WO2024213767A1

WO2024213767A1 - Engraftment of mesenchymal stromal cells engineered to stimulate immune infiltration in tumors

Info

Publication number: WO2024213767A1
Application number: PCT/EP2024/060081
Authority: WO
Inventors: Pierre Guermonprez; Fillipe Luiz ROSA DO CARMO; Stéphanie HUGUES
Original assignee: Universite de Geneve; Centre National de la Recherche Scientifique CNRS; Institut National de la Sante et de la Recherche Medicale INSERM; Universite Paris Cite
Current assignee: Universite de Geneve; Centre National de la Recherche Scientifique CNRS; Institut National de la Sante et de la Recherche Medicale INSERM; Universite Paris Cite
Priority date: 2023-04-14
Filing date: 2024-04-12
Publication date: 2024-10-17
Anticipated expiration: 2025-10-14

Abstract

Dendritic cells (DCs) control the activation and infiltration of CD8+ T cells within the tumor microenvironment (TME). This process supports the immune control of tumors and explain the efficiency of immune checkpoint blockade immunotherapy. However, DC infiltration in tumors is typically low and controlled by immunosuppressive mechanisms activated by tumors. Here, the inventors propose a new approach to promote DC infiltration in solid tumors, increase anti- tumor specific T cell responses and achieve the activation of anti-tumor immunity. This approach is based on the intra-tumoral engraftment within solid tumors of autologous engineered stromal cells delivering factors favoring DC infiltration. Specifically, fibroblasts engineered to express the membrane bound form of FLT3L (eMSC-FLT3L) induce anti-tumor immunity when combined with adjuvant like the poly(I:C) double stranded RNA mimic. DC infiltration, T cell activation and the therapeutic anti-tumor effect is uniquely dependent on the presence of both eMSC-FLT3L and poly(I:C). Mechanistic experiments demonstrate that poly(I:C)-induced chemokine (CXCR3 and CCR5 ligands) are required for the therapeutic effect of eMSC-FLT3L + poly(I:C). Finally, stromal cell-based delivery of chemokines (CXCR3, CCR5 or XCR1 ligands) together with membrane bound form of FLT3L is sufficient to activate anti-tumor immunity in the absence of poly(I:C). Altogether, these data support the therapeutic potential of intra-tumoral engraftment of autologous mesenchymal stromal cells to induce anti-tumor immunity.

Description

ENGRAFTMENT OF MESENCHYMAL STROMAL CELLS ENGINEERED TO STIMULATE IMMUNE INFILTRATION IN TUMORS

FIELD OF THE INVENTION:

The present invention is in the field of medicine, in particular immunology and oncology.

BACKGROUND OF THE INVENTION:

The development of efficient immunotherapies calls for a better understanding of “immune landscape” of the tumor microenvironment (TME)¹. Components of the TME include lymphocytes, natural killer cells (NK cells), dendritic cells (DCs) and macrophages. But this infiltration is dependent of the tumor type ². The main challenges of cancer immunotherapy consist in triggering immune cells infiltration within the TME and preventing tumor-associated immunosuppression³. The recruitment of DCs inside the tumor is a great approach considering the essential role of dendritic cells as Antigen Presenting Cell (APC) coordinating the activation of both innate and adaptive immunity.

Human DCs are sentinel cells of the immune system specialized in controlling T cell function^4- ⁶. Murine and human DCs are partly evolutionary conserved between human and mice^7-10. DCs have a short half-life (few days) and are continuously replenished by a developmental process originating in hematopoietic stem cells¹⁰ and involving circulating precursors in both mice and humans^11-15. DC development is critically dependent on the activation the Flt3 receptor tyrosine kinase by its cognate ligand FLT3L in both mice and humans ⁷’^16-18. Circulating DC precursors (pre-DCs) seed secondary lymphoid organs (SLOs) and non-lymphoid tissues (NLTs). Upon activation of terminal differentiation, DCs upregulate CCR7 chemokine receptor and undergo a migration process towards the T cell zones of SLOs^{8-10 19} thereby mediating the delivery of tumor antigens at T cell priming sites^20-22. DCs are equipped with specific machineries enabling antigen presentation by both MHCI and MHCII molecules ensuring the presentation of tumor antigens by MHCII and MHCI (cross presentation) ⁸-¹⁰49,23,24 p_reciseiy, DCs consist in two major subtypes. Type 1 DCs or DC1 (CD141⁺XCRl⁺Clec9A⁺IRF8⁺ DCls in humans and XCRl⁺Clec9A⁺IRF8⁺ DCls in mice) are conserved between mouse and human, and share the expression of specific surface markers such as Clec9A ²⁵ and XCR1²⁶ as well as the transcription factor (TF) IRF8, which is essential for the development of murine DC I^25-30. Type 2 DCs or DC2s (CDlc⁺CleclOA⁺SIRP-a⁺IRF4⁺ DC2s in humans and SIRP-a⁺IRF4⁺ DC2s in mice) express the IRF4 TF³¹, which controls their development in the murine model ^31,32. Human IRF4⁺ SIRP-a⁺DCs include DC2 (CDlc⁺CD14 BTLA⁺CD5⁺CD88 )^{14, 33-35} but also other DC subtypes like DC3 (CDlc⁺CD14^+/‘ CD88'CD163⁺)³⁶ or inflammatory DCs (CDlc⁺CD14⁺CD88^+/')^14,33-35. Murine IRF4⁺ SIRP-a⁺ are heterogeneous^37-41.

Despite their poor infiltration within tumors⁴², multiple line of evidence supports the notion that DCs, and DC1 in particular, are major controllers of the priming and recruitment of CD8⁺ TILs. DC Is are essential for the rejection of intrinsically immunogenic tumors ^43,44. Recent studies show that murine DC Is are actually required for the induction of immune responses activated by immune checkpoint blockers (ICB)^22,45. In addition to their priming function, intra-tumoral DC Is represent a major source of chemokines CXCL9 which are essential to recruit circulating CXCR3⁺ CD8⁺ effector T cells^46,47. This process is activated by IFN-y provided by CCR5⁺ effector T cells recruited by tumor-derived CCL5 ⁴⁸. The CXCL9-CXCR3 axis is required for the efficiency of anti-PDl ICB in immunogenic tumors⁴⁷. ICB immunotherapy also triggers a rapid activation of intra-tumoral DCs through IFN-y signalling enhancing IL-12 secretion⁴⁹. The strong immunogenic activity of DC1 can be activated by in vivo delivery of FLT3L together with agonists of DC maturation like anti-CD40 antibodies or TLR3 agonists^23,50,51. As a result, DC1 expansion is sufficient to support ICB response in non-responsive tumor models^23,50,51. FLT3L-based approaches for DC1 expansion within tumors could rely on the strong proliferative expansion of DC-committed progenitors in the bone marrow⁷² and/or limited proliferation of DC1 at peripheral, tumor sites^16,52. However, these approaches do not directly impact on the chemotactic processes underlying DC1 infiltration.

Both murine pre-DCls and pre-DC2s express high levels of the CCR5 chemokine receptor⁵³. Murine pre-DCsl express CXCR3 and respond to the IFN-inducible CXCL9 and CXCL1O⁵³. Once recruited, murine pre-DCls terminally differentiate into DC1 and gain or increase expression of the XCR1 chemokine receptor^27,54,55. XCR1 is selectively expressed in murine and human DCsl play an important role in the maintenance of recruited pre-DCsl and their positioning^56,57. NK cells and effector CD8⁺ T cells secrete high amounts of the XCR1 ligands XCL1 and XCL2, together with CCR5 ligands including CCL3, CCL4 and CCL5 ^56-58. The efficiency of DC infiltration in tumors is curtailed by tumor-orchestrated immunosuppressive mechanisms. COX1/2 enzymes expressed in tumors limit NK-dependent recruitment of DCs within tumors⁵⁷. Activation of the beta-catenin pathways in Braf^l60W'IPten¹' melanoma curtails CCL4-dependent recruitment of DCls⁵⁹. In multiple tumor types, the abundance of the DC1- associated signatures is correlated to protective outcomes⁵⁷. Importantly, Barry et al. have shown that DC1 infiltration predicts the responsiveness to PD1/PDL1 ICB in melanoma⁶⁰. Collectively, these data support the notion that DC1 recruitment within tumor is a pillar of an efficient anti -turn or immune response by ICB.

Recent works have contributed to the identification of pre-DCs circulating progenitors, their regulation by FLT3L engaging the FLT3 receptor tyrosine kinase at homeostasis and during immune responses ⁿ’¹⁶’¹⁹’⁶¹. [_n particular, the Guermonprez lab is addressing how inflammatory factors control the development of DCs subsets. Recently, new methodologies to promote DC infiltration in non-lymphoid tissues using synthetic niches formed by the engraftment of engineered mesenchymal SCs delivering hematopoietic growth factors (eMSCs) I matrigel implants ⁶². This study has evidenced that eMSCs delivering 2 hematopoietic and 1 chemotactic factors (FLT3L, KITL and CXCL12) support the development of human DCs subsets after engraftment in the dermis of immunodeficient mice. This provides a unique proof-of-principle for the implementation of SCs in manipulating DCs population in vivo.

DC-promoting intervention have already been explored by means of FLT3L delivery for instance. It is well established that systemic administration of FLT3L in mice²⁹ and humans Flt3L³⁰’³¹ dramatically expands non-activated DCs. Administration of FLT3L is being considered and clinically tested as an immunotherapeutic option (clinical trials n°NCT02839265 and NCT01976585). However, limitations associated to systemic treatment with FLT3L include repeated injections and systemic undesired effects such as alteration of hematopoiesis (anemia) and systemic expansion of Tregs. There is thus a room for improving the safety and efficiency of FLT3L delivery so as to stimulate anti -tumor immunity.

SUMMARY OF THE INVENTION:

The present invention relates the intra-tumoral engraftment of mesenchymal stromal cells engineered for expressing FLT3L for stimulating dendritic cell infiltration and thus sparking T cell-dependent anti-tumor immunity.

DETAILED DESCRIPTION OF THE INVENTION:

Dendritic cells (DCs) control the activation and infiltration of CD8+ T cells within the tumor microenvironment (TME). This process supports the immune control of tumors and explain the efficiency of immune checkpoint blockade immunotherapy. However, DC infiltration in tumors is typically low and controlled by immunosuppressive mechanisms activated by tumors. Here, the inventors propose a new approach to promote DC infiltration in solid tumors, increase antitumor specific T cell responses and achieve the activation of anti-tumor immunity. This approach is based on the intra-tumoral engraftment, within solid tumors, of autologous engineered stromal cells delivering factors favoring DC infiltration. Specifically, mesenchymal stromal cells (isolated from bone marrow, adipose, and other tissue source) engineered (eMSC) to express the membrane bound form of FLT3L (eMSC-FLT3L) induce anti -tumor immunity when combined with adjuvant like the poly(I:C) double stranded RNA mimic or agonistic CD40 antibody. DC infiltration, T cell activation and the therapeutic anti-tumor effect is dependent on the presence of both eMSC-FLT3L and immuno-modulators, such as anti-CD40 Abs and TLR agonists. Mechanistic experiments demonstrate that poly(I:C)-induced chemokine (CXCR3 and CCR5 ligands) are required for the therapeutic effect of eMSC-FLT3L + poly(I:C). Furthermore, eMSC-FLT3L and poly(I:C) increases the frequency of tumorspecific IFNg-producing T cells but does not modify CD4+Treg populations thereby promoting anti-tumor immunity. Finally, stromal cell-based delivery of chemokines (CXCR3, CCR5 or XCR1 ligands) together with membrane bound form of FLT3L is sufficient to activate antitumor immunity in the absence of poly(I:C). Altogether, these data support the therapeutic potential of intra-tumoral engraftment of autologous mesenchymal stromal cells to induce antitumor immunity.

Main definitions:

As used herein, the terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component. Polypeptides when discussed in the context of gene therapy refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, which retains the desired biochemical function of the intact protein.

As used herein, the term “polynucleotide” refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

As used herein, the expression “derived from” refers to a process whereby a first component (e.g., a first polypeptide), or information from that first component, is used to isolate, derive or make a different second component (e.g., a second polypeptide that is different from the first one).

