Method to capture circulating tumor extracellular vesicles

Abstract

The present invention relates to a method to capture circulating tumor extracellular vesicles. Here the inventors describe a novel approach inspired by Chimeric Antigen Recognition (CAR)-like strategy, to selectively capture and locally accumulate circulating tumor EVs towards engrafted trapping cells, or endogenous trapping tissues of choice. Focusing on Prostate Cancer (PCa)-derived EVs as a proof of concept, they combined the pHluorin- genetic tagging of tumor EVs with the expression on target cells of the EV-Trap, a chimeric surface mCD8α receptor exposing various nanobodies directed against EVs' surface antigens (synthetic or tumor associated). They demonstrate how the simultaneous presence of PCa- derived CD63-pHluorin+, CD9-pHluorin+ or PSMA+ EVs in a system presenting GFP-TRAP or PSMA-TRAP accessible cell surfaces, results in significant accumulation and rerouting of PCa-derived pHluorin and PSMA EVs towards the EV-TRAP sites, both in vitro and in vivo in the zebrafish embryo. This approach has the potential to shed new lights on the role of EVs in various physiopathological processes, in particular on pre-metastatic niches formation. Thus, the present invention relates to a fusion protein comprising or consisting of an antigen binding compound, the mCD8α and optionally a domain which induce endocytosis (or internalisation).

Description

[0001] METHOD TO CAPTURE CIRCULATING TUMOR EXTRACELLULAR VESICLES

[0002] FIELD OF THE INVENTION:

[0003] The present invention relates to a fusion protein comprising or consisting of an antigen binding compound, the mCD8a and optionally a domain which induce endocytosis (or internalisation).

[0004] BACKGROUND OF THE INVENTION:

[0005] Extracellular Vesicles (EVs) are recognized nowadays as key mediators in various fields of biology, including cellular and molecular biology of tumors. Tumor-derived EVs play pivotal roles in pre-metastatic niches formation, in tumor migration, survival and proliferation, as well as in tumor immune-escape and chemoresistance transfer. Yet, the field is still facing major gaps in our understanding of the role of EVs in vivo. Several approaches have been recently developed to live-track the dynamic and fate of tumor derived EVs at a single-particle scale in vivo, notably in the zebrafish embryo. However, tools to selectively guide and manipulate tumor EVs dissemination and targeting in vivo are still lacking in the field, complicating efforts to clarify their relative contributions to the overall intercellular communication in pathological development. Here, the inventors reasoned how manipulating tumor EVs biodistribution in vivo by forcing the spatial rerouting of tumor EVs towards a chosen cellular target might result in a better comprehension of their biological roles, for instance in pre-metastatic niches formation.

[0006] SUMMARY OF THE INVENTION:

[0007] Here the inventors describe a novel approach inspired by Chimeric Antigen Recognition (CAR)-like strategy, to selectively capture and locally accumulate circulating tumor EVs towards engrafted trapping cells, or endogenous trapping tissues of choice. Focusing on Prostate Cancer (PCa)-derived EVs as a proof of concept, they combined the pHluorin-genetic tagging of tumor EVs with the expression on target cells of the EV-Trap, a chimeric surface mCD8a receptor exposing various nanobodies directed against EVs’ surface antigens (synthetic or tumor associated). They then assessed the capacity of this EV-Trap to divert EVs from their natural target and accumulate PCa-derived EVs at the location of their choice. They demonstrate how the simultaneous presence of PCa-derived CD63-pHluorin+, CD9-pHluorin+ or PSMA+ EVs in a system presenting GFP-TRAP or PSMA-TRAP accessible cell surfaces, results in significant accumulation and rerouting of PCa-derived pHluorin and PSMA EVs towards the EV-TRAP sites, both in vitro and in vivo in the zebrafish embryo. This approach has the potential to shed new lights on the role of EVs in various physiopathological processes, in particular on pre-metastatic niches formation.

[0008] Thus, the present invention relates to a fusion protein comprising or consisting of an antigen binding compound, the mCD8a and optionally a domain which induce endocytosis (or internalisation).

[0009] Particularly, the invention is defined by its claims.

[0010] DETAILED DESCRIPTION OF THE INVENTION:

[0011] Fusion protein of the invention

[0012] The inventors generated a fusion protein (also called “chimeric surface receptor”) which they called EV-Trap for selectively capture and locally accumulate circulating tumor EVs.

[0013] Thus, a first aspect of the invention relates to a fusion protein comprising or consisting of an antigen binding compound, the mCD8a and optionally a domain which induce endocytosis (or internalisation).

[0014] Particularly, the invention relates to fusion protein consisting of an antigen binding compound, the mCD8a and optionally a domain which induce endocytosis (or internalisation). In a particular embodiment, the fusion protein also comprises a reporter compound.

[0015] In a particular embodiment, the fusion protein also comprises in option a tag compound.

[0016] In a particular embodiment, the fusion protein also comprises in option a loading compound.

[0017] In a particular embodiment, the fusion protein of the invention, further comprising a linker connecting the compound or domain of the fusion protein. Particularly, the linker can be a domain of an inducible photodimerization system (see for example Benedetti Lorena, eLife. 2020).

[0018] According to the invention, the antigen binding compound, the mCD8a, the reporter compound, the tag compound and the loading compound can be fused together by their C- terminus or N-terminus. The antigen binding compound

[0019] In one embodiment and according to the invention, the antigen binding compound is an antibody.

[0020] As used herein the term "antibody" or "immunoglobulin" have the same meaning and will be used equally in the present invention. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. molecules that contain an antigen binding site that immunospecifically binds an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments. In natural antibodies, two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (1) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody binding site and the antigenic determinant. Antibody binding sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FR) can participate to the antibody binding site or influence the overall domain structure and hence the binding site. Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L- CDR2, L- CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. An antigen-binding site, therefore, typically includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs. As used herein, the term “specificity” refers to the ability of an antibody to detectably bind an epitope presented on an antigen while having relatively little detectable reactivity with non-antigen proteins or structures. Specificity can be relatively determined by binding or competitive binding assays, using, e.g. Biacore instruments, as described elsewhere herein. Specificity can be exhibited by, e.g. an about 10: 1, about 20: 1, about 50: 1, about 100: 1, 10.000: 1 or greater ratio of affinity / avidity in binding to the specific antigen versus non-specific binding to other irrelevant molecules.

[0021] The term “affinity”, as used herein, means the strength of the binding of an antibody to an epitope. The affinity of an antibody is given by the dissociation constant Kd, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen. The affinity constant Ka is defined by 1 / Kd. Preferred methods for determining the affinity of mAbs can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which references are entirely incorporated herein by reference. One preferred and standard method well known in the art for determining the affinity of mAbs is the use of Biacore instruments.

[0022] The terms "monoclonal antibody", "monoclonal Ab", "monoclonal antibody composition", "mAb", or the like, as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0023] The antibodies of the present invention are produced by any technique known in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination. Typically, knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said antibodies, by standard techniques for production of polypeptides. For instance, they can be synthesized using well-known solid phase method, preferably using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, California) and following the manufacturer’s instructions. Alternatively, antibodies of the present invention can be synthesized by recombinant DNA techniques well-known in the art. For example, antibodies can be obtained as DNA expression products after incorporation of DNA sequences encoding the antibodies into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired antibodies, from which they can be later isolated using well-known techniques. In one embodiment, the monoclonal antibody of the invention is a chimeric antibody, particularly a chimeric mouse / human antibody.

[0024] According to the invention, the term "chimeric antibody" refers to an antibody that comprises a VH domain and a VL domain of a non-human antibody, and a CH domain and a CL domain of a human antibody.

[0025] In some embodiments, the human chimeric antibody of the present invention can be produced by obtaining nucleic sequences encoding VL and VH domains as previously described, constructing a human chimeric antibody expression vector by inserting them into an expression vector for animal cell having genes encoding human antibody CH and human antibody CL, and expressing the coding sequence by introducing the expression vector into an animal cell. As the CH domain of a human chimeric antibody, it may be any region that belongs to human immunoglobulin, but those of IgG class are suitable and any one of subclasses belonging to IgG class, such as IgGl, IgG2, IgG3 and IgG4, can also be used. Also, as the CL of a human chimeric antibody, it may be any region which belongs to Ig, and those of kappa class or lambda class can be used. Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques are well known in the art (See Morrison SL. et al. (1984) and patent documents US5,202,238; and US5,204, 244).

[0026] In another embodiment, the monoclonal antibody of the invention is a humanized antibody. In particular, in said humanized antibody, the variable domain comprises human acceptor frameworks regions, and optionally human constant domain where present, and non- human donor CDRs, such as mouse CDRs.

[0027] In one embodiment, the humanized antibody can be derived from a chimeric antibody.

[0028] In another embodiment, the monoclonal antibody of the invention is a caninized or primatized based on the same methods of humanization.

[0029] According to the invention, the term "humanized antibody" refers to an antibody having variable region framework and constant regions from a human antibody but retains the CDRs of a previous non-human antibody.

[0030] The humanized antibody of the present invention may be produced by obtaining nucleic acid sequences encoding CDR domains, as previously described, constructing a humanized antibody expression vector by inserting them into an expression vector for animal cell having genes encoding (i) a heavy chain constant region identical to that of a human antibody and (ii) a light chain constant region identical to that of a human antibody, and expressing the genes by introducing the expression vector into an animal cell. The humanized antibody expression vector may be either of a type in which a gene encoding an antibody heavy chain and a gene encoding an antibody light chain exists on separate vectors or of a type in which both genes exist on the same vector (tandem type). In respect of easiness of construction of a humanized antibody expression vector, easiness of introduction into animal cells, and balance between the expression levels of antibody H and L chains in animal cells, humanized antibody expression vector of the tandem type is preferred. Examples of tandem type humanized antibody expression vector include pKANTEX93 (WO 97 / 10354), pEE18 and the like. Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (See, e. g., Riechmann L. et al. 1988; Neuberger MS. et al. 1985). Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO91 / 09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA. et al. (1994)), and chain shuffling (U.S. Pat. No.5, 565, 332). The general recombinant DNA technology for preparation of such antibodies is also known (see European Patent Application EP 125023 and International Patent Application WO 96 / 02576).

[0031] In some embodiments the antibody of the invention is a human antibody.

[0032] As used herein the term "human antibody is intended to include antibodies having variable and constant regions derived from human immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0033] Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, cur. Opin. Pharmacol. 5; 368-74 (2001) and lonberg, cur. Opin. Immunol. 20; 450-459 (2008). Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, or which are present extra-chromosomally or integrated randomly into the animal’s chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat.Biotech. 23; 1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™ technology; U.S. Patent No. 5,770,429 describing HUMAB® technology; U.S. Patent No. 7,041,870 describing K-M MOUSE® technology, and U.S. Patent Application publication No. US 2007 / 0061900, describing VELOCIMOUSE® technology. Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region. Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 13: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86(1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005). Fully human antibodies can also be derived from phagedisplay libraries (as disclosed in Hoogenboom et al., 1991, J. Mol. Biol. 227:381; and Marks et al., 1991, J. Mol. Biol. 222:581). Phage display techniques mimic immune selection through the display of antibody repertoires on the surface of filamentous bacteriophage, and subsequent selection of phage by their binding to an antigen of choice. One such technique is described in PCT publication No. WO 99 / 10494. Human antibodies described herein can also be prepared using SCID mice into which human immune cells have been reconstituted such that a human antibody response can be generated upon immunization. Such mice are described in, for example, U.S. Patent Nos. 5,476,996 and 5,698,767 to Wilson et al.

[0034] In one embodiment, the antibody of the invention is an antigen-biding fragment selected from the group consisting of a Fab, a F(ab)’2, a single domain antibody, a ScFv, a Sc(Fv)2, a diabody, a triabody, a tetrabody, an unibody, a minibody, a maxibody, a small modular immunopharmaceutical (SMIP), minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody as an isolated complementary determining region (CDR), and fragments which comprise or consist of the VL or VH chains.

[0035] The term “antigen-binding fragment” of an antibody, as used herein, refers to one or more fragments of an intact antibody that retain the ability to specifically binds to a given antigen. Antigen-binding functions of an antibody can be conferred by fragments of an intact antibody. Examples of binding-fragments encompassed within the term antigen bindingfragment of an antibody include a Fab fragment, a monovalent fragment consisting of the VL,VH,CL and CHI domains; a Fab’ fragment, a monovalent fragment consisting of the VL,VH,CL,CH1 domains and hinge region; a F(ab’)2 fragment, a bivalent fragment comprising two Fab’ fragments linked by a disulfide bridge at the hinge region; an Fd fragment consisting of VH domains of a single arm of an antibody; a single domain antibody (sdAb) fragment (Ward et al., 1989 Nature 341 :544-546), which consists of a VH domain or a VL domain; and an isolated complementary determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by an artificial peptide linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (ScFv); see, e.g., Bird et al., 1989 Science 242:423-426; and Huston et al., 1988 proc. Natl. Acad. Sci. 85:5879-5883). "dsFv" is a VH:VL heterodimer stabilised by a disulfide bond. Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)2. Such single chain antibodies include one or more antigen-binding portions or fragments of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as are intact antibodies. A unibody is another type of antibody fragment lacking the hinge region of IgG4 antibodies. The deletion of the hinge region results in a molecule that is essentially half the size of traditional IgG4 antibodies and has a univalent binding region rather than the bivalent biding region of IgG4 antibodies. Antigen-binding fragments can be incorporated into single domain antibodies, SMTP, maxibodies, minibodies, intrabodies, diabodies, triabodies and tetrabodies (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). The term "diabodies" “tribodies” or “tetrabodies” refers to small antibody fragments with multivalent antigen-binding sites (2, 3 or four), which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Antigen-binding fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CHl-VH-CHl) which, together with complementary light chain polypeptides, form a pair of antigen-binding regions (Zapata et al., 1995 Protein Eng. 8(10); 1057-1062 and U.S. Pat. No. 5,641,870).

[0036] The Fab of the present invention can be obtained by treating an antibody that specifically reacts with a protease, papaine. Also, the Fab can be produced by inserting DNA encoding Fab of the antibody into a vector for prokaryotic expression system, or for eukaryotic expression system, and introducing the vector into a procaryote or eucaryote (as appropriate) to express the Fab.

[0037] The F(ab')2 of the present invention can be obtained by treating an antibody that specifically reacts with a protease, pepsin. Also, the F(ab')2 can be produced by binding Fab' described below via a thioether bond or a disulfide bond.

[0038] The Fab' of the present invention can be obtained by treating F(ab')2 that specifically reacts with a reducing agent, dithiothreitol. Also, the Fab' can be produced by inserting DNA encoding Fab' fragment of the antibody into an expression vector for prokaryote, or an expression vector for eukaryote, and introducing the vector into a prokaryote or eukaryote (as appropriate) to perform its expression.

[0039] The scFv of the present invention can be produced by obtaining cDNA encoding the VH and VL domains as previously described, constructing DNA encoding scFv, inserting the DNA into an expression vector for prokaryote, or an expression vector for eukaryote, and then introducing the expression vector into a prokaryote or eukaryote (as appropriate) to express the scFv. To generate a humanized scFv fragment, a well-known technology called CDR grafting may be used, which involves selecting the complementary determining regions (CDRs) from a donor scFv fragment, and grafting them onto a human scFv fragment framework of known three-dimensional structure (see, e. g., W098 / 45322; WO 87 / 02671; US5,859,205; US5,585,089; US4,816,567; EP0173494).

[0040] Domain Antibodies (dAbs) are the smallest functional binding units of antibodies - molecular weight approximately 13 kDa - and correspond to the variable regions of either the heavy (VH) or light (VL) chains of antibodies. Further details on domain antibodies and methods of their production are found in US 6,291,158; 6,582,915; 6,593,081; 6,172,197; and 6,696,245; US 2004 / 0110941; EP 1433846, 0368684 and 0616640; WO 2005 / 035572, 2004 / 101790, 2004 / 081026, 2004 / 058821, 2004 / 003019 and 2003 / 002609, each of which is herein incorporated by reference in its entirety.

[0041] UniBodies are another antibody fragment technology, based upon the removal of the hinge region of IgG4 antibodies. The deletion of the hinge region results in a molecule that is essentially half the size of a traditional IgG4 antibody and has a univalent binding region rather than a bivalent binding region. Furthermore, because UniBodies are about smaller, they may show better distribution with potentially advantageous efficacy. Further details on UniBodies may be obtained by reference to WO 2007 / 059782, which is incorporated by reference in its entirety.

[0042] In one embodiment and according to the invention, the antigen binding compound is a single domain antibody.

[0043] As used herein the term “single domain antibody” has its general meaning in the art and refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals, which are naturally devoid of light chains. Such single domain antibody are also called VHH or “nanobody®”. For a general description of (single) domain antibodies, reference is also made to the prior art cited above, as well as to EP 0 368 684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21(11):484- 490; and WO 06 / 030220, WO 06 / 003388. The nanobody® has a molecular weight approximately one-tenth that of a human IgG molecule, and the protein has a physical diameter of only a few nanometers. One consequence of the small size is the ability of camelid nanobodies to bind to antigenic sites that are functionally invisible to larger antibody proteins, i.e. camelid nanobodies are useful as reagents to detect antigens that are otherwise cryptic using classical immunological techniques, and as possible therapeutic agents. Thus, yet another consequence of small size is that a nanobody® can inhibit as a result of binding to a specific site in a groove or narrow cleft of a target protein, and hence can serve in a capacity that more closely resembles the function of a classical low molecular weight drug rather than that of a classical antibody. The low molecular weight and compact size further result in nanobodies being extremely thermostable, stable to extreme pH and to proteolytic digestion, and poorly antigenic. Another consequence is that nanobodies readily move from the circulatory system into tissues, and even cross the blood-brain barrier and can treat disorders that affect nervous tissue. Nanobodies can further facilitate drug transport across the blood brain barrier. See U.S. patent application 20040161738 published August 19, 2004. These features combined with the low antigenicity to humans indicate great therapeutic potential. The amino acid sequence and structure of a single domain antibody can be considered to be comprised of four framework regions or "FRs", which are referred to in the art and herein as "Framework region 1" or "FR1 "; as "Framework region 2" or "FR2"; as "Framework region 3 " or "FR3"; and as "Framework region 4" or “FR4” respectively; which framework regions are interrupted by three complementary determining regions or "CDRs", which are referred to in the art as "Complementarity Determining Region for "CDR1”; as "Complementarity Determining Region 2" or "CDR2” and as "Complementarity Determining Region 3" or "CDR3", respectively. Accordingly, the single domain antibody can be defined as an amino acid sequence with the general structure : FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4 respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3. In the context of the invention, the amino acid residues of the single domain antibody are numbered according to the general numbering for VH domains given by the International ImMunoGeneTics information system aminoacid numbering (http: / / imgt.cines.fr / ).

