Tim-3 decoy secreted by engineered cells to improve immunotherapy

Abstract

The present invention refers to a cell, preferably a T cell, secreting a soluble fusion protein comprising the extracellular domain of TIM-3.

Description

[0001] Tim-3 decoy secreted by engineered cells to improve immunotherapy TECHNICAL FIELD

[0002] The present invention relates to the field of medicine. Particularly, the present invention relates to the field of therapies against cancer.

[0003] BACKGROUND ART

[0004] Immunotherapy is being recognized as one of the most promising treatments for cancer. However, the success rate of these treatments has been low and a large number of cancers, such as CD19+ B-cell malignancies, remain refractory. In particular, CAR-T-cell therapy has emerged as an effective therapeutic approach for patients with cancer, and a number of emerging strategies hold promise for improving CAR-T-cell function and persistence. These include optimizing the design of CAR constructs, increasing the proportion of memory T cells, altering the TME through cytokine signaling, and overcoming immune checkpoint inhibition with immune checkpoint blockers, such as T cell immunoglobulin and mucin domain-containing protein 3 (TIM3).

[0005] TIM3 is a member of the TIM family of immunoregulatory proteins. These are characterized by a common structural organization consisting of an amino-terminal immuno-globulin variable domain (V domain) with five non-canonical cysteines, a mucin stalk, a transmembrane domain and a cytoplasmic tail. Members of the TIM family are encoded by three genes in humans (HAVCR1, HAVCR2 and TIMD4, encoding TIM1, TIM3 and TIM4, respectively).

[0006] Of the TIM family, TIM3 has received the most attention because of its association with the regulation of immune responses in autoimmunity and cancer. Although it was originally identified as a molecule expressed by interferon-* (IFN*)-producing CD4+ and CD8+ T cells, many other cell types, including regulatory T cells (Treg cells), myeloid cells, natural killer (NK) cells and mast cells, have been shown to express TIM3. Tim-3 plays a key role in inhibiting Th1 responses and the expression of cytokines such as TNF and INF-*.

[0007] A major mechanism of cancer relapse is the suppression of T cells through the activation of immune checkpoint receptor (ICRs). Additionally, escape mechanisms of many cancers rely on the ability to upregulate immune checkpoint (IC) ligands in other cell types within the tumor microenvironment (TME), thus leading to T-cell exhaustion and loss of immunosurveillance. However, despite their importance in the development of more effective chemotherapeutics and immunotherapies, the expression of ICR in T cells and their corresponding ligands on blast cells and MSCs within the TME throughout cancer progression remains poorly characterized. In addition, CAR-T cells also express ICRs and are susceptible to T-cell intrinsic immunoregulatory exhaustion signals.

[0008] Decoys occur naturally in humans and are very useful for targeting receptors / ligands with redundant functions. Indeed, receptor-ligand promiscuity is a hallmark of the chemokine / cytokine network, but is also evident for ICRs, such as TIM-3, where the same receptor has multiple ligands that can bind to the same or distinct sites within the extracellular domain. However, the effect of the TIM-3 decoys in the context of CAR-T cells also remains unexplored, and new therapies that improve the efficacy of immunotherapy are needed.

[0009] BRIEF DESCRIPTION OF DRAWINGS

[0010] Fig. 1: Increased expression of the TIM-3-galectin-9 axis throughout disease progression in B-ALL. (A) Cartoon of the immune checkpoint receptors (ICRs) and their ligands expressed in cells. (B) Heatmap showing the median protein expression (analyzed by flow cytometry) of ICRs in CD4+and CD8+T cells (left panels) and ICR ligands in leukemic blasts and mesenchymal stromal cells (MSCs) (right panels) obtained from the BM of patients with B-ALL at diagnosis (Dx, n=47) and relapse (Rel, n=38), normalized to age-matched non-leukemic / healthy donor (HD) BM (HD, n=20). (C) Individual FACS mean fluorescence intensity (MFI) of TIM-3 expression in CD4+and CD8+T cells and its ligand galectin-9 in leukemic blasts and MSCs for each B-ALL / HD BM sample analyzed. Pediatric and adult patients are color-coded. The Mann-Whitney U-test was used to compare the expression of ICRs and ligands between patients and HDs. *p»0.05, **p* 0.01, ***p*0.001.

[0011] Fig.2: Galectin-9 impairs CAR19-T cells. (A) Cartoon depicting the in vitro experimental design of co-cultures of CAR19-T cells with B-ALL cells. (B) Expression levels of TIM-3 in CAR19-T cells (left panel) and galectin-9 in CAR19-T cell-resistant B-ALL cells (right panel) determined by FACS after 24h of CAR-T cell: B-ALL co-culture. (C, D) Apoptosis assay (Annexin-V / 7-AAD) and T cell proliferation measured by an eFluor 670 retention assay, after incubation of CAR19-T cells with or without rhGalectin-9 (1 • g / mL) for 48h. Left panels show representative FACS profiles. Right panels show average data. (E) CAR19-T cell-mediated cytotoxicity against SEM, REH and NALM6 B-ALL cells at 1:2 E: T ratio in the presence / absence of rhGalectin-9 (1 • g / mL) for 48h.

[0012] (F) Western blot detection of galectin-9 in the indicated non-transduced and galectin-9-overexpressing B-ALL cells (SEMGal9, REHGal9and NALM6Gal9). GAPDH was used as a housekeeping control. (G) CAR19-T cell-mediated cytotoxicity against non-transduced and galectin-9-overexpressing SEM, REH and NALM6 B-ALL cells at 1:8 E: T ratio for 48h. All data are shown as mean±SEM. Three independent experiments were performed with T cells from different donors. *p* 0.05, **p* 0.01, ***p* 0.001. Fig. 3: A TIM-3-Fc decoy secreted by primary T cells enhances CAR19-T cell efficacy and persistence in vivo. (A) Cartoon depicting the experimental strategy to block galectin-9 binding to TIM-3 on CAR19-T cells through a TIM-3-Fc decoy secreted by co-administered non-CAR-T cells. (B) Representative fluorescence microscopy image of activated untransduced (UT) and TIM-3-Fc transduced (dTomato+) primary T cells. (C) TIM-3 expression, quantified by FACS, on UT and TIM-3-Fc-T cells 7 days after transduction. (D) Proliferation of UT and TIM-3-Fc-T cells.

[0013] (E) Experimental design to compare CAR19 and CAR19+TIM-3-Fc-T cell persistence in vivo. NSG mice were injected i.v. with 1 x106B-ALL PDX cells (galectin-9+) and, 4 days later, a single i.v. injection of 2x106T cells was administered in mice (UT, TIM3Fc, CAR19 and CAR19+TIM-3-Fc, n=6 mice / group). 0.5x106CAR19+or / and 0.5x106TIM-3-Fc+cells were administered. Leukemia and T cell expansion were monitored over time. In vivo CAR19 and CAR19+TIM-3-Fc T cells were rechallenged with fresh B-ALL cells on day 25. (F) Representative FACS analysis of human B-ALL (hCD19+) and T cell (hCD3+) engraftment. (G) Follow-up of leukemic and T cell persistence in PB of treated mice over time. (H) Endpoint T cell quantification in PB, spleen and BM in the different groups of treated mice. (I) Experimental design to compare CAR19- and CAR19+TIM-3-Fc-T cell efficacy and persistence in vivo under stress conditions (1 x106T cells comprising 0.175x106CAR19+or / and 0.325x106TIM-3-Fc+, n=6 mice / group). Mice were followed up for 3 weeks. (J, K) Endpoint quantification of blasts (J) and T cells (K) in PB, spleen and BM in the indicated groups of treated mice. All data are expressed as mean±SEM. *p» 0.05, **p» 0.01.

[0014] Fig. 4: In vitro comparison of the T cell phenotype and cytotoxic activity of CAR19- and “all-in-one” CAR19-TIM-3-Fc-T cells. (A) Cartoon depicting the all-in-one bicistronic CAR19-F2A-TIM-3-Fc lentiviral construct used to block galectin-9 binding to TIM-3 on CAR19-T cells. (B) Transduction efficiencies (GFP+) in untransduced (UT), CAR19- and CAR19-TIM-3-Fc-T cells.

[0015] (C) TIM-3-Fc decoy released into the supernatants of UT, CAR19- and CAR19-TIM-3-Fc-T cells.

[0016] (D) Proliferation rate of UT, CAR19- and CAR19- TIM-3-Fc-T cells over time. (E, F) CD4 and CD8 distribution (E) and naive (CCR7+CD45RA+), central memory (CM, CCR7+CD45RA-), effector memory (EM, CCR7-CD45RA-) and terminally differentiated effector memory (EMRA, CCR7-CD45RA+) distribution (F) on UT and transduced (pos) and untransduced (neg) CAR19- and CAR19-TIM-3-Fc-T cells. (G, H) Expression of the ICRs TIM-3, LAG-3, and PD-1 (G) and the activation marker CD69 (H) on transduced (GFP+) CAR19 and CAR19-TIM-3-Fc CD4+and CD8+T cells 5 days post-transduction. (I) Galectin-9 expression by FACS on 4 independent primary B-ALL samples. (J) Representative FACS gating strategy to analyze cytotoxicity assays with UT, CAR19- or CAR19-TIM-3-Fc-T cells and primary B-ALL cells. (K) Absolute numbers of blasts, GFP+CAR19- and CAR19-TIM-3-Fc-T cells and total T cells as well as IFN* release after cytotoxicity assays with four different primary B-ALL cells at 1:8 E: T ratio during 48h. All data are shown as mean±SEM and n=3 independent experiments were performed with T cells from different donors. *p» 0.05, **p» 0.01, ****p.0.0001.

[0017] Fig. 5: Enhanced in vivo anti-leukemic efficacy of all-in-one CAR19-TIM-3-Fc-T cells in a B-ALL PDX stress model. (A) Experimental design to compare CAR19 and all-in-one bicistronic CAR19-TIM-3-Fc-T cell efficacy and persistence in vivo under stress conditions (1.5x106T cells comprising 0.175x106CAR19+or CAR19-TIM-3-Fc+T cells, n=5 mice / group). Leukemia engraftment and T cell expansion were monitored weekly for 4 weeks. (B) Mouse weight during the follow-up. (C) Detection of TIM-3-Fc decoy in the plasma of animals treated with CAR19- or bicistronic CAR19-TIM-3-Fc-T cells. (D) Endpoint quantification of blasts in PB, spleen and BM in the different groups of mice. (E) Total transduced (GFP+) T cells at endpoint in PB of animals treated with CAR19- or all-in-one CAR19-TIM-3-Fc-T cells. All data are shown as mean±SEM. *p« 0.05, ***p* 0.001, ns: not significant.

[0018] Fig. 6: T cell characterization of in vivo-expanded CAR19- and CAR19-TIM-3-Fc-T cells by spectral flow cytometry. (A) Cartoon of the experimental design for the identification of overexpanded T cell subpopulations by spectral flow cytometry in mice treated with CAR19- and CAR19-TIM-3-Fc-T cells. (B) UMAP visualization shows a total of 1,917,663 T cells, including both transduced (GFP+) and non-transduced (GFP ), identified in BM and spleen from all mice (n=10) treated with CAR19-TIM-3-Fc-T cells (n=5) and CAR19-T cells (n=5). Color-coded UMAPs identifying major T cell subsets, innate T cells, and functional and maturation-associated T cell subsets. (C) Volcano plots identifying T cell subpopulations differentially expanded in vivo between CAR19TIM3Fc and CAR19 groups in spleen (left panels) and BM (right panels) for transduced (GFP+) and non-transduced (GFP ) T cells. (D) Venn diagram identifies bystander (GFP ) MAIT CD4+T cells and memory CD27+CD4+CD8+T cells significantly expanded in vivo in both spleens and BM from mice treated with CAR19-TIM-3-Fc-T cells (represented in bold in panel C).

[0019] Fig.7: Representative FACS gating strategy for the detection of IC ligands in B cells (red cells) and ICRs in CD4+ (light blue) and CD8+ (blue) T cells from HD and B-ALL patients. Percentages of marker-positive cells are indicated.

[0020] Fig. 8: Representative FACS gating strategy for the detection of IC ligands in BM MSCs (magenta).

[0021] Fig. 9: Representative dot plots showing fluorescence minus one (FMO) FACS staining controls. Red, light blue and dark blue cells represent B-ALL cells, CD4+ and CD8+ T-cells, respectively. Fig. 10: Representative FACS gating strategy used to analyze in vitro the expression of TIM-3 and Galectin-9 in T-cells and B-ALL cells, respectively, after CAR-T cell cytotoxicity assays. Doublets were removed from the analysis. Alive target cells were identified as CD3-GFP-CD10+. CAR19-T cells were identified as CD3+GFP+.