As used herein, the “percent identity” between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions/total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below. The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology. 48 (3): 443-53.). The percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle may be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5. In general, the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For instance, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%. The % identity is typically determined over the whole length of the query sequence on which the analysis is performed. Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical irrespective of any chemical and/or biological modification. According to the invention, a first amino acid sequence having at least 80% of identity with a second amino acid sequence means that the first sequence has 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence.

As used herein, the term “mutation” has its general meaning in the art and refers to a substitution, deletion or insertion. In particular, the term "substitution" means that a specific amino acid residue at a specific position is removed and another amino acid residue is inserted into the same position. Within the specification, the mutation are references according to the standard mutation nomenclature.

As used herein, the term “expression” of a polynucleotide sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.

As used herein, the term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as, for example, a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "polynucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase “polynucleotide sequence that encodes a protein or a RNA” may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

As used herein, the term "membrane polypeptide" refers to a membrane-associated polypeptide of which at least one portion is embedded within the phospholipid cell membrane. For example, integral membrane proteins may be nearly fully contained within the cell membrane, or may have extracellular portions, cytoplasmic portions, or both. Anchored membrane proteins are characterized as having at least one hydrophobic "anchor" portion that is embedded within the cell membrane, and either a cytoplasmic or extracellular portion. Other membrane protein types, and specific examples thereof, are readily appreciated by those skilled in the art.

As used herein, the term “FLT3L” has its general meaning in the art and refers to the Fms- related tyrosine kinase 3 ligand encoded by FLT3LG gene. Flt3 ligand (FL) is a hematopoietic four helical bundle cytokine. It is structurally homologous to stem cell factor (SCF) and colony stimulating factor 1 (CSF-1). In synergy with other growth factors, Flt3 ligand stimulates the proliferation and differentiation of various blood cell progenitors. In particular, it is a major growth factor stimulating the growth of dendritic cells. An exemplary amino acid sequence of FLT3L is shown as SEP ID NO:1 In particular, FLT3L comprises an extracellular domain that ranges from the amino acid residue at position 27 to the amino acid residue at position 184 in SEQ ID NO: 1, a transmembrane domain that ranges from the amino acid residue at position 185 to the amino acid residue at position 205 in SEQ ID NO: 1 and a cytoplasmic domain that ranges from the amino acid residue at position 206 to the amino acid residue at position 235 in SEQ ID NO: 1.

SEQ ID NO : 1 >sp | P49771 | FLT3L HUMAN Fms-related tyrosine kinase 3 ligand 0S=Homo sapiens OX=9606 GN=FLT3LG PE=1 SV=1 MTVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPI SSDFAVKIRELSDYLLQDYPVTVASNLQDEEL CGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNI SRLLQETSEQL VALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQPPLLLLLLLPVGLLLLAAAWCLHW QRTRRRTPRPGEQVPPVPSPQDLLLVEH

As used herein, the term “FLT3L membrane polypeptide” refers to a membrane polypeptide that derives from FTL3L.

As used herein, the “GM-CSF” has its general meaning in the art and refers to the granulocytemacrophage colony-stimulating factor encoded by the CSF2 gene. The term is also known as Colony-stimulating factor (CSF), Molgramostin, or Sargramostim. GM-CSF stimulates the growth and differentiation of hematopoietic precursor cells from various lineages, including granulocytes, macrophages, eosinophils. An exemplary amino acid sequence for GM-CSF is shown as SEQ ID NO:2. SEQ ID NO : 2 >sp | P04141 | CSF2 HUMAN Granulocyte-macrophage colonystimulating factor OS=Homo sapiens OX=9606 GN=CSF2 PE=1 SV=1 MWLQSLLLLGTVACSI SAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVI SEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQI ITF ESFKENLKDFLLVI PFDCWEPVQE

As used herein, the term “CXCR3” has its general meaning in the art and refers to the chemokine receptor CXCR3 that is a Gai protein-coupled receptor in the CXC chemokine receptor family. Other names for CXCR3 are G protein-coupled receptor 9 (GPR9) and CD183. There are three isoforms of CXCR3 in humans: CXCR3-A, CXCR3-B and chemokine receptor 3 -alternative (CXCR3-alt) CXCR3-A binds to the CXC chemokines CXCL9 (MIG), CXCL10 (IP-10), and CXCL11 (I-TAC) whereas CXCR3-B can also bind to CXCL4 in addition to CXCL9, CXCL10, and CXCL11.

As used herein, the term “CXCL9” has its general meaning in the art and refers to the C-X-C motif chemokine 9. The term is also known as gamma-interferon-induced monokine, monokine induced by interferon-gamma (HuMIG; MIG), small-inducible cytokine B9, CMK, MIG, an SCYB9. An exemplary amino acid sequence of CXCL9 is shown as SEQ ID NO:3. 125 0S=Homo sapiens OX=9606

TPWRKGRCS CI STNQGTIH LQSLKDLKQF APSPSCEKIE I IATLKNGVQ TCLNPDSADV KELIKKWEKQ VSQKKKQKNG KKHQKKKVLK VRKSQRSRQK KTT

As used herein, the term “CXCL11” has its general meaning in the art and refers to the C-X-C motif chemokine 11. The term is also known as Beta-Rl, H174, Interferon gamma-inducible protein 9 (IP-9), Interferon-inducible T-cell alpha chemoattractant (I-TAC), Small-inducible cytokine Bl 1, IT AC, SCYB11, and SCYB9B. An exemplary amino acid sequence for CXCL11 is shown as SEQ ID NO:4.

SEQ ID NO : 4 >sp | 014625 | CXL11_HUMAN | 22-94 0S=Homo sapiens OX=9606 GN=CXCL11 PE=1 SV=1 FPMFKRGRCL CIGPGVKAVK VADIEKASIM YPSNNCDKIE VI ITLKENKG QRCLNPKSKQ ARLI IKKVER KNF

As used herein, the term “CCR5” has its general meaning in the art and refers to the C-C chemokine receptor type 5, that is also known as CCR5 or CD 195. CCR5 is a protein on the surface of white blood cells that is involved in the immune system as it acts as a receptor for chemokines. CCR5's cognate ligands include CCL3, CCL4 (also known as MIP la and ip, respectively), and CCL3L1. CCR5 furthermore interacts with CCL5 (a chemotactic cytokine protein also known as RANTES).

As used herein, the term “CCL5” has its general meaning in the art and refers to the chemokine (C-C motif) ligand 5 that is encoded by the CCL5 gene. The term is also known as EoCP, Eosinophil chemotactic cytokine, SIS-delta, Small-inducible cytokine A5, T cell-specific protein P228 (TCP228), T-cell-specific protein RANTES, D17S136E, and SCYA5. An exemplary amino acid sequence for CCL5 is shown as SEP ID NO:5. sapiens OX=9606 GN=CCL5

SPYSSDTTPC CFAYIARPLP RAHIKEYFYT SGKCSNPAW FVTRKNRQVC ANPEKKWVRE YINSLEMS

As used herein, the term “CCL11” has its general meaning in the art and refers to the chemokine (C-C motif) ligand 11 that is encoded by the CCIA 1 gene. The term is also known as Eotaxin, C-C motif chemokine 11, Eosinophil chemotactic protein, Small-inducible cytokine Al l, and SCYA1 1. An exemplary amino acid sequence for CCL5 is shown as SEQ ID NO:6. OS=Homo sapiens OX=9606

GPASVPTTCC FNLANRKI PL QRLESYRRIT SGKCPQKAVI FKTKLAKDIC ADPKKKWVQD SMKYLDQKSP TPKP

As used herein, the term “XCR1” has its general meaning in the art and refers to the chemokine receptor XCR1. XCR1 is the receptor for XCL1 and XCL2 (or lymphotactin-1 and -2). XCR1 is also known as GPR5.

As used herein, the term “XCL1” has its general meaning in the art and refers to Chemokine (C motif) ligand. The term is also known as Lymphotactin, AT AC, C motif chemokine 1, Cytokine SCM-1, Lymphotaxin or SCM-l-alpha. An exemplary amino acid sequence is shown as SEQ ID NO:7 114 OS=Homo sapiens OX=9606

VGSEVSDKRT CVSLTTQRLP VSRIKTYTIT EGSLRAVI FI TKRGLKVCAD PQATWVRDW RSMDRKSNTR NNMIQTKPTG TQQSTNTAVT LTG As used herein, the term “CXCL12” has its general meaning in the art and refers to the C-X-C motif chemokine 12. The term is also known as Intercrine reduced in hepatomas (IRH; hIRH) or Pre-B cell growth-stimulating factor (PBSF). An exemplary amino acid sequence for CXCL12 is shown as SEP ID NO:8

SEQ ID NO : 8 >sp | P48061 | SDF1_HUMAN Stromal cell-derived factor 1 OS=Homo sapiens OX=9606 GN=CXCL12 PE=1 SV=1 MNAKWWLVLVLTALCLSDGKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIV ARLKNNNRQVCIDPKLKWIQEYLEKALNKRFKM

As used herein, the term “SCF” has its general meaning in the art and refers to the KIT ligand. The term is also known as Mast cell growth factor (MGF), Stem cell factor (SCF), and c-Kit ligand. An exemplary amino acid sequence for SCF is shown as SEQ ID NO:9. Kit ligand OS=Homo sapiens OX=9606

MKKTQTWILTCIYLQLLLFNPLVKTEGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPG

MDVLPSHCWI SEMWQLSDSLTDLLDKFSNI SEGLSNYSI IDKLVNIVDDLVECVKENSS

KDLKKSFKSPEPRLFTPEEFFRI FNRSIDAFKDFWASETSDCWSSTLSPEKDSRVSVT

KPFMLPPVAASSLRNDSSSSNRKAKNPPGDSSLHWAAMALPALFSLI IGFAFGALYWKKR

QPSLTRAVENIQINEEDNEI SMLQEKEREFQEV

As used herein, the term “CD40” has its general meaning in the art and refers to human CD40 polypeptide receptor. In some embodiments, CD40 is the isoform of the human canonical sequence as reported by UniProtKB-P25942 (also referred as human TNR5).

As used herein, the term “CD40L” has its general meaning in the art and refers to human CD40L polypeptide, for example, as reported by UniProtKB-P25942, including its CD40- binding domain of SEQ ID NO:1Q.

