Short-CSF1-associated extracellular vesicles and uses thereof

Abstract

The invention relates to methods and pharmaceutical compositions for the treatment of cancer. The inventors investigate the role and specific contribution of extracellular vesicles (EVs) in cancer environment, particularly the role of Short-CSF1-associated extracellular vesicle (S-CSF1-associated EV) in immunotherapy using different models of cancer. The inventors demonstrate the proof of concept of anti-tumor effect of S-CSF1-associated EVs in vivo in syngeneic mice bearing a breast carcinoma or a lung carcinoma and that S-CSF1- associated EV induce strong reduction of tumor growth upon intratumoral injection. The inventors unravel an unexpected ability of S-CSF1-EVs to promote a tumor microenvironment associated with tumor shrinkage. The inventors have also shown in vitro that the S-CSF1-EVs induce differentiation of monocytes into macrophages which do not secrete some of the cytokines associated with pro-tumoral phenotype. Altogether, the present invention highlights the role of this specific S-CSF1-associated EV and provides in vitro and in vivo evidences towards its use in the treatment of cancer. Thus, the present invention relates to S-CSF1-associated EVs and their use in the treatment of cancer.

Description

[0001] SHORT-CSF1-ASSOCIATED EXTRACELLULAR VESICLES AND USES

[0002] THEREOF

[0003] FIELD OF THE INVENTION:

[0004] The invention relates to the field of immunotherapy. The invention relates to methods and pharmaceutical compositions for the treatment of cancer.

[0005] BACKGROUND OF THE INVENTION:

[0006] Different populations of immune cells infiltrate tumors. Tumor-associated macrophages (TAMs) favor tumor progression, promoting cancer cell invasion and metastasis in mouse models (Cassetta and Pollard, 2018). Recent single-cell analyses of human cancers revealed the heterogeneity of macrophage populations, thus challenging our understanding of TAM biology (Azizi et al., 2018; Chevrier et al., 2017; Lavin et al., 2017). TAMs derive from circulating monocytes that are recruited into the tumor via the CCL2-CCR2 chemokine signaling pathway (Franklin et al., 2014; Qian et al., 2011). The fate of monocytes is not predetermined and largely depend on the microenvironmental cues they encounter (Goudot et al., 2017). Specifically, the identity of tumor-derived factors that contribute to TAM heterogeneity and the mechanisms underlying intratumoral monocyte differentiation remain unclear.

[0007] Cytokines and chemokines are well-known factors among the signals that can influence TAM differentiation and activation (Cassetta et al., 2019). Extracellular vesicles (EVs), however, represent novel candidates. EVs are complex vehicles of intercellular communication and were suggested to have an impact on macrophage activation (Chow et al., 2014; Haderk et al., 2017; Wu etal., 2016; Ying etal., 2016). EVs, such as exosomes, ectosomes, microvesicles, oncosomes, are membrane-enclosed structures that contain proteins and nucleic acids and can be released into the extracellular environment by all cell types, including cancer cells (Tkach and Thery, 2016; Van Niel et al., 2018). Once released, EVs can interact with recipient cells and modulate their function (Cocozza et al., 2020). Particularly, EVs released by cancer cells play an important role in shaping the tumor immune microenvironment. EVs were shown to modulate lymphocytes and myeloid cell functions in cancer by triggering either pro-tumor or anti-tumor immune responses (Kugeratski and Kalluri, 2021; Robbins and Morelli, 2014), which may depend on numerous factors, such as the cancer type, stage, or EV subtype analyzed (Tkach et al., 2018).

[0008] The inventors have previously demonstrated that a macrophage-specific growth and survival factor, CSF1 (also known as M-CSF), when it is expressed by tumor cells, can associate to their EVs, and be exposed on tumor EV surface (Tkach et al., 2022). The tumor CSF1- associated EV drove differentiation of macrophages with features of inflammatory macrophages that could promote a tumor immune microenvironment associated with a favorable prognosis in Triple-Negative Breast Cancer (TNBC) patients.

[0009] In many cancers, CSF1 has been shown to enhance growth and aggressiveness of tumors, so it is often viewed as a pro-tumor cytokine (Tang et al., 1992; Kacinski et al., 1997; Lin et al., 2001; Lin et al., 2006; Tamimi et al., 2008). However, there are studies showing the antitumor properties of a membrane bound form of CSF1. In these studies, different tumor cells (human U251 glioma, rat T9 / 9L glioma, rat MADB106 breast cancer and hepatocellular carcinoma) that express mM-CSF have failed to form either subcutaneous or intracranial tumors in rodents. By contrast, tumor cells transduced with the secreted form of M-CSF were not killed by monocytes / macrophages in vitro and were capable of forming tumors in immunocompetent animals (Jadus et al., 1998; Graf et al., 1999; Dan et al., 2001; Williams et al., 2001; Jadus et al., 2003; Chen et al., 2002; Sanchez et al., 2002).

[0010] The inventors developed a non-tumor-derived EV-based CSF1 carrier, for use as injected therapeutic tool. Several forms of CSF1 generated by alternative splicing of the CSF1 RNA are described in the literature (Douglass et al., 2008). All these forms contain the extracellular N-terminal 223 amino acid CSF1 sequence, which binds to the CSFl-receptor to induce signaling, fused to a transmembrane and cytosolic domain. However, proteins encoded by the long and intermediate RNAs are quickly cleaved intracellularly by either ex- or P- convertases to release the soluble active CSF1, while the protein encoded by the short RNA lacks these two cleavage sequences and remains associated to the producing cell membrane for a longer period, although it might be eventually released after cleavage at a juxta-membrane site by other enzymes. In addition, the long RNA encodes for a protein with O-glycosylation sites and the juxta-membrane cleavage site, thus leading to a large highly glycosylated form of the protein.

[0011] The inventors demonstrated that the short (S)-CSFl is present on EVs and that S-CSF1- EVs induce surprisingly strong reduction of tumor growth upon intratumoral injection, and a different tumor microenvironment, with fewer macrophages and more NK cells. The inventors investigated the role of S-CSFl-EVs in immunotherapy using models of breast and lung cancer that are highly infiltrated with macrophages (Lin et al., 2001; Zhou et al., 2024). The inventors unravel an unexpected ability of S-CSFl-EVs to promote a tumor microenvironment associated with tumor shrinkage. This effect differs from induction of adaptive immune system activation against the tumor (Douglas et al., 2008). Since the tumor must expose the S-CSF1 on its surface for the anti-tumor activity to work, there is no obvious mechanistic reason for S-CSFl-EVs to play an anti-tumor role, since it is not specifically associated to the surface of tumor cells.

[0012] The inventors demonstrated the proof of concept of anti-tumor effect of Short-CSFl- associated extracellular vesicles (S-CSFl-associated EVs) in vivo in syngeneic mice bearing breast carcinoma or lung carcinoma. The inventors have shown in vitro that the S-CSFl-EVs induce differentiation of monocytes into macrophages which do not secrete at least one, in particular some, of the cytokines associated with pro-tumoral phenotype. There is no disclosure in the art of the anti-tumoral role of S-CSFl-associated EVs, and their use in the treatment of cancer.

[0013] SUMMARY OF THE INVENTION:

[0014] The invention relates to methods and pharmaceutical compositions for the treatment of cancer. In particular, the invention is defined by the claims.

[0015] DETAILED DESCRIPTION OF THE INVENTION:

[0016] The inventors investigate the role and specific contribution of extracellular vesicles (EVs) in cancer environment, particularly the role of Long-CSFl -associated extracellular vesicle (L-CSF1 -associated EV) and Short-CSFl -associated extracellular vesicle (S-CSFl- associated EV). The inventors demonstrated that long (L) highly glycosylated CSF1 efficiently binds to EVs. When comparing the modifications within tumoral environment treated by injecting either L-CSFl-EVs or S-CSFl-EVs, the inventors observed that L-CSFl-EVs did not induce the same anti-tumoral-microenvironment as the one provided when the S-CSF-l-EVs were administered. Surprisingly, while slightly less abundant on EVs than the long CSF1, S- CSFl-EVs induce strong reduction of tumor growth upon intratumoral injection, and a different tumor microenvironment, with fewer macrophages of a different phenotype, and more NK cells. The inventors investigated the role of S-CSFl-associated EVs in immunotherapy using models of cancer that are highly infiltrated with macrophages (Lin et al., 2001; Zhou et al., 2024) and demonstrated the proof of concept of anti-tumor effect of S-CSFl-associated EVs in vivo in syngeneic mice bearing a breast carcinoma or a lung carcinoma. The inventors unravel an unexpected ability of S-CSFl-EVs to promote a tumor microenvironment associated with tumor shrinkage. The inventors have also shown in vitro that the S-CSFl-EVs induce differentiation of monocytes into macrophages which do not secrete some of the cytokines associated with pro-tumoral phenotype. Altogether, the present invention highlights the role of this specific S-CSFl-associated EV, its use in the treatment of cancer, in particular in the treatment of lung cancer, more particularly lung carcinoma, and breast cancer, more particularly breast carcinoma.

[0017] Accordingly, the invention relates to S-CSFl-associated EV, and its use in the treatment of cancer.

[0018] S-CSFl-associated EV and S-CSFl-EV-induced macrophase

[0019] In a first aspect, the invention relates to a Short-CSFl -associated extracellular vesicle (S-CSFl-associated EV), which can be isolated and / or modified, or combined with another compound like but not limited to a therapeutic compound.

[0020] Thus, in some embodiments, the invention relates to an isolated or modified S-CSFl- associated EV.

[0021] In some embodiments, the invention relates to a composition, in particular a pharmaceutical composition, comprising the S-CSFl-associated EV (EV, which can be isolated and / or modified and / or combined).

[0022] In another aspect, the present invention relates to S-CSFl-associated EV comprising or expressing on its surface an antigen-recognizing receptor or an antigen-recognizing domain such as an antigen-recognizing receptor or an antigen-recognizing domain that specifically or preferentially binds to a tumor-associated antigen or a TAM- associated antigen.

[0023] In some embodiments, the invention relates to a S-CSFl-associated EV comprising an antigen-recognizing receptor.

[0024] In some embodiments, the S-CSFl-associated EV comprises an antigen-recognizing receptor or an antigen-recognizing domain that specifically or preferentially binds to a tumor- associated antigen.

[0025] In some embodiments, the S-CSFl-associated EV comprises an antigen-recognizing receptor or an antigen-recognizing domain that specifically or preferentially binds to a TAM- associated antigen.

[0026] In some embodiments, the invention relates to a composition comprising the S-CSFl- associated EV comprising on its surface an antigen-recognizing receptor.

[0027] In some embodiments, the invention relates to a composition comprising the S-CSFl- associated EV comprising on its surface an antigen-recognizing receptor or an antigenrecognizing domain that specifically or preferentially binds to a tumor- associated antigen or a T AM-associated antigen. The S-CSFl-associated EV and the composition of the present invention are each suitable in in vitro uses or ex vivo uses, for research and experimental applications, and in vivo uses, for therapeutic applications and in adoptive cell immunotherapy.

[0028] The term “CSF1” has its general meaning in the art and refers to a Colony Stimulating Factor 1, more particularly to human Colony Stimulating Factor 1, a cytokine that controls the production, differentiation, and function of macrophages. CSF1 may correspond to the protein referenced as the Uniprot reference No. P09603 and its isoforms (1 to 3). CSF1 is a cytokine that plays an essential role in the regulation of survival, proliferation and differentiation of hematopoietic precursor cells, especially mononuclear phagocytes, such as macrophages and monocytes. CSF1 promotes the release of proinflammatory chemokines.