[0044] Camel Ig can be modified by genetic engineering to yield a small protein having high affinity for a target, resulting in a low molecular weight antibody-derived protein known as a "nanobody®" or “VHH”. See U.S. patent number 5,759,808 issued June 2, 1998; see also Stijlemans, B. et al. , 2004 J Biol Chem 279: 1256-1261 ; Dumoulin, M. et al. , 2003 Nature 424: 783-788; Pleschberger, M. et al. 2003 Bioconjugate Chem 14: 440- 448; Cortez- Retamozo, V. et al. 2002 Int J Cancer 89: 456-62; and Lauwereys, M. et al. 1998 EMBO J 17: 3512-3520. Engineered libraries of camelid antibodies and antibody fragments are commercially available, for example, from Ablynx, Ghent, Belgium. In certain embodiments herein, the camelid antibody or nanobody® is naturally produced in the camelid animal, i.e. is produced by the camelid following immunization with an antigen or a peptide fragment thereof, using techniques described herein for other antibodies. Alternatively, the antigen-binding camelid nanobody® is engineered, i.e. produced by selection for example from a library of phage displaying appropriately mutagenized camelid nanobody® proteins using panning procedures with an antigen as a target.

[0045] In some embodiments, the single domain antibody is a “humanized” single domain antibody.

[0046] As used herein the term “humanized” refers to a single domain antibody of the invention wherein an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain has been "humanized", i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH sequence (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional chain antibody from a human being. Methods for humanizing single domain antibodies are well known in the art. Typically, the humanizing substitutions should be chosen such that the resulting humanized single domain antibodies still retain the favorable properties of single domain antibodies of the invention. The one skilled in the art is able to determine and select suitable humanizing substitutions or suitable combinations of humanizing substitutions.

[0047] Engineered antibodies of the invention include those in which modifications have been made to framework residues within VH and / or VL, e.g. to improve the properties of the antibody. Typically, such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations can be "backmutated" to the germline sequence by, for example, site-directed mutagenesis or PCR- mediated mutagenesis. Such "backmutated" antibodies are also intended to be encompassed by the invention. Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent Publication No. 20030153043 by Carr et al.

[0048] In some embodiments, the glycosylation of an antibody is modified. Glycosylation can be altered to, for example, increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[0049] In some embodiments, some mutations are made to the amino acids localized in aggregation “hotspots” within and near the first CDR (CDR1) to decrease the antibodies susceptibility to aggregation (see Joseph M. Perchiacca et al., Proteins 2011; 79:2637-2647).

[0050] The antibody of the present invention may be of any isotype. The choice of isotype typically will be guided by the desired effector functions. IgGl and IgG3 are isotypes that mediate such effectors functions as ADCC (Antibody-dependant cellular cytotoxicity) or CDC (Complement-dependent cytotoxicity), when IgG2 and IgG4 don’t or in a lower manner. Either of the human light chain constant regions, kappa or lambda, may be used. If desired, the class of a monoclonal antibody of the present invention may be switched by known methods. Typical, class switching techniques may be used to convert one IgG subclass to another, for instance from IgGl to IgG2. Thus, the effector function of the monoclonal antibodies of the present invention may be changed by isotype switching to, e.g. an IgGl, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody for various therapeutic uses.

[0051] In one embodiment, the antigen binding compound according to the invention is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).

[0052] In one embodiment and according to the invention, the antigen binding compound is a polypeptide (glycosylated or not) that will bind to an antigen.

[0053] The polypeptide of the invention may be produced by any suitable means, as will be apparent to those of skill in the art. In order to produce sufficient amounts of polypeptide or functional equivalents thereof for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention. Preferably, the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. When expressed in recombinant form, the polypeptide is preferably generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host is E coli. In specific embodiments, it is contemplated that polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution. In example adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters.

[0054] A strategy for improving drug viability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water- soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain. Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications. Those of skill in the art are aware of PEGylation techniques for the effective modification of drugs. For example, drug delivery polymers that consist of alternating polymers of PEG and tri -functional monomers such as lysine have been used by VectraMed (Plainsboro, N.J.). The PEG chains (typically 2000 daltons or less) are linked to the a- and e-amino groups of lysine through stable urethane linkages. Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain. The reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules. These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer. The molecular weight of the PEG segments affects the spacing of the drug / linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading). In general, increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory half-life of the conjugate. Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the threshold-limiting glomular filtration (e.g., less than 60 kDa). In addition, to the polymer backbone being important in maintaining circulatory half-life, and biodistribution, linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue. For example, this type of tissue activated drug delivery is particularly useful where delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of pathology. Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery.

[0055] In one embodiment and according to the invention, the antigen binding compound is an artificial protein with the capacity to bind antigens mimicking that of antibodies, also termed herein antigen-binding antibody mimetic. Such proteins comprise affitins and anticalins. Affitins are artificial proteins with the ability to selectively bind antigens. They are structurally derived from the DNA binding protein Sac7d, found in Sulfolobus acidocaldarius, a microorganism belonging to the archaeal domain. By randomizing the amino acids on the binding surface of Sac7d, e.g. by generating variants corresponding to random substitutions of 11 residues of the binding interface of Sac7d, an affitin library may be generated and subjecting the resulting protein library to rounds of ribosome display, the affinity can be directed towards various targets, such as peptides, proteins, viruses and bacteria. Affitins are antibody mimetics and are being developed as tools in biotechnology. They have also been used as specific inhibitors for various enzymes (Krehenbrink et al., 2008). The skilled person may readily develop affitins with the required binding properties using methods know in the art, in particular as disclosed in patent application W02008068637 or WO2007139397 and the above-cited publication, in particular the generation of phage display and / or ribosome display libraries and their screening using an antigen as disclosed herein. Anticalins are artificial proteins that are able to bind to antigens, either to proteins or to small molecules. They are antibody mimetic derived from human lipocalins which are a family of naturally binding proteins. Anticalins are about eight times smaller with a size of about 180 amino acids and a mass of about 20 kDa (Skerra, 2008). Anticalin phage display libraries have been generated which allow for the screening and selection, in particular of anticalins with specific binding properties. The skilled person may readily develop affitins with the required binding properties using methods know in the art, in particular as disclosed in EP patent EP1270725 Bl, US patent U.S. Pat. No. 8,536,307 B2, (Schlehuber and Skerra, 2002) and the above-cited publication, in particular the generation of phage display and / or ribosome display libraries and their screening using an antigen as disclosed herein. Anticalins and affitins may both be produces in a number of expression system comprising bacterial expression systems. Thus, the invention provides affitins, anticalins and other similar antibody mimetics with the features of the antibodies described herein, in particular with regard to binding to CD 127, non-induction and / or inhibition of CD 127 internalization, maturation of DCs, all of which are contemplated as macromolecules of the invention. Humanization

[0056] Particularly, the antigen binding compound of the invention recognizes a cancerous antigen like the PSMA (Prostate-specific membrane antigen), EpCam (Epithelial cell adhesion molecule) Cellular adhesion, EGFR (Epidermal growth factor receptor), CD20, MUC1 (Mucin 1), HER2 (Human epidermal growth factor receptor 2), CEA (Carcinoembryonic antigen), CD22 (Siglec-2) CD33 (Siglec-3), LEWIS Y, TAG-72 (Tumor-associated glycoprotein 72), CD30 (TNFRSF1), CD19, CD44V6, CD56 (NCAM), GD2 ganglisoside, GD3 ganglisoside (see for example Even-Desrumeaux Klervi, Cancers 2011).

[0057] Particularly, the antigen binding compound is a nanobody which recognize the PSMA. mCD8a

[0058] According to the invention, the CD8alpha is a cell surface glycoprotein found on most cytotoxic T lymphocytes that mediates efficient cell-cell interactions within the immune system. The CDalpha, acting as a coreceptor, and the T-cell receptor on the T lymphocyte recognize antigen displayed by an antigen-presenting cell (APC) in the context of class I MHC molecules. The mCD8alpha is the alpha chain of the CB8. The mCD8alpha (mCD8a) corresponds to the alpha membrane chain of the CD8. the Entrez (NCBI) number access of the CD8 is 925.

[0059] The domain which induces endocytosis According to the invention, a domain which induce endocytosis (or the inducible endocytosis domain) denotes a molecule or compound inducing the process of endocytosis which means that some molecules or compounds will reach the intracellular space of a cell.

[0060] A domain which induces endocytosis of the invention included but is not limited clathrin, FEME, caveolin and cavin proteins, CLIC / GEEC.

[0061] In a particular embodiment, the inducible endocytosis domain is a chemically inducible endocytosis domain. For example, a chemically inducible endocytosis domain can be associated to the mCD8a plasma membrane anchor (CD8-mCherry-FRB) and a clathrin hook of the AP2 beta subunit (FKBP-P2-GFP). When the rapamycin is added, it causes the heterodimerization of FKBP and FRB domains allowing the recruitment of clathrin and the internalization of the whole complex.

[0062] The reporter compound

[0063] According to the invention, the reporter compound can be a fluorescent protein and is used to tag components in the cell for studies using fluorescence spectroscopy and fluorescence microscopy.

[0064] The reporter compound of the invention included but is not limited to monomeric red fluorescent proteins like mCherry, GFP, YFP, RFP, mScarlet.

[0065] The tag compound

[0066] According to the invention, the tag compound is a peptide or a protein grafted onto a recombinant protein here the fusion protein of the invention. Tags can be added to either end of the target protein, so they are either C-terminus or N-terminus specific or are both C-terminus and N-terminus specific. Some tags are also inserted at sites within the protein of interest; they are known as internal tags.

[0067] The tag compound of the invention included but is not limited to HA-tag, ALFA-tag, AviTag, C-tag, Calmodulin-tag, iCapTag™ (intein Capture Tag), polyarginine tag, E-tag, FLAG-tag, His-tag, Gly-His-tag, Myc-tag, NE-tag, RholD4-tag, S-tag, SBP-tag, Softag 1, Softag 3, Spot-tag, Strep-tag, T7-tag, TC tag, Ty tag, V5 tag, VSV-tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, DogTag, SdyTag, SNAP-Tag or Halo-Tag.

[0068] The loading compound

[0069] According to the invention, the loading compound is the moeitie of a photodimerizers grafted onto a recombinant protein here the fusion protein of the invention. Loading compound can be added to either end of the target protein, so they are either C-terminus or N-terminus specific or are both C-terminus and N-terminus specific. Photodimerizer include eMagnet, Lov2 or CIBN / Cry2.

[0070] According to the invention, any therapeutic agent can be charged to the loading compound (see below for the listing of the therapeutic agent).

[0071] Nucleic acid sequence

[0072] Accordingly, a further object of the invention relates to a nucleic acid molecule encoding an antigen binding compound according to the invention, the mCD8a according to the invention, a domain inducing endocytosis according to the invention and / or the fusion protein according to the invention.

[0073] The nucleic acid of the invention can be DNA or RNA.

[0074] As used herein, the terms “coding sequence” or “a sequence which encodes a particular protein” or “encoding nucleic acid” or “nucleic acid molecule encoding”, denotes a nucleic acid sequence which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5’ (amino) terminus and a translation stop codon at the 3’ (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences.

[0075] Vectors

[0076] Typically, said nucleic acid is a DNA or RNA molecule, which may be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector. As used herein, the terms “vector”, “cloning vector” and “expression vector” mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the Introduced sequence. So, a further aspect of the invention relates to a vector comprising a nucleic acid of the invention. Such vectors may comprise regulatory elements, such as a promoter, enhancer, terminator and the like, to cause or direct expression of said antibody upon administration to a subject. Examples of promoters and enhancers used in the expression vector for animal cell include early promoter and enhancer of SV40 (Mizukami T. et al. 1987), LTR promoter and enhancer of Moloney mouse leukemia virus (Kuwana Y et al. 1987), promoter (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983) of immunoglobulin H chain and the like. Any expression vector for animal cell can be used, so long as a gene encoding the human antibody C region can be inserted and expressed. Examples of suitable vectors include pAGE107 (Miyaji H et al. 1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984), pKCR (’’Hare K et al. 1981), pSGl beta d2-4-(Miyaji H et al. 1990) and the like. Other examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like. Other examples of viral vector include adenoviral, retroviral, herpes virus and AAV vectors. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, gPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO 95 / 14785, WO 96 / 22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94 / 19478.

[0077] Thus, the invention relates to a vector comprising a nucleic acid molecule according to the invention.

[0078] Non-viral vectors

[0079] In a preferred embodiment, the vector use according to the invention is a non-viral vector. Typically, the non-viral vector may be a plasmid encoding a complex according to the invention.

[0080] Particularly, a non-viral vector can be an exosome.

[0081] Non-viral vectors mainly comprise chemical systems that are not of viral origin and generally include chemical methods such as cationic liposomes and polymers. Non-viral vectors useful in the practice of the present invention are very well known in the art. According to the invention, non-viral vectors include but are not limited to liposomes such as cationic liposomes, solid-lipid nanoparticles (SLNs or LNPs) such as [(4- hydroxybutyl)azanediyl]di(hexane-6, 1-diyl) bis(2-hexyldecanoate)-based nanoparticles; niosomes; polymers such as cationic polymers; polymers-based nanoparticles such polyethylenimine(PEI)-based nanoparticles; lipopeptides-based nanoparticles such as lipid 1,2- dilinoleyloxy-3 -dimethylaminopropane (dLin-DMA)-based nanoparticles, dilinoleylmethyl-4- dimethylaminobutyrate (dLin-MC3-DMA)-based nanoparticles, ALC-0315-based nanoparticles, ALC-0159-based nanoparticles SM-102-based nanonparticles and ; and chitosans as described in Toualbi L, et al. International Journal of Molecular Sciences, Maier.M et al. Molecular Therapy (2013), Shriane D et al.Biol Pharm Bull (2018). Non-viral vectors according to the invention include also the non-viral vectors described in patent WO2017049245 and W02018081480.

[0082] In some embodiments, the method according to the invention, wherein the non-viral vector is a cationic polymers-based nanoparticle, and more particularly is a polyethylenimine (PEI)-based nanoparticle.

[0083] In some embodiments, the method according to the invention, wherein the vector is a non-viral vector comprising ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) encoding a complex according to the invention.

[0084] In some embodiments, the method according to the invention, wherein the vector is a non-viral vector comprising messenger ribonucleic acid (mRNA) encoding a complex according to the invention.

[0085] Viral vectors

[0086] Gene delivery viral vectors useful in the practice of the present invention can be constructed utilizing methodologies well known in the art of molecular biology. Typically, viral vectors carrying transgenes are assembled from polynucleotides encoding the transgene, suitable regulatory elements and elements necessary for production of viral proteins which mediate cell transduction.

[0087] The terms “Gene transfer” or “gene delivery” refer to methods or systems for reliably inserting foreign DNA into host cells. Such methods can result in transient expression of nonintegrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e. g. , episomes), or integration of transferred genetic material into the genomic DNA of host cells.

[0088] Examples of viral vector include adenoviral, retroviral, lentiviral, herpesvirus and adeno-associated virus (AAV) vectors.

[0089] Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO95 / 14785, WO96 / 22378, US5,882,877, US6,013,516, US4,861,719, US5,278,056 and WO94 / 19478.

[0090] In a particular embodiment, adeno-associated viral (AAV) vectors are employed.

[0091] In another particular embodiment, the AAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO and all variants of AAV9, including AAV PHP.B (see for example the patent application WO2015038958), AAVPHP.eB, AAV-PHP.N", and "AAV-PHP.B- DGT (see the patent application W02017100671 or Chan Y Ken, Nat Neurosci. 2017 Aug;20(8): 1172-1179.), AAV3B, AAV-2i8, Rh74, AAV capBlO, AAVMacPNSl or AAVMacPNS2 or any other serotypes of AAV that can infect human, monkeys or other species.

[0092] By an "AAV vector" is meant a vector derived from an adeno-associated virus serotype, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV6, AAV9 etc. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and / or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences are necessary for the rescue, replication and packaging of the AAV virion. Thus, an AAV vector is defined herein to include at least those sequences required in cis for replication and packaging (e.g. functional ITRs) of the virus. The ITRs need not be the wildtype nucleotide sequences, and may be altered, e.g. by the insertion, deletion or substitution of nucleotides, so long as the sequences provide for functional rescue, replication and packaging. AAV expression vectors are constructed using known techniques to at least provide as operatively linked components in the direction of transcription, control elements including a transcriptional initiation region, the DNA of interest (i.e. the CYP46A1 gene) and a transcriptional termination region.

[0093] The control elements are selected to be functional in a mammalian cell. The resulting construct that contains the operatively linked components is bounded (5'and Y) with functional AAV ITR sequences. By "adeno-associated virus inverted terminal repeats " or "AAVITRs" is meant the art-recognized regions found at each end of the AAV genome which function together in cis as origins of DNA replication and as packaging signals for the virus. AAV ITRs, together with the AAV rep coding region, provide for the efficient excision and rescue from, and integration of a nucleotide sequence interposed between two flanking ITRs into a mammalian cell genome. The nucleotide sequences of AAV ITR regions are known. See, e. g., Kotin, 1994; Berns, KI "Parvoviridae and their Replication" in Fundamental Virology, 2nd Edition, (B. N. Fields and D. M. Knipe, eds. ) for the AAV-2 sequence. As used herein, an "AAV ITR" does not necessarily comprise the wild-type nucleotide sequence, but may be altered, e. g., by the insertion, deletion or substitution of nucleotides. Additionally, the AAV ITR may be derived from any of several AAV serotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, etc. Furthermore, 5' and 3'ITRs that flank a selected nucleotide sequence in an AAV vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i.e. to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the heterologous sequence into the recipient cell genome when AAV Rep gene products are present in the cell. Additionally, AAV ITRs may be derived from any of several AAV serotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, etc. Furthermore, 5' and 3'ITRs that flank a selected nucleotide sequence in an AAV expression vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i.e., to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the DNA molecule into the recipient cell genome when AAV Rep gene products are present in the cell.

[0094] Particularly preferred are vectors derived from AAV serotypes having tropism for and high transduction efficiencies in cells of the mammalian CNS, particularly neurons. A review and comparison of transduction efficiencies of different serotypes is provided in Cearley CN et al., 2009. In one preferred example, AAV-2 -based vectors have been shown to direct long-term expression of transgenes in CNS, preferably transducing neurons. In other nonlimiting examples, preferred vectors include vectors derived from AAVrhlO serotype, which have also been shown to transduce cells of the CNS and particularly deep grey nucleus and diffusely cortex and white matter.