SEQ ID NO : 10> CD40L binding domain

MQKGDQNPQIAAHVI SEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNR

EASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHG TGFTSFGLLKL

As used herein, the term “mesenchymal stromal cell” or “MSC” refers to cells that meet the definition set in 2006 by The International Society for Cellular Therapy (ISCT): (1) adherence to plastic, (2) expression of CD73, CD90, and CD105 antigens, while being CD14, CD34, CD45, and HLA-DR negative, and (3) ability to differentiate to osteogenic, chondrogenic and adipogenic lineage (Dominici et al. (2006) Cytotherapy, 8:315-317). MSC have been traditionally defined as spindle-shaped or fibroblast-like plastic adherent cells. Although originally isolated from bone marrow, MSC have now been isolated from a variety of tissues including bone periosteum, trabecular bone, adipose tissue, synovium, skeletal muscle, dental pulp and cord blood. MSCs can be derived from any tissue including, but not limited to, bone marrow, adipose tissue, amniotic fluid, endometrium, trophoblast-derived tissues, cord blood, Wharton jelly, and placenta. In particular, the term includes cells that are CD34 positive upon initial isolation from tissue but satisfy the ISCT criteria after expansion. The term also include cells that are isolated from tissues using cell surface markers selected from the list comprised of NGF-R, PDGF-R, EGF-R, IGF-R, CD29, CD49a, CD56, CD63, CD73, CD105, CD106, CD140b, CD146, CD271, MSCA-1, SSEA4, STRO-1 and STRO-3 or any combination thereof, and satisfy the ISCT criteria either before or after expansion. More particularly, the term includes cells described in the literature as bone marrow stromal stem cells (BMSSC), marrow- isolated adult multipotent inducible cells (MIAMI) cells, multipotent adult progenitor cells (MAPC), mesenchymal adult stem cells (MASCS), MULTISTEM® (Athersys, Inc., Cleveland, Ohio), PROCHYMAL® (Osiris Therapeutics, Inc., Columbia, Md.), remestemcel-L, Mesenchymal Precursor Cells (MPCs), Dental Pulp Stem Cells (DPSCs), PLX cells, PLX- PAD, ALLOSTEM® (Allosource, Centennial, Colo.), ASTROSTEM® (Osiris Therapeutics, Inc., Columbia, Md.), Ixmyelocel-T, MSC-NTF, NurOwn™ (Brainstorm Cell Therapeutics Inc., Hackensack, N. J.), STEMEDYNE™-MSC (Stemedica Cell Technologies Inc., San Diego, Calif.), STEMPEUCEL® (Stempeudics Research, Bangalore, India), StempeucelCLI, StempeucelOA, HiQCell, Hearticellgram-AMI, REVASCOR® (Mesoblast, Inc., Melbourne, Australia) CARDIOREL® (Reliance Life Sciences, Navi Mumbai, India), CARTISTEM® (Medipost, Rockville, Md ), PNEUMOSTEM® (Medipost, Rockville, Md ), PROMOSTEM® (Medipost, Rockville, Md.), Homeo-GH, AC607, PDA001, SB623, CX601, AC607, Endometrial Regenerative Cells (ERC), adipose-derived stem and regenerative cells (ADRCs) obtained with the CELUTION® System (Cytori Therapeutics, Inc., San Diego, Calif.), perivascular-derived cells, and pericyte-derived cells. Finally, the term includes cells that only satisfy one or more of the ISCT criteria when cultured under one set of conditions but satisfy the full set of ISCT criteria when cultured on plastic tissue culture flasks in the presence of tissue culture medium containing 10% fetal bovine serum.

As used herein, the term "population" refers to a population of cells, wherein the majority (e.g., at least about 50%, preferably at least about 60%, more preferably at least about 70%, and even more preferably at least about 80%) of the total number of cells have the specified characteristics of the cells of interest and express the markers of interest (e.g. a population of mesenchymal stromal cells comprises at least about 50%, preferably at least about 60%, more preferably at least about 70%, and even more preferably at least about 80% of cells which have the highly immune functions).

As used herein, the term “engineered” refers to an aspect of having been manipulated and altered by the hand of man. In particular, the term “engineered cell” refers to a cell that has been subjected to a manipulation, so that its genetic, epigenetic, and/or phenotypic identity is altered relative to an appropriate reference cell such as otherwise identical cell that has not been so manipulated. In some embodiments, the manipulation is or comprises a genetic manipulation. In some embodiments, a genetic manipulation is or comprises one or more of (i) introduction of a polynucleotide not present in the cell prior to the manipulation (i.e., of a heterologous polynucleotide); (ii) removal of a polynucleotide, or portion thereof, present in the cell prior to the manipulation; and/or (iii) alteration (e.g., by sequence substitution) of a polynucleotide, or portion thereof, present in the cell prior to the manipulation. In some embodiments, a an engineered cell is one that has been manipulated so that it contains and/or expresses a particular agent of interest (e.g., a protein, a polynucleotide, and/or a particular form thereof) in an altered amount and/or according to altered timing relative to such an appropriate reference cell. Those of ordinary skill in the art will appreciate that reference to an “engineered cell” herein may, in some embodiments, encompass both the particular cell to which the manipulation was applied and also any progeny of such cell.

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 patient at risk of contracting the disease or suspected to have contracted the disease as well as patients 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 patient 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 patient 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 patient 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 patient during treatment of an illness, e.g., to keep the patient 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 interval, 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., pain, disease manifestation, etc.]).

As used herein, the expression “therapeutically effective amount” is an amount sufficient to effect a beneficial or desired clinical result upon treatment. An effective amount can be administered to a subject in one or more doses. In terms of treatment, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease. The effective amount is generally determined by the physician on a case-by-case basis and is within the skill of one in the art. Several factors are typically taken into account when determining an appropriate dosage to achieve an effective amount. These factors include age, sex and weight of the subject, the condition being treated, the severity of the condition and the form and effective concentration of the mesenchymal stromal cells administered.

As used herein, the term “pharmaceutical composition” refers to a composition described herein, or pharmaceutically acceptable salts thereof, with other agents such as carriers and/or excipients. The pharmaceutical compositions as provided herewith typically include a pharmaceutically acceptable carrier.

As used herein, the term “pharmaceutically acceptable carrier” includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical-Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof.

Engineered cells of the present invention:

The first object of the present invention relates to a mesenchymal stromal cell that is engineered to express a FLT3L membrane polypeptide.

In some embodiments, the FLT3L membrane polypeptide of the present invention comprises an amino acid sequence having at least 80% of identity with the amino acid sequence that ranges from the amino acid residue at position 27 to the amino acid residue at position 184 in SEQ ID NO: 1.

In some embodiments, the FLT3L membrane polypeptide of the present invention comprises an amino acid sequence having at least 80% of identity with the amino acid sequence that ranges from the amino acid residue at position 27 to the amino acid residue at position 184 in SEQ ID NO: 1 wherein the proline residue at position 173 is mutated and/or the leucine residue at position 174 is mutated and/or the glutamic acid residue at position 175 is mutated. In some embodiments, the mutation is particularly suitable for avoiding the cleavage by the large family of transmembrane metalloproteases (ADAM) cleavage such as TNFa converting enzyme (TACE). In some embodiments, the proline residue at position 173 is substituted by a threonine residue. In some embodiments, the leucine residue at position 174 is substituted by a serine residue. In some embodiments, the glutamic acid residue at position 175 is substituted by a threonine residue.

In some embodiments, the FLT3L membrane polypeptide of the present invention comprises the amino acid sequence that ranges from the amino acid residue at position 27 to the amino acid residue at position 184 in SEQ ID NO: 11.

SEQ ID NO : 11 >

MTVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPI SSDFAVKIRELSDYLLQDYPVTVASNLQDEEL CGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNI SRLLQETSEQL VALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRTSTATAPTAPQPPLLLLLLLPVGLLLLAAAWCLHW QRTRRRTPRPGEQVPPVPSPQDLLLVEH In some embodiments, the FLT3L membrane polypeptide of the present invention comprises an amino acid sequence having at least 80% of identity with the amino acid sequence that ranges from the amino acid residue at position 27 to the amino acid residue at position 205 in SEQ ID NO: 1.

In some embodiments, the FLT3L membrane polypeptide of the present invention comprises an amino acid sequence having at least 80% of identity with the amino acid sequence that ranges from the amino acid residue at position 27 to the amino acid residue at position 205 in SEQ ID NO: 1 wherein the proline residue at position 173 is mutated and/or the leucine residue at position 174 is mutated and/or the glutamic acid residue at position 175 is mutated. In some embodiments, the proline residue at position 173 is substituted by a threonine residue. In some embodiments, the leucine residue at position 174 is substituted by a serine residue. In some embodiments, the glutamic acid residue at position 175 is substituted by a threonine residue.

In some embodiments, the FLT3L membrane polypeptide of the present invention comprises the amino acid sequence that ranges from the amino acid residue at position 27 to the amino acid residue at position 205 in SEQ ID NO: 11.

In some embodiments, the FLT3L membrane polypeptide of the present invention comprises an amino acid sequence having at least 80% of identity with the amino acid sequence that ranges from the amino acid residue at position 27 to the amino acid residue at position 235 in SEQ ID NO: 1.

In some embodiments, the FLT3L membrane polypeptide of the present invention comprises an amino acid sequence having at least 80% of identity with the amino acid sequence that ranges from the amino acid residue at position 27 to the amino acid residue at position 235 in SEQ ID NO: 1 wherein the proline residue at position 173 is mutated and/or the leucine residue at position 174 is mutated and/or the glutamic acid residue at position 175 is mutated. In some embodiments, the proline residue at position 173 is substituted by a threonine residue. In some embodiments, the leucine residue at position 174 is substituted by a serine residue. In some embodiments, the glutamic acid residue at position 175 is substituted by a threonine residue.

In some embodiments, the FLT3L membrane polypeptide of the present invention comprises the amino acid sequence that ranges from the amino acid residue at position 27 to the amino acid residue at position 235 in SEQ ID NO: 11. According to the present invention the mesenchymal stromal cell is not engineered to express a CXCL12 polypeptide nor a SCF polypeptide. Thus, in some embodiments, the mesenchymal cell of the present invention is not engineered to express a CXCL12 polypeptide that comprises an amino acid having at least 80% of identity with the amino acid sequence as set forth in SEQ ID NO:8. In some embodiments, the mesenchymal cell of the present invention is not engineered to express a SCF polypeptide that comprises an amino acid having at least 80% of identity with the amino acid sequence as set forth in SEQ ID NO:9.

In some embodiments, the mesenchymal stromal cell of the present invention is further engineered to express a GM-CSF polypeptide. In some embodiments, the mesenchymal stromal cell of the present invention is engineered to express the GM-CSF polypeptide that comprises an amino acid sequence having at least 80% of identity with the amino acid sequence as set forth in SEQ ID NO:2.

In some embodiments, the mesenchymal stromal cell of the present invention is further engineered to express at least one chemokine selected from the group consisting of CXCR3 ligands, CCR5 ligands, and XCR1 ligands.

In some embodiments, the mesenchymal stromal cell of the present invention is further engineered to express a CXCL9 polypeptide. In some embodiments, the mesenchymal stromal cell of the present invention is engineered to express the CXCL9 polypeptide that comprises an amino acid sequence having at least 80% of identity with the amino acid sequence as set forth in SEQ ID NO:3.

In some embodiments, the mesenchymal stromal cell of the present invention is further engineered to express a CXCL11 polypeptide. In some embodiments, the mesenchymal stromal cell of the present invention is engineered to express the CXCL11 polypeptide that comprises an amino acid sequence having at least 80% of identity with the amino acid sequence as set forth in SEQ ID NO:4.

In some embodiments, the mesenchymal stromal cell of the present invention is further engineered to express a CCL5 polypeptide. In some embodiments, the mesenchymal stromal cell of the present invention is engineered to express the CCL5 polypeptide that comprises an amino acid sequence having at least 80% of identity with the amino acid sequence as set forth in SEQ ID NO:5.

In some embodiments, the mesenchymal stromal cell of the present invention is further engineered to express a CCL11 polypeptide. In some embodiments, the mesenchymal stromal cell of the present invention is engineered to express the CCL11 polypeptide that comprises an amino acid sequence having at least 80% of identity with the amino acid sequence as set forth in SEQ ID NO:6.

In some embodiments, the mesenchymal stromal cell of the present invention is further engineered to express a XCL1 polypeptide. In some embodiments, the mesenchymal stromal cell of the present invention is engineered to express the XCL1 polypeptide that comprises an amino acid sequence having at least 80% of identity with the amino acid sequence as set forth in SEQ ID NO:7.

In some embodiments, the mesenchymal stromal cell of the present invention is further engineered to express a CD40L polypeptide. In some embodiments, the mesenchymal stromal cell of the present invention is engineered to express the CD40L polypeptide that comprises an amino acid sequence having at least 80% of identity with the amino acid sequence as set forth in SEQ ID NO: 10.

In some embodiments, the mesenchymal stromal cell of the present invention is further engineered to express a CCL5 polypeptide and a CXCL9 polypeptide. In some embodiments, the mesenchymal stromal cell of the present invention is engineered to express the CCL5 polypeptide that comprises an amino acid sequence having at least 80% of identity with the amino acid sequence as set forth in SEQ ID NO: 5 and the CXCL9 polypeptide that comprises an amino acid sequence having at least 80% of identity with the amino acid sequence as set forth in SEQ ID NO:3.