[0029] The term “Short-CSFl” or “S-CSF1” has its general meaning in the art and refers to an isoform of Colony Stimulating Factor 1 that is shorter than the canonical isoform of CSF-1 that has 554 amino acid residues, in particular the CSF-1 corresponding to the Uniprot sequence referenced P09603.1. In an embodiment, a short-CSFl corresponds to a CSF-1 protein comprising the extracellular domain of a full-length CSF-1, or at least a portion of the extracellular domain of a full-length CSF-1, and further comprising the transmembrane domain of-a full-length CSF-1. In an embodiment, a short-CSFl corresponds a protein having the sequence of the extracellular domain of a Short-CSFl as defined herein, and comprising a transmembrane domain, that being either the transmembrane domain of a S-CSF1 as described herein, or a transmembrane domain issued from another protein. Full-length SCF1 may correspond to the protein having the sequence set forth in SEQ ID No. 4. S-CSF1 may correspond to a protein encoded by the nucleic acid SEQ ID NO. 1. It corresponds to the Uniprot reference No. P09603.3 encoding a S-CSF1, with the exception of a single substitution (D91Y), for example found in the rs 1064527 variant. The S-CSF-1 may correspond to a protein having the sequence set forth in SEQ ID No. 2 (which is derived from the S-CSF-1 protein referenced in Uniprot database under number P09603-3; it bears the substitution D91Y as compared to the P09603-3 protein). Alternatively, The S-CSF-1 may correspond to a protein having the sequence set forth in Seq ID No. 3 (which corresponds to the S-CSF-1 protein referenced in Uniprot database under number P09603-3).

[0030] >SEQ ID NO:1 ATGACCGCGCCGGGCGCCGCCGGGCGCTGCCCTCCCACGACATGGCTGGGCTCCCTGC TGTTGTTGGTCTGTCTCCTGGCGAGCAGGAGTATCACCGAGGAGGTGTCGGAGTACTGTAGC CACATGATTGGGAGTGGACACCTGCAGTCTCTGCAGCGGCTGATTGACAGTCAGATGGAGAC CTCGTGCCAAATTACATTTGAGTTTGTAGACCAGGAACAGTTGAAAGATCCAGTGTGCTACC TTAAGAAGGCATTTCTCCTGGTACAATACATAATGGAGGACACCATGCGCTTCAGAGATAAC ACCCCCAATGCCATCGCCATTGTGCAGCTGCAGGAACTCTCTTTGAGGCTGAAGAGCTGCTT CACCAAGGATTATGAAGAGCATGACAAGGCCTGCGTCCGAACTTTCTATGAGACACCTCTCC AGTTGCTGGAGAAGGTCAAGAATGTCTTTAATGAAACAAAGAATCTCCTTGACAAGGACTGG AATATTTTCAGCAAGAACTGCAACAACAGCTTTGCTGAATGCTCCAGCCAAGGCCATGAGAG GCAGTCCGAGGGATCCTCCAGCCCGCAGCTCCAGGAGTCTGTCTTCCACCTGCTGGTGCCCA GTGTCATCCTGGTCTTGCTGGCCGTCGGAGGCCTCTTGTTCTACAGGTGGAGGCGGCGGAGC CATCAAGAGCCTCAGAGAGCGGATTCTCCCTTGGAGCAACCAGAGGGCAGCCCCCTGACTCA GGATGACAGACAGGTGGAACTGCCAGTGTAG

[0031] > SEQ ID N0:2 (variant of P09603-3 CSF1 isoform with D-to-Y substitution)

[0032] MTAPGAAGRCPPTTWLGSLLLLVCLLASRS ITEEVSEYCSHMIGSGHLQSLQRLIDSQ METSCQITFEFVDQEQLKDPVCYLKKAFLLVQYIMEDTMRFRDNTPNAIAIVQLQELSLRLK SCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQG HERQSEGSSSPQLQESVFHLLVPSVILVLLAVGGLLFYRWRRRSHQEPQRADSPLEQPEGSP LTQDDRQVELPV

[0033] > SEQ ID NO:3 (P09603-3 CSF1 isoform)

[0034] MTAPGAAGRCPPTTWLGSLLLLVCLLASRS ITEEVSEYCSHMIGSGHLQSLQRLIDSQ METSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLK SCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQG HERQSEGSSSPQLQESVFHLLVPSVILVLLAVGGLLFYRWRRRSHQEPQRADSPLEQPEGSP LTQDDRQVELPV

[0035] The term “Long-CSFl” or “L-CSF1” has its general meaning in the art and refers to a Long-Colony Stimulating Factor 1 variant. L-CSF1 may correspond to the protein referenced as the Uniprot reference No. P09603-1 CSF1 isoform XI, and conservative variants thereof. L- CSF1 can have the amino sequence set forth in SEQ ID No. 4. A “variant” may refer to a polypeptide that has an amino acid sequence that differs from one of the sequences selected from SEQ ID NO: 2 or SEQ ID No. 3 by less than 20, 10, 5, 4, 3, 2 or 1 substitutions, insertions or deletions. In another particular embodiment, a variant refers to a polypeptide that has an amino acid sequence that differs from one of the sequences selected from SEQ ID NO: 2 or SEQ ID No. 3 by less than 50, 40, 30, 20, 10, 5, 4, 3, 2 or 1 substitutions, the substitutions being conservative substitutions. The term "conservative substitution" as used herein refers to the replacement of one amino acid residue with another, without altering the conformation or enzymatic activity of the polypeptide so modified, including, but not limited to, the replacement of one amino acid with another having similar properties (such as, for example, polarity, hydrogen bonding potential, acidity, basicity, shape, hydrophobicity, aromaticity and the like). Examples of conservative substitutions are found in the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (methionine, leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and small amino acids (glycine, alanine, serine and threonine).

[0036] The term “Extracellular vesicle” or “EV” has its general meaning in the art and refers to complex vehicles of intercellular communication, such as but not limited to exosomes, ectosomes, microvesicles, oncosomes and membrane-enclosed structures or particles such as virus-like particles (VLP) (Chow et al., 2014; Haderk et al., 2017; Wu et al., 2016; Ying et al., 2016). The term “Extracellular vesicle” or “EV” also refers to membrane-enclosed structures that contain proteins and nucleic acids, and can be released into the extracellular environment by any cell type, including cancer cells (Tkach and Thery, 2016; van Niel et al., 2018). Once released, EVs can interact with recipient cells and modulate their function (Cocozza et al., 2020).

[0037] The term “CSFl-associated EV” refers to CSFl-bearing EVs. The term “CSF1- associated EV” refers to an EV characterized by the presence of CSF1 on its surface. The term “CSFl-associated EV” also refers to an EV comprising CSF1. The term “CSFl-associated EV” also refers to CSFl-associated EV isolated and characterized such as described in the examples.

[0038] The term “S-CSFl-associated EV” refers to S-CSFl-bearing EVs. The term “S-CSF1- associated EV” refers to an EV characterized by the presence of S-CSF1 on its surface. The term “S-CSFl-associated EV” also refers to an EV comprising S-CSF1. The term “S-CSF1- associated EV” also refers to S-CSFl-associated EV isolated and characterized such as described in the examples.

[0039] The term “L-CSF1 -associated EV” refers to L-CSF1 -bearing EVs. The term “L-CSF1- associated EV” refers to an EV characterized by the presence of L-CSF1 on its surface. The term “L-CSF1 -associated EV” also refers to an EV comprising L-CSF1. The term “L-CSF1- associated EV” also refers to L-CSFl-associated EV isolated and characterized such as described in the examples.

[0040] The term “macrophages” has its general meaning in the art and refers to a type of leukocyte of the immune system which are mononuclear phagocytes. Macrophages play a critical role in innate and adaptative immunity, as well as in tissue-homeostasis. Macrophages differentiate from embryonic precursors or from circulating monocytes and remain in different tissues including tumors. Macrophages residing in healthy tissues are named Tissue-resident macrophages (TRM). Macrophages infiltrating tumors are named Tumor-associated macrophages or TAM. Macrophages may be defined by various combination of markers as disclosed in the present invention. The term “macrophage” also relates to a monocyte-derived macrophage (MDM). Monocyte-derived macrophages (MDMs), can be generated for example upon CSF1 (M-CSF) or GM-CSF treatment of monocytes.

[0041] In another aspect, the present invention relates to the macrophages which are activated by the S-CSFl-associated EV. Said macrophages are also named in the present invention S- CSFl-EV-induced macrophages.

[0042] Accordingly, the invention also relates to an isolated or modified macrophage, tumor- associated macrophages (TAM) or a progenitor thereof, wherein said macrophage or progenitor thereof has been co-cultured in vitro with S-CSFl-associated EV to generate S-CSF1-EV- induced macrophages.

[0043] Said macrophages may particularly comprise S-CSFl-associated EVs.

[0044] So, the invention also relates to an isolated or modified macrophage, tumor-associated macrophages (TAM) or a progenitor thereof, comprising S-CSFl-associated EV.

[0045] In one embodiment, the invention relates to an isolated or modified activated macrophage comprising S-CSFl-associated EV. In another embodiment, the invention further relates to an isolated or modified immunoresponsive macrophage, tumor-associated macrophages (TAM) or a progenitor thereof, activated by S-CSFl-associated EV.

[0046] In a further embodiment, the invention relates to an isolated or modified immunoresponsive macrophage, tumor-associated macrophages (TAM) or a progenitor thereof, comprising S-CSFl-associated EV.

[0047] In another aspect, the invention relates to an isolated or modified macrophage, tumor- associated macrophages (TAM), or a progenitor thereof, encoding an antigen-recognizing receptor, wherein said macrophage, tumor-associated macrophages (TAM) or progenitor thereof has been further co-cultured in vitro with S-CSFl-associated EV to generate S-CSF1- EV-induced macrophages encoding an antigen-recognizing receptor.

[0048] In one embodiment, the invention relates to an isolated or modified macrophage, tumor- associated macrophages (TAM), or a progenitor thereof, encoding an antigen-recognizing receptor, wherein said macrophage, tumor-associated macrophages (TAM) or progenitor thereof comprises S-CSFl-associated EV.

[0049] Said isolated or modified macrophage, tumor-associated macrophages (TAM), or a progenitor thereof, encoding an antigen-recognizing receptor and comprising S-CSFl- associated EV is an S-CSFl-EV-induced macrophage.

[0050] In one embodiment, the invention relates to an isolated or modified macrophage, tumor- associated macrophages (TAM), or a progenitor thereof, encoding a chimeric antigen receptor (CAR), wherein said macrophage, tumor-associated macrophages (TAM) or progenitor thereof has been further co-cultured in vitro with S-CSFl-associated EV to generate S-CSF1-EV- induced CAR-macrophages.

[0051] In one embodiment, the invention relates to an isolated or modified macrophage, tumor- associated macrophages (TAM), or a progenitor thereof, encoding a chimeric antigen receptor (CAR), wherein said macrophage, tumor-associated macrophages (TAM) or progenitor thereof comprises S-CSFl-associated EV.

[0052] Said isolated or modified macrophage, tumor-associated macrophages (TAM), or a progenitor thereof, encoding a chimeric antigen receptor (CAR), and comprising S-CSFl- associated EV is an S-CSFl-EV-induced CAR-macrophage. In some embodiments, the invention relates to a composition comprising the isolated or modified macrophage, tumor-associated macrophages (TAM) or progenitor thereof of the invention.

[0053] In some embodiments, the invention relates to a composition comprising the S-CSF1- EV-induced macrophages or the S-CSFl-EV-induced CAR-macrophages of the invention.

[0054] The isolated or modified macrophage, tumor- associated macrophage (TAM) or a progenitor thereof co-cultured in vitro with S-CSFl-associated EV are also named S-CSF1- EV-induced macrophage. Particularly, said isolated or modified macrophage, tumor-associated macrophage (TAM) or a progenitor thereof may comprise S-CSFl-associated EV.

[0055] The term “S-CSFl-EV-induced macrophage” refers to activated and immunoresponsive macrophages activated by S-CSFl-associated EV and associated with NK cell infiltration, or T cell infiltration or NK cell and T cell infiltration. The term “S-CSFl-EV-induced macrophage” also refers to macrophages activated by S-CSFl-associated EV. The activation of macrophages refers to an induction of a signal transduction or changes in gene expression in the cell resulting in initiation of an immune response. For example, activation of a macrophage may involve activation of an intracellular cascade inducing detectable cell proliferation and / or leading to the initiation of effector functions. S-CSFl-EV-induced macrophages can thus be associated with T cell infiltration, induced cytokine production, phagocytosis, cell signalling, target cell killing, or antigen processing and presentation. Typically, in response to ligand biding to an antigenrecognizing receptor, a signal transduction cascade is produced. In certain embodiments, when a recombinantly expressed CAR binds to an antigen, a transduction cascade is activated such that an immune response is initiated. The term “S-CSFl-EV-induced macrophage” also refers to S-CSFl-EV-induced macrophage isolated, cultured and characterized such as described in the examples.

