Engineered antigen presenting cells and uses thereof

Abstract

The present invention relates to engineered receptors that have the ability to enhance uptake, processing, and presentation of tumor-associated antigens to T cells by a modified antigen-presenting cell, triggering its activation. It further relates to vectors or antigen presenting cells expressing said receptors, composition and uses thereof for the prevention and / or treatment of a cancer.

Description

[0001] ENGINEERED ANTIGEN PRESENTING CELLS AND USES THEREOF

[0002] Field of the Invention

[0003] The present invention relates to the delivery of immunostimulatory cells expressing extracellular vesicle-internalizing receptors and their use in the immunotherapy for prevention and / or treatment of cancer.

[0004] Background of the Invention

[0005] Directing or enabling the immune system to recognize and eliminate cancer cells has become a clinically validated approach for treating cancer. Effective induction of anti-tumor immunity requires antigen-presenting cells (APCs), including dendritic cells (DCs), to acquire, process and present tumor antigens to T cells. Adoptive transfer of DCs loaded with tumor lysates or defined tumor antigens and subsequently matured ex vivo may help to boost anti-tumor immunity in patients with cancer. This method, called “DC vaccine”, has been tested in clinical trials with mixed results (Perez and De Palma, 2019, Nature Communications. Springer US, 10(1), 1 10. doi: 10.1038 / s41467-019-13368-y; Heras-Murillo et al., 2024, Nature Reviews Clinical Oncology. Springer US, 21(April). doi: 10.1038 / s41571-024-00859-1).

[0006] In addition to clinically approved immune checkpoint blockade (ICB) targeting the PD-L1 / PD-1 and CTLA4 pathways, cell therapies based on genetically modified T cells are gaining traction in the clinical arena (Morotti et al., 2021, British Journal of Cancer. Springer US, 124(11), 1759-1776. doi: 10.1038 / s41416-021-01353-6). DCs have also received significant attention due to their ability to present tumor-associated antigens (TAAs) and orchestrate immune responses against cancer. Personalized vaccines utilizing patient-derived DCs pulsed with TAAs have been extensively explored, yet their clinical efficacy has often fallen short despite demonstrating safety and clear proof of immunogenicity in a multitude of clinical trials (Perez andDe Palma, 2019, Nature Communications. Springer US, 10(1), 1 10. doi: 10.1038 / s41467-019-13368-y; Heras-Murillo et al., 2024, Nature Reviews Clinical Oncology. Springer US, 21 (April), doi: 10.1038 / s41571-024-00859-l). This discrepancy is attributed in part to inherent limitations of traditional DC vaccine formulations, including suboptimal antigen presentation and activation of the manufactured cell product (Perez and De Palma, 2019, supra). Efforts to bolster the efficacy of DC-based therapies have recently focused on developing cell engineering strategies for improving treatment efficacy (Perez and De Palma, 2019, supra). Leveraging immunologically competent DCs is paramount for effective vaccination. Indeed, DCs stimulated by danger signals upregulate co-stimulatory molecules essential for activating T cells and migrate to lymph nodes, where they initiate immune responses. Various maturation cocktails have been employed in clinical settings to induce the desirable DC maturation, with promising outcomes observed in select trials (Gu et al., 2020 Acta Pharmacologica Sinica. Springer US, (May), doi: 10.1038 / s41401-020-0415-5).

[0007] Traditional DC preparations typically involve patient-derived DCs - generally monocyte-derived DCs (moDCs) - loaded ex vivo with defined TAAs or patient-derived tumor lysates; this step is then followed by a maturation step aimed to promote activation of DCs and antigen presentation (Heras-Murillo et al., 2024, supra).

[0008] An alternative approach involves leveraging TAA uptake after administration of the DCs, allowing for the presentation of patient-specific TAAs directly in the body, without the need for a tumor biopsy or knowledge of specific antigens. Tumor-derived extracellular vesicles (EVs) and other tumor-derived particles may provide a source of relevant TAAs (Wolfers et al., 2001, Nature Medicine, 7(3), pp. 297- -303. doi: 10.1038 / 85438). In pursuit of this, DCs engineered to express a non-signaling, chimeric antigen receptor (CAR)-like molecule aimed at enhancing the uptake of tumor-derived EVs displaying a known, cancer-specific surface antigen, referred to as the "bait antigen" were developed (Squadrito et al., 2018, Nature Methods. Nature Publishing Group, 15(3), 183-186. doi: 10.1038 / nmeth.4579). Upon binding of the single-chain antibody fragment (scFv) to the bait antigen, the EV-internalizing receptor (EVIR) facilitated EV internalization and subsequent presentation of a wide range of TAAs, termed "prey antigens", found inside or on the surface of the EVs (Squadrito etal, 2018, Nature Methods. Nature Publishing Group, 15(3), 183-186. doi: 10.1038 / nmeth.4579). Priming of T cells by these EVIR-engineered DCs occurred primarily through "cross-dressing", wherein preformed major histocompatibility class I (MHCI)-TAA complexes were horizontally transferred from the cancer cells to the DCs via tumor-derived EVs, rather than being cross-presented on endogenous MHCI. While pre-formed, tumor-derived MHCI-antigen complexes may encompass immunologically relevant TAAs (Martinez-Usatorre and De Palma, 2022, EMBO Molecular Medicine, 14(10), 12-15. doi: 10.15252 / emmm.202216523), the observed lack of cross-presentation of EV-associated TAAs was a potential limitation of the EVIR strategy (Squadrito et al, 2018, supra), likely due to the fact EV uptake and internalization by the DCs was not associated with proper DC activation. Indeed, the EVIR lacks intracellular signaling domains, being based on a truncated low-affinity truncated (non-signaling) nerve growth factor receptor (ALNGFR) (Squadrito et al., 2018, supra). WO 2019 / 126358 describes the design of single chain chimeric agent receptors for expression in T cells (CAR-T cells) comprising a first intracellular signaling domain selected from DAP- 10 and DAP- 12, as second intracellular signaling domain selected from a large number of proteins without indication of substantial differences of effects between those and an immunoreceptor tyrosine-based activation domain, in particular comprising a cytoplasmic signaling sequence from CD3^.

[0009] Therefore, to address some of the limitations of traditional DC vaccines, there is still a need for developing anti-tumor vaccines able to induce strong and broad T-cell responses that are specific for multiple known and unknown TAAs on a personalized manner, but at the same time applicable to a broad range of patients.

[0010] Summary of the Invention

[0011] The invention is based on the design of new extracellular vesicle-internalizing receptors (EVIRs) that enable the DCs to (i) recognize a surface molecule present on cancer cells and their secreted EVs, (ii) phagocytose cancer cells or internalize EVs and other tumor-derived particles, which are sources of TAAs, and (iii) undergo immunostimulatory activation to prime antigen-specific T cells. One of the applications of this invention was to trigger the expression of immune-stimulatory molecules on demand, i.e. upon encountering cancer cells or tumor-derived EVs / particles. For these properties, the new EVIRs are termed “instructive EVIRs”, or iEVIRs.

[0012] It was shown that these new iEVIRs advantageously convert melanoma-derived EVs from immunosuppressive to stimulatory cues for DCs. Further, a ubiquitination-resistant iEVIR was designed and it was shown that DCs modified to express this ubiquitination-resistant iEVIR expanded antigen-specific T cells and delayed tumor growth in an immunotherapy -resistant melanoma model. DCs expressing those new iEVIRs provide a useful tool for antigen-agnostic cancer cell therapy by coupling antigen uptake with DC activation, without the need for ex vivo antigen loading or cell stimulation and maturation.

[0013] One aspect of the invention relates to a recombinant iEVIR directed against at least one cancercell surface molecule (called bait antigen).

[0014] Another aspect of the invention relates to an isolated nucleic acid molecule encoding an iEVIR according to the invention.

[0015] In another aspect, the invention provides a recombinant vector comprising a nucleic acid molecule encoding an EVIR according to the invention.

[0016] Another aspect of the invention relates to an isolated cell expressing at least one iEVIR of the invention, in particular an antigen-presenting cell (APC), such as DCs, monocytes, macrophages, or other hematopoietic cells, and compositions thereof. Another aspect of the invention provides an ex vivo method (i.e., in culture) of inducing expression of at least one iEVIR of the invention in an APC comprising the step of transfecting or transducing ex vivo said cell with a gene transfer vector according to the invention.

[0017] Another aspect of the invention relates to an isolated iEVIR-expressing APC obtainable by a method according to the invention.

[0018] Another aspect of the invention relates to an iEVIR-expressing APC or a recombinant vector according to the invention for use as a medicament.

[0019] Another aspect of the invention provides a pharmaceutical composition comprising cells of the invention or at least one recombinant vector according to the invention and at least one pharmaceutically acceptable carrier, diluent or excipient thereof.

[0020] Another aspect of the invention relates to an iEVIR-expressing APC or a recombinant vector according to the invention for use in the prevention and / or treatment of a cancer.

[0021] Another aspect of the invention relates to a use of an isolated iEVIR-expressing APC for the preparation of a pharmaceutical composition for the prevention and / or treatment of a cancer. Another aspect of the invention provides a method of inducing in vivo the expression of at least one iEVIR of the invention in an APC or a stem / progenitor cell thereof in a subject in need thereof, said method comprising a step of: administering a vector encoding an iEVIR according to the invention to said subject under suitable conditions for inducing transfection or transduction of the subject’s APCs, or stem / progenitor cell thereof, in vivo with said vector. Another aspect of the invention provides a method of preventing and / or treating a cancer comprising administering effective amounts of iEVIR-expressing APCs, or prospective stem / progenitor cells thereof, or at least one recombinant vector according to the invention in a subject in need thereof.

[0022] Another aspect of the invention provides a kit for carrying out methods according to the invention comprising at least one iEVIR, or at least one recombinant expression vector, or at least one iEVIR-expressing cell according to the invention.

[0023] Description of the figures

[0024] Figure 1 illustrates the screening of signaling EVIRs for antigen-directed DC activation as described in Example 1 for the design of EVIRs of the invention compared to a parental, nonsignaling EVIR (ALNGFR-EVIR or simply EVIR). A: The schematics illustrate the engineering of new EVIRs wherein the ligand-binding domains of the indicated DC receptors are replaced with an anti-HER2 scFv. B: Flow cytometry analysis of surface expression of the newly designed EVIRs in DCs (MutuDCs) using either anti-NGFR (above) or anti -Fab staining (below). A representative experiment is shown. C: Expression of HER2 in MC38 cancer cells (MC38-HER2). D: Expression of CD86 in MutuDCs after co-culture with MC38-HER2 cancer cells, measured by flow cytometry (mean± SEM; n = 3 independent cell cultures). Statistical analysis by one-way ANOVA.

[0025] Figure 2 illustrates an iEVIR according to the invention, which coordinates antigen binding and DC activation without tonic signaling as described in Example 2. A: Schematic representation of the iEVIR (“instructive EVIR”, or mutCD8 / CD40-FcRy-ITAM-EVIR); B:

[0026] Surface expression of the iEVIR or parental EVIR measured by anti-Fab flow cytometry analysis. C: Expression of the indicated DC activation markers measured by flow cytometry in MutuDCs transduced with iEVIR or EVIR and treated with cancer cells or EVs as indicated (mean± SEM; n= 3-4 independent cell cultures). MFI, mean fluorescence intensity. Statistical analysis by one-way ANOVA.

[0027] Figure 3 illustrates that an anti-GD2 iEVIR of the invention promotes DC activation specifically in response to GD2+ EVs as described in Example 3. Al & A2: Surface expression of the anti-GD2 iEVIR in MutuDCs, measured by anti-Fab staining using flow cytometry (Al), and surface expression of GD2 in cancer cells (A2). A representative experiment is shown for each. B-C: CD86 or MHCII expression in MutuDCs transduced and treated as indicated, measured by flow cytometry (mean ± SEM; n= 4 independent cell cultures). FACS dot plots in (B) show representative samples. Statistical analysis by one-way ANOVA. D: Cytokine quantification in medium conditioned by MutuDCs transduced and treated as indicated, measured with LEGENDplex™ kit (mean± SEM; n= 3 independent cell cultures). Statistical analysis by one-way ANOVA. E-G: Bulk RNA-seq analysis of MutuDCs transduced and treated with or without EVs as indicated (n= 3 independent cell cultures). The data show Hallmark pathways significantly deregulated (adjusted P-value < 0.05) in the indicated comparison.

[0028] Figure 4 illustrates that an iEVIR of the invention promotes antigen-dependent DC activation conducive to T-cell activation as described in Example 4. Al & A2: Flow cytometry analysis of OT-I cell proliferation in response to co-culture with MutuDCs. Al: Histogram plots of peaks of T cell proliferation cycles revealed by dilution of cell trace violet; representative samples are shown. A2: Quantification of T cells that performed 4 or 5 rounds of cell division (mean ± SEM; n= 3 independent cell cultures). B: Flow cytometry analysis of intracellular TNF in OT-I cells co-cultured with MutuDCs transduced and treated with EVs as indicated (mean± SEM; n= 3 independent cell cultures). Cl & C2: Flow cytometry analysis of OT-II cell proliferation in response to co-culture with MutuDCs transduced as indicated and co- cultured with MC38-HER2 / OVA cells. Cl: Histogram plots of peaks of T cell proliferation cycles revealed by dilution of cell trace violet; representative samples are shown. C2: T cells that performed 4 or 5 rounds of cell division (mean ± SEM; n= 3-4 independent cell cultures). Statistical analysis by one-way ANOVA. D: Flow cytometry analysis of intracellular IFNγ in OT-II cells co-cultured with MutuDCs transduced as indicated and co-cultured with MC38-HER2 / 0VA cells (mean± SEM; n= 3 independent cell cultures). Statistical analysis by oneway ANOVA.

[0029] Figure 5 illustrates that an iEVIR of the invention promotes antigen-dependent transactivation of an exogenous gene in DCs as described in Example 5. A: The schematic on the top shows the structure of the inducible promoter designed to couple expression of an exogenous gene (here FLAG-CD40L) to iEVIR-mediated DC activation. Two, 4 or 7 NFkB response elements were alternatively tested. The FACS dot plots on the bottom show the basal expression of FLAG-CD40L in MutuDCs measured by flow cytometry. B: The schematic on the top shows the structure of the inducible promoter with 2 NFkB response elements, designed to couple expression of an exogenous gene (here FLAG-CD40L) to iEVIR-mediated DC activation. The flow cytometry analysis on the bottom shows FLAG-CD40L expression in MutuDCs treated with CpG or left untreated. C-D: Flow cytometry analysis of FLAG-CD40L in MutuDCs cotransduced with the inducible FLAG-CD40L expression cassette and the indicated iEVIRs either against HER2 or GD2. Data show dot plots show representative samples (Cl & DI) and quantification of FLAG-CD40L expression (C2 & D2) (mean ± SEM; n= 3 independent cell cultures). Statistical analysis by one-way ANOVA.