Any type of isolated mesenchymal stromal cells may be suitable for the purposes of the present invention. Such mesenchymal stromal cells may be isolated from a variety of organisms. Preferably the MSCs are isolated from murine or human sources. Most preferably, the MSCs are isolated from human sources. The MSCs may be isolated from a variety of tissue types. MSCs are cells found in bone marrow, blood, dental pulp cells, adipose tissue, skin, spleen, pancreas, brain, kidney, liver, heart, retina, brain, hair follicles, intestine, lung, lymph node, thymus, bone, ligament, tendon, skeletal muscle, dermis, and periosteum. The presence of MSCs in culture colonies may be verified by specific cell surface markers which are identified with monoclonal antibodies. See U.S. Pat. Nos. 5,486,359 and 7,153,500. MSCs are characterized by their ability to adhere to plastic tissue culture surfaces (Friedenstein et. al., reviewed in Owen & Friedenstein, 1988), and by being an effective feeder layers for hematopoietic stem cells (Eaves et. al., 2001). Mesenchymal stem cells (MSCs) may be purified using methods known in the art (Wakitani et. al., 1995; Fukuda and Yuasa, 2006; Woodbury et al., 2000; Deng et. al., 2001; Kim et. al., 2006; Mareschi et. al., 2006; Krampera et. al., 2007).

Methods for preparing the mesenchymal stromal cells of the present invention

The mesenchymal stromal cell of the present invention is preparing by any conventional method well known in the art.

Thus, a further object of the present invention relates to a method of preparing a mesenchymal stromal cell of the present invention, comprising the step consisting of introducing into a mesenchymal stromal cell a polynucleotide that encodes for the FLT3L membrane polypeptide of the present invention and optionally one or more polynucleotide(s) that encode(s) for a GM- CSF polypeptide and/or at least one chemokine selected from the CXCR3 ligands, CCR5 ligands, and XCR1 ligands, and optionally one or more polynucleotide(s) that encodes a CD40L polypeptide.

It is contemplated that a polynucleotide can be introduced into the mesenchymal stromal cells as naked DNA or in a suitable vector.

Naked DNA generally refers to the DNA contained in a plasmid expression vector in proper orientation for expression. Physical methods for introducing a polynucleotide construct into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, nucleofection, and the like. Other means can be used including colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. In some embodiments, the polynucleotide is introduced into the mesenchymal stromal cell by a viral vector that is an adeno-associated virus (AAV), a retrovirus, lentivirus, bovine papilloma virus, an adenovirus vector, a vaccinia virus, a polyoma virus, or an infective virus. In some embodiments, the vector is a retroviral. Retroviruses may be chosen as gene delivery vectors due to their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and for being packaged in special cell- lines. In order to construct a retroviral vector, the polynucleotide of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line is constructed containing the gag, pol, and/or env genes but without the LTR and/or packaging components. When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media. The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. The higher complexity enables the virus to modulate its life cycle, as in the course of latent infection. Some examples of lentivirus include the Human Immunodeficiency Viruses (HIV 1, HIV 2) and the Simian Immunodeficiency Virus (SIV). Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe. Lentiviral vectors are known in the art, see, e.g. U.S. Pat. Nos. 6,013,516 and 5,994,136, both of which are incorporated herein by reference. In general, the vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign polynucleotide, for selection and for transfer of the polynucleotide into a host cell. The gag, pol and env genes of the vectors of interest also are known in the art. Thus, the relevant genes are cloned into the selected vector and then used to transform the target cell of interest. Recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Pat. No. 5,994,136, incorporated herein by reference. This describes a first vector that can provide a polynucleotide encoding a viral gag and a pol gene and another vector that can provide a polynucleotide encoding a viral env to produce a packaging cell. Introducing a vector providing a heterologous gene into that packaging cell yields a producer cell which releases infectious viral particles carrying the foreign gene of interest. The env preferably is an amphotropic envelope protein which allows transduction of cells of human and other species.

Typically, the vector of the present invention includes "control sequences'", which refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.

Another polynucleotide sequence, is a "promoter" sequence, which is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3 '-direction) coding sequence. Transcription promoters can include "inducible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters”. To increase the expression, polynucleotides of the present invention may be operably linked to strong promoters, such as retroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murine stem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), P-actin, ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter, elongation factor (EF)-la and the spleen focus-forming virus (SFFV) promoter.

In some embodiments, the sequence of the polynucleotides is codon optimized for expression in a mammalian cell. Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons that code for the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows an identical polypeptide to be encoded by a variety of nucleotide sequences. A variety of codon optimization methods is known in the art, and include, e.g., methods disclosed in at least U.S. Pat. Nos. 5,786,464 and 6,114,148. Use of polycistronic expression cassettes that can both express the FLT3L membrane polypeptide and/or the GM-CSF polypeptide and/or at least one chemokine selected from the CXCR3 ligands, CCR5 ligands, and XCR1 ligands, and/or a CD40L polypeptide. Typically the polycistronic expression cassettes comprise various viral and non-viral Internal Ribosome Entry Sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-KB IRES, RUNX1 IRES, p53 IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, aphthovirus IRES, picomavirus IRES, poliovirus IRES and encephalomyocarditis virus IRES) and/or cleavable linkers (e.g., 2A peptides, e.g., P2A, T2A, E2A and F2A peptides). Thus use of polycistronic polynucleotides or vectors encoding for the polypeptides of interest are particularly suitable for preparing the mesenchymal stromal cells of the present invention.

Once the population of mesenchymal stromal cells is obtained, functionality of the cells may be evaluated according to any standard method which typically include a suppressive assay. Cell surface phenotype of the cells with the appropriate binding partners can also be confirmed. Quantifying the secretion of various cytokines may also be performed. Methods for quantifying secretion of a cytokine in a sample are well known in the art. For example, any immunological method such as but not limited to ELISA, multiplex strategies, ELISPOT, immunochromatography techniques, proteomic methods, Western blotting, FACS, or Radioimmunoassays may be applicable to the present invention.

Methods of therapy:

A further object of the present invention relates to a method of therapy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the population of mesenchymal stromal cells of the present invention.

In particular, the method of the present invention is particularly suitable for the treatment of cancer.

As used herein, the term "cancer" has its general meaning in the art and includes, but is not limited to, solid tumors and blood borne tumors. The term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels. The term "cancer" further encompasses both primary and metastatic cancers. Examples of cancers that may be treated by methods and compositions of the present invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

Typically the patient subjected to the above method may suffer from a solid cancer. As used herein, the term “solid cancer” has its general meaning in the art and refers to an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors may be benign (not cancer), or malignant (cancer). Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias (cancers of the blood) generally do not form solid tumors. In some embodiments, the solid cancer is selected from the group consisting adrenal cortical cancer, anal cancer, bile duct cancer (e.g. periphilar cancer, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, bone cancer (e.g. osteoblastoma, osteochrondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of the bone, chordoma, multiple myeloma), brain and central nervous system cancer (e.g. meningioma, astocytoma, oligodendrogliomas, ependymoma, gliomas, medulloblastoma, ganglioglioma, Schwannoma, germinoma, craniopharyngioma), breast cancer (e.g. ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating lobular carcinoma, lobular carcinoma in situ, gynecomastia), cervical cancer, colorectal cancer, endometrial cancer (e.g. endometrial adenocarcinoma, adenocanthoma, papillary serous adnocarcinoma, clear cell), esophagus cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g. choriocarcinoma, chorioadenoma destruens), Kaposi's sarcoma, kidney cancer (e.g. renal cell cancer), laryngeal and hypopharyngeal cancer, liver cancer (e.g. hemangioma, hepatic adenoma, focal nodular hyperplasia, hepatocellular carcinoma), lung cancer (e.g. small cell lung cancer, non-small cell lung cancer), mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer (e.g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (e.g. melanoma, nonmelanoma skin cancer), stomach cancer, testicular cancer (e.g. seminoma, nonseminoma germ cell cancer), thymus cancer, thyroid cancer (e.g. follicular carcinoma, anaplastic carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma), vaginal cancer, vulvar cancer, and uterine cancer (e.g. uterine leiomyosarcoma).

In some embodiments, the patient suffers from a primary cancer. In some embodiments, the patient suffers from a locally advanced cancer. In some embodiments, the patient suffers from a stage II TNM cancer. In some embodiments, the patient suffers from a stage III TNM cancer. In some embodiments, the patient suffers from a metastatic cancer. In some embodiments, the patient suffers from a stage IV TNM cancer.

Advantageously, the mesenchymal stromal cell of the present invention is particularly suitable for the treatment of cold tumors. As used herein, the term “cold tumor” has its general meaning in the art and describes a tumor that is not likely to trigger a strong immune response. Cold tumors tend to be surrounded by cells that are able to suppress the immune response and keep T cells (a type of immune cell) from attacking the tumor cells and killing them. Cold tumors usually do not respond to immunotherapy. Most cancers of the breast, ovary, prostate, pancreas, and brain (glioblastoma) are considered cold tumors.

In particular, the mesenchymal stromal cell of the present invention is particularly suitable to turn cold tumor into hot tumor by improving dendritic cell infiltration and subsequently T-cell infiltration of the tumor. As used herein, the term “hot tumor” has its general meaning in the art and describes a tumor that is likely to trigger a strong immune response. Hot tumors often have many molecules on their surface that allow T cells (a type of immune cell) to attack and kill the tumor cells. Hot tumors usually respond to immunotherapy). For example, the mesenchymal stromal cell of the present invention for increasing the density of dendritic cell by more than about 20%, preferably with at least about 30%, at least about 40%, at least about 50%.

In particular, the mesenchymal stromal cell is particularly suitable for the treatment of cancer characterized by a low tumor infiltration of CD8+ T cells. Typically the tumor-infiltration of dendritic cell and/or CD8+ T cells is determined by any convention method in the art. For example, said determination comprises quantifying the density of dendritic cells and/or CD8+ T cells in a tumor sample obtained from the subject.

In some embodiments, the population of mesenchymal stromal cells of the present invention is administered to the patient in combination with one or more adjuvant(s).

As used herein, the term “adjuvant” refers to a compound that can induce and/or enhance the immune response against an antigen when administered to a subject or an animal. It is also intended to mean a substance that acts generally to accelerate, prolong, or enhance the quality of specific immune responses to a specific antigen. In the context of the present invention, the term "adjuvant" means a compound, which enhances both innate immune response by affecting the transient reaction of the innate immune response and the more long-lived effects of the adaptive immune response by activation and maturation of the antigen-presenting cells (APCs) especially Dentritic cells (DCs).

In some embodiments, the adjuvant is a Toll-Like Receptor (TLR) agonist that is selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, and TLR8, TLR9 agonists. More particularly, the adjuvant is a TLR3 agonist.

In some embodiments, the adjuvant is Polyinosinic-polycytidylic acid (poly (I:C)) or polyinosinic-polycytidylic acid and poly-L-lysine (poly-ICLC).

In some embodiments, adjuvant activates the STING (Stimulator of Interferon Genes) pathway, which is an important signaling pathway that leads to the production of type I interferons and other immune mediators. Agonists that can activate the STING pathway include but are not limited to i) cGAMP (cyclic GMP-AMP): cGAMP is a small molecule that is produced by the enzyme cGAS (cyclic GMP-AMP synthase) in response to cytoplasmic DNA. cGAMP binds to and activates STING, leading to the production of type I interferons and other immune mediators. Synthetic cGAMP analogs, such as ADU-S100 and MK-1454; ii) DMXAA (5,6- dimethylxanthenone-4-acetic acid); iii) STING-activating cyclic dinucleotides (CDNs): CDNs are small molecules that can bind to and activate STING. Synthetic CDNs, such as ADU-S100 and MIW815 and iv) GSK-3745417 and VT1021.

Even more particularly, the mesenchymal stem cell of the present invention is particularly suitable for enhancing the potency of immunotherapy administered to a patient suffering from a cancer as part of a treatment regimen.

Thus a further object of the present invention relates for a method of enhancing the potency of immunotherapy administered to a patient suffering from a cancer as part of a treatment regimen, the method comprising administering to the subject a therapeutically effective amount of a population of mesenchymal stromal cells of the present invention in combination with immunotherapy.

As used herein, the expression “enhancing the potency of immunotherapy” refers to the ability of the population of mesenchymal stromal cells to increase the ability of immunotherapy to enhance the proliferation, migration, persistence and/or cytotoxic activity of CD8+ T cells.