[0056] The term “antigen-recognizing receptor” as used herein refers to a receptor that is capable of activating a macrophage immunoresponsive cell in response to its binding to an antigen such as tumor- associated antigen or TAM-associated antigen. Non-limiting examples of antigen-recognizing receptors include chimeric antigen receptors (“CARs”), and antigenrecognizing receptors that specifically or preferentially bind to a tumor-associated antigen or a TAM-associated antigen (tumor antigen-recognizing receptors or TAM antigen-recognizing receptors). The terms "tumor-associated antigen” or “TAA” refer to tumor antigen or cancer cell antigen. The term "tumor-associated antigen” refers to peptides, proteins, glycoproteins or carbohydrates that are specifically or preferentially expressed by cancer cells.

[0057] The term “chimeric antigen receptor” or “CAR” as used herein refers to a molecule comprising an extracellular antigen-binding domain that is fused to an intracellular signalling domain that is capable of activating or stimulating a macrophage as herein defined, and a transmembrane domain. In certain embodiments, the extracellular antigen-binding domain of a CAR comprises a scFv. The scFv can be derived from fusing the variable heavy and light regions of an antibody. Alternatively or additionally, the scFv may be derived from Fab’s (instead of from an antibody, e.g., obtained from Fab libraries). In certain embodiments, the scFv is fused to the transmembrane domain and then to the intracellular signalling domain. In certain embodiments, the CAR is selected to have high binding affinity or avidity for the antigen.

[0058] The term "antibody" used herein should be intended in the broadest sense and includes polyclonal and monoclonal antibodies, including full antibodies and functional (antigenbinding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, variable heavy chain (VH) regions capable of specifically binding the antigen, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. Unless otherwise stated, the term "antibody" should thus be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgGl, IgG2, IgG3, IgG4, IgM, IgE, IgA, and IgD and any origin (such as human camelid or other). In some embodiments the antibody comprises a heavy chain variable region and a light chain variable region. The term “antibody” encompasses whole native antibodies but also recombinant and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. In certain embodiments, an antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant (CH) region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant CL region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further sub-divided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL IS composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g ., effector cells) and the first component (Cl q) of the classical complement system.

[0059] As used herein, “CDRs” are defined as the complementarity determining region amino acid sequences of an antibody which are the hypervariable regions of immunoglobulin heavy and light chains. See, e.g., Rabat et ak, Sequences of Proteins of Immunological Interest, 4th U. S. Department of Health and Human Services, National Institutes of Health (1987). Generally, antibodies comprise three heavy chain and three light chain CDRs or CDR regions in the variable region. CDRs provide the majority of contact residues for the binding of the antibody to the antigen or epitope. In certain embodiments, the CDRs regions are delineated using the Rabat system (Rabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, ET.S. Department of Health and Human Services, NIH Publication No. 91-3242).

[0060] An "antibody fragment" refers herein to a molecule other than a full antibody that comprises a portion of a full antibody that binds the antigen to which the full antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; variable heavy chain (VH) regions, VHH antibodies, single-chain antibody molecules such as scFvs and single-domain VH single antibodies; and multispecific antibodies formed from antibody fragments. In particular embodiments, the antibodies are single-chain antibody fragments comprising a variable heavy chain region and / or a variable light chain region, such as scFvs.

[0061] As used herein, the term “single-chain variable fragment” or “scFv” is a fusion protein of the variable regions of the heavy (VH) and light chains (VE) of an immunoglobulin covalently linked to form a VH: :VE heterodimer. The VH and VE are either joined directly or joined by a peptide-encoding linker (e.g., 10, 15, 20, 25 amino acids), which connects the N- terminus of the VH with the C-terminus of the VE, or the C-terminus of the VH with the N- terminus of the VE. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility. Despite removal of the constant regions and the introduction of a linker, scFv proteins retain the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies can be expressed from a nucleic acid including VH - and VL -encoding sequences as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See also U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs having inhibitory activity have been described (see, e.g., Zhao et al., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife eta., J Clin Invst 2006 116(8):2252- 61; Brocks et al., Immuno technology 1997 3(3): 173-84; Moosmayeret al., Ther Immunol 1995 2(10:31-40). Agonistic scFvs having stimulatory activity have been described (see, e.g., Peter et al., J Bioi Chem 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71 ; Ledbetter et al., Crit Rev Immunoll997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 2003 1638(3):257-66).

[0062] The term “Single-domain antibodies” as used herein are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human singledomain antibody.

[0063] The term "antigen" or "Ag" as used herein meant a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunoresponsive cells, or both. It must be understood that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Thus, any DNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid. The term "tumor antigen" as used herein refers to any polypeptide expressed by a tumor that is capable of inducing an immune response.

[0064] As used herein, the term “affinity” is meant a measure of binding strength. Affinity can depend on the closeness of stereochemical fit between antibody combining sites and antigen determinants, on the size of the area of contact between them, and / or on the distribution of charged and hydrophobic groups. As used herein, the term “affinity” also includes “avidity”, which refers to the strength of the antigen-antibody bond after formation of reversible complexes. Methods for calculating the affinity of an antibody for an antigen are known in the art, including, but not limited to, various antigen -binding experiments, e.g., functional assays (e.g., flow cytometry assay).

[0065] By "specifically binds" is meant a polypeptide or fragment thereof that recognizes and binds a polypeptide of interest, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.

[0066] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0067] In some embodiments, the cells of the invention express one or more antigenrecognizing receptors on the surface. The cells thus may comprise one or more nucleic acids that encode one or more antigen- specific receptors, optionally operably linked to a heterologous regulatory control sequence. Typically such antigen-specific receptors bind the target antigen with a Kd binding affinity of 10-6M or less, 10-7 M or less, 10-8 M or less, 10-9 M or less, 10- 10 M or less, or 10-11 M or less (lower numbers indicating greater binding affinity).

[0068] Typically, the nucleic acids are heterologous, (i.e., for example which are not ordinarily found in the cell being engineered and / or in the organism from which such cell is derived). In some embodiments, the nucleic acids are not naturally occurring, including chimeric combinations of nucleic acids encoding various domains from multiple different cell types. The nucleic acids and their regulatory control sequences are typically heterologous. For example, the nucleic acid encoding the antigen- specific receptor may be heterologous to the immune cell and operatively linked to an endogenous promoter of the T-cell receptor such that its expression is under control of the endogenous promoter.

[0069] Among the antigen- specific receptors as per the invention are chimeric antigen receptors (CAR).

[0070] In some embodiments, the engineered antigen-specific receptors comprise chimeric antigen receptors (CARs), including activating or stimulatory CARs, costimulatory CARs (see WO2014 / 055668), and / or inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013)).

[0071] Chimeric antigen receptors (CARs), (also known as Chimeric immunoreceptors, Chimeric T cell receptors, Artificial T cell receptors) are engineered antigen- specific receptors, which graft an arbitrary specificity onto an immune effector cell. Typically, these receptors are used to graft the specificity of a monoclonal antibody onto an immune cell (e.g. an immunoresponsive cell as defined herein), with transfer of their coding sequence facilitated by viral vectors (typically retroviral vector).

[0072] CARs generally include an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components, in some aspects via linkers and / or transmembrane domain(s). Such molecules typically mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and / or a signal through a costimulatory receptor alone.

[0073] The CAR may include:

[0074] (a) an extracellular antigen-binding domain,

[0075] (b) a transmembrane domain,

[0076] (c) optionally a co-stimulatory domain, and

[0077] (d) an intracellular signaling domain.

[0078] In some embodiments, the CAR is constructed with a specificity for a particular antigen (or marker or ligand), such as an antigen expressed in a particular cell type to be targeted by adoptive cell therapy, such as a cancer marker. The CAR typically includes in its extracellular portion one or more antigen binding molecules, such as one or more antigen-binding fragment, domain, or portion of an antibody, typically one or more antibody variable domains. For example, the extracellular antigen-binding domain may comprise a light chain variable domain and a heavy chain variable domain, typically as an scFv.

[0079] The moieties used to bind to antigen include three general categories, either single-chain antibody fragments (scFvs) derived from antibodies, Fab’s selected from libraries, or natural ligands that engage their cognate receptor (for the first- generation CARs). Successful examples in each of these categories are notably reported in Sadelain M, Brentjens R, Riviere I. The basic principles of chimeric antigen receptor (CAR) design. Cancer discovery. 2013; 3(4):388-398 (see notably table 1) and are included in the present application.

[0080] Antibodies include chimeric, humanized or human antibodies, and can be further affinity matured and selected as described above. Chimeric or humanized scFv’s derived from rodent immunoglobulins (e.g. mice, rat) are commonly used, as they are easily derived from well-characterized monoclonal antibodies. Humanized antibodies contain rodent-sequence derived CDR regions; typically the rodent CDRs are engrafted into a human framework, and some of the human framework residues may be back-mutated to the original rodent framework residue to preserve affinity, and / or one or a few of the CDR residues may be mutated to increase affinity. Fully human antibodies have no murine sequence, and are typically produced via phage display technologies of human antibody libraries, or immunization of transgenic mice whose native immunoglobin loci have been replaced with segments of human immunoglobulin loci. Variants of the antibodies can be produced that have one or more amino acid substitutions, insertions, or deletions in the native amino acid sequence, wherein the antibody retains or substantially retains its specific binding function. Conservative substitutions of amino acids are well known and described above. Further variants may also be produced that have improved affinity for the antigen.

[0081] Typically, the CAR includes an antigen-binding portion or portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb).

[0082] In some embodiments, the CAR comprises an antibody heavy chain variable domain that specifically binds the antigen, such as a cancer marker or cell surface antigen of a cell or disease to be targeted, such as a tumor cell or a cancer cell, such as any of the target antigens described herein or known in the art.

[0083] In some embodiments, the CAR contains an antibody or an antigen-binding fragment (e.g. scFv) that specifically recognizes an antigen, such as an intact antigen, expressed on the surface of a cell.

[0084] In some embodiments, the CAR contains a TCR-like antibody, such as an antibody or an antigen-binding fragment (e.g. scFv) that specifically recognizes an antigen, such as a tumor- associated antigen, presented on the cell surface as a MHC-peptide complex. In some embodiments, an antibody or antigen-binding portion thereof that recognizes an MHC-peptide complex can be expressed on cells as part of a recombinant receptor, such as an antigen- specific receptor. Among the antigen- specific receptors are functional non-TCR antigen- specific receptors, such as chimeric antigen receptors (CARs). Generally, a CAR containing an antibody or antigen-binding fragment that exhibits TCR-like specificity directed against peptide-MHC complexes also may be referred to as a TCR-like CAR.

[0085] In some aspects, the antigen- specific binding, or recognition component is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the CAR includes a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, the transmembrane domain that is naturally associated with one of the domains in the CAR is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0086] The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD154, ICOS or a GITR), the y subunit of Fc receptor. The transmembrane domain can also be synthetic. In some embodiments, the transmembrane domain is derived from CD28, CD8, CD3-zeta, or the y subunit of Fc receptor.

[0087] In some embodiments, a short oligo- or polypeptide linker, for example, a linker of between 2 and 10 amino acids in length, is present and forms a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.