[0095] The selected nucleotide sequence is operably linked to control elements that direct the transcription or expression thereof in the subject in vivo. Such control elements can comprise control sequences normally associated with the selected gene.

[0096] Alternatively, heterologous control sequences can be employed. Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, the phosphoglycerate kinase (PKG) promoter, CAG, neuronal promoters, promoter of Dopamine- 1 receptor and Dopamine-2 receptor, the SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like. In addition, sequences derived from nonviral genes, such as the murine metallothionein gene, will also find use herein. Such promoter sequences are commercially available from, e. g. , Stratagene (San Diego, CA). For purposes of the present invention, both heterologous promoters and other control elements, including but not limited to CNS-specific and inducible promoters, enhancers and the like, will be of particular use. Examples of heterologous promoters include the CMV promoter. Examples of CNS- specific promoters include those isolated from the genes from myelin basic protein (MBP), glial fibrillary acid protein (GFAP), and neuron specific enolase (NSE).

[0097] Examples of inducible promoters include DNA responsive elements for ecdysone, tetracycline, hypoxia andaufin.

[0098] The AAV expression vector which harbours the DNA molecule of interest bounded by AAV ITRs, can be constructed by directly inserting the selected sequence(s) into an AAV genome which has had the major AAV open reading frames ("ORFs") excised therefrom. Other portions of the AAV genome can also be deleted, so long as a sufficient portion of the ITRs remain to allow for replication and packaging functions. Such constructs can be designed using techniques well known in the art. See, e. g. , U. S. Patents Nos. 5,173, 414 and 5,139, 941; International Publications Nos. WO 92 / 01070 (published 23 January 1992) and WO 93 / 03769 (published 4 March 1993); Lebkowski et al., 1988 ; Vincent et al., 1990; Carter, 1992 ; Muzyczka, 1992 ; Kotin, 1994; Shelling and Smith, 1994 ; and Zhou et al., 1994. Alternatively, AAV ITRs can be excised from the viral genome or from an AAV vector containing the same and fused 5' and 3' of a selected nucleic acid construct that is present in another vector using standard ligation techniques. AAV vectors which contain ITRs have been described in, e.g. U. S. Patent no. 5,139, 941. In particular, several AAV vectors are described therein which are available from the American Type Culture Collection ("ATCC") under Accession Numbers 53222,53223, 53224,53225 and 53226. Additionally, chimeric genes can be produced synthetically to include AAV ITR sequences arranged 5' and 3' of one or more selected nucleic acid sequences. Preferred codons for expression of the chimeric gene sequence in mammalian CNS cells (notably neurons or astrocytes) can be used. The complete chimeric sequence is assembled from overlapping oligonucleotides prepared by standard methods. See, e. g., Edge, 1981 ; Nambair et al., 1984 ; Jay et al., 1984. In order to produce rAAV virions, an AAV expression vector is introduced into a suitable host cell using known techniques, such as by transfection. A number of transfection techniques are generally known in the art. See, e. g. , Graham et al., 1973;, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis etal. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al., 1981. Particularly suitable transfection methods include calcium phosphate co-precipitation (Graham et al., 1973), direct microinjection into cultured cells (Capecchi, 1980), electroporation (Shigekawa et al., 1988), liposome mediated gene transfer (Mannino et al., 1988), lipid-mediated transduction (Feigner et al., 1987), and nucleic acid delivery using high-velocity microprojectiles (Klein et al., 1987). Immune cells and extracellular vesicles

[0099] A further aspect of the invention relates to an immune cell which has been transfected, infected or transformed by a nucleic acid and / or a vector according to the invention or which express the fusion protein of the invention.

[0100] The term "transformation" means the introduction of a "foreign" (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to an immune cell, so that the immune cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence here the fusion protein. An immune cell that receives and expresses introduced DNA or RNA bas been "transformed".

[0101] The nucleic acids of the invention may be used to produce a fusion protein of the present invention in a suitable expression system. The term "expression system" means an immune cell and compatible vector under suitable conditions, e.g. for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the immune cell.

[0102] In a particular embodiment, the immune cell of the invention will express at its surface the fusion protein of the invention. An immune cell expressing at its surface a fusion protein of the invention is called EV-Trap.

[0103] Thus, the invention also relates to an immune cell expressing the fusion protein according to the invention.

[0104] According to the invention, the term “immune cell” denotes dendritic cell, macrophage, microglia, monocyte, neutrophil, NK cell, lymphocyte like B lymphocyte or T lymphocyte like CD4+ T lymphocyte or CD8+ T lymphocyte.

[0105] Surprisingly, the inventor discovered that the immune cell of the invention which express the fusion protein of the invention, secretes extracellular vesicles which also express at their surfaces the fusion protein of the invention.

[0106] Thus, the invention also relates to all subpopulations of extracellular vesicles (extracellular vesicles, ectosomes, apoptotic bodies) which express the fusion protein of the invention.

[0107] Thus, the invention also relates to extracellular vesicles expressing the fusion protein according to the invention.

[0108] Applications of the EV-trap of the invention In another aspect, the invention relates to an immune cell expressing the fusion protein according to the invention or to extracellular vesicles expressing the fusion protein according to the invention for use in the treatment of a cancer in a subject in need thereof.

[0109] According to the invention, the immune cell expressing the fusion protein according to the invention or the extracellular vesicles expressing the fusion protein would be useful when the antigen recognized by the fusion protein is associated with a diseases (for example cancerous antigen like the PSMA (Prostate-specific membrane antigen), EpCam (Epithelial cell adhesion molecule) Cellular adhesion, EGFR (Epidermal growth factor receptor), CD20, MUC1 (Mucin 1), HER2 (Human epidermal growth factor receptor 2), CEA (Carcinoembryonic antigen), CD22 (Siglec-2) CD33 (Siglec-3), LEWIS Y, TAG-72 (Tumor- associated glycoprotein 72), CD30 (TNFRSF1), CD19, CD44V6, CD56 (NCAM), GD2 ganglisoside, GD3 ganglisoside (see for example Even-Desrumeaux Klervi, Cancers 2011)) and when the immune cell express the fusion protein according to the invention at their outer surface or when the extracellular vesicles express the fusion protein according to the invention at their outer surface.

[0110] According to the invention, the cancer may be a liquid or a solid cancer.

[0111] In one embodiment, the cancer may be a cancer selected from the group consisting in adrenal cortical cancer, anal cancer, bile duct cancer (e.g. perihilar cancer, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, bone cancer (e.g. osteoblastoma, osteochrondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of the bone, chordoma), brain and central nervous system cancer (e.g. meningioma, astocytoma, oligodendrogliomas, ependymoma, gliomas, medulloblastoma, ganglioglioma, Schwannoma, germinoma, craniopharyngioma), breast cancer (e.g. ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating lobular carcinoma, lobular carcinoma in situ, gynecomastia), Castleman disease (e.g. giant lymph node hyperplasia, angiofollicular lymph node hyperplasia), cervical cancer, colorectal cancer, endometrial cancer (e.g. endometrial adenocarcinoma, adenocanthoma, papillary serous adenocarcinoma, clear cell), esophagus cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g. choriocarcinoma, chorioadenoma destruens), Hodgkin's disease, Kaposi's sarcoma, kidney cancer (e.g. renal cell cancer), laryngeal and hypopharyngeal cancer, liver cancer (e.g. hemangioma, hepatic adenoma, focal nodular hyperplasia, hepatocellular carcinoma), lung cancer (e.g. small cell lung cancer, non-small cell lung cancer), mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer (e.g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (e.g. melanoma, nonmelanoma skin cancer), stomach cancer, testicular cancer (e.g. seminoma, nonseminoma germ cell cancer), thymus cancer, thyroid cancer (e.g. follicular carcinoma, anaplastic carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma,), vaginal cancer, vulvar cancer, uterine cancer (e.g. uterine leiomyosarcoma), leukaemia (like acute myeloid leukaemia, acute lymphoid leukaemia, chronic myelomonocytic leukemia (CMML)...), lymphoma and myelodysplastic syndrome (MDS).

[0112] According to the invention, the cancer can be a metastatic cancer.

[0113] In a particular embodiment, the immune cell expressing the fusion protein of the invention or the extracellular vesicles expressing the fusion protein of the invention will target extra-vesicles (EV) expressing a cancerous antigen. In other word, the immune cell expressing the fusion protein of the invention or the extracellular vesicles expressing the fusion protein of the invention will target tumor-derived EV i.e. EV secreted by tumoral cells and particularly metastatic tumoral cells.

[0114] More particularly, thanks to the immune cells of the invention or the extracellular vesicles of the invention, an opsonisation phenomenon can be induced or a stimulation of tumoral antigen presentation can be expected.

[0115] As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, a subject according to the invention is a human. More particularly, the subject is suffering from a cancer.

[0116] As used herein, the terms "treatment" and "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

[0117] Accordingly, one object of the present invention relates to a method of treating a cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an immune cell expressing the fusion protein according to the invention or of extracellular vesicles expressing the fusion protein according to the invention.

[0118] Therapeutic composition

[0119] In another aspect, the invention relates to a therapeutic composition comprising an immune cell expressing the fusion protein according to the invention or extracellular vesicles expressing the fusion protein according to the invention.

[0120] Thus, the invention also relates to a therapeutic composition comprising an immune cell expressing the fusion protein according to the invention or extracellular vesicles expressing the fusion protein according to the invention for use in the treatment of a cancer in a subject in need thereof.

[0121] According to the invention, the fusion protein according to the invention or extracellular vesicles expressing the fusion protein according to the invention or the therapeutic composition of the invention are administrated in a therapeutically effective amount. Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

[0122] As used herein, the term "therapeutically effective amount" or “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of the inhibitor or the composition of the present invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the inhibitor or the composition of the present invention to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the inhibitor or the composition are outweighed by the therapeutically beneficial effects. The efficient dosages and dosage regimens for the inhibitor or the composition of the present invention depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician having ordinary skill in the art may readily determine and prescribe the effective amount of the inhibitor or the composition of the invention required. For example, the physician could start doses of the inhibitor or the composition of the present invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable dose of the inhibitor or the composition of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. Such an effective dose will generally depend upon the factors described above. For example, a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease. Typically and for example, the ability of a compound to inhibit cancer may, for example, be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, this property of a composition may be evaluated by examining the ability of the compound to induce cytotoxicity by in vitro assays known to the skilled practitioner. A therapeutically effective amount of a therapeutic compound may decrease tumor size, or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is about 0.1-100 mg / kg, such as about 0.1-50 mg / kg, for example about 0.1- 20 mg / kg, such as about 0.1-10 mg / kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg / kg, about 5 mg / kg or about 8 mg / kg. An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is 0.02-100 mg / kg, such as about 0.02-30 mg / kg, such as about 0.05-10 mg / kg or 0.1-3 mg / kg, for example about 0.5-2 mg / kg. Administration may e.g. be intravenous, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time. In some embodiments, the efficacy may be monitored by visualization of the disease area, or by other diagnostic methods described further herein, e.g. by performing one or more PET-CT scans, for example using a labeled antibody of the present invention, fragment or mini-antibody derived from the antibody of the present invention. If desired, an effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more subdoses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In some embodiments, the immune cell expressing the fusion protein according to the invention or extracellular vesicles expressing the fusion protein according to the invention are administered by slow continuous infusion over a long period, such as more than 24 hours, in order to minimize any unwanted side effects. An effective dose of an antibody of the present invention may also be administered using a weekly, biweekly or triweekly dosing period. The dosing period may be restricted to, e.g., 8 weeks, 12 weeks or until clinical progression has been established. As non-limiting examples, treatment according to the present invention may be provided as a daily dosage of an antibody of the present invention in an amount of about 0.1- 100 mg / kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg / kg, per day, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.

[0123] Administration may be intravenous, intramuscular, intraperitoneal, intratumoral or subcutaneous, and for instance administered proximal to the site of the target. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time. In some embodiments, the efficacy may be monitored by visualization of the disease area, or by other diagnostic methods described further herein, e.g. by performing one or more PET-CT scans. If desired, an effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In some embodiments, the oligomers of the present invention are administered by slow continuous infusion over a long period, such as more than 24 hours, in order to minimize any unwanted side effects. An effective dose of the CAR-T cells of the present invention may also be administered using a weekly, biweekly or triweekly dosing period. The dosing period may be restricted to, e.g., 8 weeks, 12 weeks or until clinical progression has been established. As non-limiting examples, treatment according to the present invention may be provided as a daily dosage of the CAR-T cells of the present invention in an amount of about 0.1-100 mg / kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45,

[0124] 50, 60, 70, 80, 90 or 100 mg / kg, per day, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,

[0125] 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,

[0126] 37, 38, 39, or 40, or alternatively, at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,

[0127] 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.

[0128] In other words, the quantity of the immune cell or the population of immune cells administered to a subject in need thereof is between 104to 109cells per kg. Particularly, the quantity of cells injected is 106or 107cells per kg. The immune cell or the population of immune cells of the invention can be administrated is 1, 2, 3, 4 or 5 times to the subject in need thereof.

[0129] "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the subject, etc.

[0130] The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like.

[0131] Particularly, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze- dried compositions. In particular, these may be in organic solvent such as DMSO, ethanol which upon addition, depending on the case, of sterilized water or physiological saline permit the constitution of injectable solutions.

[0132] In addition, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.

[0133] In each of the embodiments of the treatment methods described herein, the CAR-T cells of the invention are delivered in a manner consistent with conventional methodologies associated with management of the disease or disorder for which treatment is sought. In accordance with the disclosure herein, an effective amount of the CAR-T cells of the invention administered to a subject in need of such treatment for a time and under conditions sufficient to prevent or treat the disease or disorder.

[0134] Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 pm) are generally designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be easily made.

[0135] Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)). MLVs generally have diameters of from 25 nm to 4 pm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations.

[0136] Combination and kit of part In another aspect of the invention, the immune cell expressing the fusion protein according to the invention or extracellular vesicles expressing the fusion protein according to the invention or the pharmaceutical composition of the invention may comprise a further therapeutic active agent. The present invention also relates to a kit comprising an inhibitor according to the invention and a further therapeutic active agent.

[0137] According to the invention, anti-cancer agents may be added to the pharmaceutical composition or used in combination with the inhibitor of the invention in the case of the treatment of a cancer.

[0138] Anti-cancer agents or conventional cancer therapies can be surgery, radiotherapy, chemotherapy, or combinations thereof.

[0139] Anti-cancer agents include other anti-cancer antibodies, cytotoxic agents, chemotherapeutic agents, anti -angiogenic agents, anti-cancer immunogens, cell cycle control / apoptosis regulating agents, hormonal regulating agents, and other agents described below.

[0140] In some embodiments, the inhibitor or composition of the present invention is used in combination with a chemotherapeutic agent. The term "chemotherapeutic agent" refers to chemical compounds that are effective in inhibiting tumor growth. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimus tine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Inti. Ed. Engl. 33:183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idanrbicin, marcellomycin, mitomycins, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomgrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophospharnide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pento statin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylarnine; trichothecenes (especially T-2 toxin, verracurin A, roridinA and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit honnone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0141] In some embodiments, the inhibitor or composition of the present invention is used in combination with a targeted cancer therapy. Targeted cancer therapies are drugs or other substances that block the growth and spread of cancer by interfering with specific molecules ("molecular targets") that are involved in the growth, progression, and spread of cancer. Targeted cancer therapies are sometimes called "molecularly targeted drugs," "molecularly targeted therapies," "precision medicines," or similar names. In some embodiments, the targeted therapy consists of administering the subj ect with a tyrosine kinase inhibitor. The term “tyrosine kinase inhibitor” refers to any of a variety of therapeutic agents or drugs that act as selective or non-selective inhibitors of receptor and / or non-receptor tyrosine kinases. Tyrosine kinase inhibitors and related compounds are well known in the art and described in U.S Patent Publication 2007 / 0254295, which is incorporated by reference herein in its entirety. It will be appreciated by one of skill in the art that a compound related to a tyrosine kinase inhibitor will recapitulate the effect of the tyrosine kinase inhibitor, e.g., the related compound will act on a different member of the tyrosine kinase signaling pathway to produce the same effect as would a tyrosine kinase inhibitor of that tyrosine kinase. Examples of tyrosine kinase inhibitors and related compounds suitable for use in methods of embodiments of the present invention include, but are not limited to, dasatinib (BMS-354825), PP2, BEZ235, saracatinib, gefitinib (Iressa), sunitinib (Sutent; SU11248), erlotinib (Tarceva; OSI-1774), lapatinib (GW572016; GW2016), canertinib (CI 1033), semaxinib (SU5416), vatalanib (PTK787 / ZK222584), sorafenib (BAY 43-9006), imatinib (Gleevec; STI571), leflunomide (SU101), vandetanib (Zactima; ZD6474), MK-2206 (8-[4-aminocyclobutyl)phenyl]-9-phenyl-l,2,4-triazolo[3,4-f][l,6]naphthyridin- 3(2H)-one hydrochloride) derivatives thereof, analogs thereof, and combinations thereof. Additional tyrosine kinase inhibitors and related compounds suitable for use in the present invention are described in, for example, U.S Patent Publication 2007 / 0254295, U.S. Pat. Nos. 5,618,829, 5,639,757, 5,728,868, 5,804,396, 6,100,254, 6,127,374, 6,245,759, 6,306,874, 6,313,138, 6,316,444, 6,329,380, 6,344,459, 6,420,382, 6,479,512, 6,498,165, 6,544,988, 6,562,818, 6,586,423, 6,586,424, 6,740,665, 6,794,393, 6,875,767, 6,927,293, and 6,958,340, all of which are incorporated by reference herein in their entirety. In some embodiments, the tyrosine kinase inhibitor is a small molecule kinase inhibitor that has been orally administered and that has been the subject of at least one Phase I clinical trial, more preferably at least one Phase II clinical, even more preferably at least one Phase III clinical trial, and most preferably approved by the FDA for at least one hematological or oncological indication. Examples of such inhibitors include, but are not limited to, Gefitinib, Erlotinib, Lapatinib, Canertinib, BMS- 599626 (AC-480), Neratinib, KRN-633, CEP-11981, Imatinib, Nilotinib, Dasatinib, AZM- 475271, CP-724714, TAK-165, Sunitinib, Vatalanib, CP-547632, Vandetanib, Bosutinib, Lestaurtinib, Tandutinib, Midostaurin, Enzastaurin, AEE-788, Pazopanib, Axitinib, Motasenib, OSI-930, Cediranib, KRN-951, Dovitinib, Seliciclib, SNS-032, PD-0332991, MKC-I (Ro- 317453; R-440), Sorafenib, ABT-869, Brivanib (BMS-582664), SU-14813, Telatinib, SU- 6668, (TSU-68), L-21649, MLN-8054, AEW-541, and PD-0325901.