As used herein, the term “immunotherapy” has its general meaning in the art and refers to the treatment that consists in administering an immunogenic agent i.e. an agent capable of inducing, enhancing, suppressing or otherwise modifying an immune response.

In some embodiments, the immunotherapy consists in administering the patient with at least one immune checkpoint inhibitor.

As used herein, the term "immune checkpoint inhibitor" has its general meaning in the art and refers to any compound inhibiting the function of an immune inhibitory checkpoint protein.

As used herein the term "immune checkpoint protein" has its general meaning in the art and refers to a molecule that is expressed by T cells in that either turn up a signal (stimulatory checkpoint molecules) or turn down a signal (inhibitory checkpoint molecules). Immune checkpoint molecules are recognized in the art to constitute immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see e.g. Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al. , 2011. Nature 480:480- 489). Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD- 1, LAG-3, TIM-3 and VISTA. Inhibition includes reduction of function and full blockade. Preferred immune checkpoint inhibitors are antibodies that specifically recognize immune checkpoint proteins. A number of immune checkpoint inhibitors are known and in analogy of these known immune checkpoint protein inhibitors, alternative immune checkpoint inhibitors may be developed in the (near) future. The immune checkpoint inhibitors include peptides, antibodies, nucleic acid molecules and small molecules. Examples of immune checkpoint inhibitor includes PD-1 antagonists, PD-L1 antagonists, PD-L2 antagonists, CTLA-4 antagonists, VISTA antagonists, TIM-3 antagonists, LAG-3 antagonists, TGIT-antagonists, IDO antagonists, KIR2D antagonists, A2AR antagonists, B7-H3 antagonists, B7-H4 antagonists, and BTLA antagonists.

In some embodiments, PD-1 (Programmed Death- 1) axis antagonists include PD-1 antagonist (for example anti-PD-1 antibody), PD-L1 (Programmed Death Ligand-1) antagonist (for example anti-PD-Ll antibody) and PD-L2 (Programmed Death Ligand-2) antagonist (for example anti-PD-L2 antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of MDX-1106 (also known as Nivolumab, MDX-1106-04, ONO-4538, BMS-936558, and Opdivo®), Merck 3475 (also known as Pembrolizumab, MK-3475, Lambrolizumab, Keytruda®, and SCH-900475), and CT-011 (also known as Pidilizumab, hBAT, and hBAT-1). In some embodiments, the PD-1 binding antagonist is AMP -224 (also known as B7-DCIg). In some embodiments, the anti-PD-Ll antibody is selected from the group consisting of YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736. MDX-1105, also known as BMS-936559, is an anti-PD-Ll antibody described in W02007/005874. Antibody YW243.55. S70 is an anti-PD-Ll described in WO 2010/077634 AL MEDI4736 is an anti-PD- Ll antibody described in WO2011/066389 and US2013/034559. MDX-1106, also known as MDX-1 106-04, ONO-4538 or BMS-936558, is an anti-PD-1 antibody described in U.S. Pat. No. 8,008,449 and W02006/121168. Merck 3745, also known as MK-3475 or SCH-900475, is an anti-PD-1 antibody described in U.S. Pat. No. 8,345,509 and W02009/114335. CT-011 (Pidizilumab), also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in W02009/101611. AMP -224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in W02010/027827 and WO2011/066342. Atezolimumab is an anti-PD-Ll antibody described in U.S. Pat. No. 8,217,149. Avelumab is an anti-PD-Ll antibody described in US 20140341917. CA-170 is a PD-1 antagonist described in W02015033301 & WO2015033299. Other anti-PD-1 antibodies are disclosed in U.S. Pat. No. 8,609,089, US 2010028330, and/or US 20120114649. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody chosen from Nivolumab, Pembrolizumab or Pidilizumab. In some embodiments, PD-L1 antagonist is selected from the group comprising of Avelumab, BMS-936559, CA-170, Durvalumab, MCLA-145, SP142, STI-A1011, STIA1012, STI-A1010, STI-A1014, Al 10, KY1003 and Atezolimumab and the preferred one is Avelumab, Durvalumab or Atezolimumab.

In some embodiments, CTLA-4 (Cytotoxic T-Lymphocyte Antigen-4) antagonists are selected from the group consisting of anti-CTLA-4 antibodies, human anti-CTLA-4 antibodies, mouse anti-CTLA-4 antibodies, mammalian anti-CTLA-4 antibodies, humanized anti-CTLA-4 antibodies, monoclonal anti-CTLA-4 antibodies, polyclonal anti-CTLA-4 antibodies, chimeric anti-CTLA-4 antibodies, MDX-010 (Ipilimumab), Tremelimumab, anti-CD28 antibodies, anti- CTLA-4 adnectins, anti-CTLA-4 domain antibodies, single chain anti-CTLA-4 fragments, heavy chain anti-CTLA-4 fragments, light chain anti-CTLA-4 fragments, inhibitors of CTLA- 4 that agonize the co-stimulatory pathway, the antibodies disclosed in PCT Publication No. WO 2001/014424, the antibodies disclosed in PCT Publication No. WO 2004/035607, the antibodies disclosed in U.S. Publication No. 2005/0201994, and the antibodies disclosed in granted European Patent No. EP 1212422 B. Additional CTLA-4 antibodies are described in U.S. Pat. Nos. 5,811,097; 5,855,887; 6,051,227; and 6,984,720; in PCT Publication Nos. WO 01/14424 and WO 00/37504; and in U.S. Publication Nos. 2002/0039581 and 2002/086014. Other anti-CTLA-4 antibodies that can be used in a method of the present invention include, for example, those disclosed in: WO 98/42752; U.S. Pat. Nos. 6,682,736 and 6,207,156; Hurwitz et al., Proc. Natl. Acad. Sci. USA, 95(17): 10067-10071 (1998); Camacho et al., J. Clin: Oncology, 22(145): Abstract No. 2505 (2004) (antibody CP-675206); Mokyr et al., Cancer Res., 58:5301-5304 (1998), and U.S. Pat. Nos. 5,977,318, 6,682,736, 7,109,003, and 7,132,281. A preferred clinical CTLA-4 antibody is human monoclonal antibody (also referred to as MDX-010 and Ipilimumab with CAS No. 477202-00-9 and available from Medarex, Inc., Bloomsbury, N.J.) is disclosed in WO 01/14424. With regard to CTLA-4 antagonist (antibodies), these are known and include Tremelimumab (CP-675,206) and Ipilimumab.

In some embodiments, the immunotherapy consists in administering to the patient a combination of a CTLA-4 antagonist and a PD-1 antagonist. Other immune-checkpoint inhibitors include lymphocyte activation gene-3 (LAG-3) inhibitors (Huo, Jin-Ling, et al. "The promising immune checkpoint LAG-3 in cancer immunotherapy: from basic research to clinical application. "Frontiers in Immunology 13 (2022)). In particular, LAG-3 inhibitors include IMP321 (Eftilagimod alpha), a soluble Ig fusion protein (Brignone et al., 2007, J. Immunol. 179:4202-4211) and anti-LAG3 monoclonal antibodies that include Relatlimab, Favezelimab, Tebotelimab, leramilimab, Fianlimab and TSR-033. Other immune- checkpoint inhibitors include B7 inhibitors, such as B7-H3 and B7-H4 inhibitors. In particular, the anti-B7-H3 antibody MGA271 (Loo et al., 2012, Clin. Cancer Res. July 15 (18) 3834). Also included are TIM-3 (T-cell immunoglobulin domain and mucin domain 3) inhibitors (Fourcade et al., 2010, J. Exp. Med. 207:2175-86 and Sakuishi et al., 2010, J. Exp. Med. 207:2187-94). Antibodies having specificity for TIM-3 are well known in the art and typically those described in WO2011155607, W02013006490 and WO2010117057. Other immune-checkpoint inhibitors include T cell immunoreceptor with Ig and ITIM domains (TIGIT) inhibitors (Ge, Zhouhong, et al. "TIGIT, the next step towards successful combination immune checkpoint therapy in cancer." Frontiers in Immunology 12 (2021): 699895.) such as anti-TIGIT monoclonal antibodies selected from the group consisting of BMS-986207, Tiragolumab, MK76-84, AB154, COM902, BGB-A1217, ASP8374, and M6223. Other immune-checkpoint inhibitors include Vista V-domain Ig suppressor of T cell activation (VISTA) inhibitors (Yum, Ji-Eun Irene, and Young-Kwon Hong. "Terminating Cancer by Blocking VISTA as a Novel Immunotherapy: Hasta la vista, baby. "Frontiers in Oncology 11 (2021): 658488) such as anti- VISTA inhibitors selected from the group consisting of CL8993 and CA-170.

A further object of the present invention relates to a method of treating cancer in a patient in need thereof comprising administering to the subject a therapeutically effective combination of immunotherapy with a therapeutically effective amount of a population of mesenchymal stromal cells of the present invention, wherein administration of the combination results in enhanced therapeutic efficacy relative to the administration of the immunotherapy alone.

As used herein, the expression "enhanced therapeutic efficacy" relative to cancer refers to a slowing or diminution of the growth of cancer cells or a solid tumor, or a reduction in the total number of cancer cells or total tumor burden. An "improved therapeutic outcome" or "enhanced therapeutic efficacy" therefore means there is an improvement in the condition of the patient according to any clinically acceptable criteria, including, for example, decreased tumor size, an increase in time to tumor progression, increased progression- free survival, increased overall survival time, an increase in life expectancy, or an improvement in quality of life. In particular, "improved" or "enhanced" refers to an improvement or enhancement of 1%, 5%, 10%, 25% 50%, 75%, 100%, or greater than 100% of any clinically acceptable indicator of therapeutic outcome or efficacy.

A further object of the present invention relates to a method of treating a cancer resistant to immunotherapy in a patient in need thereof comprising administering to the subject a therapeutically effective amount of a population of mesenchymal stromal cells of the present invention.

A further object of the present invention relates to a method of preventing resistance to immunotherapy in a patient suffering from a cancer comprising administering to the subject a therapeutically effective amount of a population of mesenchymal stromal cells of the present invention.

As used herein, the term “immunotherapy resistance” refers to an acquired resistance of a cancer to the immune response induced by the immunotherapy. Therefore, a resistant tumor or tumor cell is more likely to escape and survive humoral and/or cellular immune defense mechanisms in a subject receiving the immunotherapy. The phrase “overcoming immunotherapy resistance” in context of the invention shall be effective if compared to a non-treated control, the tumor or tumor cell becomes more sensitive to an immune response induced by immunotherapy. In particular, the patient become a responder. As used herein the term “responder” in the context of the present disclosure refers to a patient that will achieve a response, i.e. a patient where the cancer is eradicated, reduced or improved after immunotherapy. According to the invention, the responders have an objective response and therefore the term does not encompass patients having a stabilized cancer such that the disease is not progressing after immunotherapy. A “non-responder” or “refractory patient” includes patients for whom the cancer does not show reduction or improvement after immunotherapy. The term “non responder” also includes patients having a stabilized cancer. Typically, the characterization of the patient as a responder or non-responder can be performed by reference to a standard or a training set. The standard may be the profile of a patient who is known to be a responder or non-responder or alternatively may be a numerical value. Such predetermined standards may be provided in any suitable form, such as a printed list or diagram, computer software program, or other media. When it is concluded that the patient is a non-responder, the physician could take the decision to administer the agent that reduces or prevents the increase in ceramide levels. More particularly, the method of the present invention is particularly suitable for preventing tumor escape in a patient treated with immunotherapy. As used herein, the term “tumor escape” refers to any mechanism by which tumors escape the host's immune system.

As used herein the term "co-administering" as used herein means a process whereby the combination of immunotherapy and the population of mesenchymal stromal cells, is administered to the same patient. The immunotherapy and the population of mesenchymal stromal cells may be administered simultaneously, at essentially the same time, or sequentially. If administration takes place sequentially, the administration of the population of mesenchymal stromal cells is administered before the immunotherapy. The immunotherapy and the population of mesenchymal stromal cells need not be administered by means of the same vehicle. The immunotherapy and the population of mesenchymal stromal cells may be administered one or more times and the number of administrations of each component of the combination may be the same or different. In addition, the immunotherapy and the population of mesenchymal stromal cells need not be administered at the same site.