[0088] The CAR generally includes at least one intracellular signaling component or components. First generation CARs typically had the intracellular domain from the CD3 chain, which is the primary transmitter of signals from endogenous TCRs. Second generation CARs typically further comprise intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB (CD28), ICOS) to the cytoplasmic tail of the CAR to provide additional signals to the T cell. Co-stimulatory domains include domains derived from human CD28, 4-1BB (CD137), ICOS-1, CD27, OX 40 (CD137), DAP10, and GITR (AITR). Combinations of two co- stimulatory domains are contemplated, e.g. CD28 and 4- IBB, or CD28 and 0X40. Third generation CARs combine multiple signaling domains, such as CD3z-CD28- 4- IBB or CD3z-CD28-OX40, to augment potency. The intracellular signaling domain can be from an intracellular component of the TCR complex, such as a TCR CD3+ chain that mediates T-cell activation and cytotoxicity, e.g., the CD3 zeta chain. Alternative well-suited intracellular signaling domains include the intracellular component of various proteins including but to limited to CD3, the y subunit of Fc receptor (such as of FcsRIy), CD64, CD32, CD32b, CD32c, CCD16, CD16a, CD16b, MEGF10, CD40, the Toll-like receptor / Interleukin (IL)-l receptor (TLR / IL-1R) superfamily, members of the BAI family of phosphatidylserine receptor, such as BAI1, or members from the TAM family of phosphatidylserine receptors, such as MerTK (Penberthy, Kristen K, and Kodi S Ravichandran. “Apoptotic cell recognition receptors and scavenger receptors.” Immunological reviews vol. 269,1 (2016): 44-59, but see also Morrissey MA, Williamson AP, Steinbach AM, Roberts EW, Kern N, Headley MB, Vale RD. Chimeric antigen receptors that trigger phagocytosis. Elife. 2018 Jun 4;7:e36688), and / or other CD transmembrane domains. The CAR can also further include a portion of one or more additional molecules such as Fc receptor y, CD8, CD4, CD25, CD16. Typically, TLR signaling domains include the Toll / interleukin receptor homology domain, TIR as well as any intracellular domain interacting with MyDDosome and / or TRIFosome clusters such as in particular with MyD88, TIRAP, TRIF and / or TRAM).

[0089] The intracellular signaling domain may also or alternatively comprise a modified CD3 zeta polypeptide lacking one or two of its three immunoreceptor tyrosine-based activation motifs (ITAMs), wherein the ITAMs are IT AMI, ITAM2 and ITAM3 (numbered from the N- terminus to the C-terminus). The intracellular signaling region of CD3-zeta is residues 22-164 of the protein. IT AMI is located around amino acid residues 61-89, ITAM2 around amino acid residues 100-128, and ITAM3 around residues 131-159. Thus, the modified CD3 zeta polypeptide may have any one of ITAM1, ITAM2, or ITAM3 inactivated. Alternatively, the modified CD3 zeta polypeptide may have any two ITAMs inactivated, e.g. ITAM2 and ITAM3, or IT AMI and ITAM2. Preferably, ITAM3 is inactivated, e.g. deleted. More preferably, ITAM2 and ITAM3 are inactivated, e.g. deleted, leaving ITAM1. For example, one modified CD3 zeta polypeptide retains only IT AMI and the remaining CD3(^ domain is deleted (residues 90-164). As another example, IT AMI is substituted with the amino acid sequence of ITAM3, and the remaining CD3 £ domain is deleted (residues 90-164). See, for example, Bridgeman et al., Clin. Exp. Immunol. 175(2): 258 - 67 (2014); Zhao et al., J. Immunol. 183(9): 5563 - 74 (2009); Maus et al., WO 2018 / 132506; Sadelain et al., WO / 2019 / 133969, Feucht et al., Nat Med. 25(l):82-88 (2019). Thus, in some aspects, the antigen binding molecule is linked to one or more cell signaling modules including but not limited to CD3 (in particular CD247, CD3z) and / or modified CD3 (notably modified CD247 or CD3z), the y subunit of Fc receptor (such as of FcsRIy), CD64, CD32, CD32b, CD32c, CD16, CD16a, CD16b, MEGF10, CD40, the Toll-like receptor / Interleukin (IL)-l receptor (TLR / IL-1R) superfamily, members of the BAI family of phosphatidylserine receptor, such as BAI1, or members from the TAM family of phosphatidylserine receptors, such as MerTK, and / or other CD transmembrane domains. The CAR can also further include a portion of one or more additional molecules such as Fc receptor y, CD8, CD4, CD25, CD16. Typically, TLR signaling domains include the Toll / interleukin receptor homology domain, TIR as well as any intracellular domain interacting with MyDDosome and / or TRIFosome clusters such as in particular with MyD88, TIRAP, TRIF and / or TRAM). These one or more signaling domains may be combined with one or more costimulatory domains include domains derived, for example, from human CD28, 4- IBB (CD137), ICOS-1, CD27, OX 40 (CD137), DAP10, GITR (AfTR), CD80, CD86, CD40, CD16, CD32 and CD64.

[0090] In some embodiments, upon ligation of the CAR, the cytoplasmic domain or intracellular signaling domain of the CAR activates at least one of the normal effector functions or responses of the corresponding non-engineered immune cell (typically a phagocytic cell such as a macrophage, a dendritic cell, a monocyte or a granulocyte). For example, the CAR can induce a function of a macrophage, a dendritic cell or a monocyte such as phagocytic activity, cytotoxic activity, or secretion of cytokines or other factors.

[0091] In some embodiments, the intracellular signaling domain(s) include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptor to initiate signal transduction following antigen- specific receptor engagement, and / or a variant of such molecules, and / or any synthetic sequence that has the same functional capability.

[0092] T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen- dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigenindependent manner to provide a secondary or co- stimulatory signal (secondary cytoplasmic signaling sequences). In some aspects, the CAR adapted for the cells according to the present invention can include one or both of such signaling components.

[0093] In some aspects, the CAR includes a primary cytoplasmic signaling sequence that regulates primary activation of the FcR or the Toll-like receptor or any one of CD40, CD64, CD32, CD32b, CD32c, CD 16a, CD16bn CD 16c, members of the BAI family of phosphatidylserine receptor, such as BAI1, or members from the TAM family of phosphatidylserine receptors, such as MerTK, either in a stimulatory way, or in an inhibitory way. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine -based activation motifs or IT AMs. Examples of IT AM containing primary cytoplasmic signaling sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, CD66d, and Jedi-1, and MegflO. In some embodiments, cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta, Jedi-1, or MegflO.

[0094] The CAR can also include a signaling domain and / or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, 0X40, DAP10, and ICOS / CD80, CD86, CD40, CD16, CD32 et and CD64. In some aspects, the same CAR includes both the activating and costimulatory components; alternatively, the activating domain is provided by one CAR whereas the costimulatory component is provided by another CAR recognizing another antigen.

[0095] The CAR or other antigen- specific receptor can also be an inhibitory CAR (e.g. iCAR) and includes intracellular components that dampen or suppress a response, such as an immune response. Examples of such intracellular signaling components are those found on immune checkpoint molecules, including PD-1, CTEA4, EAG3, BTEA, 0X2R, TIM-3, TIGIT, EAIR- 1, PGE2 receptors, EP2 / 4 Adenosine receptors including A2AR. In some aspects, the engineered cell includes an inhibitory CAR including a signaling domain of or derived from such an inhibitory molecule, such that it serves to dampen the response of the cell. Such CARs are used, for example, to reduce the likelihood of off-target effects when the antigen recognized by the activating receptor, e.g, CAR, is also expressed, or may also be expressed, on the surface of normal cells.

[0096] Among the antigens targeted by the antigen- specific receptors are those expressed in the context of a disease, condition, or cell type to be targeted via the adoptive cell therapy. Among the diseases and conditions are proliferative, neoplastic, and malignant diseases and disorders, more particularly cancers. Among the cancer that can be targeted, treatment of breast cancer, in particular breast carcinoma, or lung cancer, in particular lung carcinoma, can be envisioned.

[0097] In some embodiments, the antigen is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on cells of the disease or condition, e.g., the tumor or pathogenic cells, as compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or is expressed on the engineered cells. In some such embodiments, a multi-targeting and / or gene disruption approach as provided herein is used to improve specificity and / or efficacy.

[0098] In some embodiments, the antigen is a universal tumor antigen. The term "universal tumor antigen" refers to an immunogenic molecule, such as a protein, that is, generally, expressed at a higher level in tumor cells than in non-tumor cells and also is expressed in tumors of different origins. In some embodiments, the universal tumor antigen is expressed in more than 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or more of human cancers. In some embodiments, the universal tumor antigen is expressed in at least three, at least four, at least five, at least six, at least seven, at least eight or more different types of tumors. In some cases, the universal tumor antigen may be expressed in non-tumor cells, such as normal cells, but at lower levels than it is expressed in tumor cells. In some cases, the universal tumor antigen is not expressed at all in non-tumor cells, such as not expressed in normal cells. Exemplary universal tumor antigens include, for example, human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P450 1B1 (CYP1B), HER2 / neu, p95HER2, Wilms' tumor gene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, pro state- specific membrane antigen (PSMA), p53 or cyclin (DI). Peptide epitopes of tumor antigens, including universal tumor antigens, are known in the art and, in some aspects, can be used to generate MHC- restricted antigen- specific receptors, such as TCRs or TCR-like CARs (see e.g. published PCT application No. WO2011009173 or WO2012135854 and published U.S. application No. US20140065708).

[0099] In some aspects, the antigen is expressed on multiple myeloma, such as CD38, CD138, and / or CS-1. Other exemplary multiple myeloma antigens include CD56, TIM-3, CD33, CD 123, and / or CD44. Antibodies or antigen-binding fragments directed against such antigens are known and include, for example, those described in U.S. Patent No. 8,153,765; 8,603477, 8,008,450; U.S. published application No. US20120189622; and published international PCT application Nos. W02006099875, W02009080829 or WO2012092612. In some embodiments, such antibodies or antigen-binding fragments thereof (e.g. scFv) can be used to generate a CAR.

[0100] In some embodiments, the antigen may be one that is expressed or upregulated on cancer or tumor cells, but that also may be expressed in an immune cell, such as a resting or activated T cell. For example, in some cases, expression of hTERT, survivin and other universal tumor antigens are reported to be present in lymphocytes, including activated T lymphocytes (see e.g., Weng et al. (1996) J Exp. Med., 183:2471-2479; Hathcock et al. (1998) J Immunol, 160:5702- 5706; Liu et al. (1999) Proc. Natl Acad Sci., 96:5147-5152; Turksma et al. (2013) Journal of Translational Medicine, 11: 152).

[0101] In some embodiments, the cancer is, or is associated, with overexpression of HER2 or p95HER2. p95HER2 is a constitutively active C-terminal fragment of HER2 that is produced by an alternative initiation of translation at methionine 611 of the transcript encoding the full- length HER2 receptor. HER2 or p95HER2 has been reported to be overexpressed in breast cancer, as well as gastric (stomach) cancer, gastroesophageal cancer, esophageal cancer, ovarian cancer, uterine endometrial cancer, cervix cancer, colon cancer, bladder cancer, lung cancer, and head and neck cancers. Patients with cancers that express the p95HER2 fragment have a greater probability of developing metastasis and a worse prognosis than those patients who mainly express the complete form of HER2. Saez et al., Clinical Cancer Research, 12:424- 431 (2006).

[0102] In some embodiments as provided herein, an immune cell, such as a T cell, can be engineered to repress or disrupt the gene encoding the antigen in the immune cell so that the expressed antigen- specific receptor does not specifically bind the antigen in the context of its expression on the immune cell itself. Thus, in some aspects, this may avoid off-target effects, such as binding of the engineered immune cells to themselves, which may reduce the efficacy of the engineered in the immune cells, for example, in connection with adoptive cell therapy.

[0103] In some embodiments, such as in the case of an inhibitory CAR, the target is an off- target marker, such as an antigen not expressed on the diseased cell or cell to be targeted, but that is expressed on a normal or non-diseased cell which also expresses a disease- specific target being targeted by an activating or stimulatory receptor in the same engineered cell. Exemplary such antigens are MHC molecules, such as MHC class I molecules, for example, in connection with treating diseases or conditions in which such molecules become downregulated but remain expressed in non-targeted cells.