[0030] Figure 6 illustrates that iEVIR-expressing DCs according to the invention provide therapeutic benefit without the need for antigen loading as described in Example 6. A: Procedure to study B16F10-GD2 / OVA tumor response to the combination of an anti-PDl monoclonal antibody (a-PDl) and transduced MutuDCs. Bl-2: B16F10-GD2 / OVA tumor growth. Graphs show mean tumor volume ± SEM (Ctrl-iEVIR + IgG, n=6; Ctrl-iEVIR + anti-PDl, n= 5; iEVIR + IgG, n=7; iEVIR + anti-PDl, n=7 tumors). Statistical analysis by two-way ANOVA. C:

[0031] Procedure to study B16F10-GD2 / OVA tumor response to the combination of an anti-PDl monoclonal antibody (a-PDl) and transduced moDCs. Dl-4: B16F10-GD2 / OVA tumor growth. The top left graph shows tumor volume in mice treated as indicated (mean± SEM; untransduced, n=5; Ctrl-iEVIR, n= 6; iEVIR, n=7 tumors). Statistical analysis by two-way ANOVA. The other graphs show the volume of individual tumors in the indicated cohorts. El-4: Flow cytometry analysis of intracellular TNF and IFNγ in tumor-derived CD8+ or CD4+ T cells after ex vivo re-stimulation (mean ± SEM; untransduced, n=5; Ctrl-iEVIR, n= 6; iEVIR, n=6). Statistical analysis by one-way ANOVA. F: Procedure to study survival of mice bearing B16F10-GD2 / OVA tumors treated as indicated. Gl: B16F10-GD2 / OVA tumor growth (mean volume ± SEM; Ctrl-iEVIR, n= 6; iEVIR, n=8), shown until day 15 (time-point when termination of the mice began). Statistical analysis by two-way ANOVA. G2: Tumor growth in individual mice (Ctrl-iEVIR, n= 6; iEVIR, n=8). H: Survival analysis of mice treated as indicated. Statistical analysis by Log-rank Mantel-Cox test.

[0032] Figure 7 illustrates that an ubiquitination-resistant iEVIR according to the invention improves antigen presentation by transduced DCs through EV internalization and cancer-cell phagocytosis, as described in Example 7. A: Schematic illustrating the iEVIRubNulldesign. B: Flow cytometry analysis of the indicated receptors in MutuDCs; representative samples are shown. Cl & C2: Flow cytometry analysis of PKH26 fluorescence, indicative of EV internalization, in MutuDCs transduced and treated with EVs as indicated (mean± SEM; Ctrl-EVIR, n= 4; EVIR, n= 5; iEVIR, n= 3; iEVIRubNull, n= 5 independent cell cultures). MFI, mean fluorescence intensity. D: Flow cytometry analysis of phagocytic moDCs, revealed by pHrodo Red dye. MoDCs transduced as indicated were co-cultured with B16F10-GD2 cells exposed to pHrodo Red (mean ± SEM; n= 5 independent cell cultures). Statistical analysis by one-way ANOVA.

[0033] Figure 8 illustrates that iEVIRubNull-expressing moDCs elicit antigen-specific T cells in tumors as described in Example 8. A: Procedure to study B16F10-GD2 / OVA tumor response to the combination of an anti-PDl monoclonal antibody (a-PDl) and transduced moDCs. B: Flow cytometry analysis of moDCs transduced as indicated. Cl-2: B16F10-GD2 / OVA tumor growth. Data show tumor volume (mean ± SEM; PBS, n=7; Ctrl-iEVIRubNull, n= 6; EVIR, n=8; iEVIR, n=9; iEVIRubNull, n=8). Statistical analysis by two-way ANOVA. The 5 cohorts are shown in two separate charts for clarity, with the PBS cohort shown in both. D: Flow cytometry analysis by dextramer staining of TRP2 or OVA-reactive CD8+ T cells (left and middle panels), or the combination of both (right panel), in tumors of mice treated as indicated (mean ± SEM; PBS, n=6; Ctrl-iEVIRubNull, n= 6; EVIR, n=8; iEVIR, n=9; iEVIRubNull, n=8). Statistical analysis by one-way ANOVA.

[0034] Detailed description

[0035] The term “a membrane-associated molecule” or “surface molecule” as used herein refers to any molecule that is physically embedded in the lipid bilayer or bound or anchored to a cell membrane permanently or transiently under specific conditions. The molecule may be associated with any membrane of the cancer cell, including the plasma membrane or intracellular membranes. These molecules could perform a variety of functions and belong to different functional groups including, but not limited to glycoproteins, lipids, membrane receptor proteins, transport proteins, membrane enzymes, cell adhesion molecules, and their mutated forms. These molecules can be expressed either on the cancer cell’s plasma membrane or any membrane associated with cancer-cell derived particles, such as extra-cellular vesicles (EVs). The term includes known and unknown cancer cell membrane-associated molecules, herein referred to as “bait antigens”. Examples of membrane-associated molecules include, but are not limited to, gangliosides (GD2, GD3), human epidermal growth factor receptor 2 (HER2), tyrosinase-related protein- 1 (TYRP1), carcinoembryonic antigen (CEA), mesothelin, PMEL (gplOO), and mucins.

[0036] The term “single-chain variable fragment (scFv) extracellular domain” refers to any scFv extracellular domain with specificity for any membrane-associated molecule expressed by a cancer cell or cancer-cell derived particle / vesicle. Examples of antibody domains according the invention include, but are not limited to: (i) anti-HER2 scFv, such as CHA21 (Zhou et al., 2011, The Journal of Biological Chemistry, 286: 31676-31683), a trastuzumab-based scFv (Morgan etal., 2010, Mol Ther., 18(4):843-51), a pertuzumab-based scFv (Franklin etal, 2004, Cancer Cell, 5(4):317-28) and a FRP5-based scFv (Ahmed et al., 2009, Mol Ther., 17(10): 1779-87} (ii) anti-GD2 scFv (Newik et al, 2016, Mol Ther Oncolytics, 68:139-152} or (iii) anti-TYRPl scFv (Saenger et al., 2008, Cancer Res, 68(23); 9884-91} among others.

[0037] An iEVIR of the invention may also contain a “signal peptide”, which refers to any protein fragment, either cellular or viral, that increases sorting of the iEVIR to the cell surface. A nonlimiting example is an IgK domain (von Heijne et al., 2006, Nat Rev Mol Cell BioL, 7:909-18), for example inserted at the N-terminus of the iEVIR.

[0038] The term “an antigen-presenting cell” or “APC” as referred to herein, refers to a cell that displays foreign antigens complexed with major histocompatibility complexes (MHCs) on its surface; this process is known as antigen presentation. Those cells are also sometimes referred to as or “accessory cell”. T cells may recognize these complexes using their T-cell receptors (TCRs), so APCs process antigens and present them to T-cells. Examples of APCs include, but are not limited to, dendritic cells (DCs), monocytes, macrophages, certain B cells, other hematopoietic cells, and certain activated epithelial cells.

[0039] The term tumor-associated antigen (TAA) refers to a molecule present in the cancer cell, herein also referred to as “prey antigen”, that may induce an immune response against the tumor. The term “EVIR-expressing cell” refers herein to an APC expressing an iEVIR according to the invention and, optionally, an iEVIR-regulated protein capable of attracting and / or activating other immune cells, which after being contacted with cancer cells and / or cancer-cell derived particles, such as EVs, has internalized the cancer cell and / or cancer-cell derived particles and processed TAAs, so that TAAs presentation was achieved in the APC expressing the iEVIR. The term “extracellular vesicles” or “EVs” refers herein to any membrane-containing particles or fragments derived from cancer cells. EVs may comprise exosomes, microvesicles, microparticles, apoptotic bodies, cell debris, membrane fragments and similar subcellular material of tumor origin that, therefore, may be associated with known and unknown tumor antigens. After fusion of the EV with the engineered APC, the EV-associated tumor antigens are presented by the engineered APCs to T cells in order to initiate an immune response against cancer. EVs can be isolated as described (Squadrito et al., 2014, Cell Rep, 8(5):1432-46; Thery et al., 2006, Curr Protoc Cell Biol, Chapter 3; Unit 3:22). Presentation of TAAs by the APCs may occur after processing and loading of the antigens on the APC’s MHCI or MHCII molecules (conventional and cross-presentation) as described in Villadangos et al., 2014, Immunity, 29(3):352-61, but also by direct presentation of EV-associated antigen / MHC complexes via cross-dressing as described in Scholzel etal., 2014, J Hepatol., 61(3):600-8 and Squadrito et al., 2018, supra.

[0040] The terms “cancers” or “tumors” as defined herewith are diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. Term “cancers” designate diseases exemplified by, but not limited to, epithelial carcinomas (such as tumors of the breast, prostate, lung, pancreas, liver, kidney, skin, esophagus, stomach and intestine), melanomas (tumors of the melanocytes), sarcomas (such as tumors of the bone and muscle), lymphomas and leukemias (tumors of the hematopoietic system), germ cell tumors (tumors of the gonads), neuroendocrine tumors (such as tumors of the lung, pancreas and intestine), and gliomas (tumors of the central nervous system).

[0041] As used herein, “treatment” and “treating” and the like generally mean obtaining a desired pharmacological and physiological effect. The effect may be prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof and / or may be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease. The term “treatment” as used herein covers any treatment of a cancer in a mammal, particularly a human, and includes inhibiting the disease, i.e., arresting its development; or relieving the disease, i.e., causing regression of the disease and / or its symptoms or conditions such as improvement or remediation of damage. In particular, the cells, methods, uses, formulations and compositions according to the invention are useful in the treatment of cancer and / or in the prevention of evolution of a cancer into an advanced or metastatic stage in patients with early stage cancer, thereby improving the cancer staging and patient prognosis. In particular, prevention and / or treatment of a cancer may include administration of cells according to the invention.

[0042] The term “efficacy” of a treatment or method according to the invention can be measured based on changes in the course of disease or condition in response to a use or a method according to the invention. For example, the efficacy of a treatment or method according to the invention can be measured by its impact on signs or symptoms of illness. A response is achieved when the patient experiences partial or total alleviation, or reduction of unwanted symptoms of illness. According to a particular embodiment, the efficacy can be measured through the measuring of the elicited immune response against cancer cells such as by analyzing tumorspecific T cells or by assessing cancer cell death and / or inhibition of tumor growth, progression and dissemination.

[0043] The term “effective amount” as used herein refers to an amount of at least one cell according to the invention, or a pharmaceutical formulation thereof, that elicits a detectable reduction of the symptoms of the disease in a subject that is being administered said cells, these symptoms can include, for instance decrease in solid tumor mass.

[0044] The term “subject” as used herein refers to mammals. For examples, mammals contemplated by the present invention include human, primates, domesticated animals such as cattle, sheep, pigs, horses, laboratory rodents, other pets and the like.

[0045] The term “variant” as used herein means a polypeptide substantially homologous to the original peptide sequence, but which has at least one an amino acid sequence different from that of the original sequence because of one or more deletions, insertions or substitutions. Substantially homologous means a variant amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the original amino acid sequences, as disclosed above. The percent identity of two amino acid sequences can be determined by visual inspection and / or mathematical calculation, or more easily by comparing sequence information using known computer program used for sequence comparison such as Clustal package version 1.83. A variant may comprise a sequence having at least one conservatively substituted amino acid, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics. Generally, substitutions for one or more amino acids present in the original polypeptide should be made conservatively. Examples of conservative substitutions include substitution of one aliphatic residue for another, such as He, Vai, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gin and Asn. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known (Kyte, et al, 1982, J. Mol. Biol, 157: 105- 131). For example, a "conservative amino acid substitution" may involve a substitution of a native amino acid residue with a non-native residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired.

[0046] EVIRs according to the invention

[0047] Instructive extracellular vesicle internalizing receptors (iEVIRs) of the invention comprise:

[0048] (i) a signal peptide;

[0049] (ii) a single-chain variable fragment (scFv) extracellular domain specific for a membrane-associated molecule of a cancer cell;

[0050] (iii) a transmembrane / hinge domain;

[0051] (iv) an intracellular CD40 activation domain; and

[0052] (v) an intracellular FcRy chain ITAM motif.

[0053] In a particular embodiment, is provided an iEVIR according to the invention wherein the signal peptide is an IgK signal peptide of SEQ ID NO: 11 or a variant thereof.

[0054] In a particular embodiment, is provided an iEVIR according to the invention wherein the singlechain variable fragment (scFv) extracellular domain specific for a membrane-associated molecule of a cancer cell comprises a sequence specific for one or more of the following: human epidermal growth factor receptor 2 (HER2), gangliosides (GD2, GD3), tyrosinase-related protein- 1 (TYRP1), carcinoembryonic antigen (CEA), mesothelin, PMEL (gplOO), and mucins. In a more particular embodiment, is provided an iEVIR according to the invention wherein the single-chain variable fragment (scFv) extracellular domain comprises a sequence of an antibody specific for an anti-human epidermal growth factor receptor 2 (HER2).

[0055] In another more particular embodiment, is provided an iEVIR according to the invention wherein the single-chain variable fragment (scFv) extracellular domain comprises a sequence of an antibody specific for a ganglioside GD2 (GD2).

[0056] In a more particular embodiment, is provided an iEVIR according to the invention wherein the sequence of the single-chain variable fragment (scFv) comprises or consists in the sequence of SEQ ID NO: 12, or a variant thereof.

[0057] In another more particular embodiment, is provided an iEVIR according to the invention wherein the single-chain variable fragment (scFv) comprises or consists in the sequence of SEQ ID NO: 13, or a variant thereof. In another more particular embodiment, is provided an iEVIR according to the invention wherein the transmembrane / hinge domain is a CD8a domain.

[0058] In another more particular embodiment, is provided an iEVIR according to the invention wherein the transmembrane / hinge domain is a mutated CD8a domain for disrupting spontaneous (ligand-independent) dimerization / multimerization of the chimeric receptor to limit potential tonic signaling. In a particular embodiment, is provided an iEVIR according to the invention wherein the CD8a domain comprises or consists in SEQ ID NO: 15 or a variant thereof.

[0059] In a particular embodiment, is provided an iEVIR according to the invention wherein the intracellular CD40 activation domain comprises or consists in SEQ ID NO: 16 or a variant thereof, in particular a variant wherein lysines are replaced by arginines.

[0060] In a particular embodiment, is provided an iEVIR according to the invention wherein the intracellular FcRy chain ITAM motif comprises or consists in SEQ ID NO: 17 or a variant thereof, in particular a variant wherein lysines are replaced by arginines.

[0061] In a particular embodiment, is provided an iEVIR according to the invention that further comprises a Strep-tag II peptide or another peptide, such HA-Tag, His-Tag or FLAG, that facilitates marking of the cells that express the iEVIR.

[0062] In a particular embodiment, is provided an iEVIR according to the invention wherein the Strep-tag II peptide comprises or consists in SEQ ID NO: 14 or a variant thereof.

[0063] In a particular embodiment, is provided an iEVIR according to the invention that further comprises an amino acid sequence that facilitates DNA engineering (e.g. a cloning site). iEVIRs according to the invention can be obtained by any known methods of molecular cloning for polypeptide expression, as described in the following examples.

[0064] According to a further particular embodiment, an iEVIR according to the invention has an amino acid sequence selected from SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 or variants thereof.

[0065] Nucleic acids of the invention

[0066] Isolated nucleic acid encoding an iEVIR according to the invention may be, for instance, natural DNA or RNA or a recombinant or synthetic DNA, RNA or LNA or a recombinant nucleic acid molecule comprising any of the nucleic acid molecules according to the invention either alone or in combination. In a particular embodiment, the nucleic acid molecules according to the invention are cDNA. In a particular embodiment, is provided an isolated nucleic acid molecule encoding an iEVIR according to the invention.

[0067] According to a further particular embodiment, is provided an isolated nucleic acid encoding iEVIRs of the invention that comprises:

[0068] (i) a nucleic acid sequence encoding a signal peptide;

[0069] (ii) a nucleic acid sequence encoding a single-chain variable fragment (scFv) extracellular domain specific for a membrane-associated molecule of a cancer cell; (iii) a nucleic acid sequence encoding a transmembrane / hinge domain;

[0070] (iv) a nucleic acid sequence encoding an intracellular CD40 activation domain; and (v) a nucleic acid sequence encoding an intracellular FcRy chain ITAM motif.

[0071] According to another further particular embodiment, is provided an isolated nucleic acid sequence encoding iEVIRs of the invention comprises:

[0072] (i) a nucleic acid sequence encoding a signal peptide;

[0073] (ii) a nucleic acid sequence encoding a single-chain variable fragment (scFv) extracellular domain specific for a membrane-associated molecule of a cancer cell; (vi) a nucleic acid sequence encoding a transmembrane / hinge domain;

[0074] (iii) a nucleic acid sequence encoding an intracellular CD40 activation domain comprises or consists in SEQ ID NO: 16 or a variant thereof; and

[0075] (iv) a nucleic acid sequence encoding an intracellular FcRy chain ITAM motif of SEQ ID NO: 17 or a variant thereof.