As used herein, the term "therapeutically effective combination" as used herein refers to an amount or dose of an immunotherapy together with the amount or dose of the population of mesenchymal stromal cells that is sufficient for treating the cancer.

The quantity of mesenchymal stromal cells to be administered will vary for the subject being treated. In some embodiments, between about 10⁴ and about IO¹⁰, between about 10⁵ and about 10⁹, or between about 10⁶ and about 10⁸ of the mesenchymal stromal cells are administered to the subject. More effective cells may be administered in even smaller numbers. In some embodiments, at least about I MO⁸, about 2* 10⁸, about 3* 10⁸, about 4* 10⁸, or about 5* 10⁸ of the mesenchymal stromal cells are administered to the subject. The precise determination of what would be considered an therapeutically effective amount may be based on factors individual to each subject, including their size, age, sex, weight, and condition of the particular subject. Dosages can be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art. Typically, the administration of the mesenchymal stromal cells of the present invention is preferably the intratumoral administration. The term “intratumoral administration” as used herein includes all techniques to deliver the population of cells to the site of a tumor including injection, or by any other means (microneedles, e.g.).

Pharmaceutical compositions:

In some embodiments, the mesenchymal stromal cells of the present invention are formulated by first harvesting them from their culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (i.e. a pharmaceutically acceptable carrier) in a treatment-effective amount.

Thus a further object of the present invention relates to a pharmaceutical composition comprising the population of mesenchymal stromal cells of the present invention and a pharmaceutically acceptable carrier.

Suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but also 5% dextrose in water or Ringer's lactate can be utilized. The infusion medium can be supplemented with human serum albumin. A treatment-effective amounts of cells in the composition is dependent on the relative representation of the mesenchymal stromal cells with the desired specificity, on the age and weight of the recipient, on the severity of the targeted condition and on the immunogenicity of the targeted Ags. These amounts of cells can be as low as approximately 10³/kg, preferably 5xlO³/kg; and as high as 10⁷/kg, preferably 10⁸/kg. The number of cells will depend upon the ultimate use for which the composition is intended, as will the type of cells included therein. For example, if cells that are specific for a particular Ag are desired, then the population will contain greater than 70%, generally greater than 80%, 85% and 90-95% of such cells. For uses provided herein, the cells are generally in a volume of a liter or less, can be 500 ml or less, even 250 ml or 100 ml or less. The clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed the desired total amount of cells.

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

Figure 1: Mesenchymal stromal cells (eMSC) engineered to express huFLT3L (eMSC- FLT3L)

(A) Plasmid to express membrane bound FLT3L and empty plasmid (eMSC-ctrl). (B) Surface expression of human membrane-bound FLT3L (huFLT3L) in mesenchymal stromal cells engineered (eMSC) engineered to express huFLT3L (eMSC-FLT3L) and control (eMSC-ctrl) assessed by flow cytometry using GFP and anti-FLT3L antibody.

Figure 2: Membrane bound FLT3L expressed in mesenchymal stromal cells supports DC differentiation from hematopoietic progenitors in vivo.

(A) Experimental strategy to assess the capability of stromal huFLT3L to drive DC differentiation in vivo. Hematopoietic stem and progenitor cells (HSPCs) isolated from CD45.1⁺ bone marrow cells were injected subcutaneously (s.c.) in CD45.2⁺ C57BL/6 mice along with eMSC-FLT3L or eMSC-ctrl or recombinant huFLT3L (rechuFLT3L, 30ug) or alone (Control) in a basement membrane extract preparation (Geltrex). Matrix plugs were harvested and analyzed at day 12 post-injection.

Quantifications of DC1 (B) and DC2 (C) in synthetic niches containing eMSC-FLT3L or eMSC-CTRL or rechuFLT3L or alone (Control) at day 12. (n = 4 plugs in one experiment. ***p < 0.001, ****p < 0.0001, one-way ANOVA test with Tukey’s multiple comparisons).

Figure 3: Membrane bound FLT3L expressed in mesenchymal stromal cells supports intra-tumoral DC differentiation from co-engrafted hematopoietic progenitors in B16- OVA-bearing mice.

(A) Experimental strategy to evaluate intra-tumoral dendritic cell differentiation HSPCs isolated from CD45.1⁺ bone marrow cells injected B16-OVA-bearing. Mice were injected subcutaneously (s.c.) with B16-OVA (5xl0⁵ cells/mice) on day 0. At day 7 and day 12 mice were injected into the tumor (i.t.) with eMSC-Ctrl (5xl0⁵ cells/mice) or eMSC-FLT3L-GFP (5xl0⁵ cells/mice) (left panel) or vehicle only (DMEM). At day 9, poly EC were injected i.t. with HSPCs isolated from CD45.1⁺ bone marrow. (B) Tumor weight at day 12. (C) frequency of CD45.1 DC1 and (D) absolute number per gram of tumor quantifications CD45.1 DC1. (n = 4-5 mice per groupe in one experiment. ***p < 0.001, ****p < 0.0001, one-way ANOVA test with Kruskal-Wallis multiple comparisons). Figure 4: Intra-tumoral engraftment of mesenchymal stromal cells expressing FLT3L alone does not impact on tumor growth.

(A) Experimental strategy. Mice were injected subcutaneously (s.c.) with B16 (5xl0⁵ cells/mice) or B16-huFL3L-GFP (5xl0⁵ cells/mice) on day 0. At day 7, eMSC-FLT3L (5xl0⁵ cells/mice) or eMSC-ctrl (5xl0⁵ cells/mice) were injected into the tumor (i.t.). (B) Tumor growth for experimental groups. Each line represents an individual mouse; n = 4-5 mice per group in one experiment; ****p < 0.0001, one-way ANOVA test with Tukey’s multiple comparisons).

Figure 5: Intra-tumoral engraftment of mesenchymal stromal cells expressing FLT3L alone does not modify DCs populations in tumors.

(A) Experimental strategy. Mice were injected subcutaneously (s.c.) with B16 (5xl0⁵ cells/mice) or B16-huFL3L-GFP (5xl0⁵ cells/mice) on day 0. At day 7, eMSC-FLT3L (5xl0⁵ cells/mice) or eMSC-ctrl (5xl0⁵ cells/mice) were injected into the tumor (i.t.). DC1 were analyzed on day 12 in the indicated organs.

(B-D) Quantification by flow cytometry of the absolute numbers of DC1 normalized to B16 group in tumor (B), skin tumor draining lymph nodes (C) and spleen (D). A line represents the mean; n = 4-5 mice per group in one experiment; ****p < 0.0001, one-way ANOVA test with Tukey’s multiple comparisons.

Figure 6: Intra-tumoral engraftment of mesenchymal stromal cells expressing FLT3L alone does not modify systemic levels of circulating FLT3L.

(A) Experimental strategy. Mice were injected subcutaneously (s.c.) with B16 (5xl0⁵ cells/mice) or B16-huFL3L-GFP (5xl0⁵ cells/mice) on day 0. At day 7, eMSC-FLT3L (5xl0⁵ cells/mice) or eMSC-ctrl (5xl0⁵ cells/mice) were injected into the tumor (i.t.).

(B) Serum huFLT3L levels were measured by ELISA on day 12. (n = 4-5 mice per group in one independent experiments. ****p < 0.0001, one-way ANOVA test with Tukey’s multiple comparisons.

Figure 7: Intra-tumoral engraftment of mesenchymal stromal cells expressing membrane bound FLT3L synergises with poly(I:C) to control tumor growth.

(A) Experimental strategy to evaluate antitumoral effects of eMSC-FLT3L and poly(I:C) approach in B16-OVA melanoma mice model. Mice were injected subcutaneously (s.c.) with B16-OVA (5xl0⁵ cells/mice) on day 0. At day 7 and day 12 mice were injected into the tumor (i.t.) with eMSC-FLT3L (5xl0⁵ cells/mice) or eMSC-ctrl (5xl0⁵ cells/mice) or vehicle only (DMEM). At day 9, mice were treated with i.t. with poly(I:C) 25pg/mouse or vehicle only.

(B) Tumor growth curve (C) Tumor size and (D) Tumor weight at day 15. Graph shows the mean tumor growth ± SD of three independent experiments (n = 10).

Figure 8: Intra-tumoral engraftment of autologous mesenchymal stromal cells expressing membrane bound FLT3L with poly(I:C)/anti-CD40 reduces primary tumor growth and lung metastasis

(A) Experimental design to compare aCD40 instead poly(I:C) in eMSC-FLT3L approach in B16-OVA melanoma mice model. Mice were injected subcutaneously (s.c.) with B16-OVA (5xl0⁵ cells/mice) on day 0. Mice were injected subcutaneously (s.c.) with B16-OVA (5xl0⁵ cells/mice) on day 0. At day 7 and day 12 mice were injected into the tumor (i.t.) with eMSC- FLT3L (5xl0⁵ cells/mice). At day 9, mice were treated with i.t. with poly(EC) 25pg/mouse or CD40 agonist 25pg/mouse. (B) Tumor growth curves and (C) tumor size at day 15. (D) Pulmonary metastasis is evaluated by counting the number of tumor nodules in the lungs (>0,5mm). Data from one experiment, n = 6-7 mice per group in one independent experiments. *p < 0.05, ***p < 0.001, one-way ANOVA test with Kruskal-Wallis for multiple comparisons.

Figure 9: Intra-tumoral engraftment of mesenchymal stromal cells expressing membrane bound FLT3L together with poly(I:C) increases survival rate.

(A-B) Experimental strategy to evaluate antitumoral effects of eMSC-FLT3L and poly(EC) approach in B16-OVA melanoma mice model. Mice were injected subcutaneously (s.c.) with B16-OVA (5xl0⁵ cells/mice) on day 0. At day 7 and day 12 mice were injected into the tumor (i.t.) with eMSC-FLT3L (5xl0⁵ cells/mice) or eMSC-ctrl (5xl0⁵ cells/mice) or vehicle only (DMEM). At day 9, mice were treated with i.t. with poly(EC) 25pg/mouse or vehicle only. For rechuFLT3L group, B16-OVA-tumor-bearing mice were injected intratumorally four times with rechuFLT3L lOpg/mice starting at day 7. (C) Tumor growth curve and survival curves. (D) Tumor weight at day 15. A line represents the mean; n = 10-17 mice per group in three experiments, p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA test with Tukey’s multiple comparisons. Overall survivals analyzed by log-rank (Mantel-Cox) test. Figure 10: Intra-tumoral engraftment of mesenchymal stromal cells expressing membrane bound FLT3L together with poly(I:C) improve the efficiency of immune checkpoint inhibition immunotherapy.

(A) Experimental strategy to evaluate synergia of eMSC-FLT3L approach associated with immune checkpoint inhibitor (ICI) therapy (anti-PDl/CTLA4). For ICI groups, B16-OVA- tumor-bearing (mice were injected intraperitoneally, every two days, with 200 pg (aPD-1 and aCTLA-4)/mice starting at day 7 (middle panel). (B) Tumor growth curve and survival curves. (C) Tumor weight at day 15. A line represents the mean; n = 7 mice per group in three experiments, p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA test with Tukey’s multiple comparisons. Overall survivals analyzed by log-rank (Mantel-Cox) test.

Figure 11: Intra-tumoral engraftment of mesenchymal stromal cells expressing membrane bound FLT3L together with poly(I:C) promotes leukocyte infiltration within tumors.

(A) Experimental design for the evaluation of the impact of eMSC-FLT3Land poly (EC) on the tumor microenvironment. Mice were injected subcutaneously (s.c.) with B16-OVA (5xl0⁵ cells/mice) on day 0. At day 7 and day 12 mice were injected into the tumor (i.t.) with eMSC- ctrl (5xl0⁵ cells/mice) or eMSC-ctrl (5xl0⁵ cells/mice) or vehicle only (DMEM). At day 9, mice were treated with i.t. with poly (EC) 25pg/mouse or vehicle only. Tumors were analyzed at day 10 or at day 15 (i.e. 24 hours after poly (EC) injection). (B-C) Quantification of the absolute numbers of Live CD45+ cells/mg of tumor at early (B) and late (C) time point. Pooled data from two independent experiments, n = 5-10 mice per group in one or two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA test with Tukey’s multiple comparisons.