[0104] In some embodiments, the engineered immune cells can contain an antigen- specific receptor that targets one or more other antigens. In some embodiments, the one or more other antigens is a tumor antigen or cancer marker. Other antigen targeted by antigen- specific receptors on the provided immune cells can, in some embodiments, include orphan tyrosine kinase receptor ROR1, tEGFR, Her2, p95HER2, Ll-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, FBP, fetal acethycholine e receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, Ll-cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D Ligands, NY- ESO-1, MART-1, gplOO, oncofetal antigen, R0R1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2 / neu, p95HER2, estrogen receptor, progesterone receptor, ephrinB2, CD 123, CS-1, c-Met, GD-2, and MAGE A3, CE7, Wilms Tumor 1 (WT-1), a cyclin, such as cyclin Al (CCNA1), and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV or other pathogens, such as gpl20 (but see also Kuhlmann AS, Peterson CW, Kiem HP. Chimeric antigen receptor T-cell approaches to HIV cure. Curr Opin HIV AIDS. 2018 Sep;13(5):446-453).

[0105] In some embodiments, the CAR binds a pathogen- specific antigen. In some embodiments, the CAR is specific for viral antigens (such as HIV, HCV, HBV, etc.), bacterial antigens, and / or parasitic antigens.

[0106] In some embodiments, the cell of the invention is genetically engineered to express two or more antigen-specific receptors on the cell, each recognizing a different antigen and typically each including a different intracellular signaling component. Such multi-targeting strategies are described, for example, in International Patent Application, Publication No.: WO 2014055668 Al (describing combinations of activating and costimulatory CARs, e.g., targeting two different antigens present individually on off-target, e.g., normal cells, but present together only on cells of the disease or condition to be treated) and Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013) (describing cells expressing an activating and an inhibitory CAR, such as those in which the activating CAR binds to one antigen expressed on both normal or nondiseased cells and cells of the disease or condition to be treated, and the inhibitory CAR binds to another antigen expressed only on the normal cells or cells which it is not desired to treat).

[0107] Example antigen-binding receptors include bispecific antibodies that are macrophageactivating antibodies or T-cell activating antibodies which bind not only the desired antigen but also an activating T-cell antigen such as CD3 epsilon.

[0108] In some contexts, the engineered cells include gene segments that cause the cells to be susceptible to negative selection in vivo, such as upon administration in adoptive cell therapy. In some contexts, overexpression of a stimulatory factor (for example, a lymphokine or a cytokine) may be toxic to a subject. Thus, in some contexts, the engineered cells include gene segments that cause the cells to be susceptible to negative selection in vivo, such as upon administration in adoptive cell therapy. For example in some aspects, the cells are engineered so that they can be eliminated as a result of a change in the in vivo condition of the patient to which they are administered. The negative selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, for example, a compound. Negative selectable genes include the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al., Cell II :223, 1977) which confers ganciclovir sensitivity; the cellular hypoxanthine phosphribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase, (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

[0109] In other embodiments of the invention, the cells of the invention are not engineered to express recombinant antigen- specific receptors, but rather include naturally occurring antigenspecific receptors specific for desired antigens, such macrophages or their progenitors cultured in vitro or ex vivo, e.g., during the incubation step(s), to promote expansion of cells having particular antigen specificity.

[0110] In vitro or ex vivo method

[0111] In another aspect, the present invention also relates to an in vitro or ex vivo method of inducing macrophage or generating S-CSFl-EV-induced macrophage, comprising the step of co-culturing macrophage, tumor-associated macrophages (TAM), or a progenitor thereof with S-CSFl-associated EV.

[0112] In some embodiments, the present invention also relates to an in vitro method of inducing macrophage encoding an antigen-recognizing receptor or generating S-CSF1-EV- induced macrophage encoding an antigen-recognizing receptor, comprising the step of coculturing macrophage, tumor-associated macrophages (TAM), or a progenitor thereof encoding an antigen-recognizing receptor with S-CSFl-associated EV.

[0113] In some embodiments, the present invention also relates to an in vitro method of inducing macrophage encoding a chimeric antigen receptor (CAR) or generating S-CSF1-EV- induced CAR-macrophage comprising the step of co-culturing macrophage, tumor-associated macrophages (TAM), or a progenitor thereof encoding a CAR with S-CSFl-associated EV.

[0114] In some embodiments, the invention relates to an in vitro method of inducing macrophage or generating S-CSFl-EV-induced macrophage, comprising the steps of: i) Providing macrophage, tumor-associated macrophages (TAM), or a progenitor thereof, ii) Providing S-CSFl-associated EV, iii) Co-culturing the macrophage, tumor-associated macrophages (TAM), or a progenitor thereof with the S-CSFl-associated EV. In some embodiments, the invention relates to an in vitro method of generating S-CSF1- EV-induced macrophage encoding an antigen -recognizing receptor, comprising the steps of: iv) Providing macrophage, tumor-associated macrophages (TAM), or a progenitor thereof encoding the antigen-recognizing receptor, v) Providing S-CSFl-associated EV, vi) Co-culturing the macrophage, tumor-associated macrophages (TAM), or a progenitor thereof encoding the antigen-recognizing receptor with the S-CSF1- associated EV.

[0115] In some embodiments, the invention relates to an in vitro method of generating S-CSF1- EV-induced CAR-macrophage, comprising the steps of: vii) Providing macrophage, tumor-associated macrophages (TAM), or a progenitor thereof encoding a chimeric antigen receptors (CAR), viii) Providing S-CSFl-associated EV, ix) Co-culturing the macrophage, tumor-associated macrophages (TAM), or a progenitor thereof encoding the CAR with the S-CSFl-associated EV.

[0116] In a further aspect, the present invention also relates to the S-CSFl-EV-induced macrophage, the S-CSFl-EV-induced macrophage encoding an antigen-recognizing receptor or the S-CSFl-induced CAR-macrophage generated by the method of the invention.

[0117] In some embodiments, the S-CSFl-EV-induced macrophage, the S-CSFl-EV-induced macrophage encoding an antigen-recognizing receptor or the S-CSFl-induced CAR- macrophage generated by the method of the invention is suitable in both in vitro uses, for research and experimental applications, and in vivo uses, for therapeutic applications and in adoptive cell immunotherapy.

[0118] In another aspect, the present invention relates to genetically engineered cell comprising the S-CSFl-associated EV of the invention.

[0119] In a further aspect, the present invention relates to a method of producing S-CSFl- associated EVs of the invention, comprising the steps of: i. Genetically engineering cells to express S-CSF1, and ii. Isolating the S-CSF1 -associated EVs.

[0120] Therapeutic method

[0121] In a further aspect, the invention relates to S-CSFl-associated EV for use as medicament or for use in therapy.

[0122] In some embodiments, the invention relates to S-CSFl-associated EV for use in the treatment of cancer. In an embodiment, the invention relates to S-CSFl-associated EV for use in the treatment of breast cancer and lung cancer. In an embodiment, the invention relates to S- CSFl-associated EV for use in the treatment of Triple Negative Breast Cancer (TNBC).

[0123] In some embodiments, the invention relates to S-CSFl-associated EV comprising an antigen-recognizing receptor for use in the treatment of cancer, in particular breast cancer and lung cancer, more particularly TNBC.

[0124] In some embodiments, the invention relates to S-CSFl-associated EV comprising an antigen-recognizing receptor or an antigen-recognizing domain that specifically or preferentially binds to a tumor-associated antigen for use in the treatment of cancer, in particular breast cancer and lung cancer, more particularly TNBC.

[0125] In some embodiments, the invention relates to S-CSFl-associated EV comprising an antigen-recognizing receptor or an antigen-recognizing domain that specifically or preferentially binds to a TAM-associated antigen for use in the treatment of cancer, in particular breast cancer and lung cancer, more particularly TNBC.

[0126] In a further aspect, the invention relates to S-CSFl-EV-induced macrophage of the invention for use as medicament or for use in therapy.

[0127] In some embodiments, the invention relates to S-CSFl-EV-induced macrophage for use in the adoptive cell immunotherapy.

[0128] In some embodiments, the invention relates to S-CSFl-EV-induced macrophage for use in the treatment of cancer, in particular breast cancer and lung cancer, more particularly TNBC.

[0129] In a further aspect, the invention relates to S-CSFl-EV-induced macrophage encoding an antigen-recognizing receptor for use in the adoptive cell immunotherapy.

[0130] In some embodiments, the invention relates to S-CSFl-EV-induced macrophage encoding a chimeric antigen receptor (CAR), herein called S-CSFl-EV-induced CAR- macrophage, for use in the adoptive cell immunotherapy. In some embodiments, the invention relates to S-CSFl-EV-induced macrophage encoding a chimeric antigen receptor (CAR) for use in the treatment of cancer, in particular breast cancer and lung cancer, more particularly TNBC.

[0131] As used herein, the terms “subject”, “individual” or “patient” are interchangeable and refer to a mammal. Typically, a subject according to the invention refers to any subject, preferably human. In some embodiment, the subject is afflicted or at risk to be afflicted with disease associated with immune dysfunction or dysregulation. In some embodiments, the term “subject” refers to a subject afflicted or at risk to be afflicted with cancer. In a particular embodiment, the term “subject” refers to a subject afflicted or at risk to be afflicted with breast cancer, triple negative breast cancer (TNBC) or lung cancer.

[0132] As used herein, the term “cancer” refers to any cancer that may affect any one of the following tissues or organs: breast; liver; kidney; heart, mediastinum, pleura; floor of mouth; lip; salivary glands; tongue; gums; oral cavity; palate; tonsil; larynx; trachea; bronchus, lung; pharynx, hypopharynx, oropharynx, nasopharynx; esophagus; digestive organs such as stomach, intrahepatic bile ducts, biliary tract, pancreas, small intestine, colon; rectum; urinary organs such as bladder, gallbladder, ureter; rectosigmoid junction; anus, anal canal; skin; bone; joints, articular cartilage of limbs; eye and adnexa; brain; peripheral nerves, autonomic nervous system; spinal cord, cranial nerves, meninges; and various parts of the central nervous system; connective, subcutaneous and other soft tissues; retroperitoneum, peritoneum; adrenal gland; thyroid gland; endocrine glands and related structures; female genital organs such as ovary, uterus, cervix uteri; corpus uteri, vagina, vulva; male genital organs such as penis, testis and prostate gland; hematopoietic and reticuloendothelial systems; blood; lymph nodes; thymus.