[0142] In some embodiments, the inhibitor or composition of the present invention is used in combination with an immunotherapeutic agent. The term "immunotherapeutic agent," as used herein, refers to a compound, composition or treatment that indirectly or directly enhances, stimulates or increases the body's immune response against cancer cells and / or that decreases the side effects of other anticancer therapies. Immunotherapy is thus a therapy that directly or indirectly stimulates or enhances the immune system's responses to cancer cells and / or lessens the side effects that may have been caused by other anti-cancer agents. Immunotherapy is also referred to in the art as immunologic therapy, biological therapy biological response modifier therapy and biotherapy. Examples of common immunotherapeutic agents known in the art include, but are not limited to, cytokines, cancer vaccines, monoclonal antibodies and noncytokine adjuvants. Alternatively the immunotherapeutic treatment may consist of administering the subject with an amount of immune cells (T cells, NK, cells, dendritic cells, B cells...). Immunotherapeutic agents can be non-specific, i.e. boost the immune system generally so that the human body becomes more effective in fighting the growth and / or spread of cancer cells, or they can be specific, i.e. targeted to the cancer cells themselves immunotherapy regimens may combine the use of non-specific and specific immunotherapeutic agents. Nonspecific immunotherapeutic agents are substances that stimulate or indirectly improve the immune system. Non-specific immunotherapeutic agents have been used alone as a main therapy for the treatment of cancer, as well as in addition to a main therapy, in which case the non-specific immunotherapeutic agent functions as an adjuvant to enhance the effectiveness of other therapies (e.g. cancer vaccines). Non-specific immunotherapeutic agents can also function in this latter context to reduce the side effects of other therapies, for example, bone marrow suppression induced by certain chemotherapeutic agents. Non-specific immunotherapeutic agents can act on key immune system cells and cause secondary responses, such as increased production of cytokines and immunoglobulins. Alternatively, the agents can themselves comprise cytokines. Non-specific immunotherapeutic agents are generally classified as cytokines or non-cytokine adjuvants. A number of cytokines have found application in the treatment of cancer either as general non-specific immunotherapies designed to boost the immune system, or as adjuvants provided with other therapies. Suitable cytokines include, but are not limited to, interferons, interleukins and colony-stimulating factors. Interferons (IFNs) contemplated by the present invention include the common types of IFNs, IFN-alpha (IFN-a), IFN-beta (IFN-P) and IFN-gamma (IFN-y). IFNs can act directly on cancer cells, for example, by slowing their growth, promoting their development into cells with more normal behavior and / or increasing their production of antigens thus making the cancer cells easier for the immune system to recognise and destroy. IFNs can also act indirectly on cancer cells, for example, by slowing down angiogenesis, boosting the immune system and / or stimulating natural killer (NK) cells, T cells and macrophages. Recombinant IFN-alpha is available commercially as Roferon (Roche Pharmaceuticals) and Intron A (Schering Corporation). Interleukins contemplated by the present invention include IL-2, IL-4, IL-11 and IL-12. Examples of commercially available recombinant interleukins include Proleukin® (IL-2; Chiron Corporation) and Neumega® (IL-12; Wyeth Pharmaceuticals). Zymogenetics, Inc. (Seattle, Wash.) is currently testing a recombinant form of IL-21, which is also contemplated for use in the combinations of the present invention. Colony-stimulating factors (CSFs) contemplated by the present invention include granulocyte colony stimulating factor (G-CSF or filgrastim), granulocyte-macrophage colony stimulating factor (GM-CSF or sargramostim) and erythropoietin (epoetin alfa, darbepoietin). Treatment with one or more growth factors can help to stimulate the generation of new blood cells in subjects undergoing traditional chemotherapy. Accordingly, treatment with CSFs can be helpful in decreasing the side effects associated with chemotherapy and can allow for higher doses of chemotherapeutic agents to be used. Various-recombinant colony stimulating factors are available commercially, for example, Neupogen® (G-CSF; Amgen), Neulasta (pelfilgrastim; Amgen), Leukine (GM-CSF; Berlex), Procrit (erythropoietin; Ortho Biotech), Epogen (erythropoietin; Amgen), Arnesp (erytropoietin). Combination compositions and combination administration methods of the present invention may also involve "whole cell" and "adoptive" immunotherapy methods. For instance, such methods may comprise infusion or re-infusion of immune system cells (for instance tumor-infiltrating lymphocytes (TILs), such as CC2+ and / or CD8+ T cells (for instance T cells expanded with tumor-specific antigens and / or genetic enhancements), antibodyexpressing B cells or other antibody-producing or -presenting cells, dendritic cells (e.g., dendritic cells cultured with a DC-expanding agent such as GM-CSF and / or Flt3-L, and / or tumor-associated antigen-loaded dendritic cells), anti-tumor NK cells, so-called hybrid cells, or combinations thereof. Cell lysates may also be useful in such methods and compositions. Cellular "vaccines" in clinical trials that may be useful in such aspects include Canvaxin™, APC-8015 (Dendreon), HSPPC-96 (Antigenics), and Melacine® cell lysates. Antigens shed from cancer cells, and mixtures thereof (see for instance Bystryn et al., Clinical Cancer Research Vol. 7, 1882-1887, July 2001), optionally admixed with adjuvants such as alum, may also be components in such methods and combination compositions.

[0143] In some embodiments, the inhibitor or composition of the present invention is used in combination with radiotherapy. Radiotherapy may comprise radiation or associated administration of radiopharmaceuticals to a patient. The source of radiation may be either external or internal to the patient being treated (radiation treatment may, for example, be in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that may be used in practicing such methods include, e.g., radium, cesium- 137, iridium- 192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodide-123, iodide-131, and indium- 111.

[0144] In some embodiments, the inhibitor or composition of the present invention is used in combination with an antibody that is specific for a costimulatory molecule. Examples of antibodies that are specific for a costimulatory molecule include but are not limited to anti- CTLA4 antibodies (e.g. Ipilimumab), anti-PDl antibodies, anti-PDLl antibodies, anti-TIMP3 antibodies, anti-LAG3 antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies or anti-B7H6 antibodies.

[0145] In some embodiments, the second agent is an agent that induces, via ADCC, the death of a cell expressing an antigen to which the second agent binds. In some embodiments, the agent is an antibody (e.g. of IgGl or IgG3 isotype) whose mode of action involves induction of ADCC toward a cell to which the antibody binds. NK cells have an important role in inducing ADCC and increased reactivity of NK cells can be directed to target cells through use of such a second agent. In some embodiments, the second agent is an antibody specific for a cell surface antigens, e.g., membrane antigens. In some embodiments, the second antibody is specific for a tumor antigen as described above (e.g., molecules specifically expressed by tumor cells), such as CD20, CD52, ErbB2 (or HER2 / Neu), CD33, CD22, CD25, MUC-1, CEA, KDR, DVD3, etc., particularly lymphoma antigens (e.g., CD20). Accordingly, the present invention also provides methods to enhance the anti-tumor effect of monoclonal antibodies directed against tumor antigen(s). In the methods of the invention, ADCC function is specifically augmented, which in turn enhances target cell killing, by sequential administration of an antibody directed against one or more tumor antigens, and an antibody of the present invention. A number of antibodies are currently in clinical use for the treatment of cancer, and others are in varying stages of clinical development. Antibodies of interest for the methods of the invention act through ADCC, and are typically selective for tumor cells, although one of skill in the art will recognize that some clinically useful antibodies do act on non-tumor cells, e.g. CD20. There are a number of antigens and corresponding monoclonal antibodies for the treatment of B cell malignancies. One popular target antigen is CD20, which is found on B cell malignancies. Rituximab is a chimeric unconjugated monoclonal antibody directed at the CD20 antigen. CD20 has an important functional role in B cell activation, proliferation, and differentiation. The CD52 antigen is targeted by the monoclonal antibody alemtuzumab, which is indicated for treatment of chronic lymphocytic leukemia. CD22 is targeted by a number of antibodies, and has recently demonstrated efficacy combined with toxin in chemotherapyresistant hairy cell leukemia. Monoclonal antibodies targeting CD20, also include tositumomab and ibritumomab. Monoclonal antibodies useful in the methods of the invention, which have been used in solid tumors, include without limitation edrecolomab and trastuzumab (herceptin). Edrecolomab targets the 17-1 A antigen seen in colon and rectal cancer, and has been approved for use in Europe for these indications. Its antitumor effects are mediated through ADCC, CDC, and the induction of an anti -idiotypic network. Trastuzumab targets the HER- 2 / neu antigen. This antigen is seen on 25% to 35% of breast cancers. Trastuzumab is thought to work in a variety of ways: downregulation of HER-2 receptor expression, inhibition of proliferation of human tumor cells that overexpress HER-2 protein, enhancing immune recruitment and ADCC against tumor cells that overexpress HER-2 protein, and downregulation of angiogenesis factors. Alemtuzumab (Campath) is used in the treatment of chronic lymphocytic leukemia; colon cancer and lung cancer; Gemtuzumab (Mylotarg) finds use in the treatment of acute myelogenous leukemia; Ibritumomab (Zevalin) finds use in the treatment of non-Hodgkin's lymphoma; Panitumumab (Vectibix) finds use in the treatment of colon cancer. Cetuximab (Erbitux) is also of interest for use in the methods of the invention. The antibody binds to the EGF receptor (EGFR), and has been used in the treatment of solid tumors including colon cancer and squamous cell carcinoma of the head and neck (SCCHN).

[0146] Another aspect of the present invention relates to i) the immune cell expressing the fusion protein according to the invention or extracellular vesicles expressing the fusion protein according to the invention, and ii) at least one further therapeutic active agent according to the invention, as a combined preparation for simultaneous, separate or sequential use in the treatment of a cancer in a subject in need thereof. As used herein, the term “simultaneous use” denotes the use of the immune cell expressing the fusion protein according to the invention or of extracellular vesicles expressing the fusion protein according to the invention inhibitor and at least one therapeutic active agent occurring at the same time.

[0147] As used herein, the term “separate use” denotes the use of the immune cell expressing the fusion protein according to the invention or of extracellular vesicles expressing the fusion protein according to the invention and at least one therapeutic active agent not occurring at the same time.

[0148] As used herein, the term “sequential use” denotes the use of the immune cell expressing the fusion protein according to the invention or of extracellular vesicles expressing the fusion protein according to the invention and at least one therapeutic active agent occurring by following an order.

[0149] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0150] FIGURES

[0151] Figure 1: GFP-TRAP and alpha- TRAP constructs and characterization.

[0152] A: schematic representation of the GFP-TRAP and alpha-TRAP

[0153] B: Time lapse of the trapping of CD63-pH expressing Extracellular vesicles (EVs) on GFP-trap expressing cells. CD63-pHluorin expressing PC3 cells were grown in EV depleted media for 48h and EV were isolated following a protocol of centrifugation, ultrafiltration and Size exclusion chromatography. HeLa cells stably expressing GFP-Trap (detected thanks to the mCherry integrated within the EV-trap constructs) were seeded for 24h. Live HeLa cells were incubated with EVs derived from CD63-pHluorin expressing PC3 cells at a ratio of 2000 EVs / receiving cell. The live cell imaging reveals a fast trapping of EVs (labeled in green thanks to the pHluorin) on GFP-Trap expressing cells in less than 10 min.

[0154] C: Specificity of the trapping. Imaging of the trapping of CD63-pH expressing Extracellular vesicles (EVs) on GFP-trap (top panel) or ALPHA-trap (bottom panel) expressing cells. CD63-pHluorin expressing PC3 cells were grown in EV depleted media for 48h and EV were isolated following a protocol of centrifugation, ultrafiltration and Size exclusion chromatography. HeLa cells stably expressing either GFP-Trap or ALPHA-trap (detected thanks to the mCherry integrated within the EV-trap constructs) were seeded for 24h. Live HeLa cells were incubated for one hour with EVs derived from CD63-pHluorin expressing PC3 cells at a ratio of 2000 EVs / receiving cell. Cells were fixed and imaged with a confocal microscope. The fluorescence imaging reveals a specific trapping of EVs (labeled in green thanks to the pHluorin) on GFP-Trap expressing cells but not on ALPHA-trap expressing cells. Of note cells note expressing either trap are still able to uptake EVs

[0155] Figure 2: EV trapping competition assays in vitro.

[0156] A: Time lapse of the trapping of CD63-pH expressing Extracellular vesicles (EVs) in a competition assay. CD63-pHluorin expressing PC3 cells were grown in EV depleted media for 48h and EV were isolated following a protocol of centrifugation, ultrafiltration and Size exclusion chromatography. HeLa cells stably expressing either GFP-Trap or ALPHA-trap (detected thanks to the mCherry integrated within the EV-trap constructs) were seeded for 24h in a dual chamber Ibidi® system. Before incubation with the EVs, the wall separating each population of HeLa cells expressing ALPHA-Trap (top left panel) or expressing either GFP- Trap or ALPHA-trap (top right panel) from each other was removed. EVs derived from CD63- pHluorin expressing PC3 cells were deposited in between the two cell populations for one hour with at various ratio for different time periods (see the quantifications below). Cells were fixed and imaged with a confocal microscope. The fluorescence imaging reveals the trapping of EVs (labeled in green thanks to the pHluorin) on GFP-Trap expressing cells.

[0157] B: Time course of the trapping competition between GFP-trap and ALPHA trap expressing cells. The level of trapping is expressed as an intensity ratio between the green fluorescence (related to EVs) and the red fluorescence (related to the EV-trap expression).

[0158] Figure 3: EV trapping competition assays in vitro (PSMA).

[0159] A: schematic representation of the PSMA-TRAP capturing EVs from LNCaP cells that express PSMA tumour antigen on EVs

[0160] B: Specificity of the trapping. Imaging of the trapping of Extracellular vesicles (EVs) from LNCaP WT cells on ALPHA-trap (top panel) or PSMA-trap (bottom panel) expressing cells. WT LNCaP cells were grown in EV depleted media for 48h and EV were isolated following a protocol of centrifugation, ultrafiltration and Size exclusion chromatography. HeLa cells stably expressing either ALPHA-trap (top panel) or PSMA-trap (bottom panel) (detected thanks to the mCherry integrated within the EV-trap constructs) were seeded for 24h. Live HeLa cells were incubated for one hour with EVs derived from WT LNCaP cells at a ratio of 2000 EVs / receiving cell. Cells were fixed and PSMA was labelled with a VHH JVZ-007 anti - PSMA nanobody coupled to green quantum dot(green fluorescence) with a confocal microscope. The fluorescence imaging reveals a specific trapping of EVs PSMA-Trap expressing cells but not on ALPHA-trap expressing cells shwong that EVs expressing PSMA can be trapped.

[0161] C: western blot analysis of the presence of PSMA in LNCaP cells and not HEK cells using a VHH JVZ-007 anti -PSMA nanobody coupled to green quantum dot for detection. The specific signal is only seen LNCaP cells proving the specificity of the nanobody.

[0162] Figure 4: GFP-Trap and PSMA-Trap assays in zebrafish embryo.

[0163] A: schematic representation of the workflow of the experiment. In brief, either EVs isolated from PC3 cells expressing CD63-pHluorin were microinjected in the vasculature of the zebrafish embryo or PC3 cells expressing CD63-pHluorin were xenografted in the perivitelline space of the zebrafish embryo allowing them to secrete CD63-pHluorin positive EVs within the vasculature of the zebrafish embryo. 3hrs later, HeLa cells expressing the GFP-Trap were xenografted in the vasculature of the zebrafish embryos.

[0164] B: Proof of concept of the trapping in vivo.

[0165] Live imaging of the trapping of Extracellular vesicles (EVs) that were isolated (following the same protocol as before) from PC3 cells expressing CD63-pHluorin (in green) and microinjected in the vasculature of the zebrafish embryo that was previously xenografted with HeLa cells expressing the GFP-Trap. The pictures show an efficient entrapment of EVs on cells expressing the GFP-Trap.

[0166] Figure 5. (A) Detection by western-blot using an Ab anti-HA tag of GFP-TRAP (Al), PSMA-TRAP (A2) and ALFA-TRAP (A3) present on extracellular vesicles isolated by size exclusion chromatography (fraction 1 to 4) and produced by HeLa cells stably transfected with the constructs expressing the corresponding TRAPs. The full chimeric proteins migrate at ~70 kDa. Equal SEC fractions volumes were charged in the lanes.

[0167] (B) Schematic protocol of the experiment shown in (C): xenograft 2dpf zebrafish embryo of EV-TRAP (GFP-Trap) expressing cells followed by microinjection of PC3 derived CD63-pHluorin positive EVs in the blood circulation. Live imaging at 4dpf of the blood circulation in the tail of the embryo.

[0168] (C) Imaging of CD63-pHluorin positive EVs interacting in vivo with EVs released from xenografted GFP-TRAP cells. Arrows point to direct interaction between EV-EV-TRAP and tumour EV.

[0169] (D) Scheme depicting the use of EV expressing EV-TRAP chimera to trap tumour EV expressing the tumour antigen targeted by the trap. Interaction between the two populations of EVs will opsonise and clear tumour derived EVs that are potentially involved in premetastatic niche formation.

[0170] EXAMPLE:

[0171] Example 1

[0172] Material & Methods

[0173] Cell cultures.

[0174] Prostate cancer (PCa) immortalized cell lines: Prostate cancer wild-type cell lines were kindly shared by Hector Peinado’s Lab (Centro Nacional de Investigaciones Oncol ogicas, Madrid) within the consortium MSCA ITN “proEVLifeCycle”. PC3 (ATCC®, Cat.#CRL- 1435™, cell line initiated from a bone metastasis of a grade IV prostatic adenocarcinoma), and LNCaP (ATCC®, Cat.#CRL-1740™, clone FCG, cell line initiated from a lymph node metastasis of prostate carcinoma) were cultivated in adhesion in filter and vent / close cap-tissue culture flasks, maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with GlutaMAX™ (Gibco™ Cat.#61870044), 10% heat-inactivated FBS and 1% penicillin / streptomycin, and incubated at 37°C, 5% CO2.

[0175] Epithelial immortalized cell lines: HeLa (ATCC® Cat.#CCL-2™, a human immortalized epithelial cell line from cervical carcinoma) and HEK 293T (ATCC® Cat.#CRL- 3216™, a human immortalized epithelial cell line from kidney) were cultivated in adhesion in filter and vent / close cap-tissue culture flasks, maintained in DMEM (Gibco™ Cat.#41965039) supplemented with 10% heat-inactivated FBS and 1% penicillin / streptomycin, and incubated at 37°C, 5% CO2.

[0176] All the cell lines were stored in cryopreservation medium (made as heat-inactivated FBS, 10% DMSO) into 1.5 mL cryogenic vials at -80°C for short-term storage, and -120°C in liquid nitrogen cans for long-term storage.

[0177] Generation of PCa stable cell lines expressing CD63-, CD81- and CD9-pHluorin,

[0178] Metastatic prostate cancer PC3, DU145 and LNCaP cells were plated in 12-well plates. When reached approximately 80% confluence, cells were infected for 24 h at MOI=5 with lentiviral particles carrying CD63-, CD81- or CD9-pHluorin sequences, a kind gift from Dr. Maarten P. Bebelman (Vrije Universiteit, Amsterdam). When possible, the transduction was performed in presence of Polybrene® (hexadimethrine bromide, Millipore Cat.#TR-1003-G, 10 mg / mL stock) diluted at 8 pg / mL, to increase the infection rate. Lentiviral particles were then washed away, and infected cells were allowed to recover for at least 24h before being further used. For the sorting, cells were detached with trypsin, centrifuged and resuspended with 10% FBS in IX PBS. They were then passed through a 70 pm cell strainer to remove aggregates. Cells were then sorted for the GFP signal using BD FACSAria™ III Cell Sorter (BD Biosciences) adapted with a 100 pm nozzle. Cells were gated the first time to remove debris, a second time to select single events, and a third time to select GFP positive events. The cell sorter’s fluidics were filled with sterile IX PBS, and the positive cells were collected in a tube containing 500 pl of heat-inactivated FBS. This step lasted 10-20 min, and we were able to collect between 3x104 and 3x105 cells, depending on the cell line. Infected and sorted polyclonal PCa cells, stably expressing CD63-, CD81- or CD9-pHluorin, were then washed and seeded back in either 12-well culture plates or T-25 tissue culture flasks, depending on the number of cells collected. No further monoclonal selection was performed on these cells.

[0179] Cells and their -derived EVs were further caracterized by confocal microscopy for detecting the intracellular sorting of transgenic tetraspanins, by western blot from whole cell protein extracts detecting for pHluorin-tag, by western blot from SEC fractions to detect pHluorin-tag on EVs, and finally by EM and dSTORM super-resolution on EVs.

[0180] EV-TRAPs lentiviral production.

[0181] For EV-TRAPs lentiviral production, we chose adherent HEK 293T as packaging cells. Cells were seeded in 10 cm -Petri dishes. When reached 50-60% confluence, HEK 293 T cells were co-transfected with a mixture solution made as follows: 15 pg of the transgene-bearer vector (either pLenti_GFP-TRAP or pLenti_PSMA-TRAP or pLenti_ALFA-TRAP vectors - see the maps in Chapter 12 -); 10 pg of psPAX2 vector (Addgene Plasmid #12260, 2nd generation lentiviral packaging plasmid from Didier Trono Lab, containing Gag, Pol, Rev and Tat-coding sequences); 5 pg of pMD2.G vector (Addgene Plasmid #12259 from Didier Trono Lab, containing VSV-G envelope-coding sequence); PEI MAX® stock solution, in proportion 3 pL : 1 pg DNA (for a PEI MAX® stock 1 mg / mL, we dissolved 200 mg of PEI MAX® - Polysciences Cat.#24765-2 - in 190 mL of sterile IX PBS; the stock solution was then adjusted to pH=7.2, filtered in sterile conditions and stored at -20°C); Opti-MEM (Gibco™ Cat.#31985070) up to 1.2 mL final volume. After transfection, cells were brought into a ML-II containment lab and incubated overnight at 37°C, 5% CO2. The subsequent day, the transfection medium was discarded and, without washing, fresh DMEM complete medium was added, so initiating the viral particles budding that we kept for 24h. The day after, the lentiviral parti cles-enriched supernatant was harvested by direct aspiration in 10 mL-syringes and then filtered through a 0.45 pm filter connected to the syringe into a 50 mL-sterile tube, to remove cell debris. A “virus precipitation solution” [prepared as 5X stock dissolving 100 g PEG 6,000 (Thermo Scientific™ Cat.#A17541.30) and 6 g NaCl in 250 mL ddH2O, warming on microwave, vortexing, adjusting to pH=7.2, filtering through a 0.2 gm Steritop™ into autoclaved bottles and storing it at -20°C] was added (at final dilution IX) to the harvested lentivirus-enriched supernatant, previously collected into a 50 mL sterile tube, then briefly mixed and kept at 4°C overnight. The day after, the lentivirus-enriched supernatant was centrifuged for 5 min at 1500 x g, 4°C. The supernatant was then removed, and the pellet resuspended in 100-300 pL of DMEM or IX PBS, depending on the pellet size. The pure lentiviral suspension was then stored at -80°C.

[0182] Generation of EV-TRAPs+ stable cell lines.

[0183] HeLa and RAW 264.7 cells were plated in 12-well plates. When reached approximately 80% confluence, cells were infected for 24 h at MOI=5 with lentiviral particles bearing GFP- TRAP mCherry HA or PSMA-TRAP_mCherry_HA or ALFA-TRAP_mCherry_HA. When possible, the transduction was performed in presence of Polybrene® (hexadimethrine bromide, Millipore Cat.#TR-1003-G, 10 mg / mL stock) diluted at 8 gg / mL, to increase the infection rate. 24 hours post-infection, we refreshed with complete medium. Antibiotic selection was started at 48h post-infection with complete DMEM medium supplemented with 4 gg / mL puromycin. After three passages, EV-TRAPs polyclonal stable cells were maintained in selection with complete DMEM supplemented with 1 gg / mL puromycin.

[0184] EVs isolation from cell cultures and characterization.

[0185] We performed EVs isolation by incubating parental adherent cells in EV-depleted medium. EV-depleted medium was prepared by supplementing 500 mL of DMEM or RPMI with 20% heat-inactivated FBS, dispensing it into 70 mL Beckman® Polycarbonate Bottle Assembly 38 x 102 mm - 6Pk tubes, loading the tubes into an ultracentrifuge rotor Type 45 Ti (part #339160), and performing ultracentrifugation overnight at 100,000 x g at 4°C into an Optima™ XPN-80 Ultracentrifuge (Beckman® Cat.#A95765), to remove FBS-associated nanoparticles (such as lipoproteins or bovine EVs). The subsequent morning, the supernatant was collected under a sterile environment, filtered through a 0.2 pm Steritop™ into new sterile bottles, and stored at 4°C. For the incubation experiments withEV-producing cells, the medium was brought to 10% FBS by adding fresh sterile medium. Antibiotics have also been added, such as 1% penicillin / streptomycin.

[0186] Parental adherent cells were seeded into large tissue culture flasks (T-150 or T-175) or Petri dishes (P-150 mm). When cells were at 80-90% confluence, their maintaining medium was removed and, after 2 washes in IX PBS, replaced with EV-depleted medium. Cells were maintained at 37°C, 5% CO2 for 48 or 72h, depending on the EVs enrichment we wanted to reach. For 1 T-150 flask, we used an incubation volume of 15 mL of EV-depleted medium. After 48 or 72h, we collected the conditioned medium from the T-150 flasks and performed differential serial centrifugations at 4°C, in the following order:

[0187] - 300 x g for 5 min, to remove cell debris and dead cells; the supernatant was then collected into a clean sterile tube, and the pellet discarded;

[0188] - 2,000 x g for 20 min, to remove apoptotic bodies; the supernatant was then collected into a clean sterile tube, and the pellet discarded.

[0189] We then applied a refined way of small EVs isolation, purification and separation from protein aggregates and contaminants, using size exclusion chromatography (SEC) columns. In particular, starting back from the point [2], we transferred the 2,000 x g-derived conditioned supernatant into 15 mL-ultrafiltration tubes (Millipore Amicon® Ultra- 15 mL centrifugal devices with Ultracel® 10,000 Dalton molecular weight cutoff filters) and centrifuged at 4°C, 5,000 x g until reaching a final volume of 500 pL. The 500 pL-ultrafiltered conditioned medium was then loaded into sepharose beads-based SEC columns (IZON® qEVoriginal / 70 nm Gen 2 Column- optimal recovery range of 70 nm to 1000 nm diameter particles, Cat.#ICO-70) for manual or automatic fractions collection. All the SEC fractions were collected into 1.5 mL or 2.0 mL low-binding polymer tubes (Sorenson BioScience™, Cat.#27210), using filtered sterile IX PBS as buffer reservoir. After fractions isolation, SEC columns were washed with 0.5 M NaOH and subsequently with filtered IX PBS, and then stored at 4°C. Each sepharose column has been used up to 5 times for the isolation of EVs from the same parental cell line. We choose to separate 13 fractions in total, of 500 pL volume each. Among the resulting 13 SEC fractions, the EVs-enriched ones were generally those going from 1 to 4, counting from the first fraction dropping out from the column. The segregation of EV enrichment / fraction was then checked by western-blotting for classical EVs markers (such as CD63, CD81, CD9, Syntenin) or EV genetic tags (like pHluorin, HA, c-Myc, or natural tags like PSMA) in each fraction. Further confirmation was obtained performing electron microscopy on SEC fractions 1 to 4.

[0190] For in cellulo and in vivo experiments, EVs-enriched SEC fractions (1 to 4) derived from T-150 tissue culture flasks (72 h incubation) were pulled together by further ultrafiltration into Millipore Amicon® Ultra-4 mL centrifugal devices with Ultracel® 10,000 Dalton molecular weight cutoff filters, towards a final volume of -200 pL.

[0191] We measured the diameter size and the concentration of the resulting EVs preparations by multi-angle dynamic light scattering (DLS) Malvern® Zetasizer Ultra pan-analytical system, using low-volume quartz batch ZEN 2112 cuvettes [see Dynamic light scattering measurements for more details]. We got a peak of particles’ diameter around 120-150 nm, so we refer to them as small EVs (sEVs). sEVs preparation were stored at -80°C.

[0192] Double-chamber EV-competition assays. EV-competition assays have been performed in 35 mm culture dishes (p-Dish) with optical transparent polymer coverslip bottom, containing a 2 chambers-silicone insert suitable for cell migration or wound healing assays (Ibidi® Cat.# 81176). A suspension of 3-7 x 105 EV-TRAP+ cells was prepared for each condition (e.g. GFP- TRAP+ cells vs ALFA-TRAP+ cells), and 70 pl of cells suspension was seeded into each chamber (so approximately 35,000 cells per chamber) the evening before the experiment.

[0193] The next day, the attached cells were washed twice within their own chambers with IX PBS to remove debris, then the silicon culture-insert was removed, and 500 pl of complete DMEM medium (10% FBS, 1% penicillin / streptomycin) containing PCa pHluorin+ EVs (in different dilutions) was used to cover the cells.

[0194] The following sEVs particles concentrations where used for our experiments:

[0195] - 1.8E+9 sEVs in 500 pL medium (ratio of 25,700 sEVs per cell),

[0196] - 6.0E+8 sEVs in 500 pL medium (ratio of 8,600 sEVs per cell),

[0197] - 3.6E+8 sEVs in 500 pL medium (ratio of 5,200 sEVs per cell).

[0198] After 30 min, Ih or 2h incubation, cells were fixed with 2% PFA diluted in their experimental medium for 10 min at room temperature, and then in 2% PFA diluted in IX PBS for other 10 min at room temperature. Fixed cells were then covered with Ibidi® mounting medium (Ibidi®, Cat.#50001) and secured with a 25 mm diameter cover glass. Images were acquired at Leica SP5 confocal microscope with a 20x oil immersion objective, using Leica LAS AF Lite 4.0 software. Lasers power were set as follows: = 488 (GFP) at 40%, and = 561 (mCherry) at 29%. Image quality was set at 18 bit. In fixed conditions we could observe both extracellular and intracellular pHluorin+ sEVs accumulation. We acquired a Z volume of 10 pm thickness for each layer singularly, processed with Leica LAS AF Lite 4.0 software. Then we recomposed the maximum projection for each layer using Icy software. The quantification of respectively mCherry (EV-TRAP) and GFP (PCa pHluorin+ sEVs) fluorescence intensity per each layer was performed in Fiji-ImageJ software. Further ratio evaluations (pHluorin / mCherry) were performed on Microsoft Excel and plotted by using GraphPad Prism software.

[0199] For real-time live-imaging experiment, videos were performed at Leica SP5 STED CW inverted confocal microscope equipped with 3 photomultiplier modules (PMT) at 37°C, 5% CO2 over 2h incubation with pHluorin+ EVs, positioning the 20x oil immersion objective right at the center of the gap between the two layers. For the time lapse film, images were acquired every 4 min. Only extracellular pHluorin sEVs dynamics could be observed by live imaging. Videos were then processed on Leica LAS AF Lite 4.0 software and quantified using Fiji- ImageJ software.

[0200] Zebrafish housing and maintenance.

[0201] In vivo experiments have been performed in zebrafish (Danio rerio) embryos from 0 to 5 dpf, in accordance with European Animal Welfare Legislation "on the protection of animals used for scientific purposes", following the 3Rs principles (Convention ETS - No. 123, Directive No. 2010 / 63ZEU) and INSERM U1266-IPNP internal Zebrafish Facility rules. The study and all the procedures were approved by CEEA Ethical Committee and French institutional organizations (agreement No. C75-14-03). All the animals were staged and cared according to standard protocols and FELASA recommendations.

[0202] Adults were maintained at a density of ~7-8 fishes / L per tank in reverse osmosis-filtered water at 28°C, with a daily photoperiod of 14h light / 10 h dark. Fishes were fed twice per day with artemia (brine shrimp)-based dry food (75 pm-, 150 pm-, 300 pm- or 400 pm-granules, depending on the developmental stage - DIETEX France).

[0203] Matings were made in groups of maximum 12 fishes (at sex ratio of 1 male : 2 females or 1 male : 3 females, to reduce the frequency of aggression events1) into polycarbonate autoclavable 1.7 L-sloped mating systems filled with aquarium water at 28°C. The mating systems were made by an external tank (Techniplast S.p.A., Cat.#ZB17BTE), a sloped internal tank with perforations designed to facilitate eggs passage to the bottom of the system (Techniplast S.p.A., Cat.#ZB17BTISLOP), a lid (Techniplast S.p.A., Cat.#ZB17BTL), and a divider allowing physical separation between males and females, but maintaining fishes' olfactory stimulus (Techniplast S.p.A., Cat.#ZB17BTD). Males and females were kept physically separated overnight, to increase sexual libido. The next morning (08:30 a.m. up to 11 :30 a.m.) the divider was taken away, initiating the typical dances and courtship behaviours leading to eggs and sperm release and further fertilization.

[0204] Zebrafish embryos were collected in Egg water-medium [see next indention for more details] into 10 cm-Petri dishes, and kept at 28°C in incubators with photoperiod, to maintain their natural circadian rhythm (14 h light / 10 h dark per day), until 6 dpf; after, they were transferred into the aquarium. All the embryos used for strains maintenance were bleached within 24 and 30 hpf, and then incubated with Egg water-medium containing Methylene blue until 6 dpf. Embryos sedation and anaesthesia was performed using 164 mg / L Tricaine mesylate at pH=7 (Ethyl 3 -aminobenzoate, MS-222, Sigma-Aldrich® Cat.#E10521) dissolved in Egg water-medium. Old adults’ euthanasia was performed by overdose of Tricaine mesylate anaesthetic dissolved in water (300 mg / L, pH=7).

[0205] Egg water-medium 10X stock was made by dissolving 3.75 g of Instant Ocean Salts Mix (Aquarium Systems, Cat.#218035) and 1 g of Calcium sulfate dihydrate (CaO4S-2H2O, Sigma-Aldrich® Cat.#C3771) in 1.25 L reverse osmosis-filtered water. The working solution was IX Egg water-medium diluted in reverse osmosis-filtered water.In general, the fishes were in good health and no specific diseases were observed during all the experiments. Fishes’ health and housing conditions were evaluated periodically (once per year) through sanitary controls headed by a veterinary doctor.

[0206] PVS cell xenografts in zebrafish embryos.

[0207] When possible, zebrafish embryos were kept in Egg-water medium supplemented with 0.003% N-Phenylthiourea (PTU) (Sigma-Aldrich® Cat.#P7629, 1% stock dissolving the powder in boiling Egg-water) from 18 hpf on, to inhibit pigmentation. All the alive embryos were separated from the undeveloped or dead ones, and put in fresh Egg-water medium at 1 dpf, to prevent toxicity due to the release of necrotic factors. At 2 dpf, chorions could be manually removed one-by-one under the stereoscope, using very sharp forceps; in alternative, embryos could be enzymatically dechorionated in bulk by incubating them in Egg-water supplemented with 2 mg / mL Pronase endo-exoproteases mix (Roche, Cat.#10165921001), at room temperature for a brief time (typically 1-10 minutes) and constantly checking the turgidity of the chorions by gently depressing them with a poker: when chorions no longer returned to a spherical shape, the Pronase treatment was considered complete.

[0208] For cell xenografts into the peri vitelline space (PVS) of zebrafish embryos at 2 dpf, the following procedure was performed: cells to transplant were brought at 90% confluence in adhesion into a T-75 flask (or a 10 cm-Petri dish) and after two washes in IX PBS and trypsin- mediated detachment, they were pelleted at 300 x g for 5 min at 4°C. The pellet was then resuspended in IX PBS and another step of centrifugation at 300 x g for 5 min at 4°C was done to remove further debris. The cell pellet was then resuspended in sterile IX PBS into a 1.5 mL- Eppendorf™ tube reaching a concentration of -0.25-0.5 xl06 cells / pL, put in an ice bucket and brought into the zebrafish facility.

[0209] In parallel, dechorionated zebrafish embryos at 2 dpf were anaesthetized into a 10 cm- Petri dish with IX Tricaine in Egg water-medium for 5 min. After anaesthesia, embryos were aligned in open air onto a 2% agarose plate bed using a homemade hairpin loop, and always ensuring that embryos didn’t dry by adding a film of IX Tricaine in Egg-water from time to time during the whole microsurgery procedure. Only well-developed embryos, with a correct separation between the head and the pericardial membrane, and without cardiac oedema, were selected for xenografts.

[0210] We pulled borosilicate capillary tips (Harvard Apparatus, 30-0019 Cat.#GC100F-10) using a Sutter Instrument® Model P-30 vertical micropipette puller (heat= 800; pull= 300).

[0211] At this point, cells were resuspended by gentle tapping the microcentrifuge tube, aspirated (around 10 pL of suspension) and back-loaded into a borosilicate capillary using a microloader tip (Eppendorf™, Cat.#5242956003) and avoiding the formation of air bubbles, as they could compromise the integrity of the embryos. Once filled with cells, the borosilicate capillary was mounted into a Narishige GJ-8 Magnetic Stand with micromanipulator (Science Products, GmbH), and oriented with the tip towards the PVS. The tip of the capillary was then cut with a blunt end using very sharp forceps. The tip was thin enough to allow the passage of cells ideally one-by-one, without clogging.

[0212] For xenograft microinjection, we used SYS-PV820 Pneumatic PicoPump (World Precision Instruments) set as follows: Hold pressure= vent (3 psi); Eject pressure= vent; Range= 100 ms. After carefully piercing in the middle of the embryo's yolk, lowering the angle of the needle and cautiously pushing until the tip of the needle reached the PVS, cells were injected by pressing the microinjector pedal, forming a solid mass located as far as possible from the heart (to prevent cardiac oedema), and with a diameter similar to the embryo’s eye as reference (around 10 nL in volume, so -200-400 cells, depending on the cells size / type). In case of cells clogging, the pressure was slightly increased and / or the capillary tip further cut.

[0213] After injection, embryos with the xenografts were first kept anaesthetized for 5-10 min in a new 10 cm-Petri dish within IX Tricaine-Egg water medium to prevent swimming movements and allow a better closure of the wound; after, they were shifted in fresh Egg-water medium and incubated at 34°C (a compromise temperature, allowing human cells xenograft growth and zebrafish development too) overnight.

[0214] The first day post injection (dpi) [3 dpf] cells xenografts were observed under a fluorescence microscope, and the ones containing few cells, presenting cardiac oedema or with cells inside the yolk were discarded. Only well-developed cell xenografts were used for further experiments or imaging.

[0215] Intravenous cell xenografts or sEVs injections in zebrafish embryos.

[0216] The preliminary steps from 0 to 2 dpf were identical to the previous indention (see PVS cell xenografts in zebrafish embryos). At 2 dpf, zebrafish embryos were first aligned onto a 35mm Ibidi® high p-Dish with optical transparent polymer coverslip bottom (Ibidi® Cat.#81156) and embedded in 0.5% low-melting agarose (Condalab, Cat.#8050) dissolved in Egg-water medium, and supplemented with 164 mg / L Tricaine (MS-222). After 10-15 min, once the agarose solidified, a suspension of 108 cells / mL cells or 2E+10 sEVs / mL in sterile IX PBS was prepared and charged into a borosilicate capillary using a microloader tip (Eppendorf™, Cat.#5242956003). The borosilicate capillary was then mounted into a Narishige GJ-8 Magnetic Stand with micromanipulator (Science Products, GmbH), and oriented with the tip towards the Duct of Cuvier of the embryo, as shown in Fig. X. The tip of the capillary was then cut to obtain a piercing oblique angled-end, using very sharp forceps. The tip was thin enough to penetrate the skin, without damaging the embryo. Once reached the Duct of Cuvier, we microinjected as follows:

[0217] - For cells intravenous xenografts (108 cells / mL suspension): injected 5-10 nL total.

[0218] - For sEVs intravenous injection (2E+10 sEVs / mL suspension): injected a range from 5 nL to 10 nL total.

[0219] Afterwards the microinjection, embryos could be either imaged right after by spinningdisk microscopy, or de-embedded from the low-melting agarose and put back in Egg-water medium into the incubator. Importantly, for cells xenografts, fishes were re-incubated at 34°C (a compromise temperature, allowing human cells xenograft growth and zebrafish development too).

[0220] Live-imaging,

[0221] Live imaging of heart-beating zebrafishes was performed at Leica DMI3000 B spinning disk XI confocal microscope, with a module W-View Gemini-2C Hamamatsu which allows to spectrally separate two wavelengths in two cameras sCMOS (Flash 4.0 v3, Hamamatsu), with 5% CO2 flux, and temperature set at 28°C. Single sEVs in the bloodflow could be observed with an Olympus 30X / 1.05 UPlanSApo silicon immersion objective (co / 0.13-0.19 / OFN22), available at INSERM IPNP. Images were acquired at 20 fpm, and films were further processed with MetaMorph® Microscopy Automation and Image Analysis Software. Another system used, in parallel, was Nikon Spinning Disk 4 CSU-W1 spinning head, with x20, x20 LWD, x40, x60, x60 WI, xlOO objectives, Nikon Eclipse Ti2 body, 408 / 441 / 490 / 561 / 639 nm lasers and prime BSI camera, available at Utrecht Universiteit.

[0222] Live imaging on cells was performed on Leica SP5 STED CW inverted confocal microscope equipped with 3 photomultiplier modules (PMT) at 37°C, 5% CO2. Images were acquired every 2 minutes for short time lapses, every 4 minutes for long time lapses (e.g. 2 h imaging in live Double-chamber EV-competition assays).

[0223] PSMA-TRAP, To generate PSMA-TRAP, we cloned the sequence of VHH JVZ007 anti-PSMA at the N-terminus extracellular side of mCD8a, with the same method described for the previous constructs.

[0224] Results

[0225] 1. Generation of Prostate Cancer cells derived Extracellular vesicles.

[0226] To obtain test the EV-trap strategy, we first started to generate the bait of these traps by developing extracellular vesicles harboring ectopical epitope at their surface. We have used mainly two cell lines of human prostate cancer cells, PC3 and LNCaP cell. Preliminary characterization of small extracellular vesicles isolated by size exclusion chromatography revealed the enrichment of CD63 and CD9 in both cell types derived EVs but also of PSMA (an EV membrane biomarker extremely useful in the diagnosis of PCa from liquid biopsies [2- 4]) on LNCaP derived sEVs (data not shown).

[0227] In order to establish a model to track and manipulate the fate of PCa-derived EVs, we generated three different PCa stable transgenic (Tg) cells lines by lentiviral infection and FACS sorting, which overexpress a chimeric CD63-pHluorin or CD9-pHluorin, with pHluorin fused in frame to the first SEL of each tetraspanin and facing the extraluminal side of the EVs, profiting from the work developed in our lab in the past few years [5-8],

[0228] We cultivated transgenic LNCaP, PC3, with the idea they could release pHluorin-tagged EVs in the extracellular environment. Of note, the presence of pHluorin (but not GFP) allows to highlight and track in live-imaging the extracellular pHluorin+ EVs only, but in fixed conditions both extracellular and intracellular pHluorin+ EVs, providing the opportunity to analyse the dynamics of PCa-derived EVs in two separate locations in a distinct manner, outside or inside the receiving cells, an assumption that will be useful for better understanding the rest of the study.

[0229] First, we characterized by confocal microscopy the expression and intracellular trafficking of CD63-pHluorin and CD9-pHluorin in LNCaP Tg stable cell lines, showing a phenotype almost identical to the WT conditions (data not shown) for both the CD63 and CD9 chimeric tetraspanins. This indicated that the presence of pHluorin fused in frame to the first SEL did not profoundly affect the normal intracellular distribution of both the constructs (data not shown). Then we performed SEC isolations from Tg CD63-pHluorin+ and CD9-pHluorin+ LNCaP conditioned media separately, and we could detect the presence of the pHluorin-tag enriched mostly in fractions 1-4 by blotting with a commercial antibody against GFP, suggesting that pHluorin-tagged sEVs might segregate again in this peculiar SEC “EV-zone” as in the WT conditions. We checked tat the main particle size of SEC-isolated EVs which reconfirmed to be again around 100-120 nm in diameter at DLS for both the Tg CD63- pHluorin+ and CD9-pHluorin+ LNCaPs (data not shown), we further characterized the SEC 1- 4-derived sEVs from CD63-pHluorin+ LNCaP cells by super-resolution dSTORM, reconstructing their 2D shape as clusters of single molecule localizations by labelling with TMIX (data not shown). Over 24,500 single clusters analyzed, we registered a general distribution in size with a peak around 100-120 nm of diameter, as represented in cloud-points diagrams of single clusters distribution and in the charts of number of clusters per diameters (data not shown), which appeared to be similar to the LNCaP WT sEVs previously analyzed (data not shown).

[0230] To go deeper on this, we next assessed the distribution of merely pHluorin single molecules this time, by labelling CD63-pHluorin+ LNCaP-derived sEVs with an antibody anti- GFP. We were able to reconstruct clusters of pHluorin, with a size comparable to that previously detected with TMIX (data not shown) but in remarkable lower count (around 50% less) if compared to the number of clusters counted with TMIX (a bona fide labelling of the total sEVs), suggesting that only a subpopulation of sEVs is actually marked with CD63- pHluorin within the total number of sEVs derived from CD63-pHluorin+ LNCaP.

[0231] We further sought confirmation whether these pHluorin clusters, previously revealed by super-resolution microscopy, were indeed in sEVs by using transmission electron microscopy. We revealed the presence of lipid bilayer sEVs tagged with pHluorin in both Tg LNCAP CD63- and CD9-pHluorin SEC [1-4] fractions isolations, by immunogold labelling using a Rabbit anti- GFP antibody coupled with protein A-gold (PAG) particles (data not shown). Importantly, as negative control, the same kind of anti-GFP PAG labelling in LNCaP WT-derived sEVs gave no staining, demonstrating the high specificity of this approach of detection and endorsing the unique genetic tagging of our homemade-generated CD63 -pHluorin and CD9-pHluorin tagged sEVs (data not shown). Interestingly enough, our sEVs populations analysed by TEM appear heterogeneously tagged with pHluorin, with some EVs displaying no pHluorin labelling at all, which is in line with our previous findings by dSTORM super-resolution showing that the number of clusters revealed with anti-GFP is considerably lower than the number of clusters revealed with TMIX from the same Tg sEVs preparations.

[0232] Subsequently, we measured the presence of PSMA in the sEVs derived from Tg CD63- and CD9-pHluorin+ LNCaP cells by western blot and dSTORM analysis. We found that the presence of PSMA in SEC-derived 1-2 sEVs fractions is in lower quantity (data not shown) if compared to the SEC-derived sEVs from WT LNCaPs by western blotting (data not shown). Further detection of PSMA and CD63 by super-resolution dSTORM revealed that Tg LNCaP- derived sEVs are very heterogeneous, with the presence of some subpopulations enriched only in PSMA, other populations mixed expressing PSMA and CD63 at different ratios, and other populations only CD63 - but not PSMA - positive (data not shown).

[0233] Finally, we completed the characterization of our homemade transgenic PCa cells producing genetically marked EVs by studying the expression of CD63-pHluorin and CD9- pHluorin in PC3 cell lines. We could observe that the expression of the two chimeric tetraspanins is comparable to that of the Tg LNCaPs described above, both in terms of intracellular sorting (data not shown) and in terms of their relative enrichment in sEVs, which we characterized by western blot, DLS and TEM from SEC isolations (data not shown).

[0234] 2. EV-TRAPs: a set of chimeric mCD8a membrane receptors exposing VHH nanobodies against transgenic or natural PCa EV-tags.

[0235] Inspired by the morphotrap tools published by Prof. Markus Affolter (Biozentrum, University of Basel) used to capture and manipulate morphogens in Drosophila melanogaster [9,10,11], and by the chimeric antigen receptor (CAR) T-cell strategies developped in the past 20 years and used to potentiate T cells to make them able to selectively recognize tumor antigens - works that earned Prof. James P. Allison (M.D. Anderson Cancer Center, University of Texas) and Prof. Tasuku Honjo (Kyoto University) the 2018 Nobel Prize in Medicine and Physiology

[0012] -, we aimed at transposing these novel concepts to tumor EV biology. We decided to produce unique chimeric proteins made as mCD8a membrane receptor fused in frame with different llama-derived VHH nanobodies facing the extracellular environment, with the intent to capture PCa EVs by the (transgenic or natural) tags expressed on their surface, and consequently manipulate and control tumor EVs path and spreading, an ambitious and never explored land of research so far to our knowledge.

[0236] The choice of using camelid VHH nanobodies for our constructs was moved by the consideration that these peculiar subtype of antibodies are the smallest in nature, with a molecular mass of about ~15 kDa, possessing incredibly high target specificity, high affinity and high structural and thermic stability [13,14], which are crucial parameters of high resistance for our prospective application in the turbolent, assorted and caotic environment of the bloodflow in vivo.

[0237] In order to selectively capture our Tg pHluorin-tagged PCa EVs (extensively described in paragraph 8.2: LNCaP and PC3 cells expressing chimeric CD63-pHluorin and CD9-pHluorin release heterogeneous subpopulations of pHluorin-tagged EVs), we generarated a GFP-TRAP by fusing mCD8a N-terminus (extracellular) part with the GBP nanotrap from Rothbauer et al.

[0015] , and we fused the C-terminus (cytosolic) part with an mCherry-tag followed by a HA- tag as receptor reporters (Fig. 1 A).

[0238] In parallel, to capture PCa EVs by exploiting their natural surface biomarkers like PSMA, we generarated a novel PSMA-TRAP by fusing mCD8a N-terminus part with the JVZ- 007 VHH nanobody from van Weerden et al.

[0016] and, similarly as before, the C-terminus part with an mCherry-tag followed by a HA-tag. (Fig. 3 A).

[0239] Lastly, we generated an ALFA-TRAP receptor with the same strategy as previously described, fusing the N-terminus of mCD8a with a VHH anti ALFA-tag (NanoTag Biotechnologies, GmbH). We used the ALFA-TRAP construct as negative control for the whole study (Fig. 1A).

[0240] The expression of the three EV-TRAPs was characterized by confocal microscopy, showing that the chimeric receptors are identical in terms of intracellular sorting, with an important membrane localization and intracellular endosomal accumulation, both in transient and stable expression in HeLa cells (data not shown). Western blot analyses from whole-cell lysates showed how all the three chimeric receptors undergo similarly important cleavage both by anti-HA and anti-mCherry blotting (data not shown), suggesting that it might exist a natural turnover of degradation and new exposure at the plasma membrane, or a stochastic cleavage happening in some receptors at their intracellular domains, potentially explaining why we observe intracellular endosomal accumulation of mCherry signal.

[0241] Notably, all the three EV-TRAPs HeLa stable cell lines release sEVs containing the GFP-TRAP, the PSMA-TRAP or the ALFA-TRAP, as observed by western-blot detection for HA-tag in SEC 1-4 fractions from the three stable EV-TRAPs cells conditioned media separately (data not shown). This aspect doesn’t appear to exert a major impact on the dynamics of entrapment of pHluorin-tagged EVs, as we will show later in the results, and at the same time might favour the neutralization of PCa EVs (and maybe of the PCa cells as well) or opsonisation events in vivo, a topic that remains fully open for further investigation.

[0242] 3. GFP-TRAP+ HeLa capture and stably retain PCa pHluorin-tagged EVs, promoting their differential spatial accumulation in competition assays in vitro.

[0243] In the initial trials, we tested different types of extracellular preparations from PC3 CD63-pHluorin+ cells on transiently expressing GFP-TRAP transfected HeLa. First, we incubated transient GFP-TRAP+ HeLa with the Tg PC3 CD63-pHluorin+ ultrafiltered supernatant from 2,000 x g centrifuge step, therefore with a diverse coarse preparation containing oncosomes, apoptotic bodies, ectosomes, exosomes and potential traces of soluble pHluorin (a condition that might be close to a possible scenario of endogenous EVs release by Tg PC3 tumor cells in vivo) at roughly 20,000 EVs / cell for Ih at 37°C. We found a very net and clear phenotypic difference in co-culture between GFP-TRAP expressing cells, which stably retained CD63-pHluorin+ coarse EVs at the plasma membrane in a saturating fashion, and untransfected cells, which physiologically uptook a limited amount of CD63-pHluorin+ EVs, without showing any evident retention at the PM (data not shown). That was for us a first exciting hint that the GFP-TRAP could have been used as a tool to forcefully capture and retain CD63-pHluorin tagged EVs towards preferential cells of choice.

[0244] Thrilled by these findings, we moved on by exploring the dynamics of capture and retention of PC3 CD63-pHluorin+ EVs isolated from 100,000 x g pellet (from differential centrifugations) by transient GFP-TRAP+ HeLa cells in live-imaging at 37°C. With a look at extracellular events only, we recorded a massive capture of PC3 CD63-pHluorin+ 100,000 x g coarse EVs (incubated at a ratio of ~20,000 EVs / cell), observing single particles going first from the frenetically moving filopodia, acting almost like “intercepting antennas”, then gradually sliding towards the entire coverage of the plasma membrane of GFP-TRAP+ HeLa cells, saturating their PM already after 12 min from the initial direct pipetting of EVs into the cell culture medium (Figure IB). Of note, we noticed some EV-TRAPping cells undergoing apoptosis after the capture, suggesting that there might be some toxicity due to high dosage of PC3-derived EVs, an argument we will better discuss later in the results (data not shown).

[0245] Next, we characterized the entrapment of pure PC3 CD63-pHluorin+ sEVs preparations from SEC 1-4 pooled fractions in similar parti cles / cell ratios as before. Differently from the incubation with the rough 2000 x g supernatant and the 100,000 x g EVs pellet, sEVs from SEC isolations were entrapped later in time in appreciable events of PM accumulation by live- imaging, from Ih incubation going on, suggesting that the size and the coarseness of these particles plays pivotal roles in their dynamics to physically reach HeLa cells in adhesion, by Brownian motion (pure / small EVs) or precipitation by gravity (coarse / large EVs). Nonetheless, we observed also here active retention of CD63-pHluorin+ sEVs at the PM of GFP-TRAP+ HeLa, but also intracellular uptaken EVs signals in PFA fixed conditions (Figure 1C top panel). This phenotype was significantly different if compared to that resulting from PC3 CD63- pHluorin+ sEVs incubation on transient ALFA-TRAP+ HeLa (the non-specific TRAP used as negative control), where we could observe generalized physiological sEVs endocytosis but not active PM accumulation at Ih (Figure 1C bottom panel). Taken together, these data showed us how the presence of a GFP-TRAP exposed on the membrane of very common cells such as HeLa confers them the power to selectively and actively capture and retain pHluorin+ EVs from prostate cancer cells at the PM, both with coarse preparations of EVs and with super pure preparations of small EVs. This simple (but mighty) tool therefore proves to be unique in its kind, as it is an ad hoc system potentially applicable for myriads of uses, and adaptable to multitudes of EVs-producing cells and EVs- receiving cell, to make an exclusive controlled system of forced cross-talk or possible spatial rerouting of selected EVs subpopulations among the complex variety of producing / receiving cells we can find for instance in vivo.

[0246] To further explore whether the GFP-TRAP expression had the potential to induce prostate cancer CD63-pHluorin+ EVs spatial rerouting and selective local spatial accumulation, we challenged our system by re-adapting a common wound healing assay into an “EVs- competition assay”, by seeding transient GFP-TRAP+ HeLa cells on one layer and transient ALFA-TRAP+ HeLa cells on the opposite layer of the chamber, and inducing an unidirectional- oriented flux of PC3 CD63-pHluorin+ EVs by pipetting them from one side of the area were cells were seeded and facing eachothers, right at the level of the central canal separating the two cell lines. We recorded films in live-imaging at 37°C showing that flowing CD63- pHluorin+ EVs were actively intercepted, retained and accumulated over 2h in a linearly increasing fashion towards the GFP-TRAP+ layer but not at the ALFA-TRAP+ layer of the chamber, where instead they were passively rolling and infiltrating between cells interstices, or following the oriented flux towards the opposite side of the chamber (Figure 2A).

[0247] Besides the insightful indications pHluorin-tagged EVs can bring us about extracellular dynamics of entrapment in live-imaging, when fixed in this EVs-competition assay, we could recover pivotal statistics on the differences of total burden of intracellular and extracellularly- retained pHluorin+ EVs per cell, as ratio of pHluorin / mCherry fluorescence intensity per layer. For instance, we tested preparations of DU145 CD63-pHluorin+ EVs and also DU145 CD9- pHluorin+ EVs derived from 100,000 x g pellet in analogs transient GFP-TRAP+ vs ALFA- TRAP+ competing assays (~20,000 EVs / cell), resulting in significantly higher DU145 pHluorin+ EVs burden at the GFP-TRAP+ layer compared to the ALFA-TRAP+ layer at Ih, for both the chimeric CD63 and CD9 tetraspanins, with in particular DU145 CD63-pHluorin+ EVs 3-folds higher in GFP-TRAP+ layer compared to ALFA-TRAP+ layer (data not shown), and DU145 CD9-pHluorin+ 2-folds higher in GFP-TRAP+ layer compared to ALFA-TRAP+ layer (a difference that might depend on a dissimilar relative percentage of pHluorin tagged EVs subpopulation per cell line) (data not shown). In brief, these data already indicated that both CD63-pHluorin and CD9-pHluorin EV- tags are suitable moi eties for the GFP-TRAP mediated capture of EVs, and that with such a kind of EVs-competition assays we in fact conceived an easy -handling chip providing fast informations about the power and efficacy of the tumor EV entrapment in fixed conditions, as well as informations about the dynamics and pace of tumor EVs capture in live-imaging. Moreover, these first data showed that our EV-TRAP strategy makes possible the formation of differential spatial zones of tumor EVs accumulation, in favour to one or another specific region of choice in vitro - now in very restricted space of actions -, and it promises to be an optimal approach to achieve tumor EVs spatial rerouting and accumulation towards a restricted anatomical site of capture in vivo.

[0248] Next, we generated HeLa (and others) stable cell lines expressing the GFP-TRAP and the ALFA-TRAP by lentiviral infection and puromycin selection (see Materials and Methods).

[0249] Focussing just on HeLa in this work, we first incubated stable GFP-TRAP+ and ALFA- TRAP+ HeLa again with PC3 CD63-pHluorin+ ultrafiltered supernatant from the 2,000 x g centrifuge step (~20,000 EVs / cell). As for transient GFP-TRAP+ HeLa, these cells showed an important retention of coarse CD63-pHluorin+ EVs at Ih incubation, with some endocytosed material (data not shown), differently from stable ALFA-TRAP+ cells which appeared undergoing to generalized endocytosis but not membrane retention in vitro (data not shown). Importantly, at this dosage of PC3 EVs, both GFP-TRAP+ and ALFA-TRAP+ HeLa cells displayed a suffering phenotype, which let us the necessity to establish a better EVs / cells ratio for our PCa EVs incubation studies in vitro (a part discussed later on MTT proliferation / cytotoxicity assays with SEC-derived PCa sEVs data not shown).

[0250] To better quantify and characterize the pace of entrapment according to the dosage of EVs and the timing of incubation, we performed a single-cell analysis of the total burden of PC3 CD63-pHluorin+ sEVs. We incubated stable GFP-TRAP+ HeLa or ALFA-TRAP+ HeLa with PC3 CD63-pHluorin+ sEVs from SEC 1-4 pooled fractions at different dosages (20, 200, 2000, 20,000 sEVs / cell) and at different time points (15 min, 30 min, Ih, 2h) separately. After fixation and confocal imaging acquisitions, we computed the ratio of the total intensity of CD63-pHluorin (membranous and intracellular) / mCherry signals per single-cell over 50 cells analysed per condition, deriving a global trend of PC3 CD63-pHluorin+ sEVs accumulation at different dosages over time on GFP-TRAP vs ALFA-TRAP HeLa. Strikingly, starting from the 200 sEVs / cell dosage, we found that the ratio values associated to each dose / time point were significantly higher for GFP-TRAP than ALFA-TRAP conditions. As a matter of the fact, timedependent accumulation of sEVs resulted with 2-fold increase already at 15 min, to reach roughly 3-fold increase at 2h in GFP-TRAP+ cells for each dosage point compared to the ALFA-TRAP+ counterpart (Figure 2B). However, the estimation of the ratio between GFP- TRAP values / ALFA-TRAP values showed a sudden decrease at 20,000 sEVs / cell (data not shown), which let us suspect a potential cytotoxicity happening in that particular condition. Prompted by this, we screened the effect of different dosages of PC3 as well as LNCaP-derived CD63-pHluorin+ and CD9-pHluorin+ sEVs on cell viability of GFP-TRAP+ and ALFA- TRAP+ HeLa at 2h incubation by MTT assays. Indeed, we could confirm that Tg LNCaP and PC3 sEVs cause significant cytotoxicity at 20,000 sEVs / cell similarly in ALFA-TRAP+ and GFP-TRAP+ HeLa cells, with Tg LNCaP sEVs causing a 40% of mortality and Tg PC3 sEVs causing 20% of mortality at this dosage (data not shown). However, and very intriguingly, Tg PC3 sEVs (but not Tg LNCaP sEVs) displayed opposite effects at relatively low doses (20, 200 and 2000 sEVs / cell), with trends looking as a slight - yet not significant - boost of proliferation on both the cell lines at 2 h (data not shown). These observations seem to be very in line with the recent findings from El Andaloussi’s team at Karolinska Institutet, showing that the cellular response to sEVs is dependent on their cell source and dosage, with sEVs (from certain particular cell types) inducing transcriptional activation at low dosages, whilst lysosomal activation at high dosages from 20,000 sEVs / cell threshold going upl7. Since approximately only a 50% subpopulation of sEVs turned out to be actually tagged with pHluorin in our Tg LNCaP sEVs (data not shown), we decided to set a compromise dosage of incubation between a decent EVs detectability by confocal microscopy and a mild cytotoxicity at 2000 sEVs / cell for further assays with Tg LNCaP and Tg PC3 sEVs in vitro.

[0251] We then exposed GFP-TRAP+ or ALFA-TRAP+ cells to a "pulse" (4°C, 30 min) with a (Rb)anti-GFP PAG-5 antibody after Ih of incubation with PC3 CD63-pHluorin+ sEVs, and a subsequent "chase" (37°C, 30 min) to also allow a possible internalization of the EVs following trapping. By applying the Tokuyasu method (see Material and Methods) we were able to demonstrate that the HeLa GFP-TRAPs actually had membrane trapping residues, with interesting pHluorin signals all accumulated as a pHluorin belt exactly in the site of forced membrane contact (data not shown). Furthermore, we observed interesting endocytosis events of entrapped sEVs, suggesting that EV-TRAP

[0252] We subsequently investigated the long-term intracellular fate (at 2h incubation with 2000 sEVs / cell) of PC3 CD63-pHluorin+ sEVs, endocytosed by ALFA-TRAP+ HeLa or by GFP-TRAP+ HeLa, by evaluating the Pearson’s co-localization coefficient (r) between intracellular pHluorin signals and the lysosomal-associated membrane protein 1 (LAMP1) detected by IF in permeabilizing conditions (so sacrificing an important amount of the membrane-associated pHluorin+ sEVs signal), to understand if PC3 sEVs could end-up their cycle in degradative organelles. Surprisingly to us, we found that in GFP-TRAP+ cells there was almost no co-localization between pHluorin residual signals and LAMP1 compartments (r <0.5) (data not shown), unlike in ALFA-TRAP+ HeLa cells where, instead, the residual pHluorin signals co-localized with a significantly higher Pearson's score within LAMP1 compartments (data not shown). Equally interesting, we analysed the residual raw signals intensity of pHluorin at 2h, which were four-times significantly higher in GFP-TRAP+ HeLa than in ALFA-TRAP+ HeLa, where they tended to zero at two hours (data not shown). This ultimately denotes that the pHluorin+ sEVs trapped and eventually internalized by GFP-TRAP undergo a non-degradative endocytic pathway which is different from that of PCa sEVs physiologically penetrating the ALFA-TRAP+ cells, as they appear to be degraded over time (data not shown) by fusion with degradative compartments (data not shown). All together these data suggest either that the forced entrapment of pHluorin+ sEVs also “protects” them from physiological degradation, or that the forced receptor-mediated internalization of PCa EVs ends-up in different non-degradative / storage compartments by default, or that it might exist a physiological limit in the endolysosomal-degradative compartments “filling” that, when it is overcome, is then followed by non-degradative storage, especially in “super-collector” (artificial or natural) cells like our GFP-TRAP+ HeLa: all these hypotheses are still fully open to further investigation.

[0253] We then recreated the conditions for the EVs-competition assays as described before, but this time by facing the stable GFP-TRAP+ vs ALFA-TRAP+ HeLa cell lines for each different types of SEC-derived small EVs previously defined, i.e. LNCaP CD63- and CD9- pHluorin+ sEVs and PC3 CD63- and CD9-pHluorin+ sEVs. We incubated the two facing layers with a concentration of 2000 sEVs / cell and compared the competition at Ih, first in live- imaging at 37°C in complete medium, to describe and quantify in real-time the membrane capture and retention capacity of pHluorin tagged EVs for each layer, and then in PF A fixed conditions, to reveal the total (intracellular and extracellular) content of pHluorin tagged EVs per cell for each layer. Over three different competition fields analysed for each Tg sEVs type, we observed and quantified the total content of sEVs at the plasma membrane in live imaging, which resulted always significantly in favour of the GFP-TRAP layer. In particular, LNCaP CD63-pHluorin+ sEVs resulted enriched in the GFP-TRAP layer with a 3 -fold increase (data not shown), while LNCaP CD9-pHluorin+ sEVs only with 1.5-fold increase (data not shown). Following, PC3 CD63-pHluorin+ sEVs were enriched at the GFP-TRAP layer with 2-fold increase (data not shown), and PC3 CD9-pHluorin+ sEVs with 1.8-fold increase (data not shown). Similarly to live-imaging measurements, in PFA-fixed conditions (revealing intracellular and membrane sEVs burden) LNCaP CD63-pHluorin+ sEVs resulted enriched in the GFP-TRAP layer with a 3 -fold increase (data not shown), LNCaP CD9-pHluorin+ sEVs with 1.5-fold increase (data not shown), PC3 CD63-pHluorin+ sEVs with 2-fold increase (data not shown), and PC3 CD9-pHluorin+ sEVs with 2-fold increase (data not shown). These similarities in the ratio values between live-imaging and PFA-fixed conditions suggest that the major contribution which determines a significant fold change in this competition assays comes from the membrane entrapment and retention of pHluorin+ sEVs rather than differential intracellular accumulations.

[0254] To confirm this, we plotted the raw values of pHluorin intensity of ALFA-TRAP layers and GFP-TRAP layers in live-imaging (extracellular, rolling / flowing interstitial sEVs [ALFA- TRAP] vs stably retained sEVs at the PM [GFP-TRAP]) and in PFA-fixed conditions (total intra- / extra-cellular pHluorin+ PCa sEVs). Here too, we observe that there are no major differences in the general trends of competition between live-imaging and PFA-fixed conditions, other than a slight increase in the signal of pHluorin in ALFA-TRAP (as expected), which however is not always significant (data not shown), suggesting that in epithelial cells like HeLa probably it does exist a physiological balance between quantity of material at the surface and endocytosed / degraded material, rather than a long-term intracellular storage (which might be instead the case for other cell lines).

[0255] On the contrary, in the GFP-TRAP layers we detected a drop in the total signal of pHluorin when fixing with PFA (data not shown), which might be due to well-known crosslinking fixatives-induced membrane artifacts, such as partial damage of the PM [18,19] or potential antigenicity loss between the GFP-TRAP and pHluorin [20,21,22,23], that ultimately cause the loss of part of pHluorin+ sEVs burden (and their associated signal) at the PM. These outcomes reinforce for us the necessity of complementing fixed conditions with live imaging conditions, especially when inspecting PM events, as with fixation we might gain information at the intracellular level but lose informations and impair membranes’ structures at the extracellular side. This also makes valuable and justifies the use of pHluorin (but not GFP) to gain information on the behaviour of sEVs acting at the level of the PM.

[0256] In addition, we suppose that - given the same number of sEVs / cell used - the relative variances in fold increase found at Ih between the different sEVs types tested might indirectly reflect the actual percentage of pHluorin-tagged sEVs from each single PCa stable cell line, rather than particular biological differences in uptake / degradation events between the different types of sEVs tested. However, direct confirmations of this assumption should arrive soon from dSTORM super-resolution quantifications performed on high numbers of sEVs analysed by TMIX vs pHluorin (GFP)-only labelling, for each PCa CD63- and CD9-pHluorin Tg stable cell line generated.

[0257] Of note, these experiments do not reflect the maximum of saturation possible for GFP- TRAP. In fact, forcing the system at high extent, we tested an incubation with something like 20,000 particles / cell from coarse 2000 x g sup. ultrafiltered medium derived from CD63- pHluorin+ LNCaP and PC3, detecting that the absorbent power of GFP-TRAP surfaces can reach 5 or even 8-fold increase, and possibly even more at higher concentrations (data not shown). This already made us thinking about the EV-TRAP as an absorbent maxi-sponge, or a maxi filter for tumor EVs, a concept which if applied in vivo can have significant implications on blocking metastatic tumor cells and EVs spreading.

[0258] 4. PSMA-TRAP+ HeLa capture and stably retain PCa PSMA-enriched EVs, promoting their differential spatial accumulation in competition assays in vitro.

[0259] As we already mentioned before, a second route we wanted to explore was to exploit one of the nanobodies with higher affinity for the extracellular domain of PSMA in the fieldl6 to generate a PSMA-TRAP, in order to capture LNCaP-derived EVs by exploiting their natural enrichment in PSMA (Figure 3 A).

[0260] In the very first trials we tested LNCaP CD63-pHluorin+ or CD9-pHluorin+ (PSMA+) 100,000 x g pellet EVs on PSMA-TRAP+ HeLa cells (~20,000 EVs / cell) in transient transfection, confident of being able to observe trapping phenomena only by the mere detection of the pHluorin-tagged EVs (as for the GFP-TRAP). However, both in live-imaging and in fixed conditions, we were able to observe only small sporadic and clustered events of entrapment of Tg LNCaP EVs by PSMA-TRAP+ HeLa cells, with plenty of non-entrapped pHluorin-tagged EVs as background (data not shown), suggesting that perhaps the detection of Tg LNCaP EVs by imaging of pHluorin did not fully correlate with the detection of PSMA- enriched EVs. This seemed to be strictly in line with our previous findings by super-resolution dSTORM on LNCaP CD63-pHluorin+ EVs, showing that it might exists a conspicuous subpopulation of Tg LNCaP PSMA-enriched EVs which is not CD63-positive (data not shown). Of note, by live imaging we could notice an important number of PSMA-TRAP+ HeLa undergoing apoptosis after the Tg LNCaP EVs entrapment (data not shown), confirming again that the EVs from this cell source present significant cytotoxicity on HeLa even at relatively low doses, as previously shown by MTT assays (data not shown). At the same time, by EVs- competition assays with PSMA-TRAP+ HeLa vs ALFA-TRAP+ HeLa, we could observe a little - yet not significant - trend of increase of the burden of accumulation of LNCAP CD9- pHluorin+ sEVs in PSMA-TRAP+ layer compared to ALFA-TRAP+ layer at Ih, suggesting again that the detection of LNCaP-derived EVs by pHluorin was not be the best strategy to detect PSMA-TRAPping events of LNCaP PSMA+ sEVs, and that our analysis might be biased also because of cell death events occurring in the system (data not shown).

[0261] Nevertheless, encouraged by the prospective of a (potential) direct applicability of the PSMA-TRAP in PCa patients, we decided to generate stable PSMA-TRAP+ cell lines (data not shown) and to continue our PSMA-TRAPping studies, but this time putting aside for a while the the pHluorin-genetic tagging strategy of LNCaP EVs. In fact, we adopted a new approach by using solely WT LNCaP (PSMA+) EVs, so remaining closer to a “natural” situation as in PCa patients, and detecting the WT EVs entrapments by labelling a posteriori with a nanobody anti-PSMA (namely the same VHH JVZ-007 previously cloned in the PSMA-TRAP), envisaging a sandwich-ELISA in cellulo on LNCaP WT EVs after entrapment to exclusively observe PSMA+ EVs (data not shown).

[0262] We first incubated stable PSMA-TRAP+ or ALFA-TRAP+ HeLa cells with LNCaP WT sEVs from SEC 1-4 fractions (~2000 sEVs / cell) for Ih, and labelled with a homemade VHH JVZ-007 conjugated with Cadmium / Selenide (CdSe) Quantum dots of 2.5 nm in diameter (Qdots2.5nm) after fixation, and in non-permeabilizing conditions for preserving the integrity of the PM of PSMA-TRAP+ cells and of LNCaP WT sEVs. Remarkably, we could observe a corona of PSMA+ EVs entrapped along the PM of PSMA-TRAP+ HeLa as very sharp single dots by TIRF angle (Figure 3B), differently from ALFA-TRAP+ HeLa which appeared nude at the PM without any evident sEVs retention but surrounded by LNCaP WT sEVs stuck on the glass slide surface (Figure 3A). We further confirmed that indeed those signals corresponded to PSMA+ EVs by labelling a posteriori with VHH JVZ-007-CdSe Qdot2.5nm in conditions without LNCaP sEVs, showing that the backgroud from VHH-Qdots was minimal and randomly associated to the cells rather then on the cover slide surface, suggesting that some unspecific interaction with the PM might happen, but in irrelevant numbers (data not shown).

[0263] In parallel, we characterized the VHH JVZ-007 conjugated with Cadmium / Selenide (CdSe) Quantum dots on LNCaP cell lysates by western-blot (Figure 3C). We detected a "nude" form around 75 kDa with a homemade llama-derived nanobody, also known as VHH JVZ-007 clone, obtained from a library screening against an extracellular epitope of PSMA by the group of Wytske van Weerden (Erasmus MC, Rotterdam) 16. VHH JVZ-007 is also able to recognize a cleaved fragment at 25 kDa in LNCaP whole-cell lysates, which is of unknown biological significance to date (Figure 3C). As remarkable difference, VHH JVZ-007 appears to recognize only the 25 kDa fragment in SEC 1-2 fractions derived from LNCaP conditioned medium, (data not shown)

[0264] Besides the high precision and the sharpness offered by Qdots detection, this type of imaging is only conceivable in very strict conditions, with a 100 X objective and in TIRF angle, set-ups which are not suitable for a wide-field observation like the EVs-competition assays imaged with a 20 X objective. To overcome this limitation, we produced another homemade VHH JVZ-007, this time conjugated with Alexa Fluor TM 488. Also in this case, the use of VHH JVZ-007-AF 488 allowed us to detect the presence of LNCaP WT sEVs trapped on the PSMA-TRAP+ HeLa’s membrane after fixation and in non-permeabilizing conditions (data not shown), this time at different magnifications and in wide fields with a 63 X objective, yet maintaining the PSMA+ sEVs precision of detection (data not shown). As a matter of the fact, wide field observations showed that only a little subpopulation of PSMA-TRAP+ cells accumulate significant amount of LNCaP WT PSMA+ sEVs at Ih incubation (data not shown). This might be due both to the scarce presence of PSMA in the small EVs derived from LNCaP SEC fractions 1-4 (data not shown), or to the possibility that the PSMA-TRAP undergoes greater cleavage than GFP-TRAP or ALFA-TRAP, as shown by western blots from whole-cell lysates (data not shown) or - similarly to Tg LNCaP-derived sEVs - to cytotoxicity induced by WT LNCaP sEVs on PSMA-TRAP+ and ALFA-TRAP+ HeLa, as demonstrated by MTT assays (data not shown). Though, single-cell analyses of the ratio of extracellular signals of PSMA / mCherry showed a slight but significant 1.5-fold increase in extracellular LNCaP WT (PSMA+) sEVs PM retention in favour of PSMA-TRAP+ cells starting at Ih up to 2h incubation (data not shown), but when quantifying at wide field in EVs-competition assays we loose informations associated to VHH JVZ-007 AF 488 signal, obtaining again a nonsignificant trend of increase in favour of the PSMA-TRAP layer (data not shown). The major issue occurring seems in fact to be that the number of single cells capturing PSMA is too low to be appreciated at large scale, at least in these conditions. Another potential limitation could be the fact that the PFA-fixation might cause antigen loss and impair the recognition by the JVZ-007, resulting in a defected signal associated to an antigen which is already poorly expressed in SEC-derived sEVs. For this reason, we are planning to repeat these experiments in the near future, but labelling for PSMA before fixation, hopefully to get more insightful data at large scale. Also, it might be wise to test larger populations of EVs derived from LNCaP, like the 2000 x g or 10,000 x g pellet, which might be more enriched in PSMA than small EVs

[0024] , For instance, we already tested the 2000 x g pellet, finding again few numbers of cells (around 5% of the total) nicely collecting PSMA+ EVs at the membrane (data not shown). Another strategy to improve the readout from this kind of PSMA-TRAP experiments might be to FACS sort the clones which better perform in PSMA entrapment and expanding them to get a selected high-performing PSMA-TRAP+ HeLa cells population, and / or in parallel to use EVs from other cell types more enriched in PSMA, or transgenic ones overexpressing PSMA

[0024] , or to generate a PSMA-pHluorin cell line for the direct detection of the epitope.

[0265] Unitedly, these data are on one hand encouraging, if we look at the single cell PSMA capture capacity, and on the other hand they must be oriented towards a better target of LNCaP EVs subpopulation more enriched in PSMA, or tested directly on EVs from PCa patients’ sera. With the possibility that there are clones that degrade the PSMA-TRAP at higher rate than others, it might be also necessary to envisage a sorting of the best performing clones and their subsequent expansion. Nevertheless, the strategy of making a cell-surface in vivo trap for specific biomarkers of tumor EVs is already in itself a novelty in the field, which can also be implemented by the association with multiple nanobodies against tumor EVs biomarkers in the same CD8 membrane receptor, a part that we will better argue in the future perspectives chapter of this thesis.

[0266] 5. EV-TRAP+ HeLa intravenous engrafts locally accumulate PCa EVs, changing their biodistribution and fate in the zebrafish embryo.

[0267] Once established a general mapping of biodistribution and fate of PCa exogenous EVs injected in the zebrafish embryo (data not shown), we next tested our EV-TRAP+ cells in vivo with the intent of locally capturing tumor PCa-derived sEVs in ectopic sites of retention, eventually reducing their spreading in the whole body (Figure 4A). Thus, we first set up a protocol previewing the engraftment of -500-800 GFP-TRAP+ HeLa cells into the Duct of Cuvier at 2 dpf, followed by the injection of -100,000-200,000 PC3 CD63-pHluorin+ sEVs into the Duct of Cuvier at one-hour post EV-TRAP+ cells engraftment (data not shown). After 10-15 min from Tg PCa sEVs injection we could already observe sparse groups of GFP-TRAP+ HeLa cells scattered in different points of the vasculature and interstices and entrapping and locally retaining PC3 flowing CD63-pHluorin+ sEVs in a stable fashion, besides EVs highspeed of circulation in the blood flow. For instance, we could capture movies in real-time of GFP-TRAP+ HeLa cells retaining PC3 CD63-pHluorin+ sEVs in the CA-CV curve (data not shown) or in the perivitelline space (PVS) (data not shown) by spinning disk live microscopy at 34°C (a compromise temperature for human HeLa cells engrafts and zebrafish embryo cells) showing that, once entrapped, PC3 CD63-pHluorin+ sEVs became so strictly associated to HeLa’s membrane that eventually followed the fate and movements of the EV-TRAPping cell (data not shown). Ultimately, we deduced that the EV-TRAPping cells act as living platforms of ectopic retention, or in other words as “living local filters” for tumor EVs. In addition to this, we compared the Pearson’s correlation coefficients of interaction between circulating PC3 CD63-pHluorin+ sEVs and ALFA-TRAP+ or GFP-TRAP+ HeLa i.v. engrafts in the vasculature by live imaging, obtaining that the interaction at the PM happens specifically with GFP-TRAP+ cells (r >0.5) but not with ALFA-TRAP+ cells (r — 0), as shown (data not shown). Preliminary data on PFA-fixed zebrafish embryos at Ih post PC3 CD63-pHluorin+ sEVs injection showed that the main sites of accumulation of PC3 CD63-pHluorin+ sEVs correspond to the sites where HeLa GFP-TRAP+ cells migrate within the vasculature (Figure 4B). In fact, comparing the whole-embryo 3D-surface plots of PC3 CD63-pHluorin+ sEVs biodistribution we detected significant changes between the GFP-TRAP+ engrafted embryos, where PC3 sEVs predominantly amassed within ectopic hotspots e.g. in the posterior caudal vein (PCV) of the trunk, or in the heart (H) of the embryo (data not shown), and the control zebrafish embryos, where instead PC3 EVs naturally tended to amass in the tip of the tail and were caught up by caudal patrolling M s, (data not shown). Similarly, preliminary data on PSMA-TRAP+ HeLa i.v. engrafts at 2 dpf show that these cells also act as local ectopic collectors of LNCaP CD9- pHluorin+ sEVs, and - again -mainly in the trunk PCV and in the heart at 1 hpi (data not shown). Importantly, in none of the EV-TRAP+ HeLa i.v. engrafts conditions we detected residual signals in the caudal DLAV, CA and CVP, denoting that this strategy effectively reduced PCa accumulation in the natural anatomical target region, as observable by comparing of the embryos maps of PCa sEVs biodistribution and their respective 3D-surface plots. This suggests that, beyond the in vitro data from EV-competition assays showing a slight (non-significant) trend of differential spatial accumulation in favour of PSMA-TRAP+ surfaces (data not shown), in the zebrafish embryo set-up the dynamics of interaction between LNCaP EVs and PSMA- TRAP+ cells might be favoured by the action of the blood pressure, so that eventually even just by detecting CD9-pHluorin we already find significant local accumulation of LNCaP sEVs in ectopic sites at 1 hpi. These observations reinforce for the entire EV community the need to implement in vitro assays with in vivo dynamical studies, as often the interaction between EVs and cells can be much more complex in vivo than in vitro and characterized by various chemi cal -physical factors such as the presence of proteins in the blood circulation, the blood pressure, the type of blood flow if laminar or turbulent under shear stress, the presence of sugars in the bloodstream, the scavenging activity of macrophages, the architecture of the vessels and much more. These data were already very encouraging to us, as they demonstrated for the first time that the presence of a biological surfaces trapping both artificial tags and natural tags of PCa EVs inside the blood vessels can act as a real filter for tumor EVs, reducing their spreading in storage target sites, and potentially impairing or diverting the formation of subsequent pre- metastatic niches.

[0268] Example 2:

[0269] Material and Methods

[0270] Generation of PCa stable cell lines expressing CD63-, CD81- and CD9-pHluorin,

[0271] Metastatic prostate cancer PC3, DU145 and LNCaP cells were plated in 12-well plates and were infected for 24 h at MOI=5 with lentiviral particles carrying CD63-, CD81- or CD9- pHluorin sequences, a kind gift from Dr. Maarten P. Bebelman (Vrije Universiteit, Amsterdam). When possible, the transduction was performed in presence of Polybrene® (hexadimethrine bromide, Millipore Cat.#TR-1003-G, 10 mg / mL stock) diluted at 8 pg / mL. Lentiviral particles were then washed away, and infected cells were allowed to recover for at least 24h before being further used.

[0272] For the sorting, cells were detached with trypsin, centrifuged and resuspended with 10% FBS in IX PBS. They were then passed through a 70 pm cell strainer to remove aggregates. Cells were then sorted for the GFP signal using BD FACSAria™ III Cell Sorter (BD Biosciences) adapted with a 100 pm nozzle. Stably expressing cells were then washed and seeded back in either 12-well culture plates or T-25 tissue culture flasks, depending on the number of cells collected. No further monoclonal selection was performed on these cells.

[0273] Generation of EV-TRAPs+ stable cell lines.

[0274] HeLa and RAW 264.7 cells were plated in 12-well plates. When reached approximately 80% confluence, cells were infected for 24 h at MOI=5 with lentiviral particles bearing GFP- TRAP mCherry HA or PSMA-TRAP_mCherry_HA or ALFA-TRAP_mCherry_HA. When possible, the transduction was performed in presence of Polybrene® (hexadimethrine bromide, Millipore Cat.#TR-1003-G, 10 mg / mL stock) diluted at 8 pg / mL, to increase the infection rate. 24 hours post-infection, we refreshed with complete medium. Antibiotic selection was started at 48h post-infection with complete DMEM medium supplemented with 4 pg / mL puromycin. After three passages, EV-TRAPs polyclonal stable cells were maintained in selection with complete DMEM supplemented with 1 pg / mL puromycin.

[0275] EVs isolation from cell cultures and characterization. We performed EVs isolation by incubating parental adherent cells in EV-depleted medium. To prepare EV-depleted medium, we supplemented 500 mL of DMEM or RPMI with 20% heat-inactivated FBS, dispensing it into 70 mL Beckman® Polycarbonate Bottle Assembly 38 x 102 mm - 6Pk tubes, loading the tubes into an ultracentrifuge rotor Type 45 Ti (part #339160), and performing ultracentrifugation overnight at 100,000 x g at 4°C into an Optima™ XPN-80 Ultracentrifuge (Beckman® Cat.#A95765), to remove FBS-associated nanoparticles (such as lipoproteins or bovine EVs). The subsequent morning, the supernatant was collected under a sterile environment, filtered through a 0.2 pm Steritop™ into new sterile bottles, and stored at 4°C. For the incubation experiments with EV-producing cells, the medium was brought to 10% FBS by adding fresh sterile medium. Antibiotics have also been added, such as 1% penicillin / streptomycin.

[0276] Parental adherent cells were seeded into large tissue culture flasks (T-150 or T-175) or Petri dishes (P-150 mm). When cells were at 80-90% confluence, their maintaining medium was removed and, after 2 washes in IX PBS, replaced with EV-depleted medium. Cells were maintained at 37°C, 5% CO2 for 48 or 72h, depending on the EVs enrichment we wanted to reach. For 1 T-150 flask, we used an incubation volume of 15 mL of EV-depleted medium. After 48 or 72h, we collected the conditioned medium from the T-150 flasks and performed differential serial centrifugations at 4°C, in the following order:

[0277] 1. 300 x g for 10 min, to remove dead cells; the supernatant was then collected into a clean sterile tube, and the pellet discarded;

[0278] 2. 2,000 x g for 20 min, to remove cell debris; the supernatant was then collected into a clean sterile tube, and the pellet discarded.

[0279] 3. Transfer the 2,000 x g-derived conditioned supernatant into 15 mL-ultrafiltration tubes (Millipore Amicon® Ultra- 15 mL centrifugal devices with Ultracel® 10,000 Dalton molecular weight cutoff filters) and centrifuged at+4°C, 4,500 x g until reaching a final volume of 500 pL. The 500 pL-ultrafiltered conditioned medium was then loaded into sepharose beads- based SEC columns (IZON® qEVoriginal / 70 nm Gen 2 Column- optimal recovery range of 70 nm to 1000 nm diameter particles, Cat.#ICO-70) for manual or automatic fractions collection. All the SEC fractions were collected into 1.5 mL or 2.0 mL low-binding polymer tubes (Sorenson BioScience™, Cat.#27210), using filtered sterile IX PBS as buffer reservoir. After fractions isolation, SEC columns were washed with 0.5 MNaOH and subsequently with filtered IX PBS, and then stored at 4°C. Each sepharose column has been used up to 5 times for the isolation of EVs from the same parental cell line. We choose to separate 13 fractions in total, of 500 pL volume each. Among the resulting 13 SEC fractions, the sEVs-enriched ones were generally those going from 1 to 4, counting from the first fraction dropping out from the column. The segregation of EV enrichment / fraction was then checked by western-blotting for classical EVs markers (such as CD63, CD81, CD9, Syntenin) or EV genetic tags (like pHluorin, HA- tag, ALFA-tag)

[0280] For in cellulo and in vivo experiments, sEVs-enriched SEC fractions (1 to 4) derived from 3 T-150 tissue culture flasks (48 h incubation) were pulled together by further ultrafiltration into Millipore Amicon® Ultra-4 mL centrifugal devices with Ultracel® 10,000 Dalton molecular weight cutoff filters, centrifuged at 4,500 x g until reaching a final volume of -200 pL.

[0281] In vivo imaging.

[0282] For in vivo imaging we set up a protocol previewing the engraftment of -500-800 GFP- TRAP+ HeLa in the perivitelline space at 2 dpf, followed at 3dpf by the injection of -100,000- 200,000 PC3 CD63-pHluorin+ sEVs into the Duct of Cuvier at one-hour post EV-TRAP+ cells engraftment. At 4dpf, we captured movies in real-time of GFP-TRAP+ HeLa cells derived EV interacting with PC3 CD63-pHluorin+ sEVs in the CA-CV curve by spinning disk live microscopy at 34°C

[0283] Results

[0284] We observe that all the three EV-TRAPs HeLa stable cell lines released sEVs containing respectively the GFP-TRAP, the PSMA-TRAP or the ALFA-TRAP, as observed by westernblot detection for HA-tag in SEC 1-4 fractions from the three stable EV-TRAPs cells conditioned media separately (Fig. 5A). We postulated that we could use them for future research as they would favour the neutralization and clearance of PCa EVs. We injected PC3 derived CD63-pHluorin EVs in the blood circulation of zebrafish embryos xenografted with HeLa Cells expressing GFP-Trap construct. Live imaging revealed the release of EV from GFP- Trap expressing cells that harboured the EV-Trap chimera in the blood flow. EV harboring EV- Trap were observed interacting with CD63-pHluorin EVs supporting their use as clearance means (Fig. 5 B and C).

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Claims

CLAIMS:

1. A fusion protein comprising or consisting of an antigen binding compound, the mCD8a and optionally a domain which induce endocytosis (or internalisation).

2. A fusion protein according to the claim 1 wherein said fusion protein also comprises a reporter compound.

3. A fusion protein according to the claims 1 or 2 wherein said fusion protein also comprises a tag compound.

4. A fusion protein according to the claims 1 to 3 wherein said fusion protein also comprises a loading compound.

5. A nucleic acid molecule encoding the fusion protein according to claims 1 to 4.

6. A vector comprising a nucleic acid molecule according to the claim 5.

7. An immune cell which has been transfected, infected or transformed by a nucleic acid according to the claim 5 and / or a vector according to the claim 6 or which express the fusion protein according to the claims 1 to 4.

8. An immune cell which comprises a nucleic acid according to the claim 5 or a vector according to the claim 6 encoding the fusion protein according to the claims 1 to 4.

9. Extracellular vesicles expressing the fusion protein according to the claims 1 to 4.

10. An immune cell expressing the fusion protein according to the claim 1 to 4 or extracellular vesicles expressing the fusion protein according to the claims 1 to 4 for use in the treatment of a cancer in a subject in need thereof.

11. A therapeutic composition comprising an immune cell expressing the fusion protein according to the claims 1 to 4 or extracellular vesicles expressing the fusion protein according to the claims 1 to 4.

12. A therapeutic composition comprising an immune cell expressing the fusion protein according to the claims 1 to 4 or extracellular vesicles expressing the fusion proteinaccording to the claims 1 to 4 for use in the treatment of a cancer in a subject in need thereof.

13. A i) immune cell expressing the fusion protein according to the claim 1 to 4 or extracellular vesicles expressing the fusion protein according to the claims 1 to 4, and ii) at least one further therapeutic active agent according to the invention, as a combined preparation for simultaneous, separate or sequential use in the treatment of a cancer in a subject in need thereof.

Images