Figure 12: Intra-tumoral engraftment of mesenchymal stromal cells expressing membrane bound FLT3L together with poly(I:C) promotes DC1 recruitment within tumors.

(A) Experimental design for the evaluation of the impact of eMSC-FLT3L and poly (EC) on the tumor microenvironment. Mice were injected subcutaneously (s.c.) with B16-OVA (5xl0⁵ cells/mice) on day 0. At day 7 and day 12 mice were injected into the tumor (i.t.) with eMSC- ctrl (5xl0⁵ cells/mice) or eMSC-ctrl (5xl0⁵ cells/mice) or vehicle only (DMEM). At day 9, mice were treated with i.t. with poly (EC) 25pg/mouse or vehicle only. Tumors were analyzed at day 15. (B) quantification of absolute numbers of DCl/g of tumor. Pooled data from two independent experiments, n = 5-10 mice per group in one or two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA test with Tukey’s multiple comparisons.

Figure 13: Intra-tumoral engraftment of mesenchymal stromal cells expressing membrane bound FLT3L together with poly(I:C) induces early DC1 activation within tumors.

(A) Experimental design for the evaluation of the impact of eMSC-FLT3L and poly (I:C) on the tumor microenvironment. Mice were injected subcutaneously (s.c.) with B16-OVA (5xl0⁵ cells/mice) on day 0. At day 7 and day 12 mice were injected into the tumor (i.t.) with eMSC- ctrl (5xl0⁵ cells/mice) or eMSC-ctrl (5xl0⁵ cells/mice) or vehicle only (DMEM). At day 9, mice were treated with i.t. with poly (I:C) 25pg/mouse or vehicle only. Tumors were analyzed at day 10 (i.e. 24 hours after poly (I:C) injection). (B-C) Mean fluorescence intensity (MFI) quantification of CD40 and CD86 expression on tumor DC1 at early time point (Day 10).

Pooled data from one independent experiment, n = 4 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA test with Tukey’s multiple comparisons. Overall survival analyzed by log-rank (Mantel-Cox) test.

Figure 14: Dendritic cells DC1 are essential to tumor control afforded by eMSC-FLT3L plus poly(I:C) immunotherapy.

(A) Experimental strategy to investigate the DC1 requirement for eMSC-FLT3L and poly (EC) immunotherapy efficacy in XC7?7^crexROSA^{loxSTOPloxDTA} (XCR1^DTA) mice, lacking DC1. (B-D) Tumor growth (B), tumor size at Day 15 (C) and overall survival (D) of B16-OVA-bearing in XCR1^DTA mice, lacking DC1, treated (orange) or not (pink). Pooled data from two independent experiments, n = 5-10 mice per group in one or two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANO VA test with Tukey’s multiple comparisons. Overall survival analyzed by log-rank (Mantel-Cox) test.

Figure 15: Intratumoral, eMSC-FLT3L delivery and poly(I:C) synergize to increase DC migration in tumor-draining lymph nodes

(A) Experimental design to evaluation of the impact of eMSC-FLT3L and poly(EC) on migratory DC1 at early time point (day 10) in tumor-draining Lymph Nodes. Mice were injected subcutaneously (s.c.) with B16-OVA (5xl0⁵ cells/mice) on day 0. At day 7 and day 12 mice were injected into the tumor (i.t.) with eMSC-FLT3L (5xl0⁵ cells/mice) or eMSC-ctrl (5xl0⁵ cells/mice) vehicle only (DMEM). At day 9, mice were treated with i.t. with poly (I:C) 25pg/mouse or vehicle only. Tumors were analyzed at day 10 (i.e. 24 hours after poly (I:C) injection). (B) Quantification of absolute numbers of migratory DC1 at early. (C-D) MFI quantification of CD40 (C) and CD86 (D) expression on migratory DC1 at early time point (DaylO). Pooled data from one independent experiment, n = 4 mice per group. *p < 0.05, **p

< 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA test with Tukey’s multiple comparisons. Overall survival analyzed by log-rank (Mantel-Cox) test.

Figure 16: Intratumoral, eMSC-FLT3L delivery and poly(I:C) triggers T cell activation in tumor-draining lymph nodes

(A) Experimental design to evaluation of the impact of eMSC-FLT3L and poly(EC) on migratory DC1 at early time point (day 10) in tumor-draining Lymph Nodes. Mice were injected subcutaneously (s.c.) with B16-OVA (5xl0⁵ cells/mice) on day 0. At day 7 and day 12 mice were injected into the tumor (i.t.) with eMSC-FLT3L (5xl0⁵ cells/mice) or eMSC-ctrl (5xl0⁵ cells/mice) or vehicle only (DMEM). At day 9, mice were treated with i.t. with poly (EC) 25pg/mouse or vehicle only. Tumors were analyzed at day 15. (B-C) Quantification (B) of Foxp3+ Regulatory T cells (Tregs) in a tumor-draining Lymph Nodes and (C) IFNy+CD8+ T cells (CD8+) at late time point (Day 15). (D) Ratio of tumor-infiltrating CD8+ T cell to Tregs cell. Line is the mean ± SD of one experiment (n = 4-5). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA test with Tukey’s multiple comparisons.

Figure 17: Intra-tumoral, eMSC-FLT3L delivery and poly(I:C) synergize to promote tumor infiltration by tumors-specific T cells

(A) Mice were injected subcutaneously (s.c.) with B16-OVA (5xl0⁵ cells/mice) on day 0. At day 7 and day 12 mice were injected into the tumor (i.t.) with eMSC-ctrl (5xl0⁵ cells/mice) or eMSC-FLT3L (5xl0⁵ cells/mice) (left panel) or vehicle only (DMEM). At day 9, mice were treated with i.t. with poly (EC) 25pg/mouse or vehicle only. Tumors were analyzed at day 10 or at day 15 (i.e. 24 hours after poly (EC) injection). (B) Quantification of the tumor-infiltrating CD8⁺ T cells at Day 15. (C) Absolute numbers quantification of intratumoral Tetramer+ cells normalized by eMSC-CTRL control group. Pooled data from two independent experiments, n = 4-8 mice per group in two independent experiments. (D) Quantification of tumor-infiltrating effector CD44⁺ IFNy⁺ CD8⁺ T cells after OVA-peptide, SIINFEKL, restimulation in presence of Brefeldin A. Line is the mean ± SD of one experiment (n = 4-5). *p < 0.05, **p < 0.01, ***p

< 0.001, ****p < 0.0001, one-way ANOVA test with Tukey’s multiple comparisons. Figure 18: Engraftments of eMSC-FLT3L synergizes with poly(I:C) to increase CD8+ effector to Treg ratio in B16-OVA tumor

(A) Experimental strategy of eMSC-FLT3L therapy in B16-OVA-bearing mice at Late Time Point. (B) Quantification of the tumor-infiltrating CD45⁺T absolute numbers per gram of tumor (C) Representative flow cytometry plots of intra-tumoral CD8+ and CD4+ activated cells (D) frequency of intra-tumoral CD8+CD44+ cells and (E) absolute numbers per gram of tumor of CD8+CD44+. (F) frequency of intra-tumoral CD8+CD44+ cells and (G) frequency ratio of intra-tumoral CD8+CD44+/ CD8+CD44+. (H) frequency of intra-tumoral CD4+Fopx3+ (Treg) and (I) frequency ratio of intra-tumoral CD8+CD44+/CD4+Fopx3+ (Treg). Data from one independent experiment, n = 4. mice per group, p < 0.05, **p < 0.01, ***p < 0.001, ****p

< 0.0001, one-way ANOVA test with Kruskal -Wallis multiple comparisons). (J) Representative flow cytometry plots of OVA-specific CD8⁺T Tetramer+ cells (K) Absolute numbers quantification normalized by eMSC-ctrl control group. (L) Ratio normalized by eMSC-ctrl control group. Pooled data from two independent experiments, n = 4-8 mice per group in two independent experiments, p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA test with Kruskal-Wallis multiple comparisons).

Figure 19: Intra-tumoral engraftment of autologous mesenchymal stromal cells expressing membrane bound FLT3L with poly(I:C) stimulates intra-tumoral infiltration of innate lymphocytes.

(A) Experimental strategy. (B) Representative flow cytometry plots of NK1.1 and ILC1 subsets in B16-OVA-bearing mice treated or not by eMSC-FLT3L + poly (I:C) approach. (C) quantification of absolute numbers per g of tumor of TCRb-NKl .1+ cells and (D) quantification of absolute numbers per g of tumor of TCRb-NKl. l+CD62L-CD49a+ ILC1 cells. Data from one independent experiment, n = 4. mice per group, p < 0.05, **p < 0.01, ***p < 0.001, ****p

< 0.0001, one-way ANOVA test with Kruskal -Wallis multiple comparisons).

Figure 20: Stromal cell therapy eMSC-FLT3L and poly(I:C) promotes the formation of memory CD44high CD8+ T cells specific for the OVA tumor antigen persisting in survivor mice.

(A) Experimental strategy to evaluate T cell memory generation. Survivor mice (from Fig.3E) that fully rejected B16-OVA tumor treated by eMSC-FLT3Land Poly(I:C) approach were challenged, 90 days after primary tumor injection, with s.c. SIINFEKL (Ipg/per mice) at the tumor site. Naive mice were injected s.c. with SIINFEKL as control. At day 3 after SIINFEKL challenge, skin-draining lymph nodes were analyzed.

(B-C) Representative flow cytometry plots (B) and quantification (C) of skin-draining Lymph nodes CD44⁺ CD8⁺ Tetramer⁺ cells after SIINFEKL OVA-peptide restimulation in vivo. (D) Representative flow cytometry plots of Tet+OVA+ CD8+ T cells (black dot) overlaid on total CD8+ T cell. Contour plot represent Effector memory T cells (Teff/EM). Line is the mean ± SD of one experiment (n = 3-6). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA test with Tukey’s multiple comparisons.

Figure 21: Antitumor effects of eMSC-FLT3L and poly(I:C) immunotherapy are dependent on CD8+ T, CD4+ T and NK cells.

(A) Experimental strategy to assess lymphocytes requirement for the therapy. B 16-OVA tumorbearing mice treated with eMSC-FLT3L and poly(LC) were injected intraperitoneally with depleting antibodies (anti-CD4, anti-CD8, or anti-NKl.l Ab or control IgG). (B) Tumor size at day 15. n = 6-7 mice per group in one independent experiments. **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA test with Tukey’s multiple comparisons).

(C) Overall survival, n = 6-7 mice per group in one independent experiments. Overall survival is analyzed by log-rank (Mantel-Cox) test.

Figure 22: Intra-tumoral engraftment of mesenchymal stromal cells expressing membrane bound FLT3L together with poly(I:C) induces intratumoral chemokine ligands for CXCR3 and CCR5 receptors.

(A) Experimental strategy: mice were injected subcutaneously (s.c.) with B16-OVA (5xl0⁵ cells/mice) on day 0. At day 7 and day 12 mice were injected into the tumor (i.t.) with eMSC- ctrl (5xl0⁵ cells/mice) or eMSC-FLT3L (5xl0⁵ cells/mice) (left panel) or vehicle only (DMEM). At day 9, mice were treated with i.t. with poly (EC) 25pg/mouse or vehicle only. Tumors were analyzed at day 10. (B) Bar graph showing the concentration of chemokines quantified on tumor homogenates by beads-based immunoassay. Individual selected chemokines of interest based on upregulated chemokine in eMSC-FLT3L + poly (I: C) found in volcano plot. Data from one independent experiment (n=4 mice per group). *p < 0.05, **p < 0.01, two-tailed Mann-Whitney test.