[0133] The term “cancer” according to the invention comprises leukemias, seminomas, melanomas, teratomas, lymphomas, non-Hodgkin lymphoma, neuroblastomas, gliomas, adenoc aminoma, mesothelioma (including pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma and end stage mesothelioma), rectal cancer, endometrial cancer, thyroid cancer (including papillary thyroid carcinoma, follicular thyroid carcinoma, medullary thyroid carcinoma, undifferentiated thyroid cancer, multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type 2B, familial medullary thyroid cancer, pheochromocytoma and paraganglioma), skin cancer (including malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi’s sarcoma, keratoacanthoma, moles, dysplastic nevi, lipoma, angioma and dermatofibroma), nervous system cancer, brain cancer (including astrocytoma, medulloblastoma, glioma, lower grade glioma, ependymoma, germinoma (pinealoma), glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors, spinal cord neurofibroma, glioma or sarcoma), skull cancer (including osteoma, hemangioma, granuloma, xanthoma or osteitis deformans), meninges cancer (including meningioma, meningio sarcoma or gliomatosis), head and neck cancer (including head and neck squamous cell carcinoma and oral cancer (such as, e.g., buccal cavity cancer, lip cancer, tongue cancer, mouth cancer or pharynx cancer)), lymph node cancer, gastrointestinal cancer, liver cancer (including hepatoma, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma and hemangioma), colon cancer, stomach or gastric cancer, esophageal cancer (including squamous cell carcinoma, larynx, adenocarcinoma, leiomyosarcoma or lymphoma), colorectal cancer, intestinal cancer, small bowel or small intestines cancer (such as, e.g., adenocarcinoma lymphoma, carcinoid tumors, Karposi’s sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma or fibroma), large bowel or large intestines cancer (such as, e.g., adenocarcinoma, tubular adenoma, villous adenoma, hamartoma or leiomyoma), pancreatic cancer (including ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors or vipoma), ear, nose and throat (ENT) cancer, breast cancer (including HER2-enriched breast cancer, luminal A breast cancer, luminal B breast cancer and triple negative breast cancer), cancer of the uterus (including endometrial cancer such as endometrial carcinomas, endometrial stromal sarcomas and malignant mixed Mullerian tumors, uterine sarcomas, leiomyosarcomas and gestational trophoblastic disease), ovarian cancer (including dysgerminoma, granulosa-theca cell tumors and Sertoli-Leydig cell tumors), cervical cancer, vaginal cancer (including squamous-cell vaginal carcinoma, vaginal adenocarcinoma, clear cell vaginal adenocarcinoma, vaginal germ cell tumors, vaginal sarcoma botryoides and vaginal melanoma), vulvar cancer (including squamous cell vulvar carcinoma, verrucous vulvar carcinoma, vulvar melanoma, basal cell vulvar carcinoma, Bartholin gland carcinoma, vulvar adenocarcinoma and erythroplasia of Queyrat), genitourinary tract cancer, kidney cancer (including clear renal cell carcinoma, chromophobe renal cell carcinoma, papillary renal cell carcinoma, adenocarcinoma, Wilm’s tumor, nephroblastoma, lymphoma or leukemia), adrenal cancer, bladder cancer, urethra cancer (such as, e.g., squamous cell carcinoma, transitional cell carcinoma or adenocarcinoma), prostate cancer (such as, e.g., adenocarcinoma or sarcoma) and testis cancer (such as, e.g., seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors or lipoma), lung cancer (including small cell lung carcinoma (SCLC), non-small cell lung carcinoma (NSCLC) including squamous cell lung carcinoma, lung adenocarcinoma (LU AD), and large cell lung carcinoma, bronchogenic carcinoma, alveolar carcinoma, bronchiolar carcinoma, bronchial adenoma, lung sarcoma, chondromatous hamartoma and pleural mesothelioma), sarcomas (including Askin's tumor, sarcoma botryoides, chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma and soft tissue sarcomas), soft tissue sarcomas (including alveolar soft part sarcoma, angiosarcoma, cystosarcoma phyllodes, dermatofibrosarcoma protuberans, desmoid tumor, desmoplastic small round cell tumor, epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant peripheral nerve sheath tumor (MPNST), neurofibrosarcoma, plexiform fibrohistiocytic tumor, rhabdomyosarcoma, synovial sarcoma and undifferentiated pleomorphic sarcoma, cardiac cancer (including sarcoma such as, e.g., angiosarcoma, fibrosarcoma, rhabdomyosarcoma or liposarcoma, myxoma, rhabdomyoma, fibroma, lipoma and teratoma), bone cancer (including osteogenic sarcoma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing’s sarcoma, malignant lymphoma and reticulum cell sarcoma, multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma, osteocartilaginous exostoses, benign chondroma, chondroblastoma, chondromyxoid fibroma, osteoid osteoma and giant cell tumors), hematologic and lymphoid cancer, blood cancer (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma and myelodysplasia syndrome), Hodgkin’s disease, non-Hodgkin’s lymphoma and hairy cell and lymphoid disorders, and the metastases thereof.

[0134] In a particular embodiment of the invention, the disease treated or prevented by using a compound according to the invention (e.g. a S-CSFl-associated EV or S-CSFl-EV-induced macrophage) is a breast cancer or a lung cancer, more particularly a triple-negative breast cancer.

[0135] As used herein, the term "treatment" or "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.]).

[0136] In a further aspect, the invention relates to a method of treating cancer comprising the step of administering to the subject a therapeutically effective amount of S-CSFl-associated EV, S-CSFl-associated EV comprising an antigen-recognizing receptor, S-CSFl-EV-induced macrophage, S-CSFl-EV-induced macrophage encoding an antigen-recognizing receptor and / or S-CSFl-EV-induced CAR- macrophage of the invention.

[0137] In some embodiments, the S-CSFl-associated EV, S-CSFl-associated EV comprising an antigen -recognizing receptor, S-CSFl-EV-induced macrophage, S-CSFl-EV-induced macrophage encoding an antigen-recognizing receptor, S-CSFl-EV-induced CAR-macrophage and / or pharmaceutical composition according to the invention is administered in combination with cancer therapies. In particular, compound and / or pharmaceutical composition of the invention may be administered in combination with targeted therapy, immunotherapy such as immune checkpoint therapy and immune checkpoint inhibitor, co-stimulatory antibodies, chemotherapy and / or radiotherapy. As used herein, the term “immunotherapy” refers to a cancer therapeutic treatment using the immune system to reject cancer. The therapeutic treatment stimulates the patient's immune system to attack the malignant tumor cells.

[0138] Immune checkpoint therapy such as checkpoint inhibitors include, but are not limited to programmed death-1 (PD-1) inhibitors, programmed death ligand-1 (PD-L1) inhibitors, programmed death ligand-2 (PD-L2) inhibitors, lymphocyte-activation gene 3 (LAG3) inhibitors, T-cell immunoglobulin and mucin-domain containing protein 3 (TIM-3) inhibitors, T cell immunoreceptor with Ig and ITIM domains (TIGIT) inhibitors, B- and T-lymphocyte attenuator (BTLA) inhibitors, V-domain Ig suppressor of T-cell activation (VISTA) inhibitors, cytotoxic T-lymphocyte-associated protein 4 (CTLA4) inhibitors, Indoleamine 2,3- dioxygenase (IDO) inhibitors, killer immunoglobulin-like receptors (KIR) inhibitors, KIR2L3 inhibitors, KIR3DL2 inhibitors and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1) inhibitors. In particular, checkpoint inhibitors include antibodies anti-PDl, anti- PD-L1, anti-CTLA-4, anti-TIM-3, anti-LAG3. Immune checkpoint therapy also includes costimulatory antibodies delivering positive signals through immune-regulatory receptors including but not limited to ICOS, CD137, CD27, OX-40 and GITR.

[0139] Example of anti-PDl antibodies include, but are not limited to, nivolumab, cemiplimab (REGN2810 orREGN-2810), tislelizumab (BGB-A317), tislelizumab, spartalizumab (PDR001 or PDR-001), ABBV-181, JNJ-63723283, BI 754091, MAG012, TSR-042, AGEN2034, pidilizumab, nivolumab (ONO-4538, BMS-936558, MDX1106, GTPL7335 or Opdivo), pembrolizumab (MK-3475, MK03475, lambrolizumab, SCH-900475 or Keytruda) and antibodies described in International patent applications W02004004771, W02004056875, W02006121168, WO2008156712, W02009014708, W02009114335, WO2013043569 and W02014047350. Example of anti-PD-Ll antibodies include, but are not limited to, LY3300054, atezolizumab, durvalumab and avelumab. Example of anti-CTLA-4 antibodies include, but are not limited to, ipilimumab (see, e.g., US patents US6,984,720 and US8,017,114), tremelimumab (see, e.g., US patents US7, 109,003 and US8, 143,379), single chain anti-CTLA4 antibodies (see, e.g., International patent applications WO1997020574 and WO2007123737) and antibodies described in US patent US8,491,895. Example of anti- VISTA antibodies are described in US patent application US20130177557. Example of inhibitors of the LAG3 receptor are described in US patent US5,773,578. Example of KIR inhibitor is IPH4102 targeting KIR3DL2. In some embodiments, the compound and / or pharmaceutical composition of the invention may be used in combination with targeted therapy. As used herein, the term “targeted therapy” refers to targeted therapy agents, drugs designed to interfere with specific molecules necessary for tumor growth and progression. For example, targeted therapy agents such as therapeutic monoclonal antibodies target specific antigens found on the cell surface, such as transmembrane receptors or extracellular growth factors. Small molecules can penetrate the cell membrane to interact with targets inside a cell. Small molecules are usually designed to interfere with the enzymatic activity of the target protein such as for example proteasome inhibitor, tyrosine kinase or cyclin-dependent kinase inhibitor, histone deacetylase inhibitor. Targeted therapy may also use cytokines. Examples of such targeted therapy include with no limitations: Ado-trastuzumab emtansine (HER2), Afatinib (EGFR (HER1 / ERBB1), HER2), Aldesleukin (Proleukin), alectinib (ALK), Alemtuzumab (CD52), axitinib (kit, PDGFRbeta, VEGFR1 / 2 / 3), Belimumab (BAFF), Belinostat (HDAC), Bevacizumab (VEGF ligand), Blinatumomab (CD19 / CD3), bortezomib (proteasome), Brentuximab vedotin (CD30), bosutinib (ABL), brigatinib (ALK), cabozantinib (FLT3, KIT, MET, RET, VEGFR2), Canakinumab (IL-1 beta), carfilzomib (proteasome), ceritinib (ALK), Cetuximab (EGFR), cofimetinib (MEK), Crizotinib (ALK, MET, ROS1), Dabrafenib (BRAF), Daratumumab (CD38), Dasatinib (ABL), Denosumab (RANKL), Dinutuximab (B4GALNT1 (GD2)), Elotuzumab (SLAMF7), Enasidenib (IDH2), Erlotinib (EGFR), Everolimus (mTOR), Gefitinib (EGFR), Ibritumomab tiuxetan (CD20), Sonidegib (Smoothened), Sipuleucel-T, Siltuximab (IL-6), Sorafenib (VEGFR, PDGFR, KIT, RAF),(Tocilizumab (IL-6R), Temsirolimus (mTOR), Tofacitinib (JAK3), Trametinib (MEK), Tositumomab (CD20), Trastuzumab (HER2), Vandetanib (EGFR), Vemurafenib (BRAF), Venetoclax (BCL2), Vismodegib (PTCH, Smoothened), Vorinostat (HDAC), Ziv-aflibercept (PIGF, VEGFA / B), Olaparib (PARP inhibitor).

[0140] In some embodiments, the compound and / or pharmaceutical composition of the invention may be used in combination with chemotherapy. As used herein, the term “antitumor chemotherapy” or “chemotherapy” has its general meaning in the art and refers to a cancer therapeutic treatment using chemical or biochemical substances, in particular using one or several antineoplastic agents or chemotherapeutic agents. Chemotherapeutic agents include, but are not limited to alkylating agents such as thiotepa and cyclo sphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (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 CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g. , calicheamicin, especially calicheamicin gammall and calicheamicin omegall ; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, 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; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; methylhydrazine derivatives including N- methylhydrazine (MIH) and procarbazine; PSK polysaccharide complex); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and doxetaxel; gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-1 1); topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine; anthracyclines, nitrosoureas, antimetabolites, epipodophylotoxins, enzymes such as L- asparaginase; anthracenediones; hormones and antagonists including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone / equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; and non-steroidal antiandrogens such as flutamide; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0141] In some embodiments, the compound and / or pharmaceutical composition of the invention is administered to the patient in combination with radiotherapy. Suitable examples of radiation therapies include, but are not limited to external beam radiotherapy (such as superficial X-rays therapy, orthovoltage X-rays therapy, megavoltage X-rays therapy, radiosurgery, stereotactic radiation therapy, Fractionated stereotactic radiation therapy, cobalt therapy, electron therapy, fast neutron therapy, neutron-capture therapy, proton therapy, intensity modulated radiation therapy (IMRT), 3-dimensional conformal radiation therapy (3D- CRT) and the like); brachytherapy; unsealed source radiotherapy; tomotherapy; and the like. Gamma rays are another form of photons used in radiotherapy. Gamma rays are produced spontaneously as certain elements (such as radium, uranium, and cobalt 60) release radiation as they decompose, or decay. In some embodiments, radiotherapy may be proton radiotherapy or proton minibeam radiation therapy. Proton radiotherapy is an ultra-precise form of radiotherapy that uses proton beams (Prezado Y, Jouvion G, Guardiola C, Gonzalez W, Juchaux M, Bergs J, Nauraye C, Labiod D, De Marzi L, Pouzoulet F, Patriarca A, Dendale R. Tumor Control in RG2 Glioma-Bearing Rats: A Comparison Between Proton Minibeam Therapy and Standard Proton Therapy. Int J Radiat Oncol Biol Phys. 2019 Jun l;104(2):266-271. doi: 10.1016 / j.ijrobp.2019.01.080; Prezado Y, Jouvion G, Patriarca A, Nauraye C, Guardiola C, Juchaux M, Lamirault C, Labiod D, Jourdain L, Sebrie C, Dendale R, Gonzalez W, Pouzoulet F. Proton minibeam radiation therapy widens the therapeutic index for high-grade gliomas. Sei Rep. 2018 Nov 7;8(1): 16479. doi: 10.1038 / s41598-018-34796-8). Radiotherapy may also be FLASH radiotherapy (FLASH-RT) or FLASH proton irradiation. FLASH radiotherapy involves the ultra-fast delivery of radiation treatment at dose rates several orders of magnitude greater than those currently in routine clinical practice (ultra-high dose rate) (Favaudon V, Fouillade C, Vozenin MC. The radiotherapy FLASH to save healthy tissues. Med Sci (Paris) 2015; 31 : 121-123. DOI: 10.105 l / medsci / 20153102002); Patriarca A., Fouillade C. M., Martin F., Pouzoulet F., Nauraye C., et al. Experimental set-up for FLASH proton irradiation of small animals using a clinical system. Int J Radiat Oncol Biol Phys, 102 (2018), pp. 619-626. doi: 10.1016 / j.ijrobp.2018.06.403. Epub 2018 Jul 11).

[0142] Pharmaceutical composition

[0143] The present invention also relates to a pharmaceutical composition, for example a therapeutic, a vaccine or a veterinary composition, comprising the S-CSFl-associated EV, S- CSFl-associated EV comprising an antigen-recognizing receptor, S-CSFl-EV-induced macrophage, S-CSFl-EV-induced macrophage encoding an antigen-recognizing receptor and / or S-CSFl-EV-induced CAR-macrophage of the invention. The S-CSF1 -associated EV, S- CSFl-associated EV comprising an antigen-recognizing receptor, S-CSFl-EV-induced macrophage, S-CSFl-EV-induced macrophage encoding an antigen-recognizing receptor and / or S-CSFl-EV-induced CAR-macrophage or the compounds of the invention may be used or prepared in a pharmaceutical composition.

[0144] In one embodiment, the invention relates to a pharmaceutical composition comprising the S-CSFl-associated EV, S-CSFl-associated EV comprising an antigen-recognizing receptor, S-CSFl-EV-induced macrophage, S-CSFl-EV-induced macrophage encoding an antigenrecognizing receptor and / or S-CSFl-EV-induced CAR-macrophage of the invention and a pharmaceutical acceptable carrier for use in the treatment of cancer in a subject of need thereof.

[0145] Typically, said pharmaceutical compositions are formulations for administration, preferably sterile compositions and formulations, such as for adoptive cell therapy. The pharmaceutical composition of the invention generally comprises a sterile pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can further be incorporated into the compositions. In some aspects, the choice of carrier in the pharmaceutical composition is determined in part by the particular engineered CAR or TCR, vector, or cells expressing the CAR or TCR, as well as by the particular method used to administer the vector or host cells expressing the CAR. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001 to about 2% by weight of the total composition.

[0146] A pharmaceutical composition is formulated to be compatible with its intended route of administration. The pharmaceutical composition can be formulated for any conventional route of administration including a parenteral, intravenous, intramuscular, subcutaneous administration and the like.

[0147] Pharmaceutical compositions of the invention may include any further compound which is used in the treatment of cancer.

[0148] In one embodiment, said additional active compounds may be contained in the same composition or administrated separately.

[0149] In another embodiment, the pharmaceutical composition of the invention relates to combined preparation for simultaneous, separate or sequential use in the treatment of cancer in a subject in need thereof.

[0150] In some embodiments, the pharmaceutical composition of the invention relates to combined preparation for simultaneous, separate or sequential use in the treatment of cancer in a subject in need thereof.

[0151] The invention also provides kits comprising the compound of the invention. Kits containing the compound of the invention find use in therapeutic methods.

[0152] The invention will be further illustrated by the following examples. However, these examples should not be interpreted in any way as limiting the scope of the present invention. FIGURES:

[0153] Figure 1 - MDA-MB-231-EVs bearing CSF1 reduce E0771 tumor growth

[0154] A) Scheme of the experimental set up. Luciferase-expressing E0771 C57B16 tumor cells were injected at dO in the mammary fat pad of C57B16 female mice. Treatments (PBS or MDA-MB- 231 EVs) were injected every 3-4 days starting at d8. B) Tumor volume measured by caliper from d8 after tumor injection in the mammary fat pad, in individual mice. For control group, mice received intratumoral PBS injection. For experimental groups, mice received 1.5xl09CSFl-EVs from the MDA-MB-231 or MDA-MB-23-CSF1 KO human triple-negative breast cancer cells. 3 independent experiments were performed.

[0155] Figure 2. CSF1 is detected both associated to EVs and as soluble protein in the secretome of HEK293 cells expressing either Short (S) or Long (L)-CSFl.

[0156] A) The conditioned medium of HEK293FlpIn cells expressing either the Short (S-CSF1 or CSFl-Short) or the Long (L-CSF1 or CSFl-Long) form of CSF1 was separated by Size Exclusion Chromatography (SEC) in EV-containing (EVs fractions) versus soluble proteincontaining fractions (Soluble fractions), intermediate (Inter) fractions and the supernatant. CSF1 was quantified by LegendPlex in EVs fractions, soluble fractions, intermediate (Inter) fractions and supernatants coming from equal initial volumes of conditioned medium, and displayed as pg / 10e6 producing cells. B) CSF1 was quantified by LegendPlex in EVs fractions as in A), number of EVs was quantified by NTA, and amount of CSF1 was displayed as pg / ml in 2.5xl0e8 EVs.

[0157] Figure 3 - HEK293-EVs bearing the L and S forms of CSF1 induce different rates of survival and different differentiation of monocytes in vitro, as evidenced by different secretomes.

[0158] A) Scheme of the experimental set up - Equal amount of long and short form of CSF1 (85 pg / ml) on EVs or soluble fractions (SF) were incubated for 5 d with freshly isolated CD 14+ monocytes from healthy donors. As positive control, cells were incubated with 100 ng / mL of rCSF-1. (B-C) Flow cytometry analysis of cells after the 5 d culture. B) Examples of the gating strategy and of results. C) live cells count (left), and quantification of CD206+CD163+ live macrophages (right). (D and E) Analysis of secreted cytokines in the conditioned medium after the 5-day culture by Cytokine Array. Two independent experiments were performed. Green arrows point to differently expressed cytokines in monocytes that were differentiated through the administration of recombinant CSF1 (rCSF) or EVs bearing either S-CSF1 or L-CSF1 (ShortEV or LongEV in E).

[0159] Figure 4 - HEK293-EVs with short-form CSF1 reduce tumor growth and metastasis, while increasing NK and T cell infiltration and decreasing neutrophils and macrophages in tumors, compared to naive HEK293-EVs.

[0160] A) Scheme of the experimental set up (top) and size of tumors (bottom). Luciferase-expressing E0771 C57B16 tumor cells were injected at dO in the mammary fat pad of C57B16 female mice. Treatments (PBS or HEK293 EVs) were injected intra-tumor every 3-4 days starting at d8 or dlO. Tumor volume measured by caliper from d8 or dlO after tumor injection is represented (each line is a mouse). At each injection, mice received 1.5xl09CSFl-EVs from S-CSF1- expressing HEK-293 cells, from naive HEK-293, or an equal volume of PBS. B) Metastasis was quantified by the luminescence signal (IVIS Lumina III imaging system) in explanted lungs of mice at endpoint (d28, d29 or d30). upper panel: representative images for each group, bottom panel: quantification of luminescence signal in each mouse. C) Flow cytometry analysis of the immune cell infiltrate in tumors at endpoint. Dot plots showing the gating strategy to quantify T cells, neutrophils, NK, and macrophages. D) Frequency of T cells, neutrophils, NK, and macrophages in CD45+ cells in tumor cell suspensions. Results of individual mice from 3 independent experiments are shown. E and F) Analysis of the immune infiltrate in tumors at endpoint by immuno-histochemistry. (Left): representative images of staining for macrophages (F4 / 80), T lymphocytes (CD3) and neutrophils (Ly6G) in one mouse of each group. Right: quantification of the density of each cell type in individual tumors.

[0161] EXAMPLE:

[0162] Material & Methods

[0163] Cell culture and transfections

[0164] MDA-MB-231 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM- Glutamax, Gibco), with 10% fetal calf serum (FCS, Gibco), penicillin-Streptomycin (Gibco) = complete medium. E0771-Luc cells were cultured in complete medium under antibiotic selection (5 pg / ml Blasticidin). CRISPR / Cas9 modified MDA-MB-231 (CSF1-KO, described in Tkach et al, PNAS 2022) were cultured in complete medium with 2pg / ml puromycin (ThermoFischer Scientific). Flp-In™ T-REx™ 293 cells overexpressing short and long CSF-1 were kept in culture in complete medium under antibiotic selection (200 pg / ml hygromycin). Generation of Stable Cell lines

[0165] The coding sequences of each of the two mouse CSF1 isoforms were cloned into the pCDNA5 / TO vector (Invitrogen, #V103320) under the control of a doxycycline-inducible CMV promoter and stable cell lines were generated into HEK293 Flpln-Trex cell line (Invitrogen, #R78007) according to manufacturer instructions. Briefly, the system allows the Flpin recombinase-driven targeted insertion of the gene of interest at an invariant transcriptionally active site within the genome and its expression is induced by addition of doxycycline (1 pg / ml) in the culture medium. Doxycycline was added to EV depleted medium for 48 hours prior to EV isolation.

[0166] Human CSF1 Long isoform (SEQ ID No. 4)

[0167] MTAPGAAGRCPPTTWLGSLLLLVCLLASRSITEEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFV DQEQLKDPVCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTF YETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQDVVTKPDCNCLYPKAIPSSDPASVSP HQPLAPSMAPVAGLTWEDSEGTEGSSLLPGEQPLHTVDPGSAKQRPPRSTCQSFEPPETPVVKDSTIGGS PQPRPSVGAFNPGMEDILDSAMGTNWVPEEASGEASEIPVPQGTELSPSRPGGGSMQTEPARPSNFLSAS SPLPASAKGQQPADVTGTALP1VGPVRPTGQDWNHTPQKTDHPSALLRDPPEPGSPRISSLRPQGLSNPS TLSAQPQLSRSHSSGSVLPLGELEGRRSTRDRRSPAEPEGGPASEGAARPLPRFNSVPLTDTGHERQSEGS SSPQLQESVFHLLVPSVILVLLAVGGLLFYRWRRRSHQEPQRADSPLEQPEGSPLTQDDRQVELPV* Human CSF1 Short isoform (SEQ ID No. 2) MTAPGAAGRCPPTTWLGSLLLLVCLLASRSITEEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFV DQEQLKDPVCYLKKAFLLVQYIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTF YETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQGHERQSEGSSSPQLQESVFHLLVPSVI LVLLAVGGLLFYRWRRRSHQEPQRADSPLEQPEGSPLTQDDRQVELPV*

[0168] EV isolation

[0169] Cell lines were cultured for 48h with EV-depleted medium (= 2x complete medium precentrifuged overnight at 100,000g in 45Ti rotor) before EVs isolation. Then, concentrated conditioned medium (CCM) was harvested by pelleting cells at 400xg for 10 min at 4 °C. Supernatant was centrifuged at 2,000 x g for 20 min at 4 °C to discard 2K pellet and then concentrated on a sterilized Sartorius Centrifugal Filter (MWCO = 10 kDa; VS2061) or Centricon Plus-70 Centrifugal Filter (MWCO = 10 kDa; Millipore). Medium was concentrated to 500 pl and overlaid on 35 nm qEV size-exclusion columns (IZON) to separate 7 to 10 as EV fractions, 11 to 14 as Intermediate and 15 to 22 as soluble fractions. Pooled fractions were then concentrated using lOKDa cut-off filters (Amicon Ultra- 15, Millipore). EVs numbers and size distribution were measured by NTA using a ZetaView (Particle Metrix).

[0170] In vivo assay in Mice injected with E0771 cells

[0171] Animal Model and Tumor Inoculation: C57B16 syngeneic mice were injected with 500,000 E0771 triple-negative mammary carcinoma cells expressing luciferase into the left fourth mammary gland, diluted in 50 pL PBS (Day 0). Tumors were allowed to develop until palpable (Day 8-10).

[0172] EV Treatment and Tumor Measurement: Mice were administered intratumoral injections of 1.5 x 10e9 EV bearing CSF1 derived from MDA-MB-231 cells or MDA-MB-231 CSF1-KO as well as either short CSFl-expressing HEK-293 cells or naive HEK-293 cells, or an equal volume of PBS (control). Five to six injections were given at 3-4 day intervals. Tumor volume was measured twice weekly using caliper-based assessments [(width2x length) / 2].

[0173] Imaging and Bioluminescence Analysis: On Days 29-30, tumor size and viability were assessed non-invasively via bioluminescence imaging using the IVIS Lumina III system (PerkinElmer). Mice were injected intraperitoneally with D-luciferin (150 mg / kg), and luminescence was measured 15 minutes post-injection. Bioluminescence data were analyzed using Living Image software, with results expressed as radiance photons from the tumor area.

[0174] Preparation of mice tumors for Flow Cytometry, and IHC: On Days 29-30, mice were sacrificed, and tumors were harvested for analysis. To evaluate immune cell infiltration by flow cytometry (FACS), tumors were cut into small pieces and incubated for 30 min at 37 °C in a digestion mix (RPMI containing 0.4 mg / ml of DNAse I (Sigma-Aldrich) and 0.5 mg / ml of collagenase D (Roche)). Tumor suspensions were then incubated with RBC lysis buffer for 5 min and filtered using 40- pm cell strainers before staining for FACS analysis.

[0175] To further validate FACS findings by IHC, formalin-fixed paraffin-embedded tumor sections were prepared by the platform of Experimental pathology of Institut Curie. Samples were subjected to Multiplexed immunohistochemistry (IHC) staining using specific antibodies for key immune markers according to the protocol developed by (Remark R and al, 2016), with some adjustment. All stains of the same sample were deconvolved into a pseudo-fluorescence signal with HALO Indicalab Deconvolution v 1.1.8 module. They were then registered using HALO registration module and fused into a single overlay in .tif format. Finaly, the overlays were cropped and exported to .ome.tiff to be analyzed with Qupath software.

[0176] In vitro assay of human monocyte differentiation

[0177] Equal amount of CSF1 (85 pg / ml) from pooled EV fractions (EV), soluble fractions (SF), intermediate (Inter) fractions, or supernatants of conditioned medium from HEK293FlpIn cells expressing either S- or L-CSF1 was incubated for 5 d with freshly isolated CD 14+ monocytes from healthy donors in the absence of any other stimuli. As control, CD 14+ cells were also incubated with 100 ng / mL of rCSF-1. Cells were then harvested for analysis by flow cytometry, and 5d conditioned medium was used for analysis of secreted cytokines Antibody array.

[0178] Flow cytometry

[0179] Monocyte-Derived Macrophages were stained in PBS containing 0.5% BSA and 2 mM EDTA for 30-45 min on ice (protected from light) using the following primary antibodies: CD 163 PE (Clone GHI / 61, BioLegend), and CD206 Alexa Fluor 647 (Clone 15-2, BioLegend). Prior to antibody staining, cells were incubated with a human Fc Blocking reagent (Miltenyi) and the LIVE / DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific). Stained cells were analyzed using FACSVerse (BD Biosciences) flow cytometer, and data were processed with Flow Jo (Flow Jo LLC).

[0180] Mouse tumor cells were stained in PBS containing bovine serum albumin (BSA) 0.5% and 2 mM EDTA for 30-45 min on ice (protected from light) with the following antibodies: CD45- FITC (Clone 30-F11, BioLegend), Ly6 C-AF700 (Clone HK1.4, BioLegend), Ly6G BV650 (Clone 1A8, BD Biosciences), CDl lc-PerCP / Cyanine5.5 (Clone N418, BioLegend), CD64- PE / Cyanine7 (Clone X54-5 / 7.1, BioLegend), XCR1 -Brilliant Violet 510 ( Clone ZET, BioLegend), CDl lb-PE-CF594 (Clone MI / 70, BD Biosciences), TCRP-BUV737 (Clone H57- 597, BD Biosciences), CD4-BUV395 (Clone RM4-5, BD Biosciences ), and NK1.1-BV480 ( Clone PK 136, BD Biosciences). Stained cells were analyzed using Aurora (Cytek) flow cytometer, and data were processed with FlowJo (FlowJo LLC).

[0181] Cytokines Quantification CSF-1 in SEC EVs, soluble fractions, inter fractions or supernatants of conditioned medium were measured using the LegendPlex multiplex assay according to the manufacturer’s instructions. Samples were acquired on a BD FACSverse and analyzed using LEGENDplex sofware (BioLegend).

[0182] Cytokine Array

[0183] Mo-mac culture supernatants were collected and centrifuged for 15 min at 300 g. Supernatants were incubated with Human XL Cytokine Array membranes (R and D Systems) according to the manufacturers’ instructions. Two independent experiments were performed. The array membrane was developed using Bio-Rad ChemiDocTouchTM system for 3-4 min. The integrated pixels density was measured using the Fiji software.

[0184] Results

[0185] Mechanisms and functions of CSF1 association to extracellular vesicles in antitumor immune responses

[0186] Extracellular Vesicles (EVs) are known to transfer their bioactive molecules to target recipient cells and thus modulate their physiology. As cargo carriers, tumor EVs (tEVs) contain various proteins, lipids, and nucleic acids, some of which are uniquely packaged, such as oncoproteins, and adhesion molecules with specific functions and metastatic tropisms. tEVs are endowed with different and specific functional abilities to either activate or inhibit anti-tumor immune responses. Recently, the inventors showed that triple negative breast cancer (TNBC) cells release EVs bearing the macrophage-colony-stimulating factor CSF1, which promote monocyte differentiation towards a distinct macrophage type with pro-inflammatory features, while the soluble form of CSF1 induce M2-like macrophages (Tkach el al., 2022). The inventors also showed that the presence of these macrophages in tumors of TNBC patients correlated with better survival and a potentially anti-tumoral immune infiltrate. Now, the inventors demonstrate that EVs from the TNBC cells reduced growth of a murine tumor in syngeneic mice when injected intratumorally, while this anti-tumoral effect was not observed if EVs from CSF1-KO tumor cells were injected (Figure 1). Based on these observations, the inventors further studied the molecular mechanism of CSF1 association to EVs and its immune consequences. The CSF1 gene encodes for a short and a long isoform: both are transmembrane molecules, one is cleaved very efficiently to shed a large extracellular molecule (long isoform), the other remains mostly attached to the membrane and exposed on the cell surface, although it can also be eventually cleaved and shed (short isoform). The inventors engineered HEK293 cells to express separately each of the two CSF1 isoform and isolated the secreted EVs, soluble factors, inter fractions and supernatants. The inventors observed both long and short forms of CSF1 associated to EVs (Figure 2), probably through different mechanisms. As illustrated in figure 2A and 2B, S-CSF1 is nonetheless 1.5 to 3 times less abundant on EVs and 1.5 to 2 times more abundant in soluble fractions than L-CSF1.

[0187] EVs bearing either the short or long form of CSF1 differently triggered the differentiation of monocytes toward macrophages in vitro (Figure 3). Indeed, S-CSFl-EVs allow less efficient survival and differentiation of monocytes than rCSFl and L-CSFl-EVs, and fewer CD206 / CD163 macrophages. Both S- and L-soluble fractions induce similar levels of monocyte survival and differentiation as S-CSFl-EVs (see Fig. 3C).

[0188] A few cytokines are differently expressed upon monocyte differentiation induced by S-CSF1 versus L-CSF1, as illustrated on Fig. 3E. In particular, S-CSFl-EVs induces macrophages secreting less Osteopontin (SPP1), but more IL16, ENA-78 and chitinase3-like than L-CSF1- EVs.

[0189] Further, EVs bearing the short form of CSF1 modified the growth and immune infiltrate of a murine mammary carcinoma when injected in vivo (Figure 4). S-CSFl-EVs induce major reduction of tumor growth (see Fig. 4A) as compared to control treatment.

[0190] Lower metastasis signal is observed in mice treated with S-CSFl-EVs than both controls (PBS and naive HEK293 EVs) (see Fig. 4B).

[0191] Tumors treated with S-CSFl-EVs display more T cells and NK cells, and fewer macrophages and neutrophils than tumors treated by naive EVs. S-CSF1-EV treatment induces more T lymphocytes and fewer macrophages than all other treatments (see Fig. 4D-4F).

[0192] The inventors investigate the role and specific contribution of extracellular vesicles (EVs) in cancer environment, particularly the role of Short-CSFl -associated extracellular vesicle (S-CSFl-associated EV) in immunotherapy using different models of cancer. The inventors demonstrate the proof of concept of anti-tumor effect of S-CSFl-associated EVs in vivo in syngeneic mice bearing a breast carcinoma (Figure 4) or a lung carcinoma (not shown) and that S-CSFl-associated EV induce strong reduction of tumor growth upon intratumoral injection, and this despite a slightly lower presence on EVs than the long form. The inventors unravel an unexpected ability of S-CSFl-EVs to promote a tumor microenvironment associated with tumor shrinkage in vivo. The inventors have also shown in vitro that the S-CSFl-EVs induce differentiation of monocytes into macrophages which do not secrete some of the cytokines associated with pro-tumoral phenotype. Altogether, the present invention highlights the role of this specific S-CSFl-associated EV and provide in vitro and in vivo evidences towards its use in the treatment of cancer.

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Claims

1. 48CLAIMS:

1. An isolated or modified Short-CSFl -associated extracellular vesicle (S-CSFl-associated EV).

2. The S-CSFl-associated EV of claim 1 wherein the S-CSF1 has an amino acid sequence SEQ ID NO:2 or SEQ ID NO:3, or a variant thereof, or is encoded by the nucleic acid sequence SEQ ID NO: 1 or a variant thereof.

3. The S-CSFl-associated EV of any one of claims 1 or 2 comprising an antigen-recognizing receptor.

4. The S-CSFl-associated EV of any one of claims 1 to 3, wherein the antigen-recognizing receptor binds to a tumor-associated antigen or a TAM-associated antigen.

5. An isolated or modified macrophage, tumor-associated macrophages (TAM) or progenitor thereof, wherein said macrophage or progenitor thereof has been co-cultured in vitro with a S- CSFl-associated EV of any one of claims 1 or 2 to generate S-CSFl-EV-induced macrophages.

6. The S-CSFl-EV-induced macrophage of claim 5, comprising S-CSFl-associated EV of any one of claims 1 or 2.

7. The S-CSFl-EV-induced macrophage according to claim 5 or 6 which further encodes an antigen-recognizing receptor.

8. The S-CSFl-EV-induced macrophage of claim 7, wherein the antigen-recognizing receptor is a chimeric antigen receptor (CAR) or binds to a tumor-associated antigen.

9. The S-CSFl-EV-induced macrophage of any one of claims 5 to 8 for use in an adoptive cell immunotherapy .

10. The S-CSFl-associated EV of any one of claims 1 to 4 and / or the S-CSFl-EV-induced macrophage of any one of claims 5 to 8 for use in therapy.

11. The S-CSFl-associated EV of any one of claims 1 to 4 and / or the S-CSFl-EV-induced macrophage of any one of claims 5 to 8 for use in the treatment of cancer.

12. The S-CSFl-associated EV of any one of claims 1 to 4 and / or the S-CSFl-EV-induced macrophage of any one of claims 5 to 8 for use in the treatment of breast cancer and lung cancer, in particular Triple Negative Breast Cancer (TNBC).

13. The S-CSFl-associated EV and / or the S-CSFl-EV-induced macrophage for use according to any one of claims 9 to 12 in combination with immunotherapy.

14. A pharmaceutical composition comprising the S-CSFl-associated EV of any one of claims 1 to 4 or the S-CSFl-EV-induced macrophage of any one of claims 5 to 8 and a pharmaceutical acceptable carrier for use in the treatment of cancer in a subject in need thereof.

15. A S-CSFl-EV-induced macrophage obtained from a macrophage, a tumor-associated macrophages (TAM) or progenitor thereof, co-cultured in vitro with a S-CSFl-associated EV.

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