[0076] According to a particular aspect, a nucleic acid sequence encoding a Strep-tag II peptide of SEQ ID NO: 14 may be added to the above coding nucleic acid sequences that facilitates marking of the cells that express the iEVIR.

[0077] In another embodiment, is provided an isolated nucleic acid molecule encoding an iEVIR according to the invention comprising a single-chain variable fragment (scFv) extracellular domain comprising a sequence specific for an anti-human epidermal growth factor receptor 2 (HER2), for example wherein the said nucleic acid molecule comprises a sequence of SEQ ID NO: 12

[0078] In another embodiment, is provided an isolated nucleic acid molecule encoding an iEVIR according to the invention comprising a single-chain variable fragment (scFv) extracellular domain comprising a sequence specific for specific for a ganglioside GD2 (GD2), wherein the said nucleic acid molecule comprises a sequence of SEQ ID NO: 13. In another embodiment, is provided an isolated nucleic acid molecule encoding an iEVIR according to the invention, further comprising nucleic acid sequence encoding an inducible system for the expression of immune-stimulatory molecules (e.g., CD40L), wherein said expression is inducible only once the said APC gets in contact with the cancer-cell surface molecule (called bait antigen)-positive cancer cells and / or cancer cell-derived particles.

[0079] According to a particular aspect, is provided an ex vivo method (i.e., in culture) of inducing expression of at least one iEVIR of the invention in an APC comprising the step of ex vivo transfecting or transducing said cell with a nucleic acid vector according to the invention to obtain engineered APCs, such as DCs, expressing at least one iEVIR of the invention on its surface.

[0080] According to a further particular embodiment, is provided an isolated nucleic acid sequence encoding an inducible system for the expression of a chimeric FLAG-CD40L protein of SEQ ID NO: 10

[0081] According to a further particular embodiment, is provided an isolated nucleic acid sequence encoding inducible expression system comprising a synthetic IL12 promoter sequence with NF-kB response elements of SEQ ID NO: 9.

[0082] According to a further particular embodiment, is provided a nucleic acid encoding an iEVIR of the invention further comprising a nucleic acid sequence encoding an inducible system for the expression of a chimeric CD40L protein of SEQ ID NO: 10 regulated by a synthetic IL12 promoter sequence with NF-kB response elements (Res, GGAATTTCC).

[0083] According to a further particular embodiment, is provided a nucleic acid encoding an iEVIR of the invention and comprising at least one of the nucleic acid sequences of SEQ ID NO: 21 to 27.

[0084] According to a further particular embodiment, is provided an engineered APC expressing at least one iEVIR of the invention that would induce a chimeric CD40L protein of SEQ ID NO: 10 regulated by a synthetic IL12 promoter sequence comprising NF-kB response elements (Res), only once the said APC gets in contact with the bait antigen-positive cancer cells and / or cancer cell-derived particles.

[0085] Vectors and methods for cell transduction

[0086] In one embodiment, the invention provides a recombinant expression vector comprising a nucleic acid molecule according to the invention, wherein the vector optionally comprises an expression controlling sequence, allowing expression in eukaryotic host cells of the encoded sequence, operably linked to said nucleic acid molecule. Different expression systems can be used, including without limitation chromosomes, episomes, plasmids, virus-derived vectors, and RNA compositions. More particularly, the recombinant vectors used can be derived from bacterial plasmids, transposons, yeast episomes, insertion elements, yeast chromosome elements, and viruses such as baculovirus, papilloma viruses such as SV40, vaccinia viruses, adenoviruses, fox pox viruses, pseudorabies viruses, herpes viruses, retroviruses, lentiviruses, and adeno-associated viruses (AAVs). These recombinant vectors can equally be made of RNA, either free or incapsulated in nanoparticles. In one embodiment, the recombinant vectors are any viral vectors selected from adenoviral vectors, adeno-associated vectors, retroviral vectors (both replication-competent and replication-defective retroviral vectors), lentiviral vectors, in particular bidirectional lentiviral vectors.

[0087] In a particular embodiment, the recombinant vector is a lentiviral vector.

[0088] In one embodiment, the invention provides a recombinant expression vector comprising nucleic acid molecules encoding for one or more than one iEVIR sequence of the invention.

[0089] In one embodiment, the invention provides a recombinant expression vector comprising at least one nucleic acid sequence encoding an inducible system for the expression of a chimeric CD40L protein regulated by a synthetic IL 12 promoter sequence with NF-kB response elements.

[0090] In one embodiment, the invention provides a recombinant expression vector comprising nucleic acid molecules encoding for one or more than one iEVIR sequence of the invention further comprising at least one nucleic acid molecule encoding an inducible system for the expression of a chimeric CD40L protein regulated by a synthetic IL12 promoter sequence with NF-kB response elements.

[0091] The nucleic acid sequence can be inserted in the recombinant expression vector by methods well known to a person skilled in the art such as, for example, those that are described in Molecular Cloning: A Laboratory Manual, Sambrook et al, 4th Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 2001.

[0092] Recombinant vectors can include nucleotide sequences that allow, control or regulate the expression and the transcription of a polynucleotide of the invention as well as the translation of an iEVIR of the invention, these sequences being selected according to the host cells that are used. For example, an appropriate secretion signal can be integrated in the recombinant vector so that the iEVIR, encoded by the nucleic acid molecule of the invention, will be directed to the membrane. In a further embodiment, is provided a host cell comprising a recombinant vector according to the invention.

[0093] The introduction of the recombinant vector in a host cell can be carried out according to methods that are well known to a person skilled in the art, such as those described in Basic Methods in Molecular Biology, Davis et al., 2nded., McGraw-Hill Professional Publishing, 1995, and Molecular Cloning: A Laboratory Manual, supra, such as transfection by calcium phosphate, transfection by DEAE dextran, transfection, microinjection, transfection by cationic lipids, electroporation, transfection by nanoparticles, transduction or infection.

[0094] In another embodiment, the invention provides a method for producing APCs capable of expressing an iEVIR, said method comprising contacting cells with a vector or a nucleic acid according to the invention.

[0095] The iEVIRs can be delivered to APCs using a lentiviral vector (or alternative viral or non-viral vector), either ex vivo on isolated APCs (or precursors thereof) or in vivo via systemic (e.g., intravenous) or local (e.g., intra-tumoral, peri-tumoral, lymphnodal, etc.) delivery of a vector of the invention encoding said iEVIRs.

[0096] In particular, the invention provides a process for producing an APC or any stem of progenitor cell thereof, expressing at least one iEVIR according to the invention, comprising contacting said APCs or stem of progenitor cell thereof, in particular DCs, monocytes or macrophages, either ex vivo or in vivo with a vector or a nucleic acid encoding said at least one iEVIR according to the invention.

[0097] iEVIR-expressing APCs

[0098] According to an embodiment, the invention provides an APC expressing at least one iEVIR according to the invention.

[0099] According to an embodiment, the invention provides a cell expressing one iEVIR according to the invention.

[0100] According to a particular embodiment, the invention provides a cell expressing at least 2, at least 3, at least 4 different iEVIRs of the invention.

[0101] According to a particular embodiment, the invention provides a cell expressing at least one iEVIR according to the invention, for example from about 1 to about 3 different iEVIRs of the invention.

[0102] According to a particular embodiment, the invention provides an APC expressing one iEVIR according to the invention further expressing at least one immune-stimulatory molecule (e.g., CD40L), wherein said expression is induced only once the said APC gets in contact with cancer-cell surface molecule (called bait antigen)-positive cancer cells and / or cancer cell-derived particles.

[0103] According to an embodiment, is provided a cell composition comprising APCs expressing at least one iEVIR of the invention, wherein at least 1%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the APCs, in particular monocytes, macrophages or DCs, express at least one iEVIR of the invention. According to a further embodiment, is provided a cell composition of the invention, wherein at least 1% of the cell population expresses at least one iEVIR of the invention.

[0104] According to an embodiment, is provided a cell composition of the invention, wherein expression of the iEVIR persists for at least several hours after delivery with a vector of the invention.

[0105] According to an embodiment, is provided a cell composition of the invention, wherein at least 1% of the cell population expresses at least one iEVIR of the invention and said expression persists for at least several hours after delivery with a vector of the invention.

[0106] According to one embodiment, the expression level and time of iEVIRs can be measured by methods such as flow cytometry, protein analysis, or nucleic acidic amplification.

[0107] According to another embodiment, the invention provides a cell according to the invention, wherein said cell is an APC.

[0108] In another embodiment, the invention provides iEVIR-expressing cells that are able to internalize EVs to the cell cytoplasm.

[0109] In another embodiment, the invention provides iEVIR-expressing cells with enhanced phagocytic activity capabilities of cancer-cell derived EVs, effectively internalizing extracellular vesicles (EVs), cancer cells or other cancer-cell derived particles, leading to improved antigen presentation as compared to the same cells not expressing an iEVIR of the invention, even when expressing a non-signalling receptor.

[0110] In another embodiment, the invention provides iEVIR-expressing cells with a further improved iEVIR that is less susceptible to degradation by ubiquitination, thereby enhancing EV uptake. According to one embodiment, the internalization level and kinetics of EVs by iEVIR-expressing cells can be measured by methods such as flow cytometry and protein analysis. Methods and uses according to the invention

[0111] In a particular embodiment, the invention provides a method of inducing expression of iEVIRs according to the invention in APCs, said method comprising the step of transfecting or transducing said cells with a vector according to the invention. Another aspect of the invention provides a method of inducing in vivo the expression of at least one iEVIR of the invention in an APC or a stem / progenitor cell thereof in a subject in need thereof, said method comprising a step of: administering a vector encoding an iEVIR according to the invention to said subject under suitable conditions for inducing transduction of the subject’s APCs or stem / progenitor cell thereof in vivo with said vector.

[0112] Another aspect of the invention provides a method of preventing and / or treating a cancer comprising administering effective amounts of iEVIR-expressing APCs or at least one recombinant vector according to the invention in a subject in need thereof.

[0113] In another embodiment, is provided a method of inducing an immune response to cancer cells in a subject, said method comprising the step of administering iEVIR-expressing vectors or cells according to the invention in a patient in need thereof, wherein said iEVIR-expressing vectors or cells are administered alone, or in combination with another anti-cancer therapy. Standard procedures used in DC vaccination procedures might be used.

[0114] In one embodiment, is provided a method of the invention comprising the step of delivering an iEVIR-expressing vector via systemic (e.g., intravenous) or local (e.g., intra-tumoral, peri-tumoral, lymph nodal, etc.) routes to a cancer subject.

[0115] In a particular embodiment, the APCs or stem / progenitor cells thereof are autologous, i.e. originating from the patient to be treated.

[0116] In a particular embodiment, the APCs or stem / progenitor cells thereof are allogenic, i.e. originating from a donor related or unrelated to the patient.

[0117] In another aspect, the invention provides a use of cells according to the invention for the preparation of a vaccine for treating and / or preventing a cancer.

[0118] According to a particular aspect, the iEVIRs of the invention significantly enhance the uptake of cancer cell-derived EVs and / or other particles and improve antigen presentation (enhanced antigen-specific T cells in the tumor), leading to a more robust activation of the immune system, compared to the parental iEVIR.

[0119] According to a particular aspect, the iEVIRs of the invention include an inducible system that allows for on-demand expression of immune-stimulatory molecules (e.g., CD40L) which adds a layer of control and precision for the treatment, enabling targeted immune activation in response to tumor-derived EVs.

[0120] According to a particular aspect, the iEVIRs of the invention can be used to develop vaccines tailored to the tumor of individual patients, provided that the patient’s tumor expresses the bait antigen (e.g., GD2, HER2, etc.) recognized by the iEVIR. Therefore, the iEVIR-expressing vectors or cells according to the invention and methods thereof are particularly advantageous over the existing approaches in immunotherapy and / or in the prevention and / or treatment of cancers.

[0121] Compositions according to the invention

[0122] Pharmaceutical compositions or formulations according to the invention may be administered as a pharmaceutical formulation, which contains iEVIR-expressing vectors or cells as described herewith.

[0123] According to a particular aspect, pharmaceutical compositions, formulations or uses according to the invention comprise the combination of an iEVIR-based treatment of the invention with therapeutic agents that enhance the adaptive immune system’s activity against the tumor, including but not limited to PD1, PDL1 and CTLA4 inhibitors.

[0124] The invention provides pharmaceutical or therapeutic cells as compositions and methods for treating a subject, preferably a mammalian subject, and most preferably a human patient who is suffering from a cancer.

[0125] Cells of the invention or formulations thereof may be administered as a pharmaceutical formulation, which can contain one or more co-agents according to the invention in any form described herein. The compositions according to the invention, together with a conventionally employed adjuvant, carrier, diluent or excipient may be placed into the form of pharmaceutical compositions and unit dosages thereof, and in such form may be employed as solids, such as tablets or filled capsules, or liquids such as solutions, suspensions, emulsions, elixirs, or capsules filled with the same, all for oral use, or in the form of sterile injectable solutions for parenteral use by injection or continuous infusion. Injectable compositions are typically based upon injectable sterile saline or phosphate-buffered saline or other injectable carriers known in the art. Such pharmaceutical compositions and unit dosage forms thereof may comprise ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended dosage range to be employed.

[0126] Compositions of this invention may be liquid formulations including, but not limited to aqueous or oily suspensions, solutions, emulsions, syrups, and elixirs. The compositions may also be formulated as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain additives including, but not limited to, suspending agents, emulsifying agents, non-aqueous vehicles and preservatives. Suspending agents include, but are not limited to, sorbitol syrup, methylcellulose, glucose / sugar syrup, gelatin, hydroxy ethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, and hydrogenated edible fats. Emulsifying agents include, but are not limited to, lecithin, sorbitan monooleate, and acacia. Preservatives include, but are not limited to, methyl or propyl p-hydroxybenzoate and sorbic acid. Dispersing or wetting agents include but are not limited to poly(ethylene glycol), glycerol, bovine serum albumin, Tween®, Span®.

[0127] Compositions of this invention may also be formulated as a depot preparation, which may be administered by implantation or by intramuscular injection.

[0128] The compounds of this invention can also be administered in sustained release forms or from sustained release drug delivery systems.

[0129] According to a particular embodiment, compositions according to the invention are for intravenous use.

[0130] According to a particular embodiment, compositions according to the invention are for intratumoral use.

[0131] According to a particular embodiment, compositions according to the invention are for subcutaneous use.

[0132] According to a particular embodiment, compositions according to the invention are for intralymphnodal use.

[0133] According to a particular aspect, compositions of the invention are vaccine compositions comprising adjuvants.

[0134] In another particular aspect, compositions according to the invention are adapted for delivery by single administration.

[0135] According to a particular embodiment, compositions of the invention are veterinary compositions.

[0136] Further materials as well as formulation processing techniques and the like are set out in Remington’s “The Science and Practice of Pharmacy”, 23rdEdition, 2020, University of the Sciences in Philadelphia. Published by Elsevier Inc., Academic Press, https: / / doi. org / 10.1016 / C2018-0-04991-9, which is incorporated herein by reference.

[0137] In another aspect, the invention provides compositions comprising vectors according to the invention.

[0138] In another aspect, the invention provides compositions comprising iEVIR-expressing cells according to the invention.

[0139] Mode of administration Cells, vectors, and formulations thereof according to this invention may be administered in any manner including parenterally, intravenously, intratumorally, subcutaneously, intra-dermally, rectally, direct tissue perfusion during surgery, or combinations thereof. Parenteral administration includes, but is not limited to, intravenous, intra-arterial, intra-peritoneal, subcutaneous and intramuscular. The compositions of this invention may also be administered in the form of an implant, which allows slow release of the compositions as well as a slow controlled i.v. infusion.

[0140] Combination

[0141] According to the invention, the vectors and cells according to the invention, and pharmaceutical formulations thereof, can be administered alone or in combination with a co-agent useful in the prevention and / or treatment of a cancer such as therapeutic antibodies that enhance the adaptive immune system’s activity against the tumor (such as anti-PDl, anti-PDLl, anti-CTLA4 antibodies), therapeutic antibodies or TLR agonists that enhance the innate immune system’s activity against the tumor, therapeutic antibodies or small molecule inhibitors that deplete endogenous monocytes, macrophages or dendritic cells (for example CSF1R inhibitors), thus favoring the engraftment of and uptake of EVs and / or other cancer cell-derived particles by iEVIR-expressing cells of the invention.

[0142] The cells according to the invention might also be combined with known chemo-, radio-therapeutics that enhance cancer cell killing and release of cancer-cell derived EVs, as defined in the application.

[0143] The invention encompasses the administration of vectors or cells, pharmaceutical formulations thereof, or composition according to the invention, wherein said vectors or cells or compositions are administered to an individual prior to, simultaneously or sequentially with other therapeutic regimens, co-agents useful in the prevention and / or treatment of a cancer, in a therapeutically effective amount.

[0144] Cells or composition according to the invention, or the pharmaceutical formulation thereof, that are administered simultaneously with said co-agents can be administered in the same or different composition(s) and by the same or different route(s) of administration.

[0145] Kits

[0146] According to another aspect of the invention, is provided a kit comprising at least one recombinant expression vector and / or at least one cell according to the invention, and optionally instructional material. According to another further embodiment, the kit according to the invention comprises at least one recombinant expression vector and further comprises at least one agent for transfecting or transducing an antigen-presenting cell or a stem / progenitor cell thereof with said recombinant expression vector.

[0147] According to another further embodiment, the kit according to the invention comprises at least one cell according to the invention and further comprises at least one agent for the preservation of said cells and / or culture of said cells with cancer cells of cancer derived EVs.

[0148] Patients

[0149] In an embodiment, patients according to the invention are suffering from any type of cancer. In an embodiment, patients according to the invention are suffering from any type of cancer at any stage, including non-metastatic and metastatic.

[0150] In a particular embodiment, patients according to the invention are suffering from carcinomas, sarcomas, melanomas, brain tumors, hematological cancers, or any pre-malignant or malignant neoplasm.

[0151] In an embodiment, patients according to the invention are suffering from melanoma.

[0152] In an embodiment, patients according to the invention are suffering from lung cancer.

[0153] In an embodiment, patients according to the invention are suffering from intestinal cancer. In an embodiment, patients according to the invention are suffering from a breast cancer. In an embodiment, patients according to the invention are suffering from glioblastoma.

[0154] In an embodiment, patients according to the invention are suffering from neuroblastoma. References cited herein are hereby incorporated by reference in their entirety. The present invention is not to be limited in scope by the specific embodiments and drawings described herein, which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention.

[0155] EXAMPLES

[0156] Example 1: Screening of intracellular activation domains for generating signaling EVIRs of the invention

[0157] Screening and selection of signaling domains

[0158] The schematics illustrate the engineering of new EVIRs of the invention comprising intracellular domains of various “DC receptors” (i.e., receptors that are naturally expressed in DCs and that regulate their activation) coupled with an extracellular anti-HER2 scFv domain, which binds human HER2 (Fig 1A).

[0159] For developing these EVIRs, a lentiviral vector (LV) expressing an anti-HER2 non-signaling EVIR was used, which contains a truncated low-affinity nerve growth factor receptor (ALNGFR) as transmembrane / intracellular domain, previously described in Squadrito et al., 2018, supra. This LV contains an expression cassette that drives transgene expression from a spleen forming focus virus (SFFV) promoter with a consensus KOZAK sequence and a BamHl restriction site upstream of the start codon. After the start codon, an IgK N-terminal signal peptide (SEQ ID NO: 11) is followed by an anti-HER2 scFv (CHA21; extracellular domain) sequence; an Agel restriction site; a sequence; a stop codon; and Mlul and Sall restriction sites (Squadrito et al., 2018'). Then, sequences derived from the DEC-205 (SEQ ID NO: 1), MRC1 (SEQ ID NO: 2), CD40 (SEQ ID NO: 3), FcyRIV (SEQ ID NO: 4), and FLT3 (SEQ ID NO: 5) (all excluding the ligand-binding domain) were codon optimized for efficient expression in mouse cells and were synthetically generated with Agel and Mlul restriction sites flanking the 5' and 3 '-end of the sequence, respectively. The Agel and Mlul sites were used to replace the ALNGFR sequence in the parental EVIR (Squadrito et al, 2018, supra) with the desired sequences in the LV described above, to obtain the new signaling EVIRs.

[0160] Additionally, sequences derived from DECTIN 1 (SEQ ID NO: 6), CLEC9A (SEQ ID NO: 7), and DC-SIGN (SEQ ID NO: 8) (all excluding the ligand-binding domain) were codon optimized for efficient expression in mouse cells and were synthetically generated. To generate signaling EVIRs encompassing these sequences, a full-length EVIR sequence containing a 5'-end BamHl, a start codon, the codon optimized receptor sequence, an Agel restriction site, the anti-HER2 scFv (CHA21), a stop codon, and a 3'-end Mlul restriction site. BamHl and Mlul restriction sites of the parental EVIR was synthetized (Squadrito et al., 2018) were then used to subclone the signaling EVIRs into the LV.

[0161] The new EVIRs were named after the name of the DC receptor used to obtain the intracellular, signaling domain of the EVIR (e.g., DEC205-EVIR).

[0162] Expression of newly designed EVIRs in MutuDCs

[0163] MutuDCs were cultured as previously described (Fuertes Marraco et al., 2012). Briefly, cells were grown in IMDM-glutamax medium supplemented with 10% FBS, 10 mM Hepes, 50 pM P-mercaptoethanol and Pen / Strep. MutuDC passage was performed non-enzymatically by treating the cells with a dissociation buffer containing 5 mM EDTA and 20 mM HEPES in PBS. LVs harboring the indicated receptors were produced using third generation LVs pseudotyped with vesicular stomatitis virus-G protein (VSV-G). Briefly, 7 million 293T cells were seeded in 150 cm tissue culture dishes in DMEM medium 24 hours before the transfection of plasmids. Two hours before transfection, the medium was replaced with fresh medium. Five plasmids were used: LV transfer plasmid (52 pg / dish), packaging pMDLg / pRRE plasmid (18.75 pg / dish), VSV-G plasmid (13.5 pg / dish), REV plasmid (9.375 pg / dish), pADVANTAGE plasmid (22.5 pg / dish). For transient transfection, the plasmid mix was incubated with CaCL for 5 minutes while rotating on a spinning wheel. Then, HEPES -buffered saline (2xHBS) was added to the plasmid mix in a dropwise manner to facilitate formation of fine precipitates. Immediately after addition of 2xHBS, the resulting transfection mixture was applied to 293 T cells. Twelve hours after transfection, the medium was replaced with fresh medium. Medium conditioned by transfected 293T cells was collected, containing LV particles, 48 hours later. The filtered (0.22 pm) medium was ultracentrifuged to concentrate the LV particles, as described (De Palma and Naldini, 2002, supra).

[0164] LVs were then titrated by transducing 293T cells with serial dilutions of LVs. Seven days posttransduction, the 293T cells were harvested and the expression of the receptors was quantified by flow cytometry using either a monoclonal antibody against NGFR (which detects the parental EVIR and its scFv-deficient control) or a polyclonal Fab antibody (for signaling EVIRs and controls). The number of LV transducing units (TU) / ml was determined as described previously (De Palma and Naldini, 2002, supra).

[0165] MutuDCs were transduced with LVs harboring the indicated receptors at a multiplicity of infection (MOI) of 20, that is 20 “transducing units” of LV for each MutuDC. Flow cytometry analysis of surface expression of the newly designed EVIRs was performed 2 days posttransduction using either APC-conjugated anti-NGFR (1:100; ME20.4-1. H4; MACS; Table 1) or AF647-conjugated anti-Fab antibody staining (1:25; polyclonal; Jackson ImmunoResearch), using an LSR II SORP apparatus. Data were analyzed using Flowjo. A representative experiment is shown. While all EVIRs were efficiently expressed, the expression level varied with the individual EVIR (Figure IB).

[0166] Newly designed EVIRs variably activate MutuDCs

[0167] To test the ability of signaling EVIRs to induce DC activation upon binding to a surface tumor antigen, MC38 colon cancer cells modified to express human HER2 were used (MC38-HER2;

[0168] Figure 1C). The example shows flow cytometry analysis of HER2 in unmodified MC38 and MC38-HER2 cancer cells. To obtain these cells, an LV encoding human HER2 under the hPGK promoter was used to generate MC38-HER2 cells, as described previously (Squadrito et al., 2018, supra). For staining of HER2 on cancer cells, the cultured cancer cells were harvested and stained with a FITC-conjugated anti-HER2 antibody. Stained cells were analyzed using an LSR II SORP apparatus. Data were analyzed using Flowjo.

[0169] Then, transduced MutuDCs co-cultured with MC38-HER2 cells at a 1:2 ratio were used. Two days post-transduction of MutuDCs with LVs encoding the indicated receptors, transduced DCs were either left untreated or were co-cultured with MC38-HER2 cancer cells. Co-culture of cancer cells with DCs was performed at a 2:1 (cancer cell: DC) ratio. After co-culture for 12 hours, cells were harvested and stained with APC-Cy7-conjuated anti-CD86 antibody (Table 1). Stained cells were analyzed using an LSR II SORP apparatus (BD Biosciences). Data were analyzed using FlowJo.

[0170] As expected, the Ctrl-EVIR and the parental non-signaling EVIR (DLNGFR) did not induce DC activation upon co-culture with MC38-HER2 cells (Figure ID). Remarkably, among signaling EVIRs, DEC205-EVIR, CLEC9A-EVIR, DC-SIGN-EVIR, MRC1-EVIR and FLT3-EVIR failed to robustly activate the DCs. However, three signaling EVIRs activated the DCs but among those, DECTIN1 -EVIR-expressing DCs exhibited high CD86 expression both in the presence and absence of MC38-HER2 cells, indicative of unwanted, tonic (ligandindependent) signaling of the receptor. Conversely, both CD40-EVIR and FcyRIV-EVIR upregulated CD86 in DCs only upon co-culture with MC38-HER2 cells, thus providing suitable receptors for further engineering. Bar graph shows mean ± SEM; n = 3 independent cell cultures. Statistical analysis by one-way ANOVA.

[0171] Example 2: Generation of a signaling EVIR (“instructive EVIR”, or iEVIR)

[0172] The properties of each of CD40-EVIR and FcyRIV-EVIR supported above were used to develop improved signaling EVIRs with antigen-binding-dependent DC activation capability.

[0173] Design of an instructive EVIR (iEVIR)

[0174] Since expression of the FcyRIV-EVIR designed in Example 1 was relatively low in transduced DCs (Figure IB) and FcyRs require assembly with the FcRy chain for both trafficking to the cell surface and signaling, it was hypothetized that lentivirally-expressed FcyRIV-EVIR might compete with endogenous FcyRs for assembly with FcRy chains, thereby limiting surface expression of the exogenous FcyRIV-EVIR. Thus, the intracellular CD40 activation domain of the CD40-EVIR was combined with the ITAM motif of the FcRy chain in the same EVIR. Additionally, the CD40 transmembrane / hinge domain was substituted with a mutated (Cys-193 to Ser-193) CD8a transmembrane / hinge domain. The mutated CD8a domain disrupts spontaneous (ligand-independent) dimerization / multimerization of the chimeric receptor to limit potential tonic signaling (Hennecke and Cosson, 1993, Journal of Biological Chemistry. A© 1993 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology., 268(35), pp. 26607- -26612. doi: 10.1016 / s0021-9258(19) 74355-5).

[0175] Thus, an EVIR according to the invention (referred to as mutCD8 / CD40-FcRy-ITAM-EVIR or instructive EVIR (iEVIRs) for brevity) was designed as follows that comprises:

[0176] (i) an IgK signal peptide as signal peptide;

[0177] (ii) an anti-HER2 scFv extracellular domain as a single-chain variable fragment (scFv) extracellular domain specific for human HER2;

[0178] (iii) a mutated (Cys-193 to Ser-193) CD8a domain comprising a mutated CD8a hinge, a CD8a transmembrane and proximal membrane sequence. The mutation disrupts spontaneous (ligand-independent) dimerization / multimerization of the chimeric receptor to limit potential tonic signaling (Hennecke and Cosson, 1993, supra),' (iv) a CD40 activation domain; and

[0179] (v) a FcRy chain ITAM motif.

[0180] A schematic representation of this construct is provided in Figure 2A. To obtain the iEVIR, the DNA coding sequence for the aforementioned molecule, flanked by 5 '-end Agel and 3 '-end Mlul restrictions sites, was codon optimized and generated synthetically. Agel and Mlul restrictions sites were then used to subclone this sequence into the parental LV containing an anti-HER2 scFv (CH21A) in place of the ALNGFR sequence described in Squadrito et al., 2018, supra. LVs were produced and titered as described in Example 1.

[0181] Surface expression of iEVIR in MutuDCs

[0182] MutuDCs were transduced with LVs harboring the new iEVIR or the parental EVIR (nonsignaling EVIR) at a multiplicity of infection (MOI) of 20, that is 20 “transducing units” of LV for each MutuDC. Flow cytometry analysis of surface expression of the EVIRs was performed 3 days post-transduction using AF647-conjugated anti-Fab antibody staining (1:25; polyclonal; Jackson ImmunoResearch), using an LSR II SORP apparatus. Data were analyzed using Flowjo. A representative experiment is shown. Both receptors (new signaling iEVIR of the invention and parental non-signaling EVIR) showed robust and comparable expression at day 3 post-transduction (Figure 2B).

[0183] Newly designed iEVIR strongly activates MutuDCs

[0184] Then, it was investigated whether the iEVIR could induce DC activation upon binding to HER2 presented by either MC38-HER2 cancer cells or their EVs. To this aim, expression of CD86, C-C chemokine receptor type 7 (CCR7; involved in DC migration), major histocompatibility complex (MHC)-II (MHCII; involved in antigen presentation), and CD40 (a co-stimulatory receptor involved in DC activation), was measured in the transduced DCs 12 hours after exposure to EVs or cancer cells as described below.

[0185] To harvest EVs from MC38 or MC38-HER2 cancer cells, the cancer cells were seeded sparsely in 15 cm tissue culture dishes in DMEM medium supplemented with 5% exosome-free FBS. Four days after, conditioned media was harvested and sequentially spun down at 500 x g (5 min), 2000 x g (10 min), 110000 x g (70 min), and 134000 x g (70 min). Pellets containing EVs were resuspended in PBS and stored at -80°C. Concentration of EVs was measured by nanoparticle tracking analysis (NTA) using a Nanosight NS300 device.

[0186] DC activation assay was carried out with the transduced MutuDCs either in co-culture with cancer cells or in the presence of EVs. The co-culture was performed by culturing the indicated cancer cells together with the transduced MutuDCs at a 2:1 (cancer cell: DC) ratio. Alternatively, EVs were added to DCs at a concentration of about 4* 106EV particles per 100 ul medium for 2 hours before washing the cells with PBS to remove EVs. Twelve hours after the treatments, MutuDCs were harvested and stained with antibodies against DC activation markers (CD86, MHCII, CCR7 and CD40).

[0187] The iEVIR robustly upregulated the aforementioned markers in the presence of HER2+ EVs or cancer cells, whereas the parental EVIR failed to do so (Figure 2C). Notably, the iEVIR did not upregulate DC activation markers when the cells were left untreated or exposed to HER2-EVs or cancer cells, indicating absence of tonic signaling and faithful ligand-dependent signaling. Flow cytometry analysis was performed using LSR II SORP. Data were analyzed using Flowjo. MFI, mean fluorescence intensity. Bar graphs show mean± SEM; n= 3-4 independent cell cultures. Statistical analysis by one-way ANOVA.

[0188] Example 3: An anti-GD2 iEVIR promotes DC activation in response to GD2+ EVs To demonstrate the versatility of the iEVIRs according to the invention, a tumor surface antigen other than HER2 was used. GD2 is a disialoganglioside preferentially expressed in some solid tumors, including neuroblastoma, glioma, and melanoma (Machy et al., 2023, Frontiers in Pharmacology, 14(August) 1-21. doi: 10.3389 / fphar.2023.1249929). GD2-targeted therapies, such as monoclonal antibodies and CAR T cells, have shown promise for cancer treatment due to the limited expression of GD2 in normal tissues (Del Bufalo et al, 2023, New England Journal of Medicine, 388(14), 1284-1295. doi: 10.1056 / nejmoa2210859).

[0189] The designed iEVIR according to the invention comprises the same elements as described in Example 2 except that an anti-GD2 scFv (14. G2a) extracellular domain as a single-chain variable fragment (scFv) extracellular domain. The scFV domain previously employed for constructing a non-signaling EVIR as described in Squadrito et al., 2018, supra was used. For these studies, an ovalbumin (OVA)-positive B16F10 melanoma cell line (B16F10-OVA) was modified to express GD2 by transducing the GD2 synthases, GD2S and GD3S, as described previously (Squadrito etal., 2018, supra), and obtained Bl 6F10-GD2 / OVA cells.

[0190] Expression of an anti-GD2 iEVIR activates MutuDCs in response to GD2+ EVs MutuDCs transduced with the Ctrl-iEVIR (lacking the scFv domain, therefore unable to bind to GD2) or the anti-GD2 iEVIR (Figure 3A1) were then exposed to GD2+EVs isolated from B16F10-GD2 / OVA melanoma cells (Figure 3A2) and analyzed 12 hours later, using methods for LV preparation, DC transduction, analysis of EVIR expression, and assessment of DC activation described in Examples 1 and 2. GD2+EVs upregulated CD86 and MHCII in iEVIR but not Ctrl-EVIR-expressing DCs 12 hours after EV treatment (Figure 3B-C). Bar graphs show mean± SEM; n= 4 independent cell cultures. Statistical analysis by one-way ANOVA. Dot plots in (B) show representative samples.

[0191] Additionally, iEVIR-expressing DCs secreted higher amounts of the T-cell chemoattractant or stimulatory cytokines CXCL9, CXCL10, IL-6, TNFa, and IL12p40 in response to GD2+ EVs, as compared with DCs expressing the Ctrl-iEVIR (Figure 3D); this response was enhanced by addition of IFNy to the cell culture medium. To measure cytokine secretion, cell supernatants were collected 12 hours after EV treatment and subjected to multiplex bead-based assays using LEGENDplex™ kits. The mouse cytokine panel 2 (Cat. No 740134), inflammation panel (Cat. No 740150), and cytokine release syndrome panel (Cat. No 741023), were used according to the manufacturer’s instructions to measure the concentration of multiple cytokines. Bar graphs show mean± SEM; n= 3 independent cell cultures. Statistical analysis by one-way ANOVA. To broaden the characterization of iEVIR-induced responses in DCs, bulk RNA sequencing (RNA-seq) of MutuDCs expressing either Ctrl-iEVIR or iEVIR, which were exposed to GD2+ EVs or left untreated was performed.

[0192] Before RNA-Seq, MutuDCs transduced with Ctrl-iEVIR or iEVIR were either treated with EVs for 2 hours and washed with PBS or left untreated. After 12 hours of further culture, total RNA was extracted from MutuDCs cells using the Promega ReliaPrep™ RNA Cell Miniprep System (Catalog number Z6011). An "Illumina stranded mRNA ligation" (ISML) prep was carried out starting from 250 ng ofRNA, according to Illumina protocol 1000000124518 v03. The libraries were quantified by qubit DNA HS and performed profile analysis by TapeStation TS4200. The libraries were sequenced on Illumina NovaSeq 6000 in a PE60 configuration. We trimmed reads for ISML (Nextera) adapters using bclconvert (v 00.000.000.3.9.3). Mapping and quantification were performed with STAR and Salmon (nf-core / maseq version 3.12.0) on the mouse genome mm 10 with reverse strand-specificity setting and default parameters. Raw counts were normalized using TMM method from EdgeR(v.4.0.16) and voom from limma (v.3.58.1). Genes were filtered out if average TPM < 1 or average counts < 5 per sample. Uncharacterized, predicted, and pseudogenes were also filtered out to keep a matrix of counts with n=10’610 genes. Differential gene expression was computed with limma. Gene set enrichment analysis was performed with clusterProfiler (v.4.10.1) applying GSEA with default parameters and 100’000 permutations to obtain P-values. The Hallmark collection from msigdbr v.7.5.1 was used. n= 3 independent cell cultures.

[0193] In Ctrl-iEVIR-transduced DCs, GD2+ EVs globally downregulated multiple inflammation-related gene pathways, including TNFa, IFNa, and IFNy pathways (Figure 3E-G). This is consistent with the potentially immunosuppressive and tolerogenic properties of tumor-derived EVs (Whiteside, 2016, Journal of Clinical Investigation, 126(4), 1216 1223. doi: 10.1172 / JCI81136). Indeed, unsupervised gene pathway analysis by Hallmark (adjusted P-value < 0.05) revealed upregulation of both type-I and II IFN signaling, TNFa signaling via NFKB, IL-6 / JAK / STAT3 signaling, and additional inflammation and immune-response pathways (e.g., “inflammatory response” and “allograft rejection”), among the most regulated pathways in iEVIR-transduced cells. Overall, these data further illustrate GD2-specific DC activation mediated by the iEVIR, which may be conducive to T-cell activation. Moreover, they indicate that the iEVIR reverses the potentially suppressive signals elicited by tumor EVs in DCs to enforce, rather than blunt, their activation.

[0194] Example 4: iEVIR promotes antigen-dependent DC activation conducive to T-cell activation

[0195] Then, it was investigated whether iEVIR-expressing DCs promote T-cell proliferation and activation in response to bait antigen-positive EVs. MutuDCs transduced with the anti-HER2 iEVIR, Ctrl-iEVIR, or the parental anti-HER2 EVIR, were used along with EVs isolated from MC38-HER2 cells modified to express OVA (MC38-HER2 / OVA) as a surrogate neoantigen for examining antigen-specific T-cell responses using with OVA-specific, MHCI-restricted CD8+ T cells (OT-I cells).

[0196] Activation of CD8+ T cells

[0197] For analysis of OT-I proliferation, OVA-reactive CD8+ T cells were isolated from the spleens of OT-I TCR transgenic female mice. Spleens were mechanically disrupted under sterile conditions and passed through a 70 pm cell strainer to obtain single-cell suspensions. The EasySep™ Mouse Naive CD8+T Cell Isolation Kit (Catalog number 19858) was employed to isolate OVA-reactive CD8+T cells from OT-I mice. T cells were then stained with cell trace violet (Invitrogen™) to measure proliferation cell cycles through dye dilution by flow cytometry. T cells were maintained in T cell medium consisting of RPMI medium supplemented with 25 pM 2-mercaptoethanol, 1 mM sodium pyruvate, MEM non-essential amino acids (Gibco™ Catalog number: 11140050), FBS, Pen / Strep, and L-Glutamate.

[0198] To study T cell proliferation in response to EVs, cancer-cell-derived EVs were added at the indicated amounts to 5*103transduced DCs. Transduced DCs were exposed to two different EV doses corresponding to either 400 or 800 EVs per DC, for 2 hours before co-culture with OT-I cells. Two hours post-EV treatment, DCs were washed with PBS to eliminate excess EVs, and 1*105OT-I T cells were added to the EV-treated DCs in T cell medium. OT-I cells were harvested for analysis after 3 days. Five hours prior to T cell staining, BD Golgi Stop™ and BD GolgiPlug™ were added to OT-I cell cultures at the manufacturer's recommended doses. Subsequently, single cell suspensions were prepared in PBS and dead cells were stained using the LIVE / DEAD Fixable Red Kit (1:500; Invitrogen; Catalog Number L34973). Cells were washed in FACS buffer and underwent surface staining for CD8 expression to identify the T cells. Cells were then fixed and permeabilized using the BD Fixation / Permeabilization Kit (Catalog Number 554714). T cell proliferation was analyzed with flow cytometry using LSR II SORP. Data were analyzed using Flowjo. Histogram plots on the left show peaks of T cell proliferation cycles revealed by dilution of cell trace violet; representative samples are shown. Data on the right show T cells that performed 4 or 5 rounds of cell division.

[0199] Three days after co-culture, EV-dependent promotion of T-cell proliferation was observed, which was enhanced by the iEVIR compared with the parental EVIR (Figure 4A1-2). This was associated with increased TNFa expression (Figure 4B), indicative of T-cell activation. Thus, the iEVIR promotes T-cell proliferation and activation more efficiently than a non-signaling EVIR in response to environmental EVs. Bar graphs show mean ± SEM; n= 3 independent cell cultures. Statistical analysis by one-way ANOVA.

[0200] Activation of CD4+ T cells

[0201] In addition to antigen presentation to CD8+T cells through MHCI, DCs can prime naive CD4+T cells by presenting peptides loaded onto MHCII molecules (Martinez-Usatorre and De Palma, 2022, supra). Because iEVIR engagement by EVs or cancer cells upregulated MHCII expression and IL12p40 secretion in DCs, it was investigated whether iEVIR-expressing MutuDCs could stimulate CD4+T-cell proliferation and IFNy production in a co-culture assay with MC38-HER2 / OVA cells and CD4+OVA-specific, MHCII-restricted T cells (OT-II). For the OT-II assay, OVA-reactive CD4+T cells were isolated from the spleens of OT-II TCR transgenic female mice. Spleens were mechanically disrupted under sterile conditions and passed through a 70 pm cell strainer to obtain single-cell suspensions. The EasySep™ Mouse CD4+T Cell Isolation Kit (Catalog number 19852) was utilized for obtaining OVA-reactive CD4+T cells from OT-II mice. Kit was used according to the manufacturer's guidelines for cell isolation from mouse spleens. OT-II T cells were then stained with cell trace violet (Invitrogen™) to measure proliferation cell cycles through dye dilution by flow cytometry. T cells were maintained in T cell medium consisting of RPMI medium supplemented with 25 pM 2 -mercaptoethanol, 1 mM sodium pyruvate, MEM non-essential amino acids (Gibco™ Catalog number: 11140050), FBS, Pen / Strep, and L-Glutamate.

[0202] For the test, 5*103transduced DCs were co-cultured with l * IO4MC38-HER2 / OVA cancer cells. Concurrently, l*105OT-II T cells were added to the DC and cancer cell co-culture in T cell medium. 5 days after the co-culture, OT-II cells were harvested. Five hours prior to T cell staining, BD Golgi Stop™ and BD GolgiPlug™ were added to OT-II cell cultures at the manufacturer's recommended doses. Subsequently, single cell suspensions were prepared in PBS and dead cells were stained using the LIVE / DEAD Fixable Red Kit (1:500; Invitrogen; Catalog Number L34973). Cells were then washed in FACS buffer and underwent surface staining for CD4 expression. Cells were then fixed and permeabilized using the BD Fixation / Permeabilization Kit (Catalog Number 554714). T cell proliferation was analyzed with flow cytometry using LSR II SORP. Data were analyzed using Flowjo. Histogram plots show peaks of T cell proliferation cycles revealed by dilution of cell trace violet; representative samples are shown. Quantitative data shows T cells that performed 4 or 5 rounds of cell division. Similar to results obtained with OT-I cells, the iEVIR greatly enhanced T-cell proliferation (Figure 4C1-2) and IFNy expression (Figure 4D) after 5 days of co-culture, compared to the non-signaling EVIR. Bar graphs show mean± SEM; n= 3-4 independent cell cultures. Statistical analysis by one-way ANOVA.

[0203] Taken together, these data indicate that the iEVIR endows DCs with the ability to acquire antigens from cancer cells and present them to both CD8+and CD4+T cells, including via bona fide cross-presentation, in the absence of other exogenous stimuli.

[0204] Example 5: iEVIR promotes antigen-dependent transactivation of an exogenous gene in DCs

[0205] Then, it was investigated whether iEVIR- and antigen-dependent signaling activation in DCs can be exploited to transactivate an exogenous gene in an inducible fashion. Construction and validation of an iEVIR-inducible gene expression cassette

[0206] To this aim, an expression cassette encompassing an inducible promoter and a coding gene was constructed (Figure 5A, top). As an inducible gene, a FLAG-tagged CD40L protein (FLAG-CD40L) was used. The inducible promoter was obtained from a 1112 promoter mutated to improve binding of NFKB (Murphy et al., 1995, Molecular and Cellular Biology, 15(10), 5258 5267. doi: 10.1128 / mcb.l5.10.5258'), to which 2, 4 or 7 additional NFKB response elements were added. The backbone of the 1112 inducible promoter was derived from a sequence spanning 200 nucleotides upstream of the TATA box in the murine IL12p40 promoter. The half-site NFKB located 123 nucleotides upstream of the TATA box was mutated into a full-site NFKB, thereby leading to a DNA sequence of the mutated IL12p40 promoter of SEQ ID NO: 9.

[0207] The backbone of the IL12 inducible promoter was then modified by adding 2, 4 or 7 NFKB response elements (REs). These promoters were generated synthetically to replace the SFFV promoter in a LV using Xhol and BamHI restriction sites.

[0208] Then, a chimeric membrane-bound CD40L was expressed under the control of the inducible promoters. The chimeric CD40L molecule was engineered by substituting the extracellular C-type lectin domain of CLEC9A with the ectodomain of CD40L. Additionally, the tyrosine residue in the intracellular CLEC9A hemilTAM was mutated to alanine to inhibit CLEC9A signaling. A FLAG tag sequence was incorporated at the extracellular C-terminus of CD40L, leading to a chimeric FLAG-CD40L of SEQ ID NO: 10.

[0209] LVs encoding the inducible promoters were produced as described above. For titration of each batch of LVs, MutuDCs were transduced with serial dilutions of LVs. Two days after transduction, DCs were split into two wells for each dilution. One cell culture was activated overnight with CpG, while the other was left untreated. MutuDCs were then harvested and stained with a monoclonal APC-conjugated anti-FLAG antibody to stain FLAG-CD40L. FLAG-CD40L expression was measured using flow cytometry for both cell cultures with and without CpG for each dilution. The LV concentration that resulted in maximal FLAG-CD40L expression in DCs upon CpG treatment, while showing the lowest basal activity in the absence of CpG, was selected for use in the experiment.

[0210] For the experiment, MutuDCs were transduced with the appropriate amounts of LVs containing the indicated promoters. Two days post transduction, MutuDCs were harvested and stained with a monoclonal APC-conjugated anti-FLAG antibody to stain FLAG-CD40L. Flow cytometry was performed using LSR Fortessa. Data were analyzed using FlowJo.

[0211] A configuration with 2 NFKB response elements was selected upon screening various configurations for acceptable basal expression (Figure 5A, bottom) and robust inducibility (Figure 5B). As shown in Figure 5B, CpG (a pro-inflammatory molecule) efficiently induced FLAG-CD40L expression in MutuDCs lentivirally transduced with the inducible expression cassette. Dot plots show flow cytometry analysis of FLAG-CD40L in MutuDCs treated as indicated.

[0212] EV-mediated, iEVIR-induced expression of an exogenous gene in DCs

[0213] Then, MutuDCs were co-transduced with the anti-HER2 iEVIR (or a control receptor lacking the anti-HER2 scFV, Ctrl -iEVIR) and the expression cassette containing the 1112 promoter with 2 additional NFKB RES. HER2+ EVs were isolated from MC38-HER2 cancer cells and GD2+ EVs were isolated from B16F10-OVA / GD2 cancer cells, as explained above. Cell activation assay with EVs was performed as explained above. Twelve hours after EV addition, DCs were harvested and stained with a monoclonal APC-conjugated anti-FLAG antibody to determine expression of CD40L. Flow cytometry was performed using LSR Fortessa. Data were analyzed using FlowJo. MutuDCs transduced with an anti-HER2 iEVIR, but not Ctrl-iEVIR, strongly upregulated FLAG-CD40L expression in response to HER2+ EVs isolated from MC38-HER2 cells (Figure 5C1 & C2). Similar results were obtained when an anti-GD2 iEVIR and GD2+ EVs isolated from B16F10-GD2 / OVA cells (Figure 5D1 & D2) were used. Bar graphs show mean± SEM; n= 3 independent cell cultures. Statistical analysis by one-way ANOVA.

[0214] These results confirm the on-demand (EV-mediated) inducibility of the iEVIR platform and demonstrate that it can be used to transactivate a transgene of interest in DCs specifically in response to tumor-derived EVs.

[0215] Example 6: iEVIR-expressing DCs sensitize tumors to PD-1 blockade without the need for antigen loading

[0216] The B16F10 melanoma model is characterized by scant T-cell infiltrates, low immunogenicity, and refractoriness to PD-1 blockade. This model was used to investigate the therapeutic potential of iEVIR-expressing DCs in combination with a PD-1 blocking antibody.

[0217] Inhibition of tumor growth by iEVIR-expressing DCs

[0218] Cancer cells were thawed and cultured for at least one week before subcutaneous injection in mice. Cells were split one day prior to injection to achieve approximately 70% confluency on the day of injection, ensuring they were in their exponential growth phase. l*106B16F10- GD2 / 0VA cancer cells in 100 pl PBS were inoculated subcutaneously in the right flank of 8-12 week-old, female C57BL / 6 mice. Two subsequent doses of 2* 106MutuDCs, transduced for 48 hours with either Ctrl-iEVIR or iEVIR were administered intravenously (via the tail vein) in tumor-bearing mice on days 11 and 14 post-tumor injection, when tumors were palpable and growing. Tumor-bearing mice received intraperitoneal (i.p.) injections twice per week of either 10 mg / kg anti-PDl antibody (rat IgG2a, clone RMPI-14, Bio X Cell InvivoMAb, Catalog number BE0146) or 10 mg / kg of an isotype rat IgG2a (clone 2 A3, Bio X Cell InvivoMAb, Catalog number BE0089), in 100 pl of PBS. All mice were sacrificed at day 18 (Figure 6A).

[0219] In a first study, it was investigated whether iEVIR-expressing DCs would sensitize established tumors (>100 mm3) to PD-1 blockade in a late intervention setting. In this advanced-tumor setting, iEVIR- but not Ctrl-iEVIR-expressing DCs detectably delayed tumor growth in combination with PD-1 blockade (Figure 6B1 & B2). Of note, this response required a combination of the iEVIR and PD-1 antibodies, as neither treatment alone had antitumoral activity in this late-treatment setting. Tumor volume was monitored and measured with a caliper throughout the experiments. The tumor volume was calculated using the formula 0.5 x d2x D, where 'd' is the shorter and 'D' is the longer tumor diameter. Mice were monitored thrice weekly to ensure their general health. The 4 cohorts are shown in two separate charts for enhanced clarity. Data show tumor volume in the indicated cohorts. Graphs show mean± SEM; Ctrl-iEVIR + IgG, n=6; Ctrl-iEVIR + anti-PDl, n= 5; iEVIR + IgG, n=7; iEVIR + anti-PDl, n=7 tumors in the indicated cohorts. Statistical analysis by two-way ANOVA.

[0220] In a second study, tumors were treated at an earlier stage of growth and used primary monocyte-derived DCs (moDCs), which provide a more physiological and preclinically relevant source of DCs for treatment trials in mice. MoDCs were generated by culturing bone marrow cells harvested from the femurs and tibiae of female C57BL / 6 mice. The bones were split to expose the bone marrow cavity. Bone marrow cells were then extracted by placing the halved bones cut-side down into centrifuge tubes and spinning briefly at a high speed to collect the cells at the bottom of the tubes. Bone marrow cells were washed and cultured in RPMI medium supplemented with 10% FBS, Pen / Strep and L-glutamine. To induce the differentiation of moDCs, GM-CSF (40 ng / ml; Peprotech) and IL-4 (40 ng / ml; Peprotech) were added to the medium and bone marrow cells were cultured in ultra-low attachment, 6-well dishes, at the density of 2x 106cells per well for 5 days (Corning™). On day 5, moDCs were transduced with LVs encoding the indicated receptors. Transduced moDCs were kept in culture for 2 days before injection.

[0221] B16F10-GD2 / OVA cells were then injected subcutaneously in mice and, on days 7 and 9 posttumor injection, intravenously administered 2xl06moDCs transduced with either Ctrl-iEVIR or iEVIR, along with PD-1 antibodies. Briefly, two subsequent doses of 2*106moDCs were administered intravenously (via the tail vein) in tumor-bearing mice. One cohort received untransduced moDCs and two other cohorts received transduced moDCs as indicated. DCs were injected when the average tumor size in all mice was around 50 mm3. Tumor-bearing mice received intraperitoneal (i.p.) injections twice per week of either 10 mg / kg anti-PDl antibody (rat IgG2a, clone RMPI-14, Bio X Cell InvivoMAb, Catalog number BE0146) or 10 mg / kg of an isotype rat IgG2a (clone 2A3, Bio X Cell InvivoMAb, Catalog number BE0089), in 100 pl of PBS. All mice were sacrificed at day 15 (Figure 6C).

[0222] This dose-regimen targets early-established tumors, which should allow sufficient time for the initiation of anti-tumor immunity by the engineered DCs. As shown in (Figure 6D1-4), iEVIR-expressing moDCs delayed tumor growth compared to moDCs either untransduced or expressing the Ctrl-iEVIR. Tumor volume was monitored and measured as explained in Figure 6B. The top left graph shows tumor volume in mice treated as indicated (mean± SEM; untransduced, n=5; Ctrl-iEVIR, n= 6; iEVIR, n=7 tumors). Statistical analysis by two-way ANOVA. The other graphs show the volume of individual tumors in the indicated cohorts (tumors in red are also indicated in panel (E) below).

[0223] Recruitment of activated T cells to the tumor by iEVIR-expressing DCs

[0224] Mouse tumors were then harvested, chopped, and treated with collagenase, then passed through a 70 pm cell strainer to obtain single-cell suspensions. The tumors were ex vivo stimulated using PMA (10 ng / ml) and lonomycin (500 ng / ml), along with treatment with BD GolgiStop and BD GolgiPlug for 5 hours at 37°C in an incubator. The stimulated tumor-derived cells were then washed and prepared in PBS and dead cells were stained using the Zombie UV Dead Cell Stain Kit (1:500; Biolegend; Catalog Number 423107). Cells were then washed in FACS buffer and underwent surface staining with the indicated antibodies. Cells were then fixed and permeabilized using the BD Fixation / Permeabilization Kit (Catalog Number 554714) and stained for intracellular markers with antibodies listed in table 1. Stained cells were analyzed using LSR Fortessa apparatus (BD Biosciences). Data were analyzed using FlowJo.

[0225] This flow cytometry analysis of the tumors at the day- 15 endpoint showed increased proportion of IFNy and TNFa+CD8+or CD4+T cells in the tumors of mice that received iEVIR- expressing moDCs (Figure 6E). Interestingly, higher proportions of IFNy+or TNFa+T cells in the tumors correlated with tumor response to iEVIR-expressing moDCs. Bar graphs show mean± SEM; untransduced, n=5; Ctrl-iEVIR, n= 6; iEVIR, n=6 tumors. Statistical analysis by one-way ANOVA.

[0226] iEVIR-expressing DCs improve survival in tumor-bearing mice

[0227] Then, a survival study was performed according to an early-intervention schedule (Figure 6F).

[0228] Cancer cells were cultured and injected as explained in Figure 6A. moDCs were harvested, cultured and transduced as explained in Figure 6C. Two subsequent doses of 2*106moDCs transduced with either Ctrl-iEVIR or iEVIR were administered intravenously (via the tail vein) in tumor-bearing mice. DCs were injected when tumors were detectable but not yet measurable. Tumor-bearing mice received intraperitoneal (i.p.) injections twice per week of either 10 mg / kg anti-PDl antibody (rat IgG2a, clone RMPI-14, Bio X Cell InvivoMAb, Catalog number BE0146) or 10 mg / kg of an isotype rat IgG2a (clone 2A3, Bio X Cell InvivoMAb, Catalog number BE0089), in 100 pl of PBS.

[0229] The mice were scheduled for termination when the tumors reached a volume of 1000 mm3. Consistent with the previous experiment, tumors of mice that received iEVIR-expressing moDCs exhibited, on average, delayed growth compared to the control group (Figure 6G1 & G2), resulting in extended survival (Figure 6H). Tumor volume was monitored and measured as explained in Figure 6B. Figure 6G1 displays B16F10-GD2 / OVA tumor growth (mean± SEM; Ctrl-iEVIR, n= 6; iEVIR, n=8 tumors), shown until day 15 (time-point when termination of the mice began). Statistical analysis by two-way ANOVA. Figure 6G2 shows tumor growth in individual mice until termination.

[0230] Taken together, these results indicate that iEVIR-expressing DCs have anti-tumoral activity in a poorly immunogenic tumor model in the absence of ex vivo antigen loading and without concurrent cytoreductive therapy, such as chemotherapy or radiotherapy.

[0231] Example 7: A ubiquitination-resistant iEVIR improves EV internalization and cancer cell phagocytosis

[0232] Generation of an iEVIR with enhanced stability

[0233] Ubiquitination of transmembrane receptors following ligand engagement and signaling directs them to the lysosome for degradation. In order to disrupt lysine ubiquitination of the iEVIR, all lysine residues were mutated into arginine in the intracellular receptor sequence and the mutated receptor was called iEVIRubNull(Figure 7A). The DNA sequence encoding iEVIRubNullwas codon optimized and generated synthetically. Agel and Mlul restrictions sites were used to subclone this sequence into the LV containing an anti-GD2 scFv (14. G2a) in place of the original (non-mutated) iEVIR sequence. LVs containing this new receptor were produced and titrated as explained in the Examples above. By suppressing receptor sorting to the lysosome (Li et al., 2020, Immunity. Elsevier Inc., 53(2), 456-470.e6. doi: 10.1016 / j.immuni.2020.07.017), ubiquitination deficiency should increase both the stability of the receptor at the cell surface and its recycling to the cell surface following internalization, thereby improving antigen presentation and, potentially, T-cell priming.

[0234] MutuDCs were transduced with LVs containing the indicated receptors. Two days after transduction, MutuDCs were harvested and either stained with APC-conjugated anti-NGFR antibody (1:100; ME20.4-1. H4; MACS) (Ctrl-EVIR and EVIR), or in a two-step process, stained with biotinylated anti-strep-tag II antibody (1:100; 5A9F9; GenScript) followed by staining with BV711 -conjugated streptavidin (iEVIR and iEVIRubNull). Flow cytometry was performed using the LSR Fortessa apparatus, and the data were analyzed using FlowJo. Representative experiments are shown. MutuDCs transduced with the iEVIRubNullshowed enhanced cell-surface expression at steady-state than cells transduced with the unmodified iEVIR or other receptors (Figure 7B), likely indicative of improved stability.

[0235] A ubiquitination-resistant iEVIR improves EV uptake

[0236] In a cellular assay with MutuDCs exposed to PKH26-labeled EVs isolated from B16F10-GD2 / OVA cells, iEVIRUbNull-expressing DCs exhibited increased EV internalization capacity compared to cells transduced with the iEVIR or parental EVIR (Figure 7C). For this experiment, EVs isolated from cancer cells were stained with PKH26 (MIDI26-1KT, Sigma, PE), following a modified version of the manufacturer's protocol. PKH26 was diluted 1:100 in diluent C and added to the EVs in a 1:1 volume ratio for 10 minutes in the dark at room temperature. Subsequently, the dye was quenched by adding 0.1% BSA in PBS. The mixture was then transferred to Vivaspin 500 (300,000 MWCO PES, VS0152 Sartorius) and centrifuged at 12,000 x g for 20 minutes at 4°C. The flow-through was discarded and the EVs were resuspended in 0.1% BSA in PBS. This centrifugation and washing cycle were repeated two additional times. Finally, the EVs were resuspended in PBS and used for the uptake assay. PKH26-stained EVs were incubated with DCs for 2 hours at defined concentrations, as indicated. After the incubation period, DCs were washed with PBS to remove residual and unbound EVs in the media. Flow cytometry was then employed to measure PKH26 fluorescence in DCs. MFI, mean fluorescence intensity. Bar graphs show mean± SEM; Ctrl- EVIR, n= 4; EVIR, n= 5; iEVIR, n= 3; iEVIRubNull, n= 5 independent cell cultures. Statistical analysis by one-way ANOVA.

[0237] A ubiquitination-resistant iEVIR improves cancer cell phagocytosis

[0238] The ability of iEVIRubNull-expressing DCs to internalize EVs more efficiently was also associated with enhanced phagocytic activity in a cell co-culture assay involving transduced moDCs and live B16F10-GD2 / OVA cells pre-stained with a fluorescent, pH-sensitive dye (Figure 7D). For this experiment, moDCs, harvested and cultured as explained in Example 6, were transduced with the indicated receptors. The pHrodo™ Red (Cat. No P35372) kit was used to measure the phagocytosis of cancer cells by DCs. Briefly, cancer cells were stained with a staining solution containing pHrodo™ AM Ester, which stains the cells' cytoplasm. This dye exhibits stronger fluorescence in the acidic pH of the lysosome than in neutral pH. After staining, the cancer cells were washed and co-cultured with moDCs. Following co-culture for 12 h, DCs were harvested and analyzed using flow cytometry to measure pHrodo™ Red fluorescence, indicative of cancer cell phagocytosis. Bar graph shows mean± SEM; n= 5 independent cell cultures. Statistical analysis by one-way ANOVA.

[0239] Example 8: iEVIRUbNull-expressing moDCs elicit antigen-specific T cells in tumors Inhibition of tumor growth by iEVIRubNull-expressing DCs

[0240] Then, it was investigated whether iEVIRubNull-expressing moDCs could elicit antigen-specific T cells in the absence of antigen loading. Mice carrying B16F10-GD2 / OVA tumors (Figure 8A) received a PD-1 antibody in combination with moDCs transduced with different EVIR constructs, including iEVIRubNulland earlier versions (Figure 8B), on days 4 and 7. For this study, moDCs, extracted and cultured as indicated in Example 6, were transduced with LVs expressing the indicated receptors. Two days after transduction, moDCs were harvested and either stained with FITC-conjugated anti-NGFR antibody (1 TOO; ME20.4-1, H4; MACS. Table 1) (EVIR) or stained with a FITC-conjugated anti-strep-tag II antibody (1:100; 5A9F9; GenScript; Table 1) (Ctrl-iEVIRubNull, iEVIR, and iEVIRubNull). Flow cytometry was performed using the LSR Fortessa apparatus, and the data were analyzed using FlowJo.

[0241] iEVIRubNull-expressing moDCs delayed tumor growth to a greater extent than moDCs transduced with other constructs (Figure 8C1 & C2). Tumor measurements were performed as explained in Figure 6B. The 5 cohorts are shown in two separate charts for clarity, with the PBS cohort shown in both. Data show tumor volume (mean ± SEM; PBS, n=7; Ctrl-iEVIRubNull, n= 6; EVIR, n=8; iEVIR, n=9; iEVIRubNull, n=8 tumors). Statistical analysis by two-way ANOVA.

[0242] At day 15, all mice were sacrificed and tumors harvested, chopped, and treated with collagenase, then passed through a 70 pm cell strainer to obtain single-cell suspensions. Single cell suspensions were prepared in PBS and dead cells were stained using the Zombie UV Dead Cell Stain Kit (1:500; Biolegend; Catalog Number 423107). Cells were then washed in FACS buffer. For MHCI dextramer staining, dextramers were added to single cell suspensions following the addition of Fc-block and prior to the staining of cell surface markers, according to the manufacturer’s instructions. Tumors were then washed and prepared in FACS buffer and stained for the indicated cell surface markers. Cells were analyzed using an LSR Fortessa apparatus (BD Biosciences). Data were analyzed using FlowJo. Bar graphs show mean ± SEM; PBS, n=6; Ctrl-iEVIRubNull, n= 6; EVIR, n=8; iEVIR, n=9; iEVIRubNull, n=8 tumors. Statistical analysis by one-way ANOVA.

[0243] In this setting of antigen-agnostic DC vaccination, the iEVIRubNullmoderately enhanced the prevalence of TRP2 (an endogenous melanoma-associated antigen) and OVA specific CD8+T cells compared to the other EVIRs, especially when the two cell populations were cumulatively assessed (Figure 8D). These results indicate that the iEVIRubNullcan promote internalization and presentation of tumor antigens to T cells.

[0244] Altogether, those data support that the iEVIR according to the invention helps the DCs to recognize, internalize, and process tiny membrane particles released by tumor cells, including extracellular vesicles (EVs), which carry many different tumor antigens. Concomitantly, expression of the iEVIR and its binding to the bait antigen (e.g., HER2 or GD2) leads to activation of the dendritic cells, specifically when they come into contact with the EVs or cancer cells that present the bait antigen. This ensures that the immune response is initiated by and targeted against cancer cells in a specific and timely manner. Additionally, the receptor allows the DCs to phagocytose and destroy cancer cells directly, further boosting their ability to fight tumors.

[0245] Further, the iEVIRs of the invention can trigger the production of an additional therapeutic gene product, making the dendritic cells even more effective.

[0246] In summary, the iEVIRs according to the invention provide a platform enhancing the body's immune response against cancer using specially engineered dendritic cells. This versatile tool can be adapted to desired surface antigens for targeting a specific type of cancer. Sequence listing

[0247] Amino acid sequence of DEC-205 protein fragment (synthetic construct)

[0248] SEQ ID NO: 1:

[0249] TKDKKLIFHVKSSKCPVAKRDGPQWVQYGGHCYASDQVLHSFSEAKQVCQELDHSATVVTIADENENK FVSRLMRENYNITMRVWLGLSQHSLDQSWSWLDGLDVTFVKWENKTKDGDGKCSILIASNETWRKVHC SRGYARAVCKIPLSPDYTGIAILFAVLCLLGLISLAIWFLLQRSHIRWTGFSSVRYEHGTNEDEVMLP SFHD

[0250] Amino acid sequence of MRC1 protein fragment (synthetic construct) SEQ ID NO: 2:

[0251] MPKIIDPVTTHSSITTKADQRKMDPQPKGSSKAAGVVTVVLLIVIGAGVAAYFFYKKRHALHIPQEAT FENTLYFNSNLSPGTSDTKDLMGNIEQNEHAII

[0252] Amino acid sequence of CD40 protein fragment (synthetic construct) SEQ ID NO: 3:

[0253] LKSRMRALLVIPVVMGILITIFGVFLYIKKVVKKPKDNEMLPPAARRQDPQEMEDYPGHNTAAPVQET LHGCQPVTQEDGKESRISVQERQVTDSIALRPLV

[0254] Amino acid sequence of FcyRIV protein fragment (synthetic construct) SEQ ID NO: 4:

[0255] LGDPGSPSMFPPWHQITFCLLIGLLFAIDTVLYFSVRRGLQSPVADYEEPKIQWSKEPQDK

[0256] Amino acid sequence of FLT3 protein fragment (synthetic construct) SEQ ID NO: 5:

[0257] PFPFIQDNISFYATIGLCLPFIVVLIVLICHKYKKQFRYESQLQMIQVTGPLDNEYFYVDFRDYEYDL KWEFPRENLEFGKVLGSGAFGRVMNATAYGISKTGVSIQVAVKMLKEKADSCEKEALMSELKMMTHLG HHDNIVNLLGACTLSGPVYLIFEYCCYGDLLNYLRSKREKFHRTWTEIFKEHNFSFYPTFQAHSNSSM PGSREVQLHPPLDQLSGFNGNLIHSEDEIEYENQKRLAEEEEEDLNVLTFEDLLCFAYQVAKGMEFLE FKSCVHRDLAARNVLVTHGKVVKICDFGLARDILSDSSYVVRGNARLPVKWMAPESLFEGIYTIKSDV WSYGILLWEIFSLGVNPYPGIPVDANFYKLIQSGFKMEQPFYATEGIYFVMQSCWAFDSRKRPSFPNL TSFLGCQLAEAEEAMYQNMGGNVPEHPSIYQNRRPLSREAGSEPPSPQAQVKIHGERS

[0258] Amino acid sequence of DECTIN1 protein fragment (synthetic construct) SEQ ID NO: 6:

[0259] MKYHSHIENLDEDGYTQLDFSTQDIHKRPRGSEKGSRAPSSPWRPIAVGLGILCFVVVVVAAVLGALA FWRHNSGRNPEEKDNFLSRNKENHKPTESSLDEKVAPSKASQTTGGFSQ

[0260] Amino acid sequence of CLEC9A protein fragment (synthetic construct) SEQ ID NO: 7:

[0261] MHAEEIYTSLQWDIPTSEASQKCQSPSKCSGAWCVVTMISCVVCMGLLATSIFLGIKFFQVSSLVLEQ QERLIQQDTALVNLTQWQRKYTLEYCQALLQRSLHSGTDASTGPVLLTSPQMVPQTLDSKETGSDCSP C

[0262] Amino acid sequence of DC-SIGN protein fragment (synthetic construct) SEQ ID NO: 8:

[0263] MSDSTEAKMQPLSSMDDDELMVSGSRYSIKSSRLRPNSGIKCLAGCSGHSQVPLVLQLLSFLFLAGLL LIILFQVSKTPNTERQKEQEKILQELTQLTDELTSRIPISQGKNESMQAKITEQLMQLKTELLSRIPI FQGQNESIQEKISEQLMQLKAELLSKISSFPVKDDSKQEKIYQQLVQMKTELFRLCRLCPWDWTFLLG NCYFFSK Nucleic acid (DNA) sequence of mutated IL12p40 promoter (synthetic construct)

[0264] SEQ ID NO: 9:

[0265] AAGTCATTTCCTCTTAACCTGGGATTTCGACGTCTATATTCCCTCTGTATGATAGATGCACTCAGGGA GGCAAGGGGGGGAGGGAGGAACTTCTTGGAATTCCCCCAGAATGTTTTGACACTAGTTTTCAGTGTTG CAATTGAGACTAGTCAGTTTCTACTTTGGGTTTCCATCAGAAAGTTCTGTAGGAGTAGAGTATATAAG CACCAGGAGCAGCCAAGGCAGCAGAAGGAACAGTGGGTGTCCAGGCACATCAGACCAGGCAGCTCGCA GCAAGGATCCGCCACC

[0266] Amino acid sequence of chimeric FLAG-CD40L fragment (synthetic construct)

[0267] SEQ ID NO: 10:

[0268] MHAEEIATSLQWDIPTSEASQKCQSPSKCSGAWCVVTMISCVVCMGLLATSIFLGIKFFQVSSLVLEQ QERLIQQDTALVNLTQWQRKYTLEYCQALLQRSLHSGTDASTGPVLLTSPQMVPQTLDSKETGSDCSP CGSFEMQRGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQV TFCSNREPSSQRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGGVFELQAGASVFVNVTE ASQVIHRVGFSSFGLLKLGSDYKDDDDK

[0269] Amino acid sequence of IgK signal peptide (synthetic construct) SEQ ID NO: 11:

[0270] MDFQVQIFSFLLISASVIMSRG

[0271] Amino acid sequence of anti-HER2 scFv extracellular domain (synthetic construct)

[0272] SEQ ID NO: 12:

[0273] DIVLTQTPSSLPVSVGEKVTMTCKSSQTLLYSNNQKNYLAWYQQKPGQSPKLLISWAFTRKSGVPDRF TGSGSGTDFTLTIGSVKAEDLAVYYCQQYSNYPWTFGGGTRLEIKRGGGGSGGGGSGGGGSGGGGSEV QLQQSGPEVVKTGASVKISCKASGYSFTGYFINWVKKNSGKSPEWIGHISSSYATSTYNQKFKNKAAF TVDTSSSTAFMQLNSLTSEDSAVYYCVRSGNYEEYAMDYWGQGTSVTVSSTGGGS

[0274] Amino acid sequence of anti-GD2 scFv extracellular domain (synthetic construct)

[0275] SEQ ID NO: 13:

[0276] EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRA TLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSSGGGGSGGGGSGGGGSDVVMTQTP LSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDF TLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELTGGGS

[0277] Amino acid sequence of Strep Tag II peptide (synthetic construct) SEQ ID NO: 14:

[0278] NWSHPQFEK

[0279] Amino acid sequence of mutated (Cys-193 to Ser-193) CD8a domain (synthetic construct)

[0280] SEQ ID NO: 15:

[0281] SHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDIYIWAPLAGTC GVLLLSLVITLYCNHRNRRRV

[0282] Amino acid sequence of CD40 activation domain (synthetic construct) SEQ ID NO: 16:

[0283] VKKPKDNEILPPAARRQDPQEMEDYPGHNTAAPVQETLHGCQPVTQEDGKESRISVQERQVTDSIAL

[0284] Amino acid sequence of FcRy chain ITAM motif (synthetic construct) SEQ ID NO: 17:

[0285] RGIQVRKAAIASREKADAVYTGLNTRSQETYETLKHEKPPQ Amino acid sequence of anti-HER2 iEVIR (synthetic construct)

[0286] SEQ ID NO: 18:

[0287] MDFQVQIFSFLLISASVIMSRGDIVLTQTPSSLPVSVGEKVTMTCKSSQTLLYSNNQKNYLAWYQQKP GQSPKLLISWAFTRKSGVPDRFTGSGSGTDFTLTIGSVKAEDLAVYYCQQYSNYPWTFGGGTRLEIKR GGGGSGGGGSGGGGSGGGGSEVQLQQSGPEVVKTGASVKISCKASGYSFTGYFINWVKKNSGKSPEWI GHISSSYATSTYNQKFKNKAAFTVDTSSSTAFMQLNSLTSEDSAVYYCVRSGNYEEYAMDYWGQGTSV TVSSTGGGSNWSHPQFEKSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTR GLDFASDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVVKKPKDNEILPPAARRQDPQEMEDYPGHN TAAPVQETLHGCQPVTQEDGKESRISVQERQVTDSIALRGIQVRKAAIASREKADAVYTGLNTRSQET YETLKHEKPPQ

[0288] Amino acid sequence of anti-GD2 iEVIR (synthetic construct)

[0289] SEQ ID NO: 19:

[0290] MDFQVQIFSFLLISASVIMSRGEVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLE WIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSSG GGGSGGGGSGGGGSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIH KVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELTGGGSNWSHP QFEKSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDIYIWAPL AGTCGVLLLSLVITLYCNHRNRRRVVKKPKDNEILPPAARRQDPQEMEDYPGHNTAAPVQETLHGCQP VTQEDGKESRISVQERQVTDSIALRGIQVRKAAIASREKADAVYTGLNTRSQETYETLKHEKPPQ

[0291] Amino acid sequence of anti-GD2 iEVIR0131”111(synthetic construct) SEQ ID NO: 20:

[0292] MDFQVQIFSFLLISASVIMSRGEVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLE WIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSSG GGGSGGGGSGGGGSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIH KVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELTGGGSNWSHP QFEKSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDIYIWAPL AGTCGVLLLSLVITLYCNHRNRRRVVRRPRDNEILPPAARRQDPQEMEDYPGHNTAAPVQETLHGCQP VTQEDGRESRISVQERQVTDSIALRPLVGIQVRRAAIASRERADAVYTGLNTRSQETYETLRHERPPQ

[0293] Nucleic sequence encoding anti-HER2 iEVIR (synthetic construct) SEQ ID NO: 21:

[0294] ATGGACTTCCAGGTGCAGATCTTTTCCTTCCTGCTGATTTCTGCATCGGTCATCATGAGCAGGGGGGA TATTGTCCTCACACAGACTCCCAGCTCCCTGCCTGTGTCCGTCGGAGAGAAAGTGACCATGACATGCA AGTCTAGTCAGACACTGCTCTACTCTAACAATCAGAAGAACTACCTCGCATGGTATCAGCAGAAACCA GGACAGAGCCCCAAGCTGCTCATCTCCTGGGCTTTCACCCGGAAATCCGGGGTGCCTGACCGCTTCAC AGGTAGCGGCTCCGGAACTGATTTTACTCTGACCATTGGATCTGTGAAGGCAGAGGACCTCGCCGTCT ACTATTGCCAGCAGTACAGTAATTATCCATGGACTTTTGGCGGAGGGACCAGGCTGGAAATCAAGAGA GGTGGAGGAGGGTCCGGTGGAGGAGGGTCTGGTGGAGGAGGGAGTGGTGGAGGAGGGTCAGAGGTGCA GCTGCAGCAGTCTGGCCCCGAAGTGGTCAAAACTGGAGCTTCAGTCAAAATCAGCTGTAAGGCATCTG GGTACAGCTTCACCGGCTACTTCATCAACTGGGTGAAGAAAAATTCAGGGAAGAGCCCTGAGTGGATC GGCCACATTTCAAGCTCCTACGCCACAAGCACTTACAACCAGAAGTTCAAAAATAAGGCCGCTTTTAC CGTGGACACATCTAGTTCAACCGCCTTCATGCAGCTGAACTCCCTCACATCTGAAGATAGTGCTGTGT ACTATTGTGTCAGGAGCGGCAACTACGAAGAATATGCTATGGATTACTGGGGGCAGGGGACCTCCGTG ACTGTCTCAAGCACCGGTGGGGGAAGCAACTGGAGCCACCCACAGTTTGAGAAGTCACACTTTGTTCC TGTGTTTCTTCCAGCCAAACCTACGACGACTCCGGCGCCGAGGCCCCCCACTCCGGCGCCAACAATTG CTTCACAGCCCCTCTCGTTACGGCCAGAGGCGAGTAGACCAGCAGCAGGAGGTGCAGTGCACACCCGA GGCCTCGACTTTGCTTCAGATATTTATATTTGGGCACCATTAGCTGGAACCTGTGGTGTTCTGCTGCT ATCTTTAGTCATCACACTGTACTGCAATCATAGAAATCGCCGGCGAGTGGTCAAGAAACCAAAGGACA ATGAAATCCTGCCTCCTGCGGCAAGGCGGCAGGACCCCCAGGAGATGGAGGACTATCCTGGGCACAAC ACTGCAGCTCCTGTCCAAGAGACACTTCATGGCTGTCAGCCAGTGACCCAGGAAGACGGAAAAGAATC TCGGATCAGCGTACAGGAGCGCCAGGTCACTGACAGCATTGCCCTGAGGGGCATCCAGGTGAGGAAAG CAGCCATTGCCTCCCGGGAAAAAGCTGACGCTGTCTACACGGGCCTCAACACAAGAAGCCAGGAGACC TATGAAACTCTGAAGCACGAGAAGCCACCTCAATGA Nucleic sequence encoding anti-GD2 iEVIR (synthetic construct) SEQ ID NO: 22:

[0295] ATGGACTTCCAGGTGCAGATCTTTTCCTTCCTGCTGATTTCTGCATCGGTCATCATGAGCAGGGGGGA AGTCCAGCTGCTCCAAAGTGGGCCAGAATTGGAGAAGCCTGGAGCCTCTGTGATGATCAGCTGCAAGG CAAGTGGGAGTTCATTTACAGGCTACAACATGAACTGGGTGCGGCAGAACATCGGAAAGAGCTTAGAG TGGATTGGGGCCATTGATCCCTATTATGGGGGCACAAGCTATAATCAGAAGTTCAAGGGACGGGCGAC CCTCACTGTGGACAAATCCTCCAGCACAGCCTACATGCACTTGAAGAGTCTTACTTCTGAAGACTCAG CTGTGTACTACTGTGTGTCAGGGATGGAGTACTGGGGCCAGGGCACCTCCGTCACTGTTAGTAGCGGT GGTGGAGGATCGGGCGGCGGAGGCTCTGGAGGGGGTGGCTCAGATGTGGTCATGACACAGACTCCCCT GTCACTGCCGGTCTCTTTGGGGGACCAGGCCAGCATATCCTGCCGCAGTTCCCAATCATTAGTTCACA GAAATGGAAATACCTATCTGCACTGGTACCTGCAGAAACCTGGGCAGTCCCCAAAACTACTCATCCAT AAAGTATCCAACAGGTTCTCTGGTGTACCTGATAGATTTTCTGGCAGCGGCAGTGGAACGGACTTTAC CTTGAAAATAAGTCGAGTGGAGGCTGAAGATCTTGGTGTTTATTTCTGTTCCCAATCTACACATGTGC CACCCCTCACCTTTGGTGCTGGAACGAAGCTGGAGCTGACCGGTGGGGGAAGCAACTGGAGCCACCCA CAGTTTGAGAAGTCACACTTTGTTCCTGTGTTTCTTCCAGCCAAACCTACGACGACTCCGGCGCCGAG GCCCCCCACTCCGGCGCCAACAATTGCTTCACAGCCCCTCTCGTTACGGCCAGAGGCGAGTAGACCAG CAGCAGGAGGTGCAGTGCACACCCGAGGCCTCGACTTTGCTTCAGATATTTATATTTGGGCACCATTA GCTGGAACCTGTGGTGTTCTGCTGCTATCTTTAGTCATCACACTGTACTGCAATCATAGAAATCGCCG GCGAGTGGTCAAGAAACCAAAGGACAATGAAATCCTGCCTCCTGCGGCAAGGCGGCAGGACCCCCAGG AGATGGAGGACTATCCTGGGCACAACACTGCAGCTCCTGTCCAAGAGACACTTCATGGCTGTCAGCCA GTGACCCAGGAAGACGGAAAAGAATCTCGGATCAGCGTACAGGAGCGCCAGGTCACTGACAGCATTGC CCTGAGGGGCATCCAGGTGAGGAAAGCAGCCATTGCCTCCCGGGAAAAAGCTGACGCTGTCTACACGG GCCTCAACACAAGAAGCCAGGAGACCTATGAAACTCTGAAGCACGAGAAGCCACCTCAATGA

[0296] Nucleic sequence encoding anti-GD2 iEVIRUbNuU(synthetic construct)

[0297] SEQ ID NO: 23:

[0298] ATGGACTTCCAGGTGCAGATCTTTTCCTTCCTGCTGATTTCTGCATCGGTCATCATGAGCAGGGGGGA AGTCCAGCTGCTCCAAAGTGGGCCAGAATTGGAGAAGCCTGGAGCCTCTGTGATGATCAGCTGCAAGG CAAGTGGGAGTTCATTTACAGGCTACAACATGAACTGGGTGCGGCAGAACATCGGAAAGAGCTTAGAG TGGATTGGGGCCATTGATCCCTATTATGGGGGCACAAGCTATAATCAGAAGTTCAAGGGACGGGCGAC CCTCACTGTGGACAAATCCTCCAGCACAGCCTACATGCACTTGAAGAGTCTTACTTCTGAAGACTCAG CTGTGTACTACTGTGTGTCAGGGATGGAGTACTGGGGCCAGGGCACCTCCGTCACTGTTAGTAGCGGT GGTGGAGGATCGGGCGGCGGAGGCTCTGGAGGGGGTGGCTCAGATGTGGTCATGACACAGACTCCCCT GTCACTGCCGGTCTCTTTGGGGGACCAGGCCAGCATATCCTGCCGCAGTTCCCAATCATTAGTTCACA GAAATGGAAATACCTATCTGCACTGGTACCTGCAGAAACCTGGGCAGTCCCCAAAACTACTCATCCAT AAAGTATCCAACAGGTTCTCTGGTGTACCTGATAGATTTTCTGGCAGCGGCAGTGGAACGGACTTTAC CTTGAAAATAAGTCGAGTGGAGGCTGAAGATCTTGGTGTTTATTTCTGTTCCCAATCTACACATGTGC CACCCCTCACCTTTGGTGCTGGAACGAAGCTGGAGCTGACCGGTGGGGGAAGCAACTGGAGCCACCCA CAGTTTGAGAAGTCACACTTTGTTCCTGTGTTTCTTCCAGCCAAACCTACGACGACTCCGGCGCCGAG GCCCCCCACTCCGGCGCCAACAATTGCTTCACAGCCCCTCTCGTTACGGCCAGAGGCGAGTAGACCAG CAGCAGGAGGTGCAGTGCACACCCGAGGCCTCGACTTTGCTTCAGATATTTATATTTGGGCACCATTA GCTGGAACCTGTGGTGTTCTGCTGCTATCTTTAGTCATCACACTGTACTGCAATCATAGAAATCGCCG GCGAGTGGTGCGCAGGCCCAGGGACAACGAGATCCTGCCACCAGCTGCTAGACGGCAGGACCCCCAGG AGATGGAGGATTACCCTGGACACAACACCGCCGCTCCAGTGCAGGAGACACTGCACGGATGCCAGCCA GTGACCCAGGAGGACGGAAGGGAGAGCAGGATCTCCGTGCAGGAGCGCCAGGTGACAGATTCCATCGC CCTGAGGCCCCTGGTGGGAATCCAGGTGAGGCGAGCAGCCATTGCCTCCAGAGAAAGAGCTGACGCTG TCTACACAGGCCTCAACACGCGCAGCCAGGAGACCTATGAAACTCTGCGGCACGAGAGGCCTCCACAA TGA Nucleic sequence encoding anti-HER2 (synthetic construct)

[0299] SEQ ID NO: 24:

[0300] GATATTGTCCTCACACAGACTCCCAGCTCCCTGCCTGTGTCCGTCGGAGAGAAAGTGACCATGACATG CAAGTCTAGTCAGACACTGCTCTACTCTAACAATCAGAAGAACTACCTCGCATGGTATCAGCAGAAAC CAGGACAGAGCCCCAAGCTGCTCATCTCCTGGGCTTTCACCCGGAAATCCGGGGTGCCTGACCGCTTC ACAGGTAGCGGCTCCGGAACTGATTTTACTCTGACCATTGGATCTGTGAAGGCAGAGGACCTCGCCGT CTACTATTGCCAGCAGTACAGTAATTATCCATGGACTTTTGGCGGAGGGACCAGGCTGGAAATCAAGA GAGGTGGAGGAGGGTCCGGTGGAGGAGGGTCTGGTGGAGGAGGGAGTGGTGGAGGAGGGTCAGAGGTG CAGCTGCAGCAGTCTGGCCCCGAAGTGGTCAAAACTGGAGCTTCAGTCAAAATCAGCTGTAAGGCATC TGGGTACAGCTTCACCGGCTACTTCATCAACTGGGTGAAGAAAAATTCAGGGAAGAGCCCTGAGTGGA TCGGCCACATTTCAAGCTCCTACGCCACAAGCACTTACAACCAGAAGTTCAAAAATAAGGCCGCTTTT ACCGTGGACACATCTAGTTCAACCGCCTTCATGCAGCTGAACTCCCTCACATCTGAAGATAGTGCTGT GTACTATTGTGTCAGGAGCGGCAACTACGAAGAATATGCTATGGATTACTGGGGGCAGGGGACCTCCG TGACTGTCTCAAGC

[0301] Nucleic sequence encoding anti-GD2 (synthetic construct)

[0302] SEQ ID NO: 25:

[0303] GAAGTCCAGCTGCTCCAAAGTGGGCCAGAATTGGAGAAGCCTGGAGCCTCTGTGATGATCAGCTGCAA GGCAAGTGGGAGTTCATTTACAGGCTACAACATGAACTGGGTGCGGCAGAACATCGGAAAGAGCTTAG AGTGGATTGGGGCCATTGATCCCTATTATGGGGGCACAAGCTATAATCAGAAGTTCAAGGGACGGGCG ACCCTCACTGTGGACAAATCCTCCAGCACAGCCTACATGCACTTGAAGAGTCTTACTTCTGAAGACTC AGCTGTGTACTACTGTGTGTCAGGGATGGAGTACTGGGGCCAGGGCACCTCCGTCACTGTTAGTAGCG GTGGTGGAGGATCGGGCGGCGGAGGCTCTGGAGGGGGTGGCTCAGATGTGGTCATGACACAGACTCCC CTGTCACTGCCGGTCTCTTTGGGGGACCAGGCCAGCATATCCTGCCGCAGTTCCCAATCATTAGTTCA CAGAAATGGAAATACCTATCTGCACTGGTACCTGCAGAAACCTGGGCAGTCCCCAAAACTACTCATCC ATAAAGTATCCAACAGGTTCTCTGGTGTACCTGATAGATTTTCTGGCAGCGGCAGTGGAACGGACTTT ACCTTGAAAATAAGTCGAGTGGAGGCTGAAGATCTTGGTGTTTATTTCTGTTCCCAATCTACACATGT GCCACCCCTCACCTTTGGTGCTGGAACGAAGCTGGAGCTG

[0304] Nucleic sequence encoding a signal peptide (synthetic construct)

[0305] SEQ ID NO: 26:

[0306] ATGGACTTCCAGGTGCAGATCTTTTCCTTCCTGCTGATTTCTGCATCGGTCATCATGAGCAGGGGG

[0307] Nucleic sequence encoding intracellular CD40 activation domain (synthetic construct) SEQ ID NO: 27:

[0308] GTCAAGAAACCAAAGGACAATGAAATCCTGCCTCCTGCGGCAAGGCGGCAGGACCCCCAGGAGATGGA GGACTATCCTGGGCACAACACTGCAGCTCCTGTCCAAGAGACACTTCATGGCTGTCAGCCAGTGACCC AGGAAGACGGAAAAGAATCTCGGATCAGCGTACAGGAGCGCCAGGTCACTGACAGCATTGCCCTG

[0309] Nucleic sequence encoding intracellular ITAM motif derived from FcRy domain (synthetic construct)

[0310] SEQ ID NO: 28:

[0311] AGGGGCATCCAGGTGAGGAAAGCAGCCATTGCCTCCCGGGAAAAAGCTGACGCTGTCTACACGGGCCT CAACACAAGAAGCCAGGAGACCTATGAAACTCTGAAGCACGAGAAGCCACCTCAA

[0312] Nucleic sequence encoding monomerized CD8 hinge (synthetic construct)

[0313] SEQ ID NO: 29:

[0314] TCACACTTTGTTCCTGTGTTTCTTCCAGCCAAACCTACGACGACTCCGGCGCCGAGGCCCCCCACTCC GGCGCCAACAATTGCTTCACAGCCCCTCTCGTTACGGCCAGAGGCGAGTAGACCAGCAGCAGGAGGTG CAGTGCACACCCGAGGCCTCGACTTTGCTTCAGATATTTATATTTGGGCACCATTAGCTGGAACCTGT GGTGTTCTGCTGCTATCTTTAGTCATCACACTGTACTGCAATCATAGAAATCGCCGGCGAGTG

Claims

Claims1. A recombinant instructive extra-cellular vesicle internalizing receptor (iEVIR) comprising:a signal peptide;a single-chain variable fragment (scFv) extracellular domain specific for a membrane- associated molecule of a cancer cell;a transmembrane / hinge domain;an intracellular CD40 activation domain; andan intracellular FcRy chain ITAM motif.

2. An iEVIR according to claim 1, wherein the intracellular CD40 activation domain comprises or consisting in a sequence of SEQ ID NO: 16 or a variant thereof.

3. An iEVIR according to any one of the preceding claims, wherein the intracellular FcRy chain ITAM motif comprises or consists in a sequence of SEQ ID NO: 17 or a variant thereof.

4. An iEVIR according to any one of the preceding claims, wherein the signal peptide is an IgK signal peptide (e.g. comprising of consisting in a sequence of SEQ ID NO: 11 or a variant thereof).

5. An iEVIR according to any one of the preceding claims, wherein the single-chain variable fragment (scFv) extracellular domain specific for a membrane-associated molecule of a cancer cell comprises a sequence specific for one or more of the following: human epidermal growth factor receptor 2 (HER2), gangliosides (GD2, GD3), tyrosinase-related protein- 1 (TYRP1), carcinoembryonic antigen (CEA), mesothelin, PMEL (gplOO), and mucins.

6. An iEVIR according to any one of the preceding claims, wherein the single-chain variable fragment (scFv) extracellular domain comprises a sequence specific for human epidermal growth factor receptor 2 (HER2) or for GD2.

7. An iEVIR according to any one of the preceding claims, comprising an amino acid sequence selected from SEQ ID NO: 18, 19 or 20, or a variant thereof.

8. An isolated nucleic acid sequence encoding an iEVIR according to any one of claims 1 to 7.

9. An isolated nucleic acid according to claim 8 comprising at least one of the nucleic acid sequences of SEQ ID NO: 21 to 30.

10. A vector system comprising at least one nucleic acid sequence of claim 8 or 9 and further comprising at least one nucleic acid sequence encoding an inducible system for the expression of a chimeric CD40L protein regulated by a synthetic IL12 promoter sequence with NF-kB response elements.

11. An ex vivo method of inducing expression of at least one iEVIR according to any of claims 1 to 7, in an antigen-presenting cell (APC), said method comprising the step of ex vivo transfecting or transducing said cell with a vector encoding at least one iEVIR according to any of any one of claims 1 to 7.

12. A method according to claim 11, wherein the vector comprises a nucleic acid sequence ofSEQ ID NO: 21 to 3013. An isolated APC expressing at least one iEVIR according to any one of claims 1 to 7.

14. An isolated APC according to claim 13, wherein said AP expresses from about 1 to about 3 different iEVIRs according to any one of claims 1 to 7.

15. A pharmaceutical composition comprising at least one vector encoding at least one iEVIR according to any of any one of claims 1 to 7 or a cell according to claim 13 or 14 or a cell obtainable from a method according to claims 11 or 12 and at least one pharmaceutically acceptable carrier, diluent or excipient thereof.

16. A pharmaceutical composition according to claim 15 for use in the prevention and / or treatment of a cancer.

17. A pharmaceutical composition for use according to claim 16, wherein said cancer is selected from suffering from melanoma, lung cancer, intestinal cancer, breast cancer, glioblastoma and neuroblastoma.

18. A method of preventing and / or treating a cancer comprising an administering effective amount of iEVIR-expressing APCs according to any one of claims 1 to 7, or prospectivestem / progenitor cells thereof, or at least one recombinant vector according to claim 10 in a subject in need thereof19. A kit comprising at least one iEVIR, or at least one recombinant expression vector according to claim 10, or at least one iEVIR-expressing cell according to any one of claims 1 to 7.

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