Figure 23: Tumour regression induced by eMSC-FL and poly(I:C) relies on CXCR3 engagement. (A) Experimental design for blocking of chemokine receptors CXCR3 (AMG487) 200 pg/mouse and/or CCR5 (Maraviroc) 500 pg/mouse mice by i.p. injection of antagonist or vehicle at days 7, 9, 12 and 13 days in B16-OVA tumor-bearing mice. (B and C) Tumor growth and tumor weight (n=9 mice/group). (D) quantification in absolute numbers per g of DC1 subset in tumor. Pooled data from two independent experiments. Data from two independent experiment (n = 8-9 mice per group), p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, p values were calculated using two-tailed Mann-Whitney test.

Figure 24: Engineering of mesenchymal stromal cells (eMSC) to express XCL1, CCL5 and CXCL9 chemokines

(A) Constructions of the huXCLl-GFP, huCCL5-GFP and huCXCL9-GFP plasmids. Inserts were cloned using the In-fusion cloning kit in a retroviral plasmid encoding the GFP fluorescent reporter. (B) Expression of GFP in stromal mesenchymal cells engineered to express human chemokines eMSC-XCLl, eMSC-CCL5 and eMSC-CXCL9 compared to control eMSC-ctrl.

(C) Quantification of huXCLl, huCCL5 and huCXCL9 chemokine by ELISA from MEF- engineered human chemokine in culture conditioned medium (24 hours after 80% confluence in 12 well plates). Data are shown as mean ± SD of triplicate.

Figure 25: Intra-tumoral, stromal cell-based delivery of FLT3L together with CXCL9, CCL5 or XCL1 chemokines induce tumor regression in the absence of poly(I:C)

(A) Evaluation of the cooperative antitumor effects of eMSC-FLT3Land eMSC-Chemokines in B 16-OVA tumor-bearing mice. Experimental design (D). Mice were inj ected subcutaneously (s.c.) with B16-OVA (5xl0⁵ cells/mice) on day 0. At day 7 and day 12 mice were injected into the tumor (i.t.) with eMSC-FLT3L (2.5xl0⁵ cells/mice) and eMSC-XCLl or eMSC-CCL5 or eMSC-CXCL9 (2.5xl0⁵ cells/mice). (B) Tumor growth and (C) tumor size at Dayl5 and overall survival (D). Data from one experiment, n = 5 mice per group in one independent experiments. *p < 0.05, ***p < 0.001, one-way ANOVA test with Tukey’s multiple comparisons. Overall survival is analyzed by log-rank (Mantel-Cox) test.

Figure 26: Intra-tumoral engraftment of autologous mesenchymal stromal cells expressing chemokines CXCL9 and CCL5 stimulates intra-tumoral infiltration of IL12- polarized T cell effectors.

(A) Schematic showing the experimental scheme IL12-Thl-CD45.1 T cells polarization and transwell migration assays. Chemokine-eMSC are culture in transwell lower compartment 24 hours before the migration assay. IL12-Thl-CD45.1 T cells polarized (IxlO⁵ cells/well) are transferred to the upper compartment of the transwell culture system, and then incubated at 37 C for 3 h. The migration index was obtained by calculating the percental number of CD4+. Or CD8+ T cells that migrated to the bottom chamber containing Chemokine-eMSC. (B) quantification of migration index CD4 + T cells and (C) quantification of migration index CD8 + T cells. Data are presented as mean and SD of one experiment three replicates, (p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA test with Kruskal-Wallis multiple comparisons). (D) Experimental design for adoptive Thl-CD45.1 T cells transfer in B16-OVA- bearing mice. Mice were injected subcutaneously (s.c.) with B16-OVA (5xl0⁵ cells/mice) on day 0. At day 7, 9 and day 12 mice were injected into the tumor (i.t.) with eMSC-Chemokine (IxlO⁶ cells/mice). At day 9, were injected i.v. with Thl-CD45.1 T cells (2xl0⁶ cells/mice).

(E) Tumors size and tumor weight at day 15. (F-G) Representative flow cytometry plots of expression on tumor and quantification in absolute numbers per g of tumor of CD45.1 CD8+ T cells (F) and CD45.1 CD4+ T (G). Data from one experiment (n=5 mice/group in one experiment; p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA test with Kruskal-Wallis multiple comparisons).

Figure 27: Engineered adipocyte mesenchymal stromal cell harboring FLT3L (eMSC- eAMSC-FLT3L) and poly(I:C) reduces tumor growth and tumor weight in B16-OVA bearing mice.

Engineered adipose derived mesenchymal stromal cell expressing membrane bound FLT3L (eMSC-eAMSC-FLT3L) and engineered fibroblasts mesenchymal stromal cell expressing membrane bound FLT3L (eMSC-eMEF-FLT3L) are challenged in B16-OVA melanoma mice model. (A) Experimental design. Mice were injected subcutaneously (s.c.) with B16-OVA (5xl0⁵ cells/mice) on day 0. At day 7 and day 12 mice were injected into the tumor (i.t.) with eMSC-eMEF-FLT3L (5xl0⁵ cells/mice) or eMSC-eAMSC-FLT3L (5xl0⁵ cells/mice). At day 9, mice were treated with i.t. with poly (I:C) 25pg/mouse or vehicle only. Tumors were analyzed at day 15. (B) Tumor growth and (C) tumor size at Day 15 and (D) tumor weight day 15. Data from one experiment, n = 5-8 mice per group in one independent experiments. *p < 0.05, ***p < 0.001, one-way ANOVA test with Kurskal-Wallis for multiple comparisons.

EXAMPLE:

Material & Methods Results

The datas are represented from figure 1 to figure 27.

Conventional type 1 dendritic cells (cDCl) cDCl infiltration in TME and tumor to lymph node trafficking correlates with favourable outcomes and clinical responses to immunotherapy. Depletion studies in preclinical models have revealed that cDCls play a key role in both CD8 + T-cell activation within the tumor microenvironment (TME). FLT3L is a growth factor for cDCls supporting their differentiation from HSCs, terminal differentiation within tissues, proliferation and survival. Here, we present a new immunotherapeutic approach based on local delivery of FLT3L within solid tumors. We show that intra-tumoral engraftment of autologous engineered mesenchymal stromal cells engineered to express membrane bound FLT3L (eMSC- FLT3L) efficiently stimulate cDCl infiltration when combined with the TLR3 agonist poly(I:C). Engraftment of eMSC-FLT3L supports sustained infiltration by DCs, promote lymph node trafficking and the cross priming of tumor-specific CD8+ T cells. As a result, eMSC- FLT3L + poly(I:C) therapy induces T cell-dependent tumor regression and circumvent resistance to CTLA4 and PD1 blockade characterizing the hard-to-treat B16 melanoma model. eMSC-FLT3L + poly(I:C) or CD40 agonist provides systemic anti-tumor immunity (metastasis control). Altogether, these data support the immunotherapeutic potential of intra-tumoral engraftment of engineered, autologous mesenchymal stromal cells to induce anti-tumor immunity.

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Claims

CLAIMS:

1. A mesenchymal stromal cell that is engineered to express a FLT3L membrane polypeptide provided that the mesenchymal stromal cell is not engineered to express a CXCL12 polypeptide nor a SCF polypeptide.

2. The mesenchymal stromal cell of claim 1 that is not engineered to express a CXCL12 polypeptide that comprises an amino acid having at least 80% of identity with the amino acid sequence as set forth in SEQ ID NO:8 and/or that is not engineered to express a SCF polypeptide that comprises an amino acid having at least 80% of identity with the amino acid sequence as set forth in SEQ ID NO:9.

3. The mesenchymal stromal cell of claim 1 according to claim 1 or 2 wherein the FLT3L membrane polypeptide comprises an amino acid sequence having at least 80% of identity with the amino acid sequence that ranges from the amino acid residue at position 27 to the amino acid residue at position 184 in SEQ ID NO: 1 or, an amino acid sequence having at least 80% of identity with the amino acid sequence that ranges from the amino acid residue at position 27 to the amino acid residue at position 205 in SEQ ID NO: 1 or, an amino acid sequence having at least 80% of identity with the amino acid sequence that ranges from the amino acid residue at position 27 to the amino acid residue at position 235 in SEQ ID NO: 1.

4. The mesenchymal stromal cell of claim 1 according to claim 3 wherein the proline residue at position 173 is mutated and/or the leucine residue at position 174 is mutated and/or the glutamic acid residue at position 175 is mutated.

5. The mesenchymal stromal cell of claim 1 according to claim 4 wherein the FLT3L membrane polypeptide comprises an amino acid sequence that ranges from the amino acid residue at position 27 to the amino acid residue at position 184 in SEQ ID NO: 11 or, an amino acid sequence that ranges from the amino acid residue at position 27 to the amino acid residue at position 205 in SEQ ID NO: 11 or, an amino acid sequence that ranges from the amino acid residue at position 27 to the amino acid residue at position 235 in SEQ ID NO: 11.

6. The mesenchymal stromal cell according to any one of claims 1 to 6 that is further engineered to express a GM-CSF polypeptide that comprises an amino acid sequence having at least 80% of identity with the amino acid sequence as set forth in SEQ ID NO:2.

7. The mesenchymal stromal cell according to any one of claims 1 to 7 that is further engineered to express at least one chemokine selected from the group consisting of CXCR3 ligands, CCR5 ligands, and XCR1 ligands.

8. The mesenchymal stromal cell according to claim 7 that is further engineered to express a CXCL9 polypeptide that comprises an amino acid sequence having at least 80% of identity with the amino acid sequence as set forth in SEQ ID NO:3.

9. The mesenchymal stromal cell according to claim 7 that is further engineered to express a CXCL11 polypeptide that comprises an amino acid sequence having at least 80% of identity with the amino acid sequence as set forth in SEQ ID NON.

10. The mesenchymal stromal cell according to claim 7 that is further engineered to express a CCL5 polypeptide that comprises an amino acid sequence having at least 80% of identity with the amino acid sequence as set forth in SEQ ID NO: 5.

11. The mesenchymal stromal cell according to claim 7 that is further engineered to express a CCL11 polypeptide that comprises an amino acid sequence having at least 80% of identity with the amino acid sequence as set forth in SEQ ID NO:6.

12. The mesenchymal stromal cell according to claim 7 that is further engineered to express a XCL1 polypeptide that comprises an amino acid sequence having at least 80% of identity with the amino acid sequence as set forth in SEQ ID NO:7.

13. The mesenchymal stromal cell according to any one of claims 7 to 12 that is further engineered to express the CD40L polypeptide that comprises an amino acid sequence having at least 80% of identity with the amino acid sequence as set forth in SEQ ID NO: 10.

14. A method of preparing the mesenchymal stromal cell according to any one of claims 1 to 13, comprising the step consisting of introducing into a mesenchymal stromal cell a polynucleotide that encodes for the FLT3L membrane polypeptide of the present invention and optionally one or more polynucleotide(s) that encode(s) for a GM-CSF polypeptide and/or at least one chemokine selected from the CXCR3 ligands, CCR5 ligands, and XCR1 ligands, and optionally one or more polynucleotide(s) that encodes a CD40L polypeptide.

15. A method of therapy in a subject in need thereof comprising administering to the patient in need thereof a therapeutically effective amount of the population of mesenchymal stromal cells according to any one of claims 1 to 13.

16. The method of claim 15 for the treatment of cancer, in particular cold tumors.

17. The method of claim 15 wherein the population of mesenchymal stromal cells is administered to the patient in combination with one or more adjuvant(s).

18. A method of enhancing the potency of immunotherapy administered to a patient suffering from a cancer as part of a treatment regimen, the method comprising administering to the subject a therapeutically effective amount of a population of mesenchymal stromal cells according to any one of claims 1 to 13 in combination with immunotherapy.

19. The method of claim 18 wherein the immunotherapy consists in administering the patient with at least one immune checkpoint inhibitor.

20. A method of treating cancer in a patient in need thereof comprising administering to the subject a therapeutically effective combination of immunotherapy with a therapeutically effective amount of a population of mesenchymal stromal cells according to any one of claims 1 to 13, wherein administration of the combination results in enhanced therapeutic efficacy relative to the administration of the immunotherapy alone.

21. A method of preventing resistance to immunotherapy in a patient suffering from a cancer comprising administering to the subject a therapeutically effective amount of a population of mesenchymal stromal cells according to any one of claims 1 to 13.

22. A pharmaceutical composition comprising the population of mesenchymal stromal cells according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier.