disulfide, Cyclohexane, Di chloromethane, Dioxane, Ethylene glycol, Ethylene glycol monobutyl acetate, Pentane, Acetonitrile, Benzene, 1 -butanol, 2- butanol, 2-butanone, t-butyl alcohol, carbon tetrachloride, chlorobenzene, cyclohexane, 1,2- dichloroethane, diethylene glycol, diethyl ether, diglyme (diethylene glycol dimethyl ether), 1,2-dimethoxy-ethane (glyme, DME), dimethyl -formamide (DMF), dimethyl sulfoxide (DMSO), 1,4-di oxane, ethyl acetate, ethylene glycol, glycerin, heptane, hexamethylphosphoramide (HMPA), hexamethylphosphorous triamide (HMPT), hexane, methyl t-butyl ether (MTBE), methylene chloride, N-methyl-2-pyrrolidinone (NMP), nitromethane, pentane, petroleum ether (ligroine), 1 -propanol, 2-propanol

[0174] The step b) and c) of the process of the invention consists in adding to the suspension the lipophilic antitumoral agent (as described above) and removing organic solvent by evaporation by incubating the mixture at a temperature between 30 to 100°C preferably 60 and 70°C, more preferably at 65°C and vortex the mixture

[0175] Typically, the organic sovent (ie chloroform) is evaporated with nitrogen, making a thin lipid film in a round-bottom flask by the removal of organic solvent. The lipid film is then reconstituted in 1 mL of filtrated PBS 1 x preheated to 65°C. Following 10 min of incubation at 65°C, the mixture is vortexed for 1 min, then after stabilization for 30 min at 65°C,

[0176] After mixing the suspension obtained from step c) with a lipophilic antitumoral agent, the step d) of the process of the invention include a step of homogenization of the resulting mixture of vesicle in order to obtain unilamellar vesicles (UVs), in particular by sonication,

[0177] Typically, the vesicles is sonicated for 5 cycles (30 sec on-off) at an amplitude of 20% to form UVs that were stabilized at 65°C for 30 min.

[0178] The step e) of the process of the invention consists in adding the antigen bidding agent directed against immunosuppressive cells just before the purification process via dialysis.

[0179] Typically, the reaction of association between the loaded liposome and the antigen bidding agent, is stopped by adding 1 mM of glycine. In order to purify the lipidic nanoparticle associated with the antigen bidding agent, two consecutive dialyses (one of two hours and another one overnight) is carried out in one liter of PBS 1 x, with 300 KDA dialysis membranes (11550970, Thermo Fisher Scientific).

[0180] The step f) of the process of the invention consists in the recovery of a suspension of the nanoparticles of the invention. These particles can then be stored at a low temperature of about 3 to 10°C, either in water, or saline solution.

[0181] Prior to its injection to a patient, the suspension of particles of the invention is kept under agitation at a temperature of 0 to 37 °C, in particular 4°C.

[0182] Another object of the invention is a suspension of nanoparticles or a particle obtained by the process according to the invention. FIGURES:

[0183] Figure 1: Val-ILs-aCD19 efficiently induce death of human CD19+cells. (A) Quantification and size of Val-ILs measured by NT A, n = 5 biological replicates. (B) HPLC- MS / MS for the quantification of Valrubicin on Val-ILs-IgG and Val-ILs-aCD19, n = 3 biological replicates. (C) Viability assessed on CD19-expressing B-ALL1 cells by XTT assay, following a single treatment, on day 0 with 1,000 or 2,000 particles / cell. The IC50 measured at 1,000 particles / cell. XTT assay performed after 72 hours of treatment, normalized to untreated, n = 5 biological replicates. (D) Viability assessed by flow cytometry on B-ALL1 cells, treated with Val-ILs-IgG or Val-ILs-aCD19 (2,000 particles / cell). Flow cytometry performed after 72 hours of treatment. Val-ILs-aCD19 efficiently affected the viability of B-ALL1 cells, but this was not the case for Val-ILs-IgG. Examples of cytometry dataset and statistics, n = 4 biological replicates. (E) Flow cytometry showing how Val-ILs-aCD19 eliminate the population of B- lymphocytes expressing CD 19 among human peripheral blood mononuclear cells (PBMC) after 48 hours of in vitro exposure with 2,000 particles / cell. Flow cytometry UMAP data frames are shown for two PBMC samples. B-lymphocytes (B-Ly), T4-lymphocytes (T4-Ly), T8- lymphocyte (T8-Ly) and the remaining hematopoietic cells (CD45+). (F) Val-ILs-aCD19 eliminate CD19+B-ALL cells isolated from human BM at diagnosis, after 48 hours of ex vivo exposure with 2,000 particles / cell. Flow cytometry dataset of one representative primary B- ALL sample. Statistics showing that Val-ILs-aCD19 efficiently eliminate >90% of the CD19+B-ALL cells, while Val-ILs-IgG had no effect. The percentage of viable cells is normalized to untreated controls, n = 13 B-ALL patients. (G) Val-ILs-aCD19 affect B-ALL cells but not the stromal mesenchymal supportive MS-5 cells. The percentage of MS-5 cells' confluence over time indicates that Val-ILs-aCD19 did not have an impact on the growth of the supportive MS- 5 cells. GFP intensity measured over time, showing that Val-ILs-otCD19 affected viability of B-ALL cells within 24 hours. Data are shown as means ± SD, n = 3 biological replicates. On this figure, data are shown as means ± SD, (A, B, C) P value measured by two-tailed unpaired Student’s t test; ****P < 0.0001; ns, non-significant. (D, F, G) P value measured by one-way Anova with Tukey’s multiple comparison test; **P < 0.01; ****P < 0.0001; ns, non-significant.

[0184] Figure 2: Treatment of PDX mice with Val-ILs-aCD19 affects the in vivo development of B-ALL. (A) Following the i.v. injection of trackable Val-ILs labeled with PKH67-Green in NSG mice, UVs were isolated from different organs, 18 hours later, to measure PKH67-Green fluorescence by flow cytometry. Data showing that Val-ILs reached BM and spleen following i.v. injections. (B) Val-ILs-aCD19 target B-ALL cells in BM and spleen of PDX mice. Val-ILs labeled with PKH67-Green were injected at 1011particles, 15 days following the transplantation of 105B-ALL cells; 18 hours later, fluorescent Val-ILs binding, on hCD45+B-ALL cells as well as endogenous murine cells, were measured by flow cytometry in BM and spleen of PDX #1 and #2. (C) Mice were treated with three injections of 1011Val-ILs on days 15, 20 and 25 post-transplantation, n = 4 mice per group for PDX #1 and n = 6 mice per group for PDX #2. Percentage of B-ALL cells in BM, spleen and blood measured by flow cytometry on day 35. Examples of cytometry dataset on BM and statistics for all organs. On the right is a picture of BM cell pellets on a 96-well plate, representative of PDX #1 and #2, showing that BM cells remained red colored following treatment with Val-ILs-aCD 19. (D) Data showing reduced splenomegaly in mice treated with Val-ILs-aCD 19. The spleen weight was assessed in relation to the body weight of the mice. (E) Mice were transplanted with bioluminescent B-ALL cells (PDX #1) and treated with 1011Val-ILs on days 15, 20 and 25 post-transplantation. Bioluminescence images were taken on days 26, 33 and 40 posttransplantation, n = 5 mice per group. Only average bioluminescence radiance over time is shown. (F) Kaplan-Meier survival plots showing that mice treated with Val-ILs-aCD 19 survived longer than untreated mice or mice treated with Val-ILs-IgG, n = 6 mice per group. On this figure, data are shown as means ± SD. (C, D, E) P value calculated against untreated condition and measured by one-way Anova with Tukey ’ s multiple comparison test; * *P < 0.01 ; ***P < 0.001; 0.0001; ns, non-significant, (F) P value calculated against untreated condition and measured by Log-Rank (Mantel-Cox) test; ***P < 0.001.

[0185] Figure 3: Val-ILs-aCD19 induce death of malignant B-ALL cells among CD34+HSC. (A) Description of the procedure to deliberately contaminate CD34+HSC isolated from human cord blood with ~1% of GFP+B-ALL cells (isolated from PDX #1 or #2). (B) After 18 hours of in vitro treatment with Val-ILs-IgG or Val-ILs-aCD 19 (2,000 particles / cell), cells were i.v. injected in NSG mice. Five weeks after the transplantation, GFP+B-ALL cells were evaluated in BM by flow cytometry, as well as the reconstitution mediated by CD34+HSC. Data showing how Val-ILs-aCD19 purged the malignant B-ALL cells among CD34+HSC in vitro, while no leukemia GFP+cells (green dots) were detected in the BM of transplanted mice. (C) Data showing that a treatment with Val-ILs-aCD 19 did not affect the human hematopoietic reconstitution mediated by CD34+HSC and characterized by hCD45+expression (red dots). Flow cytometry gating. . Figure 4: Development of Val-ILs loaded with «CD7 or aCD33 for T-ALL and AML therapies. (A) Flow cytometry plot showing the high expression of CD7 on Jurkat cells, and the low expression on RPMI-8402 cells. Treatment of these T-ALL cell lines with Val-ILs- aCD7 affects viability after three days, means of n = 4 biological replicates. (B) Flow cytometry plot showing CD33 expression on HL60 and THP1 cells. Treatment of these AML cell lines with Val-ILs-aCD33 affects viability after three days, means of n = 4 biological replicates. (C) T-ALL PDX mice were treated with three injections of 1011Val-ILs on days 20, 25 and 30 posttransplantation, data are shown as means ± SD, n = 5 mice per group. On day 45, the percentage of T-ALL cells (hCD45+hCD7+) measured by flow cytometry in spleen, BM and blood. Examples of cytometry dataset and statistics. (D) AML PDX mice were treated with three injections of 1011Val-ILs on days 15, 20 and 25 post-transplantation, data are shown as means ± SD, n = 6 mice per group. On day 35, the percentage of AML cells (hCD45+hCD33+) measured by flow cytometry in spleen, BM and blood. Examples of cytometry dataset and statistics. Splenomegaly was reduced for T-ALL PDX mice treated with Val-ILs-aCD7 (E), as well as for AML PDX mice treated with Val-ILs-aCD33 (F). Statistics of the spleen weight was assessed in relation to the body weight of the mice. On this figure, (A, B) P value measured by two-tailed unpaired Student’s t test; *7’< 0.05; ***P < 0.001; ****p < 0.0001. (C, D, E, F) P value calculated against the untreated condition and measured by one-way Anova with Tukey’s multiple comparison test; ** < 0.01; ***7’ < 0.001; **** < 0.0001; ns, nonsignificant.

[0186] Figure 5: Val-ILs target immunosuppressive cells for the immunotherapy of lymphoma. (A) C57BL / 6 mice were subcutaneously injected with 106EL4 cells, on the body side. When tumors reached 50 mm3, mice were treated with Val-ILs i.v. injected into the tail vein on days 6 and 9 (arrows), at a dose of 1011particles per injection. Different Val-ILs loaded with further antibodies were tested in order to target different populations of immune cells. Tumor growth volumes were assessed over time. Data showing that Val-ILs-aCDl lb and Val- ILs-a.CD223 efficiently attenuated tumor development in mice. Pictures of some tumors extracted from mice on day 12 are shown. (B) Tumor volumes measured on day 12, with all the tested Val-ILs, n = 3 mice per group. (C) Val-ILs-aCDl lb efficiently affect the tumor volume over time, means of n = 4 mice per group. (D) Val-ILs-aCD223 significantly affect the tumor volume over time, means of n = 5 mice per group. On this figure, data are shown as means ± SD, P values were calculated against untreated controls and measured by one-way Anova with Tukey’s multiple comparison test; *P< 0.05; **7?< 0.01; ***P < 0.001; 0.0001; ns, non-significant.

[0187] Figure 6: Val-ILs-aCDllb target MDSC which activates T8-Ly. (A) Six days following transplantation of EL4 cells, Val-ILs labeled with PKH67-Green were i.v. injected in mice at a dose of 1011particles. Eighteen hours later, the binding of Val-ILs on CD1 lb+cells and on MDSC immune cells was detected by flow cytometry in spleens but not in tumors. (B) Val-ILs-aCDl lb were i.v. injected in mice at the dose of 1011particles per injection, on days 6 and 9. Example of flow cytometry dataset on immune cells on day 12, showing the reduction in myeloid-derived suppressor cells (MDSC) in the spleen of a mouse treated with Val-ILs- aCDl lb. Flow cytometry UMAP data frame displaying a decrease in the number of MDSC (blue dots) in spleen. On the right is shown statistics of the immune system, in spleens and tumors, n = 4 mice per group. Dendritic cells (DC), T4 lymphocytes (T4-Ly) and T8 lymphocytes (T8-Ly). (C) Data showing the activation of T8-Ly measured by flow cytometry, in spleens and tumors, on day 12, following treatment with Val-ILs-aCDl lb. Statistics on the expression of markers involved in T8-Ly activation. Granzyme B (GzB), interleukin 2 (IL2), tumor necrosis factor alpha (TNFoc), and the proliferation marker Ki67, n = 4 mice per group. On this figure, data are shown as means ± SD, P values were calculated against untreated controls and measured by one-way Anova with Tukey’s multiple comparison test; *P < 0.05; ** < 0.01; ***P < 0.001; 0.0001. No statistic is shown when the P value is nonsignificant.

[0188] Figure 7: Val-ILs-«CD223 target CD223+T4-Ly which activate T8-Ly. (A) Six days following transplantation of EL4 cells, Val-ILs labeled with PKH67-Green were i.v. injected in mice at a dose of 1011particles per injection. Then, 18 hours later, green fluorescent Val-ILs- aCD223 were detected by flow cytometry, showing binding on CD223+T4-Ly in the spleen, but not in the tumor. (B) Val-ILs-aCD223 were i.v. injected in mice, at a dose of 1011particles, on days 6 and 9. Example of flow cytometry dataset on immune cells isolated from spleens on day 12, showing the reduction of CD223+T4-Ly in the spleen of a mouse treated with Val-ILs- aCD223. (C) Flow cytometry UMAP data frame showing a decrease in CD223+T4-Ly (orange dots) in spleens and tumors following treatment with Val-ILs-aCD223. The other populations of immune cells were not affected, such as DC (dark blue dots), T8-Ly (green dots) or myeloid CD1 lb+cells (clear blue dots). Statistics of the immune system analyzed by UMAP in spleens and tumors, n = 5 mice per group. (D) Flow cytometry UMAP data frame showing decrease in T4-Ly regulatory cells expressing CD25 and FoxP3 (CD223+Treg,), Thl7 T4-Ly expressing IL17 (CD223+Thl7), as well as the remaining CD223+T4-Ly (CD223+,), in spleens and tumors on day 12, following treatment with Val-ILs-aCD223. Other populations of immune cells are shown. Statistics of the CD223+T4-Ly populations analyzed by UMAP, in spleens and tumors, n = 5 mice per group. (E) Data showing the activation of T8-Ly measured by flow cytometry, in spleens and tumors, on day 12 following treatment with Val-ILs-aCD223. Example of flow cytometry data set and statistics on the expression of markers involved in T8-Ly activation. Granzyme B (GzB), interleukin 2 (IL2), tumor necrosis factor alpha (TNFoc), and the proliferation marker Ki67, n = 5 mice per group. On this figure, data are shown as means ± SD, P values were calculated against untreated controls and measured by one-way Anova with Tukey’s multiple comparison test; *P < 0.05; **7?< 0.01; ***p < 0.001. No statistic is shown when the P value is non-significant.

[0189] Figure 8: Combo Val-ILs reduce lymphoma development in vivo. C57BL / 6 mice were subcutaneously injected with 106EL4 cells, on the body side. When tumors reached 50 mm3, mice were treated with Val-ILs-aCDl lb, Val-ILs-aCD223, or a combination of Val-ILs- aCDl lb and Val-ILs-aCD223 (Combo), at a dose of 2x lOnparticles, on days 6 and 9. (A) Flow cytometry UMAP data frame and statistics showing that a treatment with Combo Val-ILs affected both MDSC (orange dots) and CD223+T4-Ly (red dots) in tumors on day 12, n = 4 mice per group. (B) On day 12, T8 lymphocytes were isolated and quantified from spleens and tumors. Data showing that treatment of mice with Combo Val-ILs does not increase the quantity of T8 lymphocytes in tumors, n = 4 mice per group. (C) On day 12, T8 lymphocytes were isolated from spleens and tumors and co-cultured with EL4 cells, at a ratio of 1 : 100 and viability assessed by XTT assay after 48 hours of treatment, n = 8 biological replicates per group. Data showing that T8 lymphocytes isolated from mice treated with Combo Val-ILs affect the viability of EL4 cells more efficiently. (D) Flow cytometry data showing that treatment with Combo Val-ILs activate the expression of PD1 (P < 0.0001) and GzB (P< 0.01) on T8 lymphocytes, n = 4 mice per group. (E) Statistics on tumor volume, n = 8 mice per group. (F) Kaplan-Meier survival plots showing that mice treated with Val-ILs-aCDl lb or Val-ILs- aCD223 survived longer than untreated mice, and the survival was even better when mice were treated with Combo Val-ILs, n = 8 mice per group. On this figure, (A, B, C, D, E) data are shown as means ± SD, P values were measured by one-way Anova with Tukey’s multiple comparison test; *P < 0.05; **P < 0.01; *** / ’< 0.001; P < 0.0001. No statistic is shown when the P value is non-significant. (G) P value measured by Log-Rank (Mantel-Cox) test; **P< 0.01; *** < 0.001.

[0190] Figure 9: Identification of Val-ILs targeting new populations of immunosuppressive cells. (A) C57BL / 6 mice were subcutaneously injected with 106EL4 cells, on the body side. When tumors reached 50 mm3, mice were treated with Val-ILs i.v. injected into the tail vein on days 6 and 9, at a dose of 1011particles per injection. Different Val-ILs loaded with further antibodies were tested in order to target different populations of immune cells. Tumor growth volumes were assessed over time. (B) Tumor volumes measured on day 12, with all the tested Val-ILs showing high response, n = 3 mice per group. The Val- ILs-Combo is a mixture of the 9 Val-ILs showing high response. (C) Val-ILs-Combo significantly affect the tumor volume over time, means of n = 3 mice per group.

[0191] Figure 10: Combination of Val-ILs and aPDl increases survival of mice. (A) Combo Val-ILs plus anti-PDl prevented the development of lymphoma. Val-ILs were i.v. injected into the tail vein, at days 6, 9 and 12, at the dose of2><1011particles per injection. aPDl or Ig control antibodies were injected, by i.p at day 6, at the dose of 200 pg per mouse. Tumors’ growth volumes were assessed over time. Data shows means, n = 8 mice per group. Two of 8 mice developed lymphoma in the group treated with the aPDl, while none of the mice treated with Combo Val-ILs plus aPDl developed lymphoma. (B) Kaplan-Meier survival plots show that mice treated with combo Val-ILs plus aPDl never developed lymphoma, n = 8 mice per group. Anti-PDl antibody (BE0146, BioXCell) or the isotype control antibody (BE0089, BioXCell) were i.p. injected, at the dose of 200 pg per mouse.

[0192] Figure 11: Val-ILs-Combo (CDllb, CD64, TIM1, CD200R3, CD204, CD49b and SIGLEC-F) and aPD-1 therapy affects growth of breast tumor cells. 1064T1 cells were transplanted through intraductal injection in the mammary gland of female BALB / c mice. When tumors reached 50 mm3, mice were treated with Val-ILs-Combo intravenously injected on days 6, 9 and 12, at a dose of 1012particles per mouse. Val-ILs-IgG was injected as a control. Mice were also intraperitoneally injected with otPD-1 immunotherapy at days 6, 9 and 12, at the dose of 200 pg per mouse. otlgG isotype was injected as a control. (A) Tumor growth volume measured overtime. The number of mice per group is indicated on figure. (B) Image of tumors isolated from the mammary gland of mice, on day 15. The number of mice per group is indicated on figure. Two pictures obtained from two independent in vivo experiments. Breast cancer 4T1 cells were resistant to ocPD-1 immunotherapy, Val-ILs-Combo was efficient to reduce the tumor sizes, and the combinatory administration of Val-ILs-Combo with aPD-1 was even more efficient. Mice that have not developed breast tumor were identified with a black cross. Tumor growth volumes measured ex vivo at day 15, and a repartition of mice free or bearing tumor is shown. On the figure, data are shown as means ± SD, P values indicated were compared to Val-ILs-IgG + oclg and calculated using one-way ANOVA with Tukey’s multiple comparisons test.

[0193] Figure 12: Val-ILs-Combo (CDllb, CD64, TIM1, CD200R3, CD204, CD49b and SIGLEC-F) and aPD-1 therapy modified the polarization of tumor associated macrophages (TAMs). Flow cytometry, on TAMs cells in the tumor microenvironment (TME) and spleen macrophages (SPMs) cells in spleen, to identify TAMs with Ml-like and M2 -like phenotypes. The number of mice analyzed per group is indicated below graphs. Data showing that the combinatory administration of Val-ILs-Combo with ocPD-1 importantly increase the switch from Ml-like to M2 -like TAMs in TME and SPMs in spleen. On the figure, data are shown as means ± SD, P values indicated were compared to Val-ILs-IgG + alg and calculated using one-way ANOVA with Tukey’s multiple comparisons test.

[0194] Figure 13: Val-ILs-Combo (CDllb, CD64, TIM1, CD200R3, CD204, CD49b and SIGLEC-F) and aPD-1 therapy importantly reduces the presence of metastatic breast cancer cells in lungs. Following lentiviral infection, 106bioluminescent 4T1 cells were transplanted through intraductal injection in the mammary gland of female BALB / c mice. When tumors reached 50 mm3, mice were treated with Val-ILs-Combo intravenously injected on days 6, 9 and 12, at a dose of 1012particles per mouse. Val-ILs-IgG was injected as a control. Mice were also intraperitoneally injected with ocPD-1 immunotherapy at days 6, 9 and 12, at the dose of 200 pg per mouse. algG isotype was injected as a control. At day, 15, using flow cytometry, Val-ILs-Combo and ocPD-1 therapy importantly reduces the presence of metastatic GFP-expressing 4T1 cells detected in lungs. On the figure, data are shown as means ± SD, P values indicated were compared to Val-ILs-IgG and calculated using one-way ANOVA with Tukey’s multiple comparisons test. Undetected CD44+GFP+tumoral 4T1 cells (ud)

[0195] EXAMPLE: Material & Methods

[0196] ILs loaded with valrubicin

[0197] L-a-phosphatidylcholine (P7443, Merck), cholesterol (C8667, Merck), DSPE-PEG- square (2000) (880136P, Merck) and DSPE-PEG-NHS (5000) (06030500706, SINOPEG) were prepared in chloroform (Merck) with a respective molar ratio of 100; 37; 1.5; 0.2, to a final volume of 26 pl. To this mix, we added 26 nmol of valrubicin (SML2516, Merck) reconstituted in DMSO (Merck). Then, chloroform was evaporated with nitrogen, making a thin lipid film in a round-bottom flask by the removal of organic solvent. The lipid film was then reconstituted in 1 mL of filtrated PBS lx preheated to 65°C. Following 10 min of incubation at 65°C, the mixture was vortexed for 1 min, then after stabilization for 30 min at 65°C, the vesicles were sonicated for 5 cycles (30 sec on-off) at an amplitude of 20% to form UVs that were stabilized at 65°C for 30 min. UVs targeting B-ALL cells, T-ALL cells or AML cells were then incubated with 2 pg of an antibody (Table 1). The reaction was stopped by adding 1 mM of glycine. In order to purify the Val-ILs, two consecutive dialyses (one of two hours and another one overnight) were carried out in one liter of PBS lx, with 300 KDA dialysis membranes (11550970, Thermo Fisher Scientific). To prove that the antibodies used to prepare Val-ILs have no cytotoxic effect on their own, anti-hCD19 (1:100), anti-hCD7 (1 :100) or anti-hCD33 (1 : 100) antibodies were reciprocally tested on BALLI, lurkat and THP1 cells, and viability was analyzed by XTT cell proliferation assay (Thermo Fisher Scientific) following 3 days of cell culture. The same protocol was followed to prepare Val-ILs used on mice developing the EL4 lymphoma. All antibodies used to prepare Val-ILs, including IgG isotypes control antibodies are described in Table 2. For PKH67+Val-ILs, 5 pL of PKH67 (MINI67-1KT, Merck) was added to lipids before sonication. After stabilization, purification was performed by ultracentrifugation at 120,000 g (Optima XE-90 ultracentrifuge, Beckman Coulter) for 90 min at 4°C. Val-ILs pellets were suspended in filtrated PBS lx and they were incubated with 2 pg of antibodies for 2 hours at room temperature under agitation. The reaction was stopped by adding 1 mM of glycine. To prepare ILs with vincristine (V8388, Merck) and dexamethasone (D1756, Merck), both compounds reconstituted in 1 mL of filtrated PBS 1 x at 1 pM each, were directly applied on the dried lipid film, then vortexed and sonicated following the same procedure. The size and concentration of Val-ILs were characterized by nanoparticle tracking analysis (NTA) using a NanoSight NS300 Instrument (Malvern Instruments, Malvern, England). The Zeta potential of Val-ILs was measured by a Zetasizer Nano-ZS (Malvern Instruments, Malvern, England). Valrubicin dosage with colorimetry was performed at 560 nm (EnVision 2104, Perkin Elmer).

[0198] Establishment of ALL and AML PDX models and treatments with Val-ILs

[0199] NOD / SCTD / yc" (NSG) mice (Charles River) were bred, raised and housed under pathogen-free conditions. To induce leukemia, we i.v. injected 105B-ALL, 105AML or 5 x 105T-ALL in a volume of 300 pL of physiological saline solution in the tail vein of non-irradiated 7-16-week-old male and female NSG mice. Males and females were randomly allocated to experimental groups and no blinding method was used for injection. Following RTqPCR and CGH array on hCD45+cells, isolated with magnetic beads, B-ALL PDX cells ^1 displayed an IGK::MYC translocation, as well as microdeletions in CDKN2A and RBI genes, B-ALL cells 2 displayed a TEL::AML1 chromosomal translocation, as well as microdeletions in IGLL5, PIK3C3 mA RBI genes. T-ALL PDX cells displayed a STIL (SCL::TAL1 interrupting locus), as well as deletions in LEF1 and CDKN2A genes. AML PDX cells displayed an MLL::AF9 chromosomal translocation. Val-ILs were i.v. injected into the tail vein, three times every five days (days 15, 20 and 25 for B-ALL and AML, and days 20, 25 and 30 for T-ALL), at a dose of 1011particles in 300 pL of physiological saline solution. After tail vein blood sampling from PDX mice, white blood cells were recovered following hemolysis (NH4C1 150 mM, KHCO3 10 mM, EDTA 0.1 mM, pH 7.4). Tibias and femurs from the two bottom legs were crushed in a mortar, in PBS lx, and total BM cells were filtered with a sterile cell strainer (70 pm). Spleens were also crushed and filtered with the sterile cell strainer in hemolysis solution, and the cells were washed with PBS lx. For flow cytometry analysis or immunohistochemistry, we recovered blood, spleen andBM cells at day 35 for B-ALL and AML, and at day 45 for T-ALL, following previously described protocols (67). We used rabbit anti-hCD19 (1:100, SAB5500047, Merck) for immunohistochemistry that was performed by the ImaFlow platform (Universite de Bourgogne, Dijon, France), under an AxioScope microscope (Zeiss). Eighteen hours after the injection of PKH67+Val-ILs in PDX mice, cells were recovered, by centrifugation at 500 g, from BM, spleen and blood to detect PKH67+Val-ILs binding, by flow cytometry. To analyze the distribution, Val-ILs were i.v. injected in NSG mice, then 18 hours later, cells were recovered by centrifugation at 500 g from BM, spleen and blood, debris were removed from the supernatant by centrifugation at 10,000 g. From the supernatant, UVs were isolated by pull-down, following previously described procedures (68) with a specific kit (15254394, Thermo Fisher Scientific), and analyzed by flow cytometry.

[0200] Bioluminescence imaging We created PDX models that developed stable bioluminescence by infecting B-ALL cells #2 with a lentivirus expressing both GFP and luciferase (67). Then, we transplanted these cells into mice that were used later for bioluminescence imaging. The lentivirus was produced in HEK293 cells after transduction with Lipofectamin 2000 (Thermo Fisher Scientific) of the pCCLc-MNDU3-Luciferase-PGK-EGFP-WPRE vector (Addgene, #89608), as well as PAX2 (Addgene, #12260) and pCMV-VSV-G (Addgene, #8454) plasmids. After two days, viral supernatants were recovered, and six-well plates were incubated for four hours with retronectin (Takara, Ozyme). Viral supernatants were then spinoculated for 30 min at 4,000 g. Cells were cultured on these plates for three days in StemMACS media (Miltenyi Biotech). Lentiviral transduced cells (GFP+) were sorted on a FACSArialll cell sorter (BD Biosciences) and transplanted in NSG mice to generate bioluminescent PDX. Animals were injected with D- luciferin potassium salt (150 mg / kg body weight). Following isoflurane-induced anesthesia, animals were imaged 15 min after D-luciferin (Merck) injection using an IVIS Lumina III system coupled with Living Image acquisition and analysis software version 4.0 (Perkin Elmer).

[0201] Establishment of in vivo EL4 lymphoma models and treatments with Val-ILs

[0202] For the murine lymphoma model, EL4 cells (TIB-39, ATCC) were cultured in DMEM media (Dominique Dutscher) supplemented with 10% fetal bovine serum (Dominique Dutscher) and Penicillin-Streptomycin-Amphotericin (PSA, Pan Biotech). For the study in vivo, 106EL4 cells were subcutaneously injected, in a volume of 100 pL of physiological saline solution, on the body side of C57BL / 6 mice (Envigo). For injection, males and females were shaved and randomly assigned to experimental groups, and no blinding method was used for injection. Mice were treated when tumors reached 50 mm3(day 6). Val-ILs were i.v. injected into the tail vein at days 6 and 9 at a dose of 1011particles, in 300 pL of physiological saline solution. Tumor growth was measured over time. In order to perform flow cytometry on immune cells isolated from spleen and tumor, mice were euthanized 12 days after the injection of EL4 cells, when the tumor reached the endpoint, >2,000 mm3, in the untreated control group. For the combinatorial study, Val-ILs were i.v. injected into the tail vein at days 6, 9 and 12, at a dose of 2 1011particles for each condition. Spleens were crushed and filtered with the sterile cell strainer in hemolysis solution, and the cells were washed with PBS lx. Tumors were chopped up into 3 to 4 mm pieces with a sterile scalpel. Tumor samples were then placed in 2.5 mL of the dissociation buffer, containing 60 U / mL of Collagenase, Type 1 (CLS-I, LS004194, Cell Systems), 30 U / mL of Collagenase, Type 2 (CLS-II, LS004174, Cell Systems), 60 U / mL of Collagenase, Type 4 (CLS-IV, LS004186, Cell Systems), and 25 pg / mL of DNAse 1 (04536282001, Merck) in filtered PBS lx. The mixture was incubated under agitation at 37°C for 45 min. Cell suspension was filtered through 30 pm separation filters (130-041-407, Miltenyi Biotech) and centrifuged at 500 g. Mice were also i.v. injected with PKH67+Val-ILs (1011particles, at day 6) and sacrificed 18 hours after to analyze, by flow cytometry, the presence of UVs in tumor and spleen, as well as the binding of fluorescent Val-ILs on immune cells. The supernatant was centrifuged at 10,000 g to remove debris, then UVs were isolated from the supernatant by pull-down with a specific kit (15254394, Thermo Fisher Scientific), and analyzed by flow cytometry.

[0203] Activity of T8 lymphocytes isolated ex vivo on EL4 cells

[0204] C57BL / 6 mice were injected with 106EL4 cells and, when tumors reached 50 mm3, mice were treated with Val-ILs. At day 12, T8 lymphocytes were recovered from tumors and spleens, with anti-CD8 magnetic beads (130-104-075, Miltenyi Biotec) and cultured in RPMI medium supplemented with 10% FBS, 1% PSA, MEM 1* (11140035, Thermo Fisher Scientific), L-glutamine 2 mM (25030149, Thermo Fisher Scientific), HEPES 10 mM (15630080, Thermo Fisher Scientific), pyruvate sodium 1 mM (11360070, Thermo Fisher Scientific) and 0-mercaptoethanol 5.5 nM (21985023, Thermo Fisher Scientific). T8 lymphocytes were enumerated and co-cultured with EL4 cells, at a ratio of 1 : 100. Viability was assessed by XTT cell proliferation assay (Thermo Fisher Scientific), following 48 hours of treatment.

[0205] Cell culture and treatment with Val-ILs

[0206] B-ALL-1 (DSMZ), Jurkat (ATCC) and RPML8402 (ATCC), THP1 (ATCC) and HL60 (ATCC) cells were cultured in RPMI- 1640 media (Dominique Dutscher) supplemented with 10% FBS and 1% PSA. For the ex vivo experiments, we used cells freshly isolated from the BM of PDX mice, cultured in StemMACS media (Miltenyi Biotec) supplemented with PSA. Murine MS-5 mesenchymal stromal cells (ACC-441, DSMZ) were cultured in IMDM media (Thermo Fisher Scientific) with 10% FBS and 1% PSA. When cell confluence reached 80%, we administered GFP+B-ALL cells in StemMACS media, and, following 5 hours of binding, 6-well plates were treated with Val-ILs-aCD19. Multiple pictures per well were taken every 12 hours using an IncuCyte S3 (Sartorius). For cell culture, cells were grown in an incubator at 37°C in a humid atmosphere and 5% CO2 pressure. Viability was assessed by XTT cell proliferation assay, measured by absorbance at 450 nm minus absorbance at 660 nm (EnVision 2104, Perkin Elmer), following previously described procedures (67). Cells were treated with different concentrations of Val-ILs, at a single dose treatment at the beginning of the experiment, and XTT cell proliferation assay was performed 72 hours after the treatment. BALL-1 and Jurkat cells were treated for 2 hours with 2,000 parti cles / cell, and then we performed fluorescence microscopy, following previously described procedures (67).

[0207] Human peripheral blood mononuclear cell (PBMC) isolation and Val-ILs treatment

[0208] PBMC were isolated following Pancoll (Pan Biotech) density gradient centrifugation. Viability, exceeding 90%, was assessed using Trypan Blue (Thermo Fisher Scientific) before the treatments, and cells were cultured in RPMI medium with 10% FBS and 1% PSA. PBMC were divided in three wells and treated with 2,000 Val-ILs / cell. Cell viability was analyzed by flow cytometry 48 hours after the treatment.

[0209] CD34+cord blood isolation, contamination with GFP+B-ALL cells, treatment with EVs and transplantation in NSG mice

[0210] Mononuclear cells were isolated from two cord blood samples following Pancoll (Pan Biotech) density gradient centrifugation. CD34+cells were recovered with magnetic beads (130-046-702, Miltenyi Biotec) and divided into 4 wells in 1 mL of StemMACS media (Miltenyi Biotec), supplemented with PSA (Pan Biotech), human stem cell factor (SCF, 25 ng / mL, 130-093-991, Miltenyi Biotec), human Interleukin 3 (IL3, 10 ng / mL, 130-093-908, Miltenyi Biotec), and human Interleukin 6 (IL6, 10 ng / mL, 130-095-365, Miltenyi Biotec). CD34+cells were deliberately contaminated with 1% of GFP+B-ALL cells (#1 or #2). The mix of CD34+and GFP+B-ALL cells were treated with 2,000 particles / cell. Eighteen hours later, cells were washed with PBS lx, then 1.5xl05cells in physiological saline solution were transplanted in NSG mice, irradiated 24 hours before the transplantation at sublethal dose of 1.5 Gray (BioMEP, Breteniere, France). After 5 weeks, development of B-ALL was monitored by GFP expression in BM. CD34+HSC reconstitutions were assessed by flow cytometry in PB and BM. We also determined the disappearance of GFP+cells in vitro by flow cytometry, 48 hours after the treatment.

[0211] Treatment of primary B-ALL samples with Val-ILs-aCD19

[0212] Cell isolated from the BM collected at diagnosis of patients with B-ALL were cultured in StemMACS medium with 1% PSA. The viability, exceeding 90%, was assessed using Trypan Blue before treatments. Cells were divided in three wells and treated with 2,000 Val- ILs / cell. Cell viability was analyzed by flow cytometry 48 hours after the treatment.

[0213] Flow cytometry ALL and AML developments in NSG mice were characterized in BM, spleen and PB by flow cytometry using antibodies described in Data not shown. These antibodies were also used to distinguish between leukemia cells and murine cells. Apoptosis was assessed following Annexin-V-FITC (556419, BD Biosciences) staining. Binding of PKH67-Green Val-ILs was detected in the green channel. We analyzed cell subsets from PBMC samples as well as the human HSC reconstitution in PB and BM of mice, using antibodies described in data not shown. Regarding the study of mice following the injection of EL4 cells, all antibodies used to analyze the different immune cells in spleens and tumors, as well as T8 lymphocyte activation ex vivo, or the activity of T8 lymphocytes isolated ex vivo on EL4 cells, are described in data not shown. Viability was assessed using Fixable Viability Stain (FVS440UV, FVS450 and FVS510, 1:1,000, BD Biosciences) or Hoechst 33342 (1 : 1,000, Thermo Fisher Scientific). Cell subsets were analyzed using an LSR-Fortessa (BD Biosciences) or an Aurora (Cytek) apparatus. Data were analyzed using FlowJo software (V10, TreeStar Inc). We used the Uniform Manifold Approximation and Projection (UMAP) FlowJo plugin for dimensionality reduction to visualize high parameter datasets in a two-dimensional space.

[0214] Statistics

[0215] All data were expressed as means ± standard deviation (SD). Differences between two groups were assessed with the two-tailed unpaired Student’ s t test or two-tailed paired Student’s / test. The one-way Anova with Tukey’ s multiple comparison test was used to assess differences between more than two groups. Differences in Kaplan-Meier survival plots were analyzed using the Log-Rank (Mantel-Cox) test. No statistical methods were used to predetermine the sample size. No animal exclusion criteria were applied. Mice were randomly allocated to experimental groups. The variance was similar between the groups that were statistically compared. Statistics were performed using Prism 8 (GraphPad), where significance is indicated on the Figures.

[0216] Results

[0217] Development of ILs incorporated with valrubicin

[0218] In our formulation, we used a mixture of L-a-phosphatidyl choline, cholesterol, DSPE- PEG-square (2000), DSPE-PEG-NHS (5000), which were prepared in chloroform with specific molar ratios of 100; 37; 1.5; 0.2. Valrubicin reconstituted in dimethyl sulfoxide (DMSO) was added at 1 mM. The liposomes were then created using a thin film hydration method in PBS lx, followed by sonication. The CD19 antigen is a transmembrane protein specific to B-cells and often utilized as a target for B-cell leukemia and lymphoma (46-48). Once the unilamellar vesicles (UVs) were stabilized, we incorporated anti-CD19 (Val-ILs-aCD19) or the IgG isotype control (Val-ILs-IgG) antibody just before the purification process via dialysis. Nanoparticle tracking analysis (NT A) found that Val-ILs, with an average diameter of 115 nm, correspond more to large UVs (ranging from 100 nm to 1 pm), and their concentration ranged from 0.8* 1012to 1.5* 1012particles per milliliter (Fig. 1A). Through Western blot analysis, we determined that approximately 50% of the antibodies were bound to the Val-ILs, and dialysis successfully removed more than 95% of the unbound antibodies (Data not shown). Additionally, the presence of the anti-CD19 antibody or IgG isotype control was confirmed through flow cytometry analysis (Data not shown). High-performance liquid chromatography coupled with mass spectrometry (HPLC-MS / MS) was used to quantify the valrubicin content in both Val-ILs-aCD19 and Val-ILs-IgG, revealing that each contained 1.52x 10"xpmol of valrubicin per particle (Fig. IB). The observation that approximately 58% of the valrubicin used in the preparation of Val-ILs was successfully incorporated is a significant finding. Additionally, the successful removal of all non-encapsulated valrubicin through dialysis confirms that the drug was completely integrated into the Val-ILs, and there was no contamination from unincorporated valrubicin during the preparation process (Data not shown). The stable zeta potentials of the Val-ILs (Data not shown) made them suitable for testing in biological samples. Transmission electron microscopy (TEM) further confirmed that Val-ILs- aCD19 had an average size of 114 ± 28 nm, with the lipid bilayer averaging 7.7 ± 3 nm in size (Data not shown).

[0219] With these detailed characterizations and promising results in hand, the study proceeded to the assessment of the efficacy of Val-ILs in order to advance a new therapeutic nanoparticle technology for the treatment of acute leukemia.

[0220] Val-ILs-«CD19 induce death of CD19-expressing cells

[0221] The in vitro evaluation of the impact of Val-ILs on the viability of B-cell acute lymphoblastic leukemia- 1 (BALL-1) cell lines yielded significant results. Utilizing the XTT cell proliferation assay, three days after a single treatment of B-ALL1 cells, the inhibitory concentration (IC50) for Val-ILs-aCD19 was determined to be 1,000 particles per cell (P < 0.0001, Fig. 1C). Notably, treatment with 2,000 particles per cell, equivalent to ~2 pL of the Val-ILs preparation per mL of culture, resulted in a significant decrease in the viability of BALL-1 cells. This finding was further substantiated through flow cytometry analysis, which confirmed the effect on cell viability (P < 0.0001, Fig. ID). Importantly, there was no effect on the T-ALL Jurkat cells, which lack CD19 expression and consequently did not bind to Val-ILs- aCD19 (Data not shown). Fluorescent immunostaining and microscopy revealed that within one hour of incubation in the in vitro culture media, Val-ILs-aCD 19 efficiently targeted the cell surface of B-ALL1 cells, while Jurkat cells remained untargeted (Data not shown). Furthermore, treatment with Val-ILs-aCD19 at 2,000 particles per cell for three days led to -95% cell death in B-ALL1 cells (P < 0.0001, compared with Val-ILs-IgG, Data not shown), without affecting the viability of Jurkat cells. These results strongly suggest that Val-ILs- aCD19 specifically induced cell death in CD19-targeted B-cells. Importantly, we noted that the Val-ILs contained only a minimal amount of valrubicin, measured at 1.52 / 108pmol per particle using HPLC-MS / MS. Consequently, the amount of valrubicin required when loaded into Val-ILs-aCD19 was 32.7-fold lower (~3>< 10'2pM) than the amount needed to achieve a similar effect on cell viability (1 pM) when the drug was directly applied to the cells. This striking difference highlights that Val-ILs are a highly efficient approach to reducing the drug concentration necessary for effective treatment.

[0222] We further assessed the effectiveness of Val-ILs-aCD19 on human peripheral blood mononuclear cells (PBMC) after 48 hours of exposure in vitro. Using flow cytometry, it was determined that Val-ILs-otCD19 induced the death of healthy B-lymphocytes expressing CD 19 at a rate exceeding 95% (Fig. IE). In contrast, other cell populations that were negative for CD 19 expression, such as T4- or T8- lymphocytes, were not sensitive to Val-ILs-aCD19. It should be noted that Val-ILs-IgG had no effect on the B-lymphocytes expressing CD 19. Thus, in PBMC isolated from blood cultured ex vivo, Val-ILs-aCD19 could specifically target and eliminate the population of cells expressing CD 19, such as B-lymphocytes, among a diverse range of hematopoietic cells. We also investigated the impact of Val-ILs-aCD19 on primary pediatric B-ALL samples isolated from the bone marrow (BM) of patients at diagnosis. The majority of living cells in these samples were CD19+B-ALL cells. While Val-ILs-IgG had no effect, treatment with Val-ILs-aCD19 for 48 hours eliminated 94 ± 6% of the B-ALL cells (P < 0.0001, Fig. IF). This indicates that Val-ILs-aCD19 effectively eradicated the population of CD19+B-ALL cells in primary B-ALL samples isolated from BM at the time of diagnosis.

[0223] These results demonstrate the potential clinical significance of Val-ILs-aCD19 in targeting and eliminating malignant B-ALL cells in pediatric patients.

[0224] Val-ILs-aCD19 target B-ALL cells in BM and spleen of PDX mice

[0225] We used a mouse model to assess the in vivo effect of VaLILs-aCD19 on primary pediatric B-ALL cells. This was achieved by transplanting the primary pediatric B-ALL cells into immunodeficient NS G mice, leading to the generation of two B-ALL PDX models referred to as #1 and #2. Thirty days after injection, -80% of the bone marrow (BM) cells recovered from the mice were B-ALL cells (hCD45+hCD19+), while the remaining 20% of cells (hCD45‘ hCD19‘) consisted of endogenous murine cells present in the BM microenvironment, in which the leukemia cells were engrafted and had expanded. Before the in vivo experiments, we had to ensure that Val-ILs-aCD19 specifically targeted hCD19+B-ALL cells. We therefore used trackable Val-ILs labeled with a green fluorescent membrane dye (PKH67) during the preparation. This labeling allowed us to verify the specific binding of Val-ILs-aCD19 to human B-ALL cells, and not to murine BM cells, 18 hours after treatment (Data not shown). We also assessed apoptosis using Annexin-V staining and observed that Val-ILs-aCD19 induced apoptosis in human B-ALL cells (P < 0.0001) while having no significant effect on murine BM cells (Data not shown). Furthermore, treatment with 2,000 particles per cell affected the viability of 100% of B-ALL cells in PDX #1 and 95% in PDX #2 (Data not shown). An additional experiment involved growing green fluorescent protein positive (GFP+) B-ALL cells on MS-5 stromal mesenchymal cells: treatment with Val-ILs-aCD19 showed a significant impact on the viability of GFP+cells within 24 hours, while the supportive MS-5 cells continued to grow (Fig. 1G). These findings support the hypothesis that Val-ILs-ciCD19 can effectively target leukemia cells while preserving mesenchymal cells, which play a supportive role for hematopoietic cells in the BM microenvironment.

[0226] After demonstrating that Val-ILs-aCD19 caused specific B-ALL cell death ex vivo, the study progressed to an assessment of whether treatment with Val-ILs-aCD19 might impact the in vivo development of B-ALL in PDX mice.

[0227] Val-ILs-aCD19 affect the development of B-ALL in PDX mice

[0228] We first aimed to characterize the distribution of Val-ILs in BM and spleen, following injection into NSG mice without prior transplantation of B-ALL cells. Ensuring that these Val- ILs were not captured by B-ALL cells was crucial. We administered Val-ILs intravenously (i.v.) through the tail vein, and, considering previous findings (49, 50) demonstrating the arrival of ILs in organs in vivo a few hours after i.v. injection, we chose to evaluate the binding of Val- ILs after an 18-hour period. Tissues were crushed and UVs were obtained through precipitation after removing cells and debris. Using flow cytometry, the presence of Val-ILs labeled with PKH67-Green was detected in the BM (20% of UVs) and spleen (50% of UVs), but not in peripheral blood (Fig. 2A).

[0229] Subsequently, we aimed to evaluate the effective binding of Val-ILs-aCD19 to B-ALL cells by transplanting 105leukemia cells into NSG mice. Fifteen days after this transplantation, Val-ILs labeled with PKH67-Green were administered via i.v. injection. After 18 hours, flow cytometry revealed the binding of Val-ILs labeled with PKH67-Green on over 60% of hCD45+B-ALL cells in both the BM and spleen, for both B-ALL PDX models (Fig. 2B). Notably, Vai- ILs-IgG did not bind to the leukemia cells, and endogenous murine cells (hCD45‘ cells) did not bind to the fluorescent Val-ILs-aCD19.

[0230] We further assessed how treatment with Val-ILs-aCD19 might affect the in vivo development of B-ALL in PDX mice. Animals received three injections of 1011Val-ILs on days 15, 20, and 25 post-transplantation. On day 35, when leukemia had reached an endpoint in the control group, mice were sacrificed to recover various tissues. Flow cytometry revealed that Val-ILs-aCD19 induced significant death in over 90% of B-ALL cells in BM (P < 0.0001), spleen (P < 0.001) and blood (P < 0.001), compared to untreated mice or mice treated with Val- ILs-IgG (Fig. 2C). Moreover, BM in the treated mice was red, suggesting the presence of red blood cells, in contrast to untreated mice or mice injected with Val-ILs-IgG, whose BM cells were white, characteristic of B-ALL development. Val-ILs-aCD19 treatment also significantly attenuated splenomegaly (P < 0.01, Fig. 2D and Data not shown). Immunohistochemistry in BM and spleen sections stained with an hCD19 antibody revealed that B-ALL cells in untreated mice or mice treated with Val-ILs-IgG had expanded considerably more than in mice treated with Val-ILs-aCD19 (Data not shown).

[0231] We next utilized lentiviral infection to create bioluminescent B-ALL cells expressing luciferase, which were then transplanted into mice. Three groups of mice were established: one group received treatment with Val-ILs-aCD19 and the other two groups received treatment with Val-ILs-IgG or physiological saline solution. The treatment was administered on days 15, 20, and 25 after transplanting 105bioluminescent B-ALL cells. Subsequently, luciferin was injected into the mice while they were asleep, allowing for the monitoring of the location of leukemia cells in living animals. We observed reduced bioluminescence in the group of mice treated with Val-ILs-aCD19 (five-fold decrease, P < 0.001, Fig. 2E). PDX mice treated with Val-ILs-aCD19 also exhibited extended survival compared to untreated PDX mice or mice treated with Val-ILs-IgG (P < 0.001, Fig. 2F).

[0232] These findings indicated that Val-ILs-aCD19 loaded with valrubicin had a substantial impact on the in vivo development of pediatric B-ALL in PDX mice, leading to improved survival rates.

[0233] Val-ILs-aCD19 eradicate malignant B-ALL cells among CD34+hematopoietic stem cells (HSC)

[0234] To address the risk of graft contamination with acute leukemia cells when using autologous peripheral blood stem cells (PBSC) for transplantation (51), we investigated the efficiency of Val-ILs in eradicating leukemia cells. This is particularly crucial for securing transplantation, and CD34+tissue purging can be an effective method for eliminating malignant cells from the graft (51). B-ALL cells from PDX #1 and #2 were transduced with a lentivirus to express green fluorescent protein (GFP). We tested the ability of Val-ILs to eradicate cancer cells when CD34+hematopoietic stem cells (HSC) isolated from human cord blood were deliberately contaminated with -1% of GFP+B-ALL cells. After 48 hours of treatment with Val-ILs-aCD19 at a concentration of 2,000 particles per cell, the B-ALL GFP+cells were completely eliminated, while the CD34+cells remained unaffected (Data not shown). This finding demonstrates that a small amount of Val-ILs (~0.4 pL diluted in one mL of cell culture) was sufficient to eradicate all malignant B-ALL cells, leaving the CD34+HSC unharmed. We further tested the effectiveness of Val-ILs-aCD19 in eradicating cancer cells when CD34+HSC were contaminated with GFP+B-ALL cells and then transplanted into NSG mice (Fig. 3A). Finally, no GFP+B-ALL cells were detected in the BM five weeks after transplantation, indicating that Val-ILs-aCD19 had successfully eradicated the leukemia cells (Fig. 3B). Additionally, the treatment did not impair the ability of CD34+HSC to reconstitute human hematopoiesis across different lineages (Fig. 3C, Data not shown).

[0235] In summary, these results suggest that Val-ILs-aCD19 could be a valuable tool for removing leukemia cells that may contaminate autologous PBSC transplantation, enhancing the safety of the transplantation procedure.

[0236] Development of Val-ILs loaded with «CD7 or «CD33 for T-ALL and AML therapies

[0237] We explored the potential of Val-ILs targeted with an antibody against CD7 on T-cell acute lymphoblastic leukemia (T-ALL) cell lines. Val-ILs-otCD7 effectively induced cell death in Jurkat cells expressing high levels of CD7, with a significant impact on cell viability achieved at a dose of 1,000 particles per cell (100% decrease, P < 0.0001). Additionally, even RPMI- 8403 cells with low CD7 expression had their viability affected by Val-ILs-aCD7 at a dose of 8,000 particles per cell (98% decrease, P < 0.0001, Fig. 4A). Val-ILs loaded with an antibody against CD33, at a dose of 4,000 particles per cell, also demonstrated efficient alterations in the viability of two acute myeloid leukemia (AML) cell lines, HL60 (68% decrease, P< 0.0001) and THP1 (100% decrease, P < 0.0001, Fig. 4B).

[0238] To further investigate the impact of these Val-ILs on in vivo T-ALL and AML development, pediatric T-ALL cells and AML cells were transplanted into NSG mice. Subsequent treatment with Val-ILs-aCD7 or Val-ILs-aCD33 involved three injections of 1011Val-ILs. Flow cytometry revealed a notable decrease in leukemia cells in the BM, spleen, and blood when compared to untreated mice or mice treated with Val-ILs-IgG. It was observed that Val-ILs-aCD7 effectively induced cell death in T-ALL PDX mice, with a significant impact on cell viability (40% decrease, P < 0.0001, Fig. 4C). Moreover, Val-ILs-aCD33 exhibited a pronounced ability to induce death of AML cells in PDX mice, resulting in a significant reduction in cell viability (60% decrease, P < 0.0001, Fig. 4D). The results showed that treatment with Val-ILs significantly reduced splenomegaly in both T-ALL (P < 0.0001, Fig. 4E) and AML (P < 0.0001, Fig. 4F) PDX mice.

[0239] Overall, this experiment demonstrated the versatility of Val-ILs in targeting and inducing cell death in specific leukemia subtypes by incorporating different antibodies, such as anti-CD7 for T-ALL and anti-CD33 for AML.

[0240] Development of Val-ILs targeting immune repressive cells for lymphoma immunotherapy

[0241] While Val-ILs-aCD19 had a significant impact on the viability of Raji lymphoma cells in vitro (Data not shown), we found that fluorescent Val-ILs-aCD19 had more difficulty reaching the lymphoma tumor in vivo (Data not shown). Consequently, the three i.v. injections of Val-ILs-aCD19 at days 15, 20, and 25 did not alter the development of the lymphoma tumor (Data not shown). To address this issue, we explored the development of nanoparticles designed to target and induce cell death in immune cells involved in cancer immunosuppression. A lymphoma mouse model was established using immunocompetent C57BL / 6 mice subcutaneously injected with EL4 cells. Various Val-ILs loaded with different antibodies were prepared to target MDSC (52-54), T4 regulatory (Treg) (55, 56) and T helper 17 (Thl7) (56) lymphocytes, as well as the immune checkpoint molecules Programmed death-ligand 1 (PD- Ll) or Programmed cell Death protein 1 (PD1) (57). Screening these Val-ILs following two i.v. injections allowed the identification of two nanoparticles loaded with aCDl lb or aCD223 antibodies as the most efficient treatment in vivo (Fig. 5A, B and Data not shown). When we repeated the experiment, Val-ILs-aCDl lb, which target myeloid cells, efficiently attenuated the development of lymphoma tumors in vivo (twenty-fold decrease, P < 0.001, Fig. 5C). In addition, Val-ILs-aCD223, which target T4 regulatory lymphocytes expressing CD223 (58, 59), were also found to effectively alter lymphoma growth in vivo (three-fold decrease, < 0.01, Fig. 5D).

[0242] In conclusion, the study revealed that while Val-ILs might not directly reach the lymphoma tumor site, it is possible to develop nanoparticles for immunotherapy by specifically targeting immune suppressive cells within the tumor microenvironment, which can contribute to combating cancer growth.

[0243] Val-ILs-aCDllb target MDSC for lymphoma immunotherapy We demonstrated that Val-ILs-aCDl lb efficiently targeted CDl lb+cells, including MDSC, within the spleen 18 hours after i.v. injection, but not in EL4 lymphoma (Fig. 6A). Following two i.v. injections of 1011particles at days 6 and 9, various immune cell populations were characterized using flow cytometry after the mice were sacrificed at day 12. The results revealed a reduced presence of MDSC in the spleens of mice treated with Val-ILs-aCDl lb (six-fold decrease, P < 0.001), as well as in tumors (two-fold decrease, P < 0.05, Fig. 6B). Additionally, T8 lymphocytes within tumors were found to be more activated, as indicated by increased expression of granzyme B (GzB, P < 0.001), interleukin 2 (IL2, P < 0.05), and tumor necrosis factor alpha (TNFa, P < 0.0001, Fig. 6C).

[0244] This experiment showed that Val-ILs-aCDl lb precisely targeted MDSC, effectively inducing the death of this population of immunosuppressive cells. This, in turn, led to the further activation of T8 lymphocytes, optimizing the anti-cancer immune response in mice developing EL4 lymphoma.

[0245] Val-ILs-aCD223 target T4 regulatory lymphocytes for lymphoma immunotherapy

[0246] We demonstrated that Val-ILs-aCD223 efficiently targeted CD223+cells within the spleen, but not within the EL4 tumor, 18 hours after i.v. injection (Fig. 7A). After two injections of 1011particles on days 6 and 9, the populations of CD223+T4 lymphocytes in both spleens and tumors were significantly affected by Val-ILs-aCD223 (Fig. 7B), with no specific effect on other immune cells (Fig. 7C). Further analysis revealed that Val-ILs-otCD223 effectively reduced the population of cells expressing FoxP3 and CD25 in tumors (three-fold decrease of Treg cells, P < 0.05) and interleukin 17 (IL17) in spleens (three-fold decrease of Thl7 cells, P < 0.05), as well as the population of CD223+cells which were not Thl7 or Treg cells in both spleens and tumors (two-fold decrease, < 0.05, Fig. 7D). Moreover, there was a notable increase in the activation of T8 lymphocytes within the tumors, as indicated by elevated expression of several markers detected by flow cytometry, including GzB (P < 0.01), IL2 P < 0.01), and TNFa P< 0.01, Fig. 7E).

[0247] In summary, Val-ILs-aCD223 effectively reduced the number of T4 lymphocytes, including Treg and Thl7 immunosuppressive cells, leading to a significant activation of T8 lymphocytes. This suggests the relevance of Val-ILs-aCD223 for immunotherapy in mice developing EL4 lymphoma.

[0248] Combination of Val-ILs targeting distinct populations of immunosuppressive cells increases survival of lymphoma mice

[0249] Anti-CDl lb and anti-CD223 antibodies had no antagonistic effect, and there was no impaired viability after ex vivo treatment (Data not shown). Furthermore, Val-ILs-aCDl lb and Val-ILs-aCD223 did not target EL4 cells (Data not shown). We investigated the combinatorial effect mediated by treatment with Val-ILs-aCDl lb and Val-ILs-aCD223 on immune cells isolated from the spleen of mice six days after the subcutaneous injection of EL4 lymphoma cells. Nanoparticles were produced, loaded with both antibodies, and administered to immune cells ex vivo. When Val-ILs were loaded with both antibodies, their ability to affect the viability of T4 lymphocytes expressing CD223 was lost (Data not shown). Therefore, it was preferable to treat mice with a combined administration ofVal-ILs-aCDl lb and Val-ILs-aCD223 (Combo Val-ILs) rather than Val-ILs loaded with both antibodies. Flow cytometry showed that a treatment with Combo Val-ILs efficiently reduced both MDSC and CD223+T4 lymphocytes in tumors (Fig. 8A). Combo Val-ILs did not further increase the amount of T8 lymphocytes detected in the tumors compared with Val-ILs-aCDl lb or Val-ILs-aCD223 alone (Fig. 8B). However, on day 12, T8 lymphocytes isolated ex vivo from the tumors and spleens of mice treated with Combo Val-ILs were significantly more effective in altering EL4 cell viability in vitro compared with T8 lymphocytes isolated from mice treated with either Val-ILs-aCDl lb or Val-ILs-aCD223 alone (P < 0.0001, Fig. 8C). This was accompanied by a marked increase in the expression of PD1 and GzB in T8 lymphocytes, indicating their activation (Fig. 8D). Treatment with Combo Val-ILs was shown to significantly reduce tumor volumes compared to treatment with Val-ILs-otCDl lb or Val-ILs-aCD223 alone (Data not shown and Fig. 8E). The survival of mice treated with Combo Val-ILs was also significantly longer (Fig. 8F), emphasizing the potential of these nanoparticles for immunotherapy in lymphoma.

[0250] Loading further antibodies to target other antigens involved in immunity, we furthermore identified 9 Val-ILs targeting populations of immunosuppressive cells, with relevant efficiency to reduce cancer tumor volume in mice (Fig. 9A-B). The most efficient Val- ILs were found to be those loaded with CDl lb, CD223, CD64, TIM1, CD200R3, CD204, CD49b, VEGFR2 and SIGLECF antibodies. Moreover, combination of these 9 Val-ILs (called Val-ILs-Combo) significantly affected the tumor volume over time (Fig. 9C).

[0251] Moreover, the combination of Val-ILs and the inhibitor checkpoint aPDl antibody prevented the development of lymphoma (Fig. 10A), and increased the survival of mice (Fig. 10B)

[0252] Val-ILs-Combo (CDllb, CD64, TIM1, CD200R3, CD204, CD49b and SIGLEC-F) and aPD-1

[0253] The Val-ILs-Combo and aPD-1 therapy affects growth of breast tumor cells (Fig. 11A and 11B) The Val-ILs-Combo and ocPD-l therapy modified the polarization of tumor associated macrophages (TAMs) (Fig. 12). Finally, the Val-ILs-Combo and aPD-1 therapy therapy importantly reduces the presence of metastatic breast cancer cells in lungs (Fig. 13A and 13B)

[0254] In summary, the study demonstrated that a combination of Val-ILs targeting and inducing death of distinct populations of immunosuppressive cells could lead to increased survival in lymphoma mice.

[0255] Conclusion:

[0256] The use of ILs, or immune liposomes, represents an innovative therapeutic approach that has been the subject of active research in preclinical models of solid cancer, showing promising results (16). Our study aimed to develop antigen-targeting ILs loaded with valrubicin, a hydrophobic analogue derivative of doxorubicin, which is an anthracy cline. Doxorubicin has been a key component of intravesical chemotherapy for over two decades, used to treat carcinoma in situ of the bladder and refractory cancer following Bacillus Calmette- Guerin treatment (41-45). Our research revealed that valrubicin, known for its lipophilic properties, readily integrated into the membrane of Val-ILs. Preclinical studies conducted on various murine models have shown that this novel nanoparticle exhibits high efficacy in the treatment of hematological malignancies. Up to this point, all the CD19-targeting ILs designed for other B-cell malignancies have utilized hydrophilic drugs, including vincristine, doxorubicin, imatinib, or rapamycin, encapsulated within the internal aqueous compartment (31-35). Patients diagnosed with B-ALL typically undergo multi-agent chemotherapy, such as vincristine and dexamethasone, as part of their treatment regimen (1-4). However, when we created Val-ILs-aCD19 loaded with these two hydrophilic compounds, we noticed that the combined efficacy of these drugs in inducing B-ALL cell death was significantly compromised once they were encapsulated within ILs, as shown in Fig. S8. These findings suggest that ILs containing hydrophilic drugs were less effective than ILs incorporating the lipophilic prodrug valrubicin. This disparity may be attributed to the ease of incorporating a lipophilic drug into the lipid membrane of ILs during nanoparticle production, while maintaining the encapsulation of hydrophilic drugs appears to be a more challenging task.

[0257] In patients with B-ALL, chimeric antigen receptor T cell (CAR-T) immunotherapy has shown a 70% response rate, but a concerning 10-20% of pediatric responders experience relapse with epitope loss (60, 61). Several mechanisms have been implicated in this phenomenon, including alternatively spliced CD19 transcripts lacking the epitope targeted by CAR-T cells (60), CD 19 mutations that result in truncated proteins (61), and CD 19 epigenetic repression contributing to antigen escape (62). Our study revealed the continued expression of CD 19 on the cell surface of residual cells in PDX mice treated with Val-ILs-aCD19, as shown in Fig. S9A. Among the 23 B-ALL PDX mice treated with Val-ILs-aCD19, only one mouse failed to respond to the treatment, as indicated in Fig. S9B. Further investigation revealed that hypermethylation of the CD19 promoter, as previously observed (62), was the underlying cause of antigen-negative escape from Val-ILs-aCD1 , but this phenomenon was observed exclusively in the non-responsive mouse.

[0258] In our preclinical study, the administration of Val-ILs did not have any noticeable impact on the weight of the mice over the course of the experiment, as demonstrated in Fig. S10A. Additionally, Val-ILs treatment did not result in significant toxicity in terms of blood parameters in the mice, except for a temporary increase in white blood cells, specifically monocytes and granulocytes, observed one day after the first i.v. injection. This effect was mitigated within a few days, despite two subsequent injections of Val-ILs.

[0259] Just as ILs loaded with a prodrug offer unique advantages, antibody-drug conjugates (ADCs) combine the precision of monoclonal antibodies with the potency of highly cytotoxic agents, and there are several ADCs currently in use for treating hematological malignancies (63, 64). Nonetheless, toxicity remains a significant concern (65). While ILs employ a strategy similar to ADCs, they offer certain advantages by modifying the pharmacokinetic properties of drugs, facilitating faster intracellular penetration and higher drug concentrations within target cells (65, 66). Numerous experiments have focused on enhancing the solubility, stability, circulation time, and drug-targeting properties of ILs (11-14). The lipophilic nature of valrubicin amplifies its cytotoxic effects by elevating intracytoplasmic drug concentrations and enabling it to traverse cell membranes with lower systemic toxicity (44). Our Val-ILs contained only a minimal amount of valrubicin, quantified at 1.52* ICT8pmol per particle using HPLC- MS / MS. Therefore, the utilization of Val-ILs loaded with valrubicin required 32.7-fold less of the drug to achieve a similar effect on cell viability compared to direct drug application. In our preclinical study involving PDX mice, we calculated that just 0.145 mg / kg of valrubicin, when incorporated into Val-ILs, was enough to reduce the expansion of leukemia cells in vivo. Val- ILs combine the precision of monoclonal antibodies with the potency of highly cytotoxic agents, potentially diminishing the severity of side effects by selectively delivering the drug to the tumor site. Traditionally, chemotherapy for leukemia involves frequent administration and is associated with severe side effects due to the non-specificity of the drugs. However, since Val-ILs contained a very low dose of the lipophilic drug and an antibody designed to specifically target the population of leukemia cells, the risk of in vivo side effects should be significantly reduced. One potential limitation of Val-ILs is that the particles loaded with antibodies targeting CD 19, CD7, or CD33 might affect not only ALL or AML cells but also healthy hematopoietic cells in BM, including B- and T-lymphocytes, as well as myeloid cells. However, this limitation is present in other therapeutic strategies including ADCs and CAR-T immunotherapies.

[0260] Another challenge detected in our preclinical study was the ability of Val-ILs to penetrate lymphoma tumors. In response, we developed Val-ILs targeting immunosuppressive cells in the spleen, including Val-ILs-aCDl lb, which target MDSC, and Val-ILs-aCD223, which target lymphocyte-activation gene 3 (LAG-3 or CD223) on T4 lymphocytes. These nanoparticles significantly influenced the development of lymphoma in vivo. Furthermore, the combination therapy involving both Val-ILs-aCDl lb and Val-ILs-aCD223 showed superior effectiveness in preventing the growth of lymphoma tumors. Interestingly, we found that treating mice with a combination of Val-ILs-aCDl lb and Val-ILs-aCD223 was more beneficial than using Val-ILs loaded with both antibodies. One possible explanation for this observation could be that Val-ILs loaded with both antibodies might be captured by CD1 Ibexpressing cells, which are more abundant among immune cells. Consequently, Val-ILs loaded with both antibodies may not effectively target the rarer population of cells, such as CD223- positive T4 lymphocytes, essential for lymphoma prevention.

[0261] In conclusion, our findings strongly indicate that immunotargeting with liposomes containing a lipophilic antitumor prodrug is a novel and effective strategy for targeting and inducing the death of leukemia cells. Furthermore, we have demonstrated that these nanoparticles serve as a valuable tool to target and induce the death of immunosuppressive cells, thus enhancing the antitumor immune response in the context of lymphoma. The interesting results obtained from our preclinical tests hold significant potential and should be further explored, serving as a stepping stone for future clinical trials in the field of hematology. This study is a promising preclinical demonstration of the effectiveness and ease of preparation of Val-ILs, which is a novel nanoparticle technology that could potentially yield effective therapies for hematological cancers based on targeted vesicle-mediated cell death. Table 1: Antibody used to prepare Val-ILs for the treatment of leukemia in PDX models. List of antibodies used to prepare Val-ILs, including IgG isotypes control antibodies.

[0262] Antigen Fluorochrome Supplier _ Reference

[0263] CD19 BV605 BD Biosciences 740394

[0264] IgGl, k BV605 BD Biosciences 562652

[0265] CD7 VioBright-FITC Miltenyi biotech 130-105-844 lgG2b VioBright-FITC Miltenyi biotech 130-104-575

[0266] CD33 PE-Cy5 BD Biosciences 551377

[0267] IgGl, k PE-Cy5 BD Biosciences 555750

[0268] Table 2: Antibody used to prepare Val-ILs for the treatment of EL4 lymphoma mice. List of antibodies used to prepare Val-ILs, including IgG isotypes control antibodies.

[0269] Antigen Fluorochrome Supplier Reference

[0270] CDllb APC-R700 BD Biosciences 564985 lgG2b, k APC-R700 BD Biosciences 564984

[0271] CD233 BV650 BD Biosciences 740560

[0272] IgGl, k BV650 BD Biosciences 563848

[0273] CD25 PE-Cy7 BD Biosciences 552880

[0274] F4 / 80 BV605 BD Biosciences 743281

[0275] Ly6c VioGreen Miltenyi biotech 130-102-207

[0276] IL- 17 PE-Cy7 Biolegend 506921

[0277] PD1 VioBlue Miltenyi biotech 130-121-437

[0278] PD-L1 BV605 Biolegend 124321

[0279] CTLA4 APC Miltenyi biotech 130-102-518

[0280] CD3E PE-Cy7 BD Biosciences 552774

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Claims

CLAIMS:

1. A composition comprising a lipid nanoparticle loaded with antitumoral agent and said lipid nanoparticle being associated with an antigen binding agent to target immunosuppressive cells.

2. The composition according to claim 1, wherein the lipid nanoparticle is a liposome.

3. The composition according to claim 1 or 2, wherein the antitumoral agent is a hydrophobic antitumoral agent.

4. The composition according to anyone of claims 1 to 3, wherein the antigen binding agent is a monoclonal antibody.

5. The composition according to claims 4, wherein the monoclonal antibody is selected from the list consisting of CD1 lb, CD223, CD25 CD127 or IL17alpha antibody6. The composition according to claims 4, wherein the monoclonal antibody is CDl lb antibody or CD223 antibody.

7. The composition according to anyone of claims 1 to 6, wherein the composition is combined with an immune checkpoint inhibitor.

8. A composition according to anyone of claim 1 to 7 for use in the prevention or the treatment of a patient affected with a cancer disease.

9. The composition for use according to claim 8 wherein the cancer disease is selected from the group consisting a solid cancer or malignant haematological disease.

10. The composition for use according to claim 9 wherein the solid cancer is lymphoma breast cancer, lung cancer, colorectal cancer.

11. The composition for use according to claim 9 wherein the malignant haematological disease is selected from the group consisting of B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), or acute myeloid leukemia (AML).

12. A composition according anyone of claim 1 to 7 for use in the treatment of drug resistant tumor or tumor relapse in a patient affected with cancer disease.

13. A pharmaceutical composition comprising a composition according to any one claims 1 to 7 and a pharmaceutically acceptable carrier.

14. The pharmaceutical composition according to claim 13 further comprising an additional therapeutic agent.

15. A method of preventing or cancer disease in a subject comprising administering to the subject a therapeutically effective amount of a composition according to any one claims 1 to 7.

16. A process for preparing a suspension of liposomes, comprising the steps of: a) Mixing a phospholipid and a lipid with a reactive moiety with an organic solvent; b) Adding a lipophilic antitumoral agent and removing organic solvent by evaporation; c) incubating the mixture at a temperature between 30 to 100°C preferably 60 and 70°C, and vortex the mixture; d) sonicated the vesicles for several cycles to obtain unilamellar vesicles (UVs); e) adding antigen bidding agent directed against immunosuppressive cells just before the purification process via dialysis; f) Recovering a suspension of liposome loaded with antitumoral agent and associated antigen bidding agent directed against immunosuppressive cells, said liposome having a diameter ranging from 100 nm to 1 pm.

Images