[0022] Fig. 11: Expression levels (MFI) determined by FACS of ICRs (BTLA, CTLA-4, LAG-3 and PD-1) in CD8+ and CD4+ T cells and IC ligands (HVEM, CD80, CD86, MHC-II, PD-L1, PD-L2 and HMGB1) in CD19+ B-cells and MSCs for each B-ALL patient at diagnostic (n=47) and relapsed (n=38) and in age-matched non-leukemic / healthy BM samples (n=20). Pediatric and adult patients are color-coded. *p• 0.05, **p• 0.01, ***p• 0.001.

[0023] Fig. 12: (A) Scheme of the pcDNA3.1 TIM-3-Fc construct (TIM-3 decoy). (B) Soluble TIM-3 detection on supernatants of 293T cells transfected with TIM-3-Fc decoy. (C) Galectin-9 binding to TIM-3-Fc decoy. BSA conditions are used as negative controls.

[0024] Fig. 13: (A) Experimental design for ex vivo cytototoxicity assays with human T cells (CD3+ ) retrieved from mice treated with CAR19 (n=3) or CAR19+TIM-3-Fc (n=3) T cells, and PDX B-ALL cells at 1:1 E: T ratio for 24h. (B) Quantification of the absolute live blasts and T cells after the ex vivo cytotoxicity assay.

[0025] Fig 14: Three-year EFS of varni-cel-treated R / R patients with B-ALL (n = 37) based on low or high TIM-3 expression in CAR+ T cells in the infusion product. “Low” denotes patients with LGALS9 / TIM-3 levels below the median, and “high” denotes above the median.

[0026] GENERAL DEFINITIONS

[0027] It must be noted that, as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Further, unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0028] The term “about” when referred to a given amount or quantity indicates that a number can vary between ± 20 % around its indicated value. Preferably "about" means ± 15 % around its value, more preferably "about" means ± 10, 8, 6, 5, 4, 3, 2 % around its value, or even "about" means ± 1 % around its value, in that order of preference. As used herein, the conjunctive term "and / or" between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by "and / or", a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term "and / or" as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term "and / or."

[0029] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or "including" or sometimes when used herein with the term "having". Any of the aforementioned terms (comprising, containing, including, having), whenever used herein in the context of an aspect or embodiment of the present invention may be substituted with the term "consisting of", though less preferred.

[0030] When used herein "consisting of" excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0031] " Percent (%) of sequence identity" with respect to proteins or polypeptides described herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference sequence (i.e., the protein or polypeptide from which it is derived), after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for example, using publicly available computer software such as BLAST. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the full-length of the sequences being compared.

[0032] Preferably, the “percentage of identity” as used herein is decided in the context of a local alignment, i.e., it is based on the alignment of regions of local similarity between nucleobase sequences, contrary to a global alignment, which aims to align two sequences across their entire span. Thus, in the context of the present invention, percentage identity is calculated preferably only based on the local alignment comparison algorithm. DESCRIPTION OF THE EMBODIMENTS

[0033] The present invention shows a therapeutic strategy based on an engineered TIM-3-Fc decoy delivered either by primary T cells co-administered with CAR19-T cells or by the CAR19-T cells themselves through a bicistronic all-in-one CAR19-TIM-3-Fc construct. The data reveals that the presence of soluble TIM-3 increases CAR19-T cell persistence through autocrine and paracrine mechanisms. Importantly, the enhanced T-cell expansion improved the cytotoxicity effect of the CAR19 construct, especially the “all in one” CAR19-TIM-3-Fc construct, which was more active than CAR19-T cells alone, conferring a therapeutic advantage in vitro and in vivo. Because CAR19-T cells target CD19+blasts, the TIM-3-Fc decoy secreted locally to the site of disease by CAR19-TIM-3-Fc-T cells is likely to enhance the anti-leukemic effect compared to TIM-3 decoy secreted by other non-CAR T cells.

[0034] The strategy using CAR19-TIM-3-Fc-T cells that continuously secrete a TIM-3 decoy in vivo improves CAR19-T-cell persistence and efficacy in pre-clinical B-ALL PDX models. Furthermore, T cells engineered to secrete the TIM-3-Fc decoy may be extended to different CAR-T cells or other malignancies involving the TIM-3-galectin-9 axis.

[0035] In view of this, a first aspect of the present invention refers to a fusion protein, also called herein the “fusion protein of the invention”, comprising the extracellular domain of TIM-3. The fusion protein of the invention is a soluble protein and is secreted by a cell, also called the “cell of the invention”.

[0036] The term “fusion protein” refers to an artificial (i.e., not natural occurring) protein created through the joining of two or more genes or coding regions. A fusion protein comprises two or more domains that are artificial or originally coded by different genes in nature. The domains of a fusion protein have been joined, one after the other, so that they are synthetized or translated as a single unit, and thus the two domains of the fusion protein are part of a single polypeptide. The term “domain” refers to distinct functional and / or structural units in a protein, and they are responsible for a particular function or interaction, contributing to the overall role of a protein.

[0037] The term “soluble protein” in the context of the fusion protein of the invention refers to a protein that is not part of the cytosol of a cell, but that is secreted from the cell of the invention to the extracellular media. Thus, the cell of the invention is capable of producing and / or secreting the fusion protein of the invention to the extracellular media.

[0038] “T-cell immunoglobulin domain and mucin domain 3” or “TIM-3” refers to a native T cell immunoglobulin mucin 3 (TIM3) protein (also known as hepatitis A virus cellular receptor 2 (HAVcr-2), kidney injury molecule-3 (KIM-3), TIM-3, Tim3, or Tim-3) from any vertebrate source, including mammals such as primates (preferably humans) and rodents (e.g., mice and rats), unless otherwise indicated. TIM-3 is a transmembrane protein composed of an N-terminal immunoglobulin variable (IgV) domain, as well as mucin stalk, transmembrane, and C-terminal cytoplasmic tail domains. The term TIM-3 as used herein encompasses naturally occurring variants of TIM3, e.g., splice variants or allelic variants.

[0039] By “extracellular domain of TIM-3” or “ECD of TIM-3” is referred herein to a soluble form of TIM-3 lacking the transmembrane and intracellular regions. Preferably, the ECD of TIM-3 refers to amino acids 22-200 of the human TIM-3 wildtype protein. More preferably, the amino acid sequence of the Extracellular Domain (ECD) of TIM3 comprises or consists of SEQ ID NO: 4, or a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 4. It is noted that here, and thorough the whole document, the positions or locations of the amino acid residues of a protein or a polypeptide sequence are numbered sequentially starting from the first amino acid residue which would then be located at position 1. For example, a protein of 137 amino acids will have those residues numbered 1 (first amino acid residue) until 137 (the last amino acid residue). Preferably, the first position or position number 1 corresponds to the first amino acid synthetized and located at the nitrogen-end (N terminal) of the polypeptide chain, corresponding to the DNA 5-prime (5') end, while the last position corresponds to the carboxyl-end (C terminal) of the protein or polypeptide. In the particular case of the ECD of TIM-3, the amino acid numbering (22-200) referred herein correspond to the mature protein sequence after the proteolysis occurring between residues 21 and 22 during secretion.

[0040] In an embodiment, the fusion protein of the invention further comprises a multimerization domain, so that the protein of the invention is capable of forming multimers with other proteins of the invention. Preferably, the multimerization domain is a dimerization domain, so that the protein of the invention is capable of forming dimers with other proteins of the invention. The ability of forming multimers, such as dimers, can be easily tested by a skilled person using techniques known in the art, such non-denaturing chromatography, or size exclusion chromatography. Preferably, the extracellular TIM-3 domain is placed in the N-terminal of the multimerization domain.

[0041] In an embodiment, the dimerization domain comprises or consists of an immunoglobulin Fc domain or a Fc fragment. The term “Fc domain” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region, and may also contain the hinge region. The terms “Fc region” or “Fc chain” or “Fc domain” as used herein is preferably meant the polypeptide comprising the CH2-CH3 domains of an IgG molecule, and preferably, inclusive of the hinge region. In Ell numbering for human IgG 1, the CH2-CH3 domain comprises amino acids 231 to 447, and the hinge region is 216 to 230. Thus the definition of “Fc domain” preferably includes both amino acids 231-447 (CH2-CH3) or 216-447 (hinge-CH2-CH3), or fragments thereof. The amino acid residue numbers of the antibody Fc domain described herein follow the Kabat EU numbering system commonly used in the art, unless specified otherwise.

[0042] An “Fc fragment” in this context can contain fewer amino acids from either or both of the N- and C-termini but still retains the ability to form a dimer with another Fc region as can be detected using standard methods, generally based on size (e.g., non-denaturing chromatography, size exclusion chromatography).

[0043] The Fc domain or fragment can be derived from any suitable class of antibody, including IgA (including subclasses lgA1 and lgA2), IgD, IgE, IgG (including subclasses lgG1, lgG2, lgG3 and lgG4), and IgM. In one embodiment, the Fc domain is a lgG1, lgG2, lgG3 or lgG4. Human IgG Fc regions are of particular use in the present invention, and can be the Fc region from human lgG1, lgG2 or lgG4. Preferably, the Fc domain is a human IgG Fc domain. In one embodiment, the Fc domain is a human IgG 1 Fc domain.

[0044] Preferably, the Fc domain comprises or consists of SEQ ID NO: 6 or a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 6.

[0045] As explained above, the Fc domain preferably comprises a hinge region, which is preferably located in the N-terminal of the Fc domain. A hinge region would normally be found between Fab and Fc regions in a naturally occurring antibody. As explained above, a Fc domain comprising a hinge spans from 216-447 amino acids (hinge-CH2-CH3 regions). The hinge can include hinge regions from other species, such as human, mouse, rat, rabbit, shark, pig, hamster, camel, llama or goat hinge regions.

[0046] In an embodiment, the Fc domain comprised in the fusion protein of the invention is a Fc domain, preferably comprising the hinge region, that has abrogated Fc receptor binding, also called herein a silent constant fragment (Fc) domain or “silent Fc domain”. Thus, the term “silent Fc domain” in the context of the present invention refers to an Fc, preferably human lgG1 Fc, domain that comprises one or more modifications such that the silent Fc domain binds to FcRn with lower affinity and avidity than the corresponding native Fc, preferably human lgG1 Fc, domain. Said modifications may be in the hinge region or in the constant region (CH2-CH3) of the silent Fc domain. Preferably, the hinge domain comprised in the silent Fc has abrogated Fc receptor binding in comparison with the corresponding native human hinge domain. Preferably, the silent Fc domain has reduced binding ability to Fc*Rllla compared to a wild-type antibody Fc domain. Silent Fc domains with "reduced", "silenced", "abrogated", “removed” or "negligible" Fc*R binding affinity and C1q binding affinity are compared to the parent polypeptide or a polypeptide comprising the corresponding wild-type Fc region domain. " Parent polypeptide" or "polypeptide comprising a wild-type Fc region" refers to a construct that does not contain amino acid modifications that cause the reduced Fc*R binding affinity. In one embodiment, the parent polypeptide is one that does not contain amino acid modifications to the hinge region and does contain modifications to the CH3 domain that promote formation of a heterodimeric Fc region.

[0047] Such silent Fc domains that exhibit reduced binding to Fc* R may retain little or no appreciable binding to Fc*R. In one embodiment, the silent Fc domain exhibits 0-10% binding to an Fc*R relative to a native IgG Fc region. In one embodiment, silent Fc domain exhibits 0-5% binding to an Fc* R compared to a native IgG Fc region. In one embodiment, silent Fc domain exhibits 0-3% binding to an Fc* R compared to a native IgG Fc region. In one embodiment, the silent Fc domain exhibits 0-1% binding to an Fc*R relative to a native IgG Fc region. Preferably, the silent Fc domain described herein has an increased in vivo half-life compared to the corresponding wildtype antibody Fc domain.

[0048] Silenced Fc domains can be obtained by mutating the Fc and / or the hinge regions, and have been described in the art. Examples of silent Fc lgG1 antibodies comprise the so-called LALA mutant comprising L234A and L235A mutation in the IgG 1 hinge amino acid sequence, according to the Kabat EU numbering system. Another example of a silent lgG1 antibody comprises the D265A mutation. Another silent lgG1 antibody comprises the N297A mutation, which results in aglycosylated / non-glycosylated antibodies. " Amino acid modification" refers to a change in the amino acid sequence of a predetermined (usually a wild-type) amino acid sequence. Exemplary modifications include amino acid substitutions, insertions and / or deletions. In certain embodiments, an amino acid modification herein is a substitution.

[0049] Preferably, the Fc domain is a silent human IgG, preferably IgG 1, Fc domain that has abrogated Fc receptor binding in comparison with the corresponding native Fc domain.

[0050] In one embodiment, the silent Fc domain comprised in the fusion protein of the invention comprises a substitution of the leucine residue at position 234 with an alanine residue (L234A), according to the Kabat EU numbering system. In one embodiment, the silent Fc domain comprised in the fusion protein of the invention comprises a substitution of the leucine residue at position 235 with an alanine residue (L235A), according to the Kabat EU numbering system. In one embodiment, the silent Fc domain comprised in the fusion protein of the invention comprises a substitution of the proline residue at position 329 with a glycine residue (P329G), according to the Kabat EU numbering system. Preferably, the silent Fc domain comprised in the fusion protein of the invention comprises LALA (L234A and L235A) mutations, and a P329G mutation (also called herein LALA PG mutation), according to the Kabat EU numbering system. Preferably, the silent Fc domain comprised in the fusion protein of the invention comprises one or more amino acid substitutions selected from the group consisting of L234A, L235A, and P329G according to the Kabat EU numbering system in the Fc domain of the wild type antibody.

[0051] Preferably, the hinge comprised in the silent Fc comprises or consists of SEQ ID NO: 7, or a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 7.

[0052] Preferably, the silent Fc domain has LALAPG mutations and thus has abrogated Fc receptor binding in comparison with the corresponding native FC domain, and comprises or consists of SEQ ID NO: 5, or a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 5. It is noted that the LALAPG mutations (L234A, L235A, and P329G mutations according to the Kabat EU numbering system) in the context of SEQ ID NO: 5 correspond to L18A, L19A and P113G mutations, respectively. Hence, in a preferred embodiment, the silent FC domain comprises or consists of SEQ ID NO: 5, or a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 5, with the proviso that the amino acid residues in position number 18, 19 and 113 in SEQ ID NO: 5, are amino acid residues Ala, Ala, and Gly, respectively.

[0053] In a preferred embodiment, the fusion protein of the invention comprises at least two domains, wherein a first domain comprises or consists of the extracellular TIM-3 domain, and the second domain comprises or consists of a silent Fc domain as defined above. Preferably, the extracellular TIM-3 domain is placed in the N-terminal of the silent Fc domain. Preferably, the fusion protein comprises, in the N-terminal to C-terminal, the extracellular (ECD) TIM-3 domain (residues 22-200), and a silent Fc domain.

[0054] As explained above, the fusion protein of the invention is soluble, i.e., it is secretable from the cell of the invention to the extracellular media. Methods to produce soluble proteins are known in the art, and include designing the fusion protein with a leader or signal peptide that leads the fusion protein throughout the cytosol and / or across intracellular membrane to the extracellular media. In an embodiment, the fusion protein comprises a leader peptide that actively transport the fusion protein of the invention out of the cell. In an embodiment, the leader peptide is the endogenous signal peptide or the TIM-3 protein, preferably human TIM-3 protein. Preferably, the signal peptide comprised in the fusion protein comprises or consists of SEQ ID NO: 1, or a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 1.

[0055] The fusion protein of the invention may further comprise other domains or polypeptides, such as tags or peptide linkers. In an embodiment, the fusion protein comprises a tag, preferably a hist tag, preferably a tag comprising or consisting of SEQ ID NO: 8, or a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 8. Preferably, the tag is placed in the C-terminal of the fusion protein of the invention.

[0056] In an embodiment, the extracellular domain TIM-3 is connected to the multimerization domain, preferably dimerization domain, more preferably Fc domain or silent FC domain, by means of a linker. Suitable nonimmunogenic linker peptides include (Gly4-Ser)n, (Ser-Gly4)n, (Gly4-S)n, Gly4(Ser-Gly4)n or Gly2(Ser-Gly2)n linker peptides, wherein n is generally a number between 1 and 10, typically between 1 and 4. In a preferred embodiment, said linker is a peptide linker consisting of the amino acid sequence selected from the group consisting of (Gly4-Ser)n, (Ser-Gly4)n, (Gly4-S)n, Gly4(Ser- Gly4)n, Gly2(Ser-Gly2)n, wherein n is a value from 1 to 10. In a more preferred embodiment, said linker is a peptide linker consisting of the amino acid sequence (Gly4-Ser)n, wherein n is a value from 1 to 10. In a yet more preferred embodiment, n is a value from 1 to 4.

[0057] In an embodiment, the linker comprises or consists of SEQ ID NO: 10, or a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 10.

[0058] Preferably, the fusion protein of the invention comprises:

[0059] - a first domain comprising or consisting of SEQ ID NO: 4, or a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 4, and

[0060] - a second domain comprising or consisting of SEQ ID NO: 5, or a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 5, with the proviso that the amino acid residues in position number 18, 19 and 113 in SEQ ID NO: 5, are amino acid residues Ala, Ala, and Gly, respectively.

[0061] Preferably, the fusion protein of the invention comprises, in the N-terminal to C-terminal direction:

[0062] - a leader peptide, preferably comprising or consisting of SEQ ID NO: 1, - a first domain comprising or consisting of SEQ ID NO: 4, or a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 4, and

[0063] - a second domain comprising or consisting of SEQ ID NO: 5, or a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 5 with the proviso that the amino acid residues in position number 18, 19 and 113 in SEQ ID NO: 5, are amino acid residues Ala, Ala, and Gly, respectively,

[0064] wherein the first and second domains are connected by a linker, preferably a linker comprising or consisting of SEQ ID NO: 10.

[0065] Preferably, the fusion protein of the invention comprises, in the N-terminal to C-terminal direction:

[0066] - a leader peptide, preferably comprising or consisting of SEQ ID NO: 1,

[0067] - a first domain comprising or consisting of SEQ ID NO: 4, or a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 4,

[0068] a linker, preferably comprising or consisting of SEQ ID NO: 10,

[0069] - a hinge, preferably comprising or consisting of SEQ ID NO: 7, and

[0070] - a second domain comprising or consisting of SEQ ID NO: 6, or a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 6.

[0071] Preferably, the fusion protein of the invention comprises or consists of SEQ ID NO: 2, or a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 2.

[0072] Preferably, the fusion protein of the invention comprises or consists of SEQ ID NO: 3, or a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 3

[0073] In an embodiment, the fusion protein of the invention comprises or consists of SEQ ID NO: 9, or a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 9.

[0074] As explained above, the cell of the invention is capable of secreting the soluble fusion protein of the invention. The cell of the invention is preferably a mammal cell, more preferably a human cell. Preferably, the cell of the invention is a cell from the human immune system, more preferably a T cell. In some embodiments, the cell is a naive T cell, memory stem T cell, effector T cell, or central memory T cell. Preferably, the cell is a mesenchymal stem cell (MSC).

[0075] In some embodiments, the cell is an autologous T cell. The term “autologous cell” refers to a cell obtained from the same patient that is to be treated using any one of the methods of the present invention.

[0076] In some embodiments, the cell is an allo-tolerant T cell. The term “allo-tolerant cell” refers to a cell that has been engineered to decrease the risk of a Graft-versus-host disease response.

[0077] In some embodiments, the T cell is a CD3-positive T cell. In some embodiments, the T cell is a CD8+ T cell.

[0078] In some embodiments, the cell is a lymphoid precursor, embryonic stem cell or an induced pluripotent stem cell with the capacity to differentiate into a mature T cell.

[0079] In an embodiment, the cell of the invention is a not naturally occurring cell that has been genetically modified to produce and secrete the fusion protein of the invention.

[0080] In an embodiment, the cell and / or the protein of the invention is administrated or used in combination with a targeting moiety capable of binding to a tumour associated antigen.

[0081] By “capable of binding a tumour associated antigen” is meant that the targeting moiety has binding affinity for a tumour associated antigen. Preferably, the binding affinity is specific, i.e., the targeting moiety is capable of specifically binding to a tumour associated antigen (which is its target molecule) in a manner which distinguishes from the binding to non-target molecules, i.e., from other molecules that are not the tumour associated antigen. Thus, the targeting moiety either does not bind to non-target molecules or exhibits negligible or substantially-reduced (as compared to the target) e. g. background, binding to nontarget molecules. Specific binding between the targeting moiety and a tumour associated antigen can be assessed by numerous techniques, such as surface plasmon resonance, flow cytometry, or ELISA assays.

[0082] Thus, a preferred embodiment of the first aspect refers to a combination therapy in which the cell and / or the fusion protein of the invention is combined with a targeting moiety capable of binding to a tumour associated antigen. The targeting moiety that is capable of binding to a tumour associated antigen can be administered simultaneously or sequentially with the cell or the fusion protein of the invention. " Combination therapy", “in combination with” or “in conjunction with” as used herein denotes any form of concurrent, parallel, simultaneous, sequential or intermittent treatment with at least two distinct treatment modalities (i.e., a CAR and the cell or fusion protein of the invention). As such, the terms refer to administration of one treatment modality before, during, or after administration of the other treatment modality to the subject. The modalities in combination can be administered in any order. The therapeutically active modalities are administered together (e.g., simultaneously in the same or separate compositions, formulations or unit dosage forms) or separately (e.g., on the same day or on different days and in any order as according to an appropriate dosing protocol for the separate compositions, formulations or unit dosage forms) in a manner and dosing regimen prescribed by a medical care taker or according to a regulatory agency. In general, each treatment modality will be administered at a dose and / or on a time schedule determined for that treatment modality. A combination therapy is also referred to as combination product. Thus, in an embodiment, the first aspect of the present invention provides a combination product comprising the cell and / or the fusion protein of invention, and a targeting moiety capable of binding to a tumour associated antigen. The products of the combination product can be administered in any form or order.

[0083] Preferably, the targeting moiety that is combined with the cell and / or the protein of the invention is an antibody, anticalin, repebody, monobody, scFv, Fab, scFab, affibody, fynomer, DARPin, nanobody, or peptide aptamer that specifically binds to a tumour associated antigen.

[0084] The term "antibody" refers to a molecule comprising at least one immunoglobulin domain that binds to, or is immunologically reactive with, a particular target. The term includes whole antibodies and any antigen binding portion or single chains thereof and combinations thereof; for instance, the term “antibody” in particular includes bivalent antibodies and bivalent bispecific antibodies. The term "bispecific antibody" refers to an antibody having two different antigenbinding regions defined by different antibody sequences. This can be understood as different target binding but includes as well binding to different epitopes in one target.

[0085] A typical type of antibody comprises at least two heavy chains (" HC") and two light chains (" LC") interconnected by disulfide bonds.

[0086] Each "heavy chain" comprises a "heavy chain variable domain" (abbreviated herein as " VH") and a "heavy chain constant domain" (abbreviated herein as " CH"). The heavy chain constant domain typically comprises three constants domains, CH1, CH2, and CH3. Each "light chain" comprises a "light chain variable domain" (abbreviated herein as " VL") and a "light chain constant domain" (" CL"). The light chain constant domain (CL) can be of the kappa type or of the lambda type. The VH and VL domains can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (" CDR"), interspersed with regions that are more conserved, termed "framework regions" (" FW"). In molecular biology, a framework region is a subdivision of the variable region (Fab) of the antibody. The variable region is composed of seven regions, four of which are framework regions and three of which are hypervariable regions. The framework region makes up about 85% of the variable region, and are responsible for acting as a scaffold for the CDR regions. These CDRs are in direct contact with the antigen and are involved in binding antigen, while the framework regions support the binding of the CDR to the antigen and may aid in maintaining the overall structure of the four variable domains on the antibody.

[0087] Each VH and VL is composed of three CDRs and four FWs, arranged from amino-terminus to carboxy-terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. The present disclosure inter alia presents VH and VL sequences as well as the subsequences corresponding to CDR1, CDR2, and CDR3.

[0088] The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991 ), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme).

[0089] The variable domains of the heavy and light chains contain a region that interacts with a binding target, and this region interacting with a binding target is also referred to as an “antigen-binding site” or “antigen binding site” herein. The constant domains of the antibodies can mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1 q) of the classical complement system. Exemplary antibodies include typical antibodies, but also bivalent fragments and variations thereof such as a F(ab’)2.

[0090] Thus, as used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, bivalent antibody fragments (such as F(ab')2), multispecific antibodies such as bispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, and any other modified immunoglobulin molecule comprising an antigen binding site.

[0091] An antibody can be of any the five major classes (isotypes) of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses thereof (e.g. lgG1, lgG2, lgG3, lgG4, lgA1 and lgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules such as therapeutic agents or diagnostic agents to form immunoconjugates.

[0092] The term “anticalin” refers to a protein that is derived from the lipocalin and that been engineered to bind to a specific target.

[0093] The term “repebody” refers to a protein that is derived from a leucine-rich repeat module and that been engineered to bind to a specific target.

[0094] The term “monobody” refers to a protein that is derived from a fibronectin type III domain that has been engineered to bind to a specific target.

[0095] The term “single-chain variable fragment” or “scFv” refers to a fusion protein comprising the variable domains of the heavy chain and light chain of an antibody linked to one another with a peptide linker. The term also includes a disulfide stabilized Fv (dsFv).

[0096] The term “antigen-binding fragment” or “Fab” refers to an antibody fragment comprising one constant and one variable domain of each of the heavy and light chain. A Fab fragment may be obtained by digesting an intact monoclonal antibody with papain.

[0097] The term “single-chain antigen-binding fragment” or “scFab” refers to a fusion protein comprising one variable and one constant domain of the light chain of an antibody attached to one variable and one constant domain of the heavy chain of an antibody, wherein the heavy and light chains are linked together through a short peptide.

[0098] The term “affibody” refers to a protein that is derived from the Z domain of protein A and that been engineered to bind to a specific target.

[0099] The term “fynomer” refers to a protein that is derived from the SH3 domain of human Fyn kinase that has been engineered to bind to a specific target.

[0100] The term “designed ankyrin repeat proteins” or “DARPin” refers to a protein that is derived from an ankyrin repeat that has been engineered to bind to a specific target.

[0101] The term “nanobody” refers to a protein comprising the soluble single antigen-binding V-domain of a heavy chain antibody, preferably a camelid heavy chain antibody. The term “peptide aptamer” refers to a short, 5-20 amino acid residue sequence that can bind to a specific target. Peptide aptamers are typically inserted within a loop region of a stable protein scaffold.

[0102] In a preferred embodiment, the targeting moiety is an antibody, F(ab’)2, Fab, preferably scFv, or scFab comprising at least a VL domain and VH domain. Preferably, the targeting moiety is a CD19-targeting moiety.

[0103] The term “CD19” refers to the Cluster of Differentiation 19 protein, which is an antigenic determinant detectable on leukemia precursor cells. CD19 is expressed on most B lineage cancers, including, e.g., acute lymphoblastic leukaemia, chronic lymphocyte leukaemia and nonHodgkin's lymphoma. Other cells with express CD19 are provided below in the definition of “disease associated with expression of CD19.” It is also an early marker of B cell progenitors. The term “CD19-targeting moiety” or “CD19 targeting moiety” refers to a substance that is able to bind CD19, preferably human CD19. Within the context of a CAR, a CD19-targeting moiety targets T cells to a CD19-positive cell, preferably a CD19+ cancer cell.

[0104] Preferably, the CD19-targeting moiety comprises or consists of the CD19-targeting moieties disclosed in PCT / EP2022 / 062374, which comprise CDRS of SEQ ID NOs 1-6 as defined in PCT / EP2022 / 062374. Preferably, the CD19-targeting moiety comprises or consists of the VL and a VH as defined by SEQ ID NOs 7 and 8, respectively, in PCT / EP2022 / 062374.

[0105] Preferably, the targeting moiety combined with the fusion protein or the cell of the invention is an antibody is a bispecific antibody, preferably an anti-CD19 / anti-CD3 bispecific antibody. An “anti-CD19 / anti-CD3 bispecific antibody” refers to a bispecific antibody comprising two different antigen-binding regions, one of which binds specifically to the antigen CD19 and one of which binds specifically to CD3. Preferably, the anti-CD19 / anti-CD3 bispecific antibody is the one defined in WO2021165248A1.

[0106] Also preferably, the targeting moiety combined with the fusion protein and / or the cell of the invention is part or is comprised in a chimeric antigen receptor (CAR). Preferably, the CAR comprises:

[0107] a) an extracellular domain comprising a tumour associated targeting moiety as defined in the first aspect or in any of the embodiments disclosed above,

[0108] b) a transmembrane domain; and

[0109] c) an intracellular signaling domain. Preferably, the extracellular domain of the CAR comprises a tumour associated targeting moiety that is a scFv comprising a VL domain and VH domain, wherein said VL domain comprises LCDR1, LCDR2 and LCDR3 polypeptides and said VH domain comprises HCDR1, HCDR2 and HCDR3 polypeptides. Preferably, the extracellular domain of the CAR comprises a CD19-targeting moiety.

[0110] The transmembrane domain of the CAR may be derived either from a natural or a synthetic source. When the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions may comprise at least the transmembrane region(s) of the α-, β- or γ- chain of CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154, or a variant thereof, wherein the variant thereof has a 95% sequence identity.

[0111] In some embodiments, the transmembrane domain comprises the transmembrane domain of CD8 or a variant thereof, wherein the variant thereof has a 98% sequence identity.

[0112] The intracellular signaling domain of the CAR provides for the activation of at least one function of the cell expressing the CAR upon binding to the ligand expressed on tumor cells. " Intracellular signaling domain" as used herein refers to all or a portion of one or more domains of a molecule (here the chimeric receptor molecule) that provides for activation of a lymphocyte. Intracellular domains of such molecules mediate a signal by interacting with cellular mediators to result in proliferation, differentiation, activation and other effector functions. Examples of intracellular signaling domains for use in a CAR of the invention include the intracellular sequences of the CD3ζ chain, and / or co-receptors that act in concert to initiate signal transduction following CAR engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.

[0113] In some embodiments, the intracellular signaling domain contains one or more intracellular signaling domains. In some embodiments, the intracellular signaling domain is a portion of and / or a variant of an intracellular signaling domain that provides for activation of at least one function of the CAR-comprising cell. In some embodiments, the intracellular signaling domain comprises the intracellular domain of CD3ζ (or CD3z), FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66b, or a variant thereof, wherein the variant thereof has a 98% sequence identity.

[0114] In some embodiments, the intracellular signaling domain comprises the intracellular domain of CD3ζ or a variant thereof, wherein the variant thereof has at least 98% sequence identity. In some embodiments, the CAR may further comprise a costimulatory signaling domain. The term “costimulatory signaling domain” refers to a signaling moiety that provides to T cells a signal which, in addition to the primary signal provided by for instance the CD3ζ chain of the TCR / CD3 complex, mediates a T cell response, including, but not limited to, activation, proliferation, differentiation, cytokine secretion, and the like. In some embodiments, the co-stimulatory signaling domain is an intracellular signaling domain that interacts with other intracellular mediators to mediate a cell response including activation, proliferation, differentiation and cytokine secretion, and the like.

[0115] In some embodiments, the costimulatory signaling domain comprises the intracellular domain of CD27, CD28, CD137 or 4-1 BB, CD134, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or CD276 or a variant thereof, wherein the variant thereof has a 95% sequence identity.

[0116] In some embodiments, the costimulatory signaling domain comprises the intracellular domain of CD137 or a variant thereof, wherein the variant thereof has a 98% sequence identity.

[0117] In an embodiment, the cell of the invention expresses or produces or comprises the targeting moiety capable of binding to a tumour associated antigen. Thus, in a preferred embodiment of the first aspect, the present invention provides a cell producing and / or secreting the fusion protein of the invention as defined in any of the embodiments above, wherein the cell also expresses or produces or comprises a targeting moiety capable of binding to a tumour associated antigen. Preferably, the cell of the invention produces or expresses or comprises a CAR, wherein the CAR comprises:

[0118] a) an extracellular domain comprising a tumour associated targeting moiety as defined above,

[0119] b) a transmembrane domain as defined above;

[0120] c) an intracellular signaling domain, as defined above, and

[0121] d) optionally, a costimulatory signaling domain as defined above.

[0122] Preferably, the cell secreting the fusion protein of the invention as defined in any of the embodiments above, also expresses or produces or comprises a CAR against CD19. Preferably, the CAR against CD19 that is combined with the fusion protein or the cell of the invention comprises or consists of SEQ ID NO: 12 as defined in PCT / EP2022 / 062374.

[0123] In an embodiment, the cell of the invention comprises a nucleic acid, called herein after the “nucleic acid of the invention”, that encodes for the fusion protein of the invention. In an embodiment, the cell of the invention comprises a nucleic acid with a sequence comprising or consisting of SEQ ID NOs: 11 or 12, or a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NOs: 11 or 12, respectively.

[0124] The cell of the invention may also comprise a nucleic acid encoding for the targeting moiety capable of binding to the tumour associated antigen, preferably CD19.

[0125] In an embodiment, the nucleic acid of the invention in the combination therapy is a bicistronic nucleic acid, and thus comprises both the sequence encoding for the fusion protein of the invention and the sequence encoding for the targeting moiety that is capable of binding to a tumour associated antigen. Hence, by “bicistronic nucleic acid” is referred herein to a polynucleotide sequence comprising:

[0126] i) a nucleic acid encoding the soluble fusion protein of the invention as defined above, and ii) a nucleic acid encoding the targeting moiety capable of binding to a tumour associated antigen defined above,

[0127] wherein both nucleic acids are preferably separated by a sequence encoding for a “2A peptide cleavage site” or by an “internal ribosome entry site region” and wherein both nucleic acids are preferably expressed under the control of the same promoter. Preferably, the promoter is the EF1a promoter. Preferably, the nucleic acid of the invention is the bicistronic nucleic acid comprising the elements represented in Fig. 4 called “CAR 19 TIM-3-Fc".

[0128] In an embodiment, the combination product or combination therapy comprises:

[0129] - the fusion protein of the invention as defined in any of the embodiments above, or a T cell expressing said fusion protein, and

[0130] - a CAR as defined in any of the embodiments above, or a T cell expressing said CAR.

[0131] In an embodiment, the combination product or combination therapy comprises:

[0132] - the fusion protein of the invention comprising or consisting of SEQ ID Nos: 2, 3, or 9, or a T cell expressing said fusion protein, and

[0133] - a CAR as defined in any of the embodiments above, or a T cell expressing said CAR.

[0134] In an embodiment, the combination product or combination therapy comprises:

[0135] - the fusion protein of the invention comprising or consisting of SEQ ID Nos: 2, 3, or 9, or a T cell expressing said fusion protein, and

[0136] ■ a CAR against CD19, or a T cell expressing said CAR.

[0137] In an embodiment, the cell expressing the fusion protein of the invention is the same cell as the cell expressing or comprising CAR. In a second aspect, the present invention provides a composition, preferably a pharmaceutical composition, comprising the protein or the cell of the invention, or a population of said cells, or the nucleic acid of the invention. This composition is also called herein the “composition of the invention”. The pharmaceutical composition of the invention can be administered in single or multiple doses.

[0138] A pharmaceutical composition as described herein may also contain other substances. These substances include, but are not limited to, cryoprotectants, surfactants, anti-oxidants, pharmaceutically acceptable carriers, diluents and stabilizing agents. As used herein, "pharmaceutically acceptable carrier" or “pharmaceutically acceptable diluent” means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The pharmaceutical composition may also comprise the combination product as defined in the first aspect or any of its embodiments.

[0139] In some embodiments, the cells 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 (a "pharmaceutically acceptable" carrier) in a therapeutically effective amount. 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.

[0140] As used herein, the term "effective amount" of an agent, e.g., a therapeutic agent such as a cell, is that amount sufficient to effect beneficial or desired results, for example, clinical results, and, as such, an "effective amount" depends upon the context in which it is being applied. For example, in the context of administering a therapeutic agent that treats cancer, an effective amount can reduce the number of cancer cells; reduce the tumor size or burden; inhibit (i.e., slow to some extent and in a certain embodiment, stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and in a certain embodiment, stop) tumor metastasis; inhibit, to some extent, tumor growth; relieve to some extent one or more of the symptoms associated with the cancer; and / or result in a favourable response such as increased progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), complete response (CR), partial response (PR), or, in some cases, stable disease (SD), a decrease in progressive disease (PD), a reduced time to progression (TTP) or any combination thereof. The term "effective amount" can be used interchangeably with "effective dose," "therapeutically effective amount," or "therapeutically effective dose". In an embodiment, the composition of the invention comprises the combination product or combination therapy as defined above, namely the cell or the fusion protein of invention, and a targeting moiety capable of binding to a tumour associated antigen, preferably a CAR.

[0141] In a third aspect, the present invention provides a kit, called herein after the kit of the invention, comprising the cell of the invention, the protein of the invention, the nucleic acid of the invention, or the composition of the invention, as defined in any of the embodiments above. In an embodiment, the kit of the invention comprises the combination product or combination therapy as defined above, namely the cell or the fusion protein or the nucleic acid of invention, and a targeting moiety capable of binding to a tumour associated antigen, preferably a CAR, more preferably a CAR against CD19. The kit may also comprise the combination product as defined in the first aspect or any of its embodiments.

[0142] In a fourth aspect, the present invention provides the cell, the fusion protein, or the nucleic acid of the invention as defined in the first aspect or any of its embodiment, the composition of the second aspect or any of its embodiments, or the kit of the third aspect of any of its embodiments, including the combination therapy or combination product defined above, for use in therapy or as a medicament. Preferably, the use is in the treatment or prevention of cancer. Thus, methods for treating or preventing cancers are also provided herein.

[0143] The terms “treatment” and “therapy”, as used in the present application, refer to a set of hygienic, pharmacological, surgical and / or physical means used with the intent to cure and / or alleviate a disease and / or symptoms with the goal of remediating the health problem. The terms “treatment” and “therapy” include preventive and curative methods since both are directed to the maintenance and / or reestablishment of the health of an individual or animal. Regardless of the origin of the symptoms, disease and disability, the administration of a suitable medicament to alleviate and / or cure a health problem should be interpreted as a form of treatment or therapy within the context of this invention.

[0144] The term “cancer” refers to a group of diseases, which can be defined as any abnormal benign or malignant new growth of tissue that possesses no physiological function and arises from uncontrolled usually rapid cellular proliferation and has the potential to invade or spread to other parts of the body.

[0145] In an embodiment, the cell, the protein, the combination product, the composition or the kit of the invention is administrated to a subject or patient in need thereof in a therapeutically effective dose. The terms “individual”, “patient” or “subject” are used interchangeably in the present application to designate a human being and are not meant to be limiting in any way. The “individual”, “patient” or “subject” can be of any age, sex and physical condition. The term “patient in need thereof” usually refers to a patient who suffers from cancer. Preferably, term “patient in need thereof” may also refer to a patient who suffers from a CD19 positive cancer.

[0146] In an embodiment, the use is in a method of treating any disease associated with CD19 expression and comprises administering a CD19 targeting moiety together with the cell, the protein, or the nucleic acid of the invention, and / or the pharmaceutical composition of the second aspect to a patient in need thereof. The phrase “disease associated with CD19 expression” includes, but is not limited to, a disease associated with expression of CD19 or condition associated with cells which express CD19 including, e.g., proliferative diseases such as a cancer or malignancy or a precancerous condition such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia; or a noncancer related indication associated with cells which express CD19.

[0147] In a preferred embodiment, the use is in a method of treating a CD19-positive cancer comprising administering a CD19 targeting moiety together with the cell, the protein, the combination product, the nucleic acid or the kit of the invention, and / or the pharmaceutical composition of the second aspect to a patient in need thereof. “CD19-positive” cancer or a “cancer associated with expression of CD19”, including a “CD19-positive” cancerous disease, is one comprising cells, which have CD19 present at their cell surface. The term “CD19-positive” also refers to a cancer that produces sufficient levels of CD19 at the surface of cells thereof.

[0148] In one embodiment, the CD19-positive cancer is a hematological cancer. In one embodiment, the hematological cancer is a lymphoma, leukemia or myeloma. In one embodiment, a cancer associated with expression of CD19 includes cancers and malignancies including, but not limited to, e.g., one or more acute leukemias including but not limited to, e.g., B-cell acute Lymphoid Leukemia (“BALL”), acute lymphoid leukemia (ALL); one or more chronic leukemias including but not limited to, e.g., Chronic Lymphoid Leukemia (CLL). In some embodiments, the CD19-positive cancer is a CD19+ B-cell malignancies, preferably B cell acute lymphoblastic leukemia.

[0149] Additional cancers or hematologic conditions associated with expression of CD19 comprise, but are not limited to, e.g., B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, Follicular lymphoma, Hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and “preleukemia” which are a diverse collection of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells, and the like.

[0150] In some embodiments, the CD19 positive cancer is relapsed / refractory CD19 positive cancer. In general, the relapse of cancer can manifest several months or years after the initial remission; however, most relapses occur within two years after the initial treatment. Refractoriness is a term that implies that the patient has no longer responded to at least one therapy strategy after a relapse.

[0151] In some embodiments, the cell, the protein, the combination product, the nucleic acid, the kit or composition of the invention is administered via one or more routes of administration using one or more of a variety of known methods. Examples of routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other general routes of administration, for example by injection or infusion. General administration can represent modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, the cell, the protein, or composition of the invention can be administered via a non-general route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

[0152] Thus, described herein are methods of treating cancer comprising administering an effective amount of a cell or protein or combination product or nucleic acid or kit of the invention, of the composition of the invention, to a subject in need thereof. For example, disclosed are methods of treating cancer, preferably a CD19-positive cancer, preferably a CD19+ B-cell malignancy, comprising administering an effective amount of the cell of the invention, preferably genetically modified to express a CAR polypeptide, preferably a CAR anti-CD19.

[0153] In some embodiments, the method and uses of the fourth aspect comprise a combination therapy. Thus, the cell, the protein, the combination product, the nucleic acid, the kit or the composition of the invention and described herein may be used in combination with other known agents and therapies, such as with a targeting moiety capable of binding to a tumour associated antigen, as explained above. Administered “in combination”, as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject’s affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery”. In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

[0154] In a preferred embodiment, the combination therapy or product comprises the protein, the cell, the nucleic acid, the kit, or the composition of the invention and a targeting moiety capable of binding to a tumour associated antigen used to obtain improved therapeutic effects in cancer patients. In a more preferred embodiment, the combination therapy or product provides synergistic therapeutic effects in cancer patients. Preferably, the tumour associated antigen is CD19 molecule, so that the targeting moiety capable of binding to a tumour associated antigen is a CD19 targeting moiety, such as the one defined above.

[0155] Preferred embodiments of the present invention include:

[0156] In a preferred embodiment, the invention provides a combination therapy comprising administering to a subject in need thereof:

[0157] 1) a soluble fusion protein comprising at least two domains, wherein:

[0158] • a first domain comprises or consists of SEQ ID NO: 4, or a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 4, and

[0159] • a second domain comprises or consists of SEQ ID NO: 5, or a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 5, and

[0160] 2) a targeting moiety, preferably a CAR, capable of binding to a tumour associated antigen, wherein the administration of 1) and 2) can be simultaneous or sequential administration.

[0161] In a preferred embodiment, the invention provides a combination therapy comprising administering to a subject in need thereof: 1) a soluble fusion protein comprising at least two domains, wherein:

[0162] • a first domain comprises or consists of SEQ ID NO: 4, and

[0163] • a second domain comprises or consists of SEQ ID NO: 5, and

[0164] 2) a targeting moiety, preferably a CAR, capable of binding to CD19 molecule, wherein the administration of 1) and 2) can be simultaneous or sequential administration.

[0165] Preferably, the invention provides a combination therapy comprising administering to a subject in need thereof:

[0166] 1) a soluble fusion protein comprising at least two domains, wherein:

[0167] • a first domain comprises or consists of SEQ ID NO: 4, or a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 4, and

[0168] • a second domain comprises or consists of SEQ ID NO: 5, or a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 5, and

[0169] 2) a targeting moiety, preferably a CAR, capable of binding to a tumour associated antigen, wherein the administration of 1) and 2) can be simultaneous or sequential administration, wherein the combination therapy is for use in a method or treating or preventing a cancer, wherein said cancer is characterized by expressing the tumour associated antigen against which the targeting moiety is designed.

[0170] Preferably, the invention provides a combination therapy comprising administering to a subject in need thereof:

[0171] 1) a soluble fusion protein comprising at least two domains, wherein:

[0172] • a first domain comprises or consists of SEQ ID NO: 4, or a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 4, and

[0173] • a second domain comprises or consists of SEQ ID NO: 5, or a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 5, and

[0174] 2) a targeting moiety, preferably a CAR, capable of binding to CD19,

[0175] wherein the administration of 1) and 2) can be simultaneous or sequential administration, wherein the combination therapy is for use in a method or treating or preventing a cancer, wherein said cancer is a CD19-positive cancer. Preferably, the invention provides a combination therapy comprising administering to a subject in need thereof:

[0176] 1) a soluble fusion protein comprising at least two domains, wherein:

[0177] • a first domain comprises or consists of SEQ ID NO: 4, and

[0178] • a second domain comprises or consists of SEQ ID NO: 5, and

[0179] 2) a CAR capable of binding to CD19,

[0180] wherein the administration of 1) and 2) can be simultaneous or sequential administration, wherein the combination therapy is for use in a method or treating or preventing a cancer, wherein said cancer is a CD19-positive cancer.

[0181] Preferably, the invention provides a combination therapy comprising administering to a subject in need thereof a cell, preferably a T cell, expressing:

[0182] 1) a soluble fusion protein comprising at least two domains, wherein:

[0183] • a first domain comprising or consisting of SEQ ID NO: 4, or a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 4, and

[0184] • a second domain comprising or consisting of SEQ ID NO: 5, or a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 5, and

[0185] 2) a targeting moiety, preferably a CAR, capable of binding to a tumour associated antigen, preferably CD19.

[0186] Preferably, the invention provides a combination therapy comprising administering to a subject in need thereof a cell, preferably T cell, expressing:

[0187] 1) a soluble fusion protein comprising at least two domains, wherein:

[0188] • a first domain comprising or consisting of SEQ ID NO: 4, or a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 4, and

[0189] • a second domain comprising or consisting of SEQ ID NO: 5, or a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 5, and

[0190] 2) a targeting moiety, preferably a CAR, capable of binding to a tumour associated antigen, wherein the administration of 1) and 2) can be simultaneous or sequential administration, and wherein the combination therapy is for use in a method or treating or preventing a cancer, wherein said cancer is characterized by expressing the tumour associated antigen against which the targeting moiety is designed. Preferably, the invention provides a combination therapy comprising administering to a subject in need thereof a cell, preferably T cell, expressing:

[0191] 1) a soluble fusion protein comprising at least two domains, wherein:

[0192] • a first domain comprising or consisting of SEQ ID NO: 4, and

[0193] • a second domain comprising or consisting of SEQ ID NO: 5, and

[0194] 2) a targeting moiety, preferably a CAR, capable of binding to CD19

[0195] wherein the administration of 1) and 2) can be simultaneous or sequential administration, and wherein the combination therapy is for use in a method or treating or preventing a cancer, wherein said cancer is a CD19-positive cancer.

[0196] In an embodiment, the combination product or combination therapy comprises:

[0197] - the fusion protein of the invention as defined in any of the embodiments above, or a T cell expressing said fusion protein, and

[0198] ■■ a CAR as defined in any of the embodiments above, or a T cell expressing said CAR, for use in a method of treatment or prevention of cancer, wherein the use comprises administering the CAR and the fusion protein, or the respective cells expressing them, either simultaneously or sequentially.

[0199] In an embodiment, the combination product or combination therapy comprises:

[0200] - the fusion protein of the invention comprising or consisting of SEQ ID Nos: 2, 3, or 9, or a T cell expressing said fusion protein, and

[0201] - a CAR as defined in any of the embodiments above, or a T cell expressing said CAR, for use in a method of treatment or prevention of cancer, wherein the use comprises administering the CAR and the fusion protein, or the respective cells expressing them, either simultaneously or sequentially.

[0202] In an embodiment, the combination product or combination therapy comprises:

[0203] - the fusion protein of the invention comprising or consisting of SEQ ID Nos: 2, 3, or 9, or a T cell expressing said fusion protein, and

[0204] - a CAR against CD19, or a T cell expressing said CAR,

[0205] for use in a method of treatment or prevention of cancer, wherein the use comprises administering the CAR and the fusion protein, or the respective cells expressing them, either simultaneously or sequentially.

[0206] In one embodiment, the fusion protein is administered prior to the CAR, followed by subsequent administration of the CAR, or conversely, the CAR is administered first, followed by the fusion protein. In another embodiment, both components of the combination product are administered simultaneously.

[0207] Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention.

[0208] SEQUENCE LISTING SEQ ID NO: 1 signal peptide TIM-3: MFSHLPFDCVLLLLLLLLTRS

[0209] SEQ ID NO: 2 fusion protein with endogenous signal peptide TIM-3:

[0210] MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFEC GNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKL VIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDL RDSGATIRIEGRMDPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL GAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0211] SEQ ID NO: 3 processed fusion protein SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRY WLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAA FPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIRIEGRMDPKSCD KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0212] SEQ ID NO: 4 extracellular domain of TIM-3 (22-200 aa) SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRY WLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAA FPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIR

[0213] SEQ ID NO: 5 Fc silent Human LALA PG IgG1 Fc with hinge PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 6 Fc silent Human PG lgG1 Fc without hinge:

[0214] SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK

[0215] SEQ ID NO: 7 hinge LALA: PKSCDKTHTCPPCPAPEAAGGP

[0216] SEQ ID NO: 8 his tag: HHHHHH

[0217] SEQ ID NO: 9 leader + fusion protein + his tag:

[0218] MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFEC GNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKL VIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDL RDSGATIRIEGRMDPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL GAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH

[0219] SEQ ID NO: 10 linker: IEGRMD

[0220] SEQ ID NO: 11 TIM31-200-silentFc-His (unprocessed):

[0221] ATGTTCAGCCATCTGCCTTTCGACTGCGTGCTGCTGCTCCTGCTTCTGCTGCTGACCAGAT CCAGCGAGGTCGAGTACAGAGCCGAAGTGGGCCAGAATGCCTACCTGCCTTGCTTCTACA CACCAGCCGCTCCAGGCAATCTGGTGCCTGTGTGTTGGGGAAAAGGCGCCTGTCCTGTGT TCGAGTGCGGCAACGTTGTGCTGAGAACCGACGAGCGGGACGTGAACTACTGGACCAGC AGATACTGGCTGAACGGCGACTTCAGAAAGGGCGACGTGTCCCTGACCATCGAGAATGTG ACACTGGCCGACAGCGGCATCTACTGCTGCAGAATCCAGATTCCTGGCATCATGAACGAC GAGAAGTTCAACCTGAAGCTGGTCATCAAGCCCGCCAAAGTGACCCCTGCTCCTACCAGA CAGAGAGACTTCACCGCCGCCTTTCCACGGATGCTGACCACAAGAGGACACGGCCCTGCC GAGACACAGACACTTGGAAGCCTGCCTGACATCAATCTGACCCAGATCAGCACCCTGGCC AACGAGCTGAGAGATAGCAGACTGGCTAATGACCTGAGAGACAGCGGCGCCACCATCAGA ATCGAGGGCAGAATGGATCCGAAGTCCTGCGACAAGACCCACACCTGTCCTCCATGTCCT GCTCCAGAAGCTGCTGGCGGCCCTTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACC CTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCTCACGAGGAC CCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAG CCTAGAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCAC CAGGATTGGCTGAATGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGGGCGCT CCCATCGAAAAGACCATCTCTAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACA CTGCCTCCAAGCAGGGACGAGCTGACCAAGAATCAGGTGTCACTGACCTGCCTGGTCAAG GGCTTCTACCCCTCTGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAAC TACAAGACAACCCCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGA CAGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAG GCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGCAAGCACCACCAC CATCACCA

[0222] SEQ ID NO: 12 TIM322-200-silentFc-His (processed):

[0223] AGCGAGGTCGAGTACAGAGCCGAAGTGGGCCAGAATGCCTACCTGCCTTGCTTCTACACA CCAGCCGCTCCAGGCAATCTGGTGCCTGTGTGTTGGGGAAAAGGCGCCTGTCCTGTGTTC GAGTGCGGCAACGTTGTGCTGAGAACCGACGAGCGGGACGTGAACTACTGGACCAGCAG ATACTGGCTGAACGGCGACTTCAGAAAGGGCGACGTGTCCCTGACCATCGAGAATGTGAC ACTGGCCGACAGCGGCATCTACTGCTGCAGAATCCAGATTCCTGGCATCATGAACGACGA GAAGTTCAACCTGAAGCTGGTCATCAAGCCCGCCAAAGTGACCCCTGCTCCTACCAGACA GAGAGACTTCACCGCCGCCTTTCCACGGATGCTGACCACAAGAGGACACGGCCCTGCCG AGACACAGACACTTGGAAGCCTGCCTGACATCAATCTGACCCAGATCAGCACCCTGGCCA ACGAGCTGAGAGATAGCAGACTGGCTAATGACCTGAGAGACAGCGGCGCCACCATCAGAA TCGAGGGCAGAATGGATCCGAAGTCCTGCGACAAGACCCACACCTGTCCTCCATGTCCTG CTCCAGAAGCTGCTGGCGGCCCTTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCC TGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCTCACGAGGACC CCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGC CTAGAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACC AGGATTGGCTGAATGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGGGCGCTC CCATCGAAAAGACCATCTCTAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACAC TGCCTCCAAGCAGGGACGAGCTGACCAAGAATCAGGTGTCACTGACCTGCCTGGTCAAGG GCTTCTACCCCTCTGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACT ACAAGACAACCCCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGA CAGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAG GCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGCAAGCACCACCAC CATCACCACGGCTCTGGCTCTAGA

[0224] The following items are also encompassed by the invention:

[0225] 1. A cell, preferably a T cell, secreting a soluble fusion protein comprising the extracellular domain of TIM-3.

[0226] 2. The cell according to item 1, wherein the soluble fusion protein further comprises a multi- merization domain, preferably a dimerization domain. 3. The cell according to item 2, wherein the multimerization domain comprises an immunoglobulin Fc domain.

[0227] 4. The cell according to item 3, wherein the immunoglobulin Fc domain is a silent IgG Fc domain with abrogated Fc receptor binding.

[0228] 5. The cell according to any one of items 2 to 4, wherein the extracellular domain of TIM-3 and the multimerization domain are connected by a linker.

[0229] 6. The cell according to any one of items 1 to 5, wherein the soluble fusion protein further comprises a signal peptide, preferably the endogenous TIM-3 signal peptide of SEQ ID NO: 1.

[0230] 7. The cell according to any one of items 2 to 6, wherein the soluble fusion protein comprises or consists of SEQ ID NO: 2, 3 or 9.

[0231] 8. The cell according to any one of items 1 to 7, wherein the cell further expresses a targeting moiety capable of binding to a tumour associated antigen.

[0232] 9. The cell according to item 8, wherein the targeting moiety capable of binding to a tumour associated antigen is a chimeric antigen receptor (CAR).

[0233] 10. The cell according to item 9, wherein the CAR is a CAR against CD19.

[0234] 11. A composition comprising a cell as defined in any of items 1 to 10, or a population of said cells.

[0235] 12. The cell as defined in any one of items 1 to 10, or the composition as defined in item 11, for use in medicine or as a medicament

[0236] 13. The cell or composition for use as defined in item 12, for use in the treatment or prevention of cancer.

[0237] 14. The cell as defined in item 10, or a composition comprising said cell, for use in the treatment or prevention of CD19+ B-cell malignancies. The invention is described below by the following examples, which must be considered as merely illustrative and in no case limiting of the scope of the present invention.

[0238] EXAMPLES

[0239] Abbreviations

[0240] 7-AAD: 7-amino actinomycin D

[0241] B-ALL: B-cell acute lymphoblastic leukemia

[0242] BM: bone marrow

[0243] CAR: chimeric antigen receptor

[0244] CR: complete remission

[0245] FBS: fetal bovine serum

[0246] HD: healthy donor

[0247] hSCF: human stem cell factor

[0248] IC: immune checkpoint

[0249] ICB: immune checkpoint blocker

[0250] ICR: immune checkpoint receptor

[0251] mAb: monoclonal antibody

[0252] MFI: mean fluorescence intensity

[0253] MSC: mesenchymal stromal cell

[0254] PB: peripheral blood

[0255] PDX: patient-derived xenograft

[0256] R / R: relapsed or refractory

[0257] scFv: single-chain variable fragment

[0258] METHODS

[0259] Primary samples and cell lines

[0260] All experimental studies using primary samples were approved by the Ethics Committee on Clinical Research of the Clinic Hospital of Barcelona (HCB / 2017 / 0781). Primary cells were obtained after written informed consent in accordance with the Declaration of Helsinki. Buffy coats were obtained from the Catalan Blood and Tissue Bank after Institutional Review Board approval (HCB / 2018 / 0030). Peripheral blood (PB) mononuclear cells (PBMCs) processing and B-ALL cell lines used are detailed in Supplemental Methods.

[0261] In vivo CAR-T cell-mediated cytotoxicity assay with PDX samples

[0262] Non-obese diabetic (NOD) Cg -PrkdcscidIl2rgim1Wjl / SzJ (NSG) mice (The Jackson Laboratory, Bar Harbor, ME) were housed under pathogen-free conditions. For all in vivo experiments, 1 x106B-ALL PDX cells were infused intravenous into sub-lethally irradiated (2-Gy) NSG mice, followed 4 days later by an i.v. infusion of 1-2x106T cells (untransduced, TIM-3-Fc, CAR19, co- administration of CAR19 and TIM-3-Fc or bicistronic all-in-one CAR19-TIM-3-Fc T cells, n=5-6 mice / group). B-ALL engraftment and T-cell expansion was monitored weekly in PB by FACS. Animals were euthanized when they had lost >20% of their weight or showed signs of disease. Plasma was collected by blood centrifugation (1500g, 10min, 4oC) obtained by intracardiac puncture. All in vivo procedures were performed in accordance with the institutional animal care committee of the Barcelona Biomedical Research Park (DAAM7393 / HRH170029). The CAR19 used in this study corresponds to the CAR defined as SEQ ID NO: 12 in PCT / EP2022 / 062374.

[0263] Statistical analysis

[0264] We used the Complex Heatmap package in R to display in heatmaps the median of ICRs and their ligands in human BM samples. Other data are presented as mean±SEM. Parametric tests were used for in vitro studies: Student’s t-test to compare two groups and one-way ANOVA to compare >3 experimental groups. Non-parametric tests were used for in vivo studies: the Mann-Whitney U-test to compare two groups and the Kruskal-Wallis test to compare >3 experimental groups. All p-values are two-tailed and statistical significance was defined as p* 0.05. All analyses were performed using Prism software v.8.0 (GraphPad Prism, San Diego, CA).

[0265] Detailed information about the immunophenotyping of samples and cell lines, generation of CAR19 and TIM-3-Fc decoy expression vectors, lentiviral production, T-cell transduction, cytotoxicity assays, soluble TIM-3 detection, galectin-9 interactions and spectral flow cytometry are provided in Supplemental Methods.

[0266] SUPPLEMENTARY METHODS

[0267] Primary samples and cell lines

[0268] B-ALL cell lines (SEM, REH and NALM6) and 293T cells were obtained from the DSMZ cell bank (Braunschweig, Germany). Galectin-9::mCherry-overexpressing B-ALL cell lines were generated by cloning galectin-9 coding sequence from the pCCL-CMV-Gal9-Myc-DDK plasmid (RC219435L1; OriGene, Rockville, MD) into the pCDH-EF1» -eFFIy-mCherry vector (kindly provided by Dr Irmela Jeremias) using standard molecular cloning techniques. The expected fragment was confirmed by Sanger sequencing. EF1* -galectin-9-mCherry lentiviruses were produced using well-established polyethylenimine (PEI)-based protocols1 2and 1 x106of the B-ALL cells SEM, REH and NALM6 were infected at a multiplicity of infection (MOI) of 5 using 5 pg / mL polybrene (Sigma-Aldrich, Madrid, Spain) and spinoculation (1200xg) for 2h at 32oC. Cells were then expanded and galectin-9-mCherry cells were sorted by FACS (>99% purity). Galectin-9 expression was confirmed by conventional western blotting using mAbs against galectin-9 (1 / 1000, OTIG3, Bio-Rad), and GAPDH (1 / 10000, 14C10, Cell Signaling Technologies, Danvers, MA), followed by incubation with an anti-mouse / rabbit IgG (Roche Chemiluminescence Western Blotting Kit). Recombinant human galectin-9 (rhGalectin-9, 2045-GA, R& D Systems) was used as a positive control (5 ng). Proteins were detected by chemiluminescence using the Lumi-Light substrate (Roche Chemiluminescence Western Blotting Kit).

[0269] Peripheral blood (PB) mononuclear cells were isolated from buffy coats of volunteer healthy donors (HDs) by Ficoll-Hypaque gradient centrifugation (GE Healthcare, Chicago, IL). Diagnosis (Dx; n=47) and relapse (Rel; n=38) BM samples from patients with B-ALL (pediatric and adult) and from HDs (n=21) were obtained from collaborating hospitals. Cytogenetic / molecular features are detailed in Table 1. Primary cells were cultured in StemSpan™ SFEM (StemCell Technologies, Vancouver, Canada) supplemented with 20% heat-inactivated foetal bovine serum (FBS; Sigma-Aldrich), penicillin / streptomycin (Gibco / lnvitrogen Corp., Carlsbad, CA), insulin-transferrin-selenium (Gibco / lnvitrogen), human Stem Cell Factor (100 ng / mL), hFMS-like tyrosine kinase 3 ligand (100 ng / mL), hlL-3 (10 ng / mL) and hlL-7 (10 ng / mL) (all from Miltenyi Biotec; Bergisch Gladbach, Germany).

[0270] Table 1. Cytogenetic and molecular features of patients with B-ALL analyzed in this study.

[0271] Total N° of patients Age

[0272] Pediatric B-ALL

[0273] Diagnosis

[0274]

[0275] 32

[0276] Hyperdiploid 6 5.5 ± 1.6

[0277] ETV6-RUNX1 10 4.9 ± 1.1

[0278] IAMP21 3 7.8 ±2.7

[0279] KMT2A-r 5 0.8 ±0.1

[0280] TCF3:: PBX1 1 9

[0281] B-other 6 9.0 ± 2.7

[0282] NA 1

[0283] Relapse 25

[0284] Hyperdiptoid 2 9.3 ± 4.3

[0285] ETV6-RUNX1 1 5,5

[0286] IA. MP21 3 11.2 + 2.9

[0287] KMT2A-r 2 4.5 + 0.5

[0288] B-other 6 15.6 ± 1.9

[0289] ETV5-ABL1 1 3

[0290] NA 10

[0291] Adult B-ALL

[0292] Diagnosis 15

[0293] BCR-ABL+ 3 41.7 + 5.0

[0294] BCR-ABL- 10 39.1 ± 6.0

[0295] NA

[0296]

[0297] 2 22.5 ± 05

[0298] Relapse 13

[0299] BCR-ABL- 11 39.6 ± 5.4

[0300] NA 2 43.5 ± 10.5

[0301] NA Not available Immunophenotyping of healthy samples, primary B-ALL samples and B-ALL cell lines The fluorochrome-labelled mAbs used for flow-cytometry analysis are listed in Table 2. Dx and Rel immunophenotyping data were generated from 85 patients with B-ALL and 20 age-matched healthy donors (HD). Table 1 summarizes the main clinico-biological features of the patients enrolled in this study. The gating strategy for the analysis of T cells, B cells and MSCs is detailed in Figures 7-9. Cytoplasmic staining for HMGB1 and CTLA-4 was performed using the Fix & Perm Reagent Kit (Nordic-Mubio, Lifespan Biosciences, Seattle, WA). All flow-cytometry data acquisition was conducted on a FACSCanto-ll cytometer equipped with FACSDiva software (BDBiosciences).

[0302] Generation of CAR19 and TIM-3-Fc decoy expression vectors

[0303] We used a clinically-validated pCCL third-generation lentiviral CD19-CAR backbone, containing the A3B1 scFv, the hinge and transmembrane domains of human CD8 and human 4-1 BB and CD3* endodomains.34CAR19 was fused to a GFP reporter gene using a 2A ribosomal skip sequence (T2A) at the C-terminal CAR sequence, allowing evaluation of transduction efficiency and CAR expression. The vector pCCL-OMSS-sTIM322-200-silentFc-His-T2A-Tto (termed TIM-3-Fc) was generated by subcloning the TIM-3-Fc coding region from pCDNA3.1(+)-OMSS-sTIM322-200-silentFc-His into the 5’ region of the pCCL-T2A-Tomato vector. The pCDNA3.1 (+)-OMss-sTIM322-200-siientFc-His vector contains a wild-type oncostatin M signal peptide (OMss) in-frame with a soluble TIM-3 (sTIM-3) Ser22-Arg200 (synthesized by GeneArt, ThermoFisher Scientific, Waltham, MA), a siientFc (including the LALA PG mutation set for abrogated Fc» receptor interactions) and a His tag. The vector pCCL-CAR19-F2A-sTIM31-200-silentFc-His-T2A-GFP (termed CAR19 TIM-3-Fc) was generated by subcloning the pCDNA3.1(+)-sTIM31-200-silentFc-His into the pCCL-CAR19-F2A-BiTE19-T2A-GFP using Xba\ digestion. The pCDNA3.1 (+)-STIM3I-2OO-siientFc-His vector contains the complete extracellular region of TIM-3, including the endogenous signal sequence, in-frame of the siientFc and His tag.

[0304] Lentiviral production, T-cell transduction, activation, and expansion

[0305] CAR19-expressing viral particles were generated by transfection of 293T cells with the pCCL third-generation lentiviral CD19-CAR vector and the packaging and envelope plasmids (VSV-G, RRE, and REV) using PEI. Supernatants were collected at 48h and 72h after transfection and were concentrated by ultracentrifugation. T-cells were activated by plate-coating with anti-CD3 (OKT3) and anti-CD28 (CD28.2) antibodies (1pg / mL, BD Biosciences, Franklin Lakes, NJ) for 2 days, and were then transduced with lentiviruses (CAR19, TIM-3-Fc or CAR19-TIM-3-Fc) at MOI of 10 in the presence of hlL-7 and hlL-15 (10 ng / mL; Miltenyi Biotec). T-cells were expanded for up to 12 days in complete RPMI-1640 medium (Gibco / lnvitrogen) containing 10% heat-inactivated FBS, penicillin / streptomycin and hlL-7 and hlL-15. T-cell transduction efficiency (tdTo+or GFP+) was analysed by FACS. During in vitro T-cell expansion, the proliferation of CAR19, TIM-3-Fc and CAR19 TIM-3-Fc T-cells was determined using the Automated Cell Countess instrument (ThermoFisher Scientific).

[0306] In vitro and ex vivo cytotoxicity assays and cytokine production of CAR19 and CAR19 TIM-3-Fc T cells

[0307] TIM-3 and galectin-9 expression was evaluated in CAR19 T-cells and blasts, respectively, after 24h of co-culture. The gating strategy used is described in Figure 10. For apoptosis and eFluor staining, CAR19 T-cells were incubated with or without 1 pg / mL of rhGalectin-9 (2045-GA, R& D Systems). After 48h, cells were labelled with 3pM eFluor 670 Cell Proliferation Dye (ThermoFisher Scientific) and incubated with a CD3 PE-Cy7 mAb. Cells were then washed and incubated for 15 mins with Annexin V-Binding Buffer and an Annexin-V PE mAb (BD Biosciences). Cells were then stained with 7-amino actinomycin D negative dye (7-AAD) and the percentage of apoptotic cells and eFluor 670 mean fluorescence intensity (MFI) was analysed on a FACSCanto-ll cytometer. The total number of cells was considered when evaluating the percentage of apoptosis, whereas only living eFluor-positive cells were assessed for eFluor fluorescence quantification.

[0308] For ex vivo cytotoxicity assays, experiments were performed using T cells pooled from liver, spleen and BM of animals treated with CAR19 T-cells (n=3) or with CAR19+TIM-3-Fc T-cells (n=3). Human T-cells were isolated by FACS and seeded with freshly thawed B-ALL PDX cells for 24h. For CAR19 T-cell cytotoxicity assays ( / n vitro or ex vivo) with B-ALL cell lines or primary B-ALL cells, the cells were stained with CD3 PE-Cy7, CD19 BV421 and a viability dye (7-AAD or DAPI). Cytotoxicity was determined by subtracting the percentage of live blasts (7AAD7DAPI-CD19+CD3 ) from 100. Absolute cell counts were determined by using Trucount beads (BD Biosciences). Interferon-* (IFN* ) was measured by enzyme-linked immunosorbent assay (ELISA) (BD Biosciences) in supernatants collected after 48h.

[0309] Soluble TIM-3 detection and analysis of galectin-9 interactions

[0310] The presence of TIM-3-Fc in 293T conditioned medium, transduced T-cell supernatants and mouse plasma was analysed by ELISA. Plasma samples were first diluted 1:4 with 1% (w / v) BSA in PBS. Briefly, flat-bottom 96-well plates (ThermoFisher Scientific) were coated with 100 • L / well of APC-conjugated AffiniPure goat anti-human IgG (109-135-098, Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:100 in PBS. After washing and blocking with 300 • L of 5% (w / v) BSA in PBS for 1 h at 37oC, 100 • L of the samples of interest were added to the wells and incubated for 1h at RT. After three washes, 100 • L of the corresponding anti-human TIM-3 mAb (1ES clone, MA5-32841, Invitrogen) was added for 1h at room temperature. Finally, HRP- conjugated goat anti-mouse IgG (115-035-003, Jackson ImmunoResearch) was added (1:2000 dilution) and the plate was washed again and developed. The interaction between human galectin-9 and TIM-3-Fc was also investigated by ELISA. rhGalectin-9 (2045-GA, R& D Systems) was coated onto wells (0.25 » g / well) in PBS. After washing and blocking with 300 • L 1% (w / v) BSA in PBS for 2h at 37oC, 100 • L of 293T conditioned medium or rhTIM-3-Fc chimera (2365-TM, R& D Systems) was added to the wells and incubated for 2h at RT. After three washes, 100 • L of the appropriate HRP-conjugated goat anti-human IgG (A0170, Sigma-Aldrich) was added (1:2000 dilution), after which the plate was washed again and developed. All optical density measurements were performed on a Multiskan FC instrument (ThermoFisher Scientific).

[0311] Spectral flow cytometry identification of in vivo expanded T cell populations

[0312] Whole BM and spleens were retrieved from B-ALL xenografted mice four weeks upon treatment with CAR19 T cells (n=5) and CAR19-TIM-3-Fc T cells (n=5). BM and spleen single cell suspension was stained with the 24-color T cell module of the Euroflow-lmmune monitoring antibody panel (Table 3) adapted for the simultaneous measurement of GFP fluorescence of both CAR19 T cells and CAR19-TIM-3-Fc T cells. Samples were labeled using the EuroFlow standard operating procedures for staining of cell surface membrane markers,5available at www. EuroFlow.org, for simultaneous immunophenotypic identification of both transduced T cells (GFP+) and bystander, non-transduced (GFP ) T cells (CD45+and CD3+), and their most relevant maturation-associated and functional T cell subsets. Stained samples were measured in a five-laser Aurora cell sorter (Aurora OS, Cytek Biosciences) using the SpectroFlo software (Cytek Biosciences). For data analysis and T cell subsetting, Infinicyt software (Cytognos SL) was used. UMAP plots of the various T cell subset profiles were built with the R (version 4.3.1) and the UWOT software packages.6 Table 2: fluorochrome-conjugated monoclonal antibodies used in the present study for FACS

[0313]

[0314] mAbs- wire studies Fiueruphure Cseee Supplier C a t Ho | ETLA APC 3168-540 BO SOWLO |. CCR?.. PE. 3D12 BO S5217S CO 18 PEGy? Hi'KM DO SE8232 CGI 3 PEGy? WM1S ED 5S1W COW BWI HIB1B EG 5S2W | GDIS RTC Him§ 8D 555412 i GDIS APGH7 EG35C1 8D 5'30177 | CD22 APC H1B22 BO 5S2SB3 I CDS RTC UCHT-i BO 5S5332 CDS- P>srCP SK7 EG MS7SS CD3 PEGy? UGHT-1 EG 533423 | CD4 PEGy? SK3 EG 5S7SS2 GD45 HTC HI3D 8D 555452 CO45PA BV51D KHSS BD 553031 I CD8S BVS18 FGSO 348335 j CD73 BVSW AD2 BO S-S3 IBS | CDS BVSW SKI BD SS3S19 J CDS' APC-OyF SKI EG 557534 | COSS PE 1387,4 ED 557227 GDSS PS 2331 BO 555355 GD8S APG 2331 BD 555555 01U-4 APC L3D1D E:> Me<s§cd 34SSGB APC SMI-3 BRM14FE WSG8 PEGy? SMI-3 SMteerM 343S1S HMGB1 PS 3E8 BESegend 851484 | HVEM PS 122 Biotegsf'd 348805 j LAG-3 PS 347-530 EG 585816 | MHS-ii PerCP L243 BD ’347402 | PD'i APS MLM4 BD 558854 | PD-L1 PE MSH-t BD S87324 PG42 PE MIHMS EG 558838 VD3-3 PE 7D3 EG 553423

[0315]

[0316] j TIM-3 BV421 703 BD 555582 Dyes

[0317]

[0318] 7-AAD BD 585815 | DAPi Sigma M 806'315 i re Ahe ■ »? wye st u d ies

[0319] COW EV431 H’BtS ED 55344-3 CD23 APC HIB22 BO 553S58 j CD3 PEGy? UCKT-1 ED 583423 j GD4S APCH7 2D1 BD 588178 I Gx^scSn-S PECy7 8M1-3 EC 348818

[0320]

[0321] | HLA'ABS’ Byaio G48-3. S BD. w. Table 3: Euroflow-immune monitoring antibody panel taking 24-color module TCRgd

[0322] TCRa7.2

[0323] CD57

[0324] CD8

[0325] CD45RA

[0326] CD4

[0327] CD127

[0328] CD3

[0329] CD45

[0330] CD279 (PD1)

[0331] CD25

[0332] CD27

[0333] TRCB1

[0334] TCRa24

[0335] CD38

[0336] NKp80

[0337] CD56

[0338] CD45 RO

[0339] CD28

[0340] CD161

[0341] CD103

[0342] CD159a

[0343] CD159c

[0344] CD185

[0345]

[0346] SUPPLEMENTARY REFERENCES

[0347] 1. Baroni ML, Sanchez Martinez D, Gutierrez Aguera F, et al. 41BB-based and CD28-based CD123-redirected T-cells ablate human normal hematopoiesis in vivo. J Immunother Cancer.

[0348] 2020;8(1):e000845.

[0349] 2. Velasco-Hernandez T, Zanetti SR, Roca-Ho H, et al. Efficient elimination of primary B-ALL cells in vitro and in vivo using a novel 4-1BB-based CAR targeting a membrane-distal CD22 epitope. J Immunother Cancer. 2020;8(2):e000896.

[0350] 3. Castella M, Boronat A, Martin-Ibanez R, et al. Development of a Novel Anti-CD19 Chimeric Antigen Receptor: A Paradigm for an Affordable CAR T Cell Production at Academic Institutions. Mol Ther Methods Clin Dev. 2018;12:134-144.

[0351] 4. Castella M, Caballero-Banos M, Ortiz-Maldonado V, et al. Point-Of-Care CAR T-Cell Production (ARI-0001) Using a Closed Semi-automatic Bioreactor: Experience From an Academic Phase I Clinical Trial. Front Immunol. 2020;11:482.

[0352] 5. Kalina T, Flores-Montero J, van der Velden VHJ, et al. EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols. Leukemia. 2012;26(9):1986— 2010.

[0353] 6. Mclnnes L, Healy J, Melville J. UMAP: Uniform Manifold Approximation and Projection for Dimension Reduction. arXiv; 2020. RESULTS

[0354] TIM-3-galecin-9 axis is upregulated in T cells, blasts and MSCs throughout B-ALL progression

[0355] ICs have been implicated in immunosurveillance and immunotherapy failure in several cancers, leading to the development and clinical use of IC blockers. However, there is limited understanding of the expression of ICRs and their ligands in B-ALL. Here, we investigated the protein expression of several IC axes in BM samples at diagnosis (Dx) and relapse (Rel) from pediatric (Dx, n=32; Rel, n=25) and adult (Dx, n=15; Rel, n=13) patients with B-ALL, and compared it with the expression in BM from HDs (n=21; Table 1). Specifically, we analyzed the ICRs BTLA, CTLA-4, LAG-3, PD-1, and TIM-3 in CD8+and CD4+T cells, while their ligands (HVEM, CD80, CD86, MHC-II, PDL-1, PDL-2, galectin-9, and HMGB1) were examined in blasts (and normal B-cells in non-leukemic BM) and MSCs (Figure 1A,7, 8, 9, -11).

[0356] Our analysis of ICR protein expression in CD4+and CD8+ T cells revealed that only CTLA-4 and TIM-3 expression was significantly higher in patients with B-ALL at diagnosis than in control BM and was further upregulated at relapse (Figure 1 B,left panels, 1C). Notably, TIM-3 expression on CD8+T cells was remarkably high at both diagnosis and relapse, and all ICRs were significantly upregulated in CD4+T cells at relapse (Figure 1B,left panels, 1C). We then examined the expression of IC ligands in normal B-cells and MSCs from non-leukemic BM, as well as in blasts and MSCs from BM of B-ALL patients. Our results indicated that galectin-9 and HMGB1 (TIM-3 ligands) were significantly more highly expressed in Dx B-ALL blasts than in normal B-cells from non-leukemic BM, and CD86 (CTLA-4’s ligand) along with galectin-9 and HMGB1 were the most significantly upregulated ligands at Rel (Figure 1B, right panels, 1C). Additionally, galectin-9 was the only IC ligand significantly upregulated in BM-MSCs from patients with B-ALL at both Dx and Rel (Figure 1 B, right panels, 1C). We found no correlation between the expression of ICRs and IC ligands and either patient age or molecular / cytogenetic subgroups (Table 1 and data not shown). Overall, this analysis revealed that the TIM-3-galectin-9 axis Is the most highly expressed IC axis in T cells, blasts, and MSCs throughout the progression of B-ALL.

[0357] Moreover, we analyzed the expression of TIM-3 in CAR19 T cells from the infusion products of 37 R / R patients with B-ALL who had received CAR19 T cells (varnimcabtagene autoleucel) at our institution. We observed a poorer event-free survival in patients with CAR19+ T cells expressing high levels of TIM-3 (Figure 14). Collectively, TIM-3-galectin-9 axis may represent a promising immunotherapeutic target for B-ALL. Galectin-9 impairs CAR19-T cell function

[0358] CAR19-T cell therapy has been transformative for patients with R / R B-ALL. However, durable CR rates at one-year post-treatment are <50% of patients,6highlighting the urgent need to better understand CAR-T cell function and molecular drivers that may improve clinical responses. Consequently, we next investigated the contribution of the TIM-3-galectin-9 axis to CAR-T cell function in B-ALL. CD19-CAR-T cells were generated according to standard protocols and were used in in vitro co-culture experiments against three primary B-ALL samples at an effector:target (E: T) ratio of 1:4 (Figure 2A, 10). The FACS analysis of TIM-3 expression on CAR19-T cells and galectin-9 expression on B-ALL blasts after 24h revealed that TIM-3 expression on CAR19-T cells was significantly higher after exposure to B-ALL cells compared to CAR19-T cells alone. Similarly, galectin-9 expression in CAR-T -cell resistant B-ALL cells was consistently much higher after coculture with CAR19-T cells (Figure 2B).

[0359] To investigate the effect of galectin-9 on CAR19-T cells, we cultured CAR19-T cells alone or with rhGalectin-9 for 48h and found that galectin-9 significantly increased apoptosis (Figure 2C) and decreased proliferation of CAR19-T cells, as measured by Annexin-V and eFluor 670 retention assay, respectively (Figure 2D). Furthermore, CAR19-T cells treated with rhGalectin-9 showed significantly reduced cytotoxic activity against SEM, REH and NALM6 B-ALL cells at an E: T ratio of 1:2 (Figure 2E). To validate these findings, we generated B-ALL cell lines overexpressing galectin-9 (SEMGal9, REHGal9and NALM6Gal9) and compared the cytotoxic effects of CAR19-T cells against wild-type and galectin-9-overexpressing B-ALL cells. The results showed that CAR19-T cell killing was consistently lower in galectin-9-overexpressing target cells than in equivalent nontransduced cells. Notably, wild-type REH cells were the most resistant to CAR19-T cell-mediated cytotoxicity (killing at E: T 1:8: 10% for REH versus 58% for SEM and 42% for NALM6), consistent with their high endogenous levels of galectin-9 (Figure 2F, G). Collectively, these findings indicate that galectin-9 has a negative effect on both the fitness and function of CAR19-T cells and suggest that inhibiting its interaction with TIM-3 in T cells may enhance the efficacy of CAR19-T-cell therapy in R / R B-ALL.

[0360] T cells secreting a TIM-3-Fc decoy enhance CAR19-T cell persistence and efficacy in vivo TIM-3 is known to have multiple ligands, and murine and human anti-TIM-3 antibodies with reported functional efficacy disrupt TIM-3 binding to PtdSer and CEACAM1 but not to galectin-9.31’32Considering this observation, we developed a strategy to block the binding of any TIM-3 ligand (including galectin-9) expressed in B-ALL cells, to the TIM-3 receptor expressed on CAR19-T cells. We generated a TIM-3 decoy to capture the TIM-3 ligands and prevent them from interacting with the receptor. The TIM-3-Fc decoy contains the extracellular domain of TIM-3 and a silent Fc domain to increase stability and in vivo half-life (Figure 3A). Following transfection into 293T cells, we identified the TIM-3-Fc decoy in the supernatant and, importantly, its binding to galectin-9 (Figure 12). We subsequently generated lentiviruses and confirmed successful transduction of activated primary T cells with the TIM-3-Fc decoy, as indicated by the red fluorescence (dTomato+,Figure 3B). Transduced T cells showed significantly increased TIM-3 expression on the membrane, likely attributed to the presence of the TIM-3-Fc molecule (Figure 3C). Interestingly, no differences in proliferation capacity were observed between untransduced and TIM-3-Fc-transduced T cells during in vitro expansion (Figure 3D).

[0361] We next tested the functionality of this strategy in vivo using a galectin-9-expressing B-ALL PDX (60% galectin-9+cells) (Figure 3E). NSG mice were injected i.v. with 1×106B-ALL cells, and 4 days later, mice were injected i.v. with 2×106of the following T cells: untransduced T cells, TIM-3-Fc-T cells (25% transduction efficiency), CAR19-T cells (25% transduction efficiency) and CAR19-T cells plus TIM-3-Fc-T cells (25% transduction efficiency for both types). PB was monitored weekly by FACS for leukemia growth and T cell persistence (Figure 3F). Analysis of blasts in PB revealed full leukemia dissemination in the control groups (untransduced and TIM-3-Fc) at week 3, while animals treated with CAR19-T cells alone or in combination with TIM-3-Fc-T cells were in CR. To functionally assess CAR19-T-cell persistence, animals were re-challenged with the same PDX and monitored for leukemia relapse. Although no blasts were detected in PB four weeks after rechallenge, there was a predominant increase in T cells in mice treated with CAR19-T+TIM-3-Fc-T cells (Figure 3G). This was confirmed in animals sacrificed at endpoint (week 7), with a consistent increase of T cells in CAR19-T+TIM-3-Fc-T-treated mice compared to CAR19-T-treated mice (Figure 3H).

[0362] We next sought to determine whether the increase in T cells observed in animals treated with CAR19-T+TIM-3-Fc T cells was responsible for the greater anti-leukemic effect in vivo in a study under stress conditions using half the dose of T cells (1×106) with lower CAR19 transduction efficiency (17.5%): untransduced T cells, TIM-3-Fc-T cells (32.5% transduction efficiency), CAR19-T cells (17.5% transduction efficiency) and CAR19-T+TIM-3-Fc-T cells (17.5% and 32.5% transduction efficiency, respectively) (Figure 31). The endpoint results showed a significantly lower tumor burden in CAR19-T+TIM-3-Fc-T-treated animals compared to CAR19-treated mice (Figure 3J). Analysis of T cell persistence revealed that CAR19-T+TIM-3-Fc-T-treated mice consistently had the highest number of T cells in PB, spleen and BM compared to other groups (Figure 3K). Finally, to assess T cell functionality, we sorted the human T cells from mice treated with CAR19-T or CAR19-T+TIM-3-Fc-T cells for an ex vivo cytotoxic assay with B-ALL cells at E: T ratio of 1:1 for 24h. The results showed that T cells from animals treated with CAR19-T+TIM-3-Fc-T cells had a greater cytotoxic effect ex vivo, with lower numbers of blasts and higher numbers of T cells, than T cells from mice treated with CAR19-T cells (Figure 13). Collectively, these data confirm that secretion of a TIM-3-Fc decoy by primary T cells enhances the persistence and efficacy of CAR19-T cells in vivo and ex vivo. TIM-3-Fc-secreting CAR19-T cells improve CAR19 activity in vitro and in vivo \Ne next sought to engineer TIM-3-Fc as a secreted decoy in CAR19-T cells. We designed a CAR19-TIM-3-Fc bicistronic construct (“all-in-one” CAR19-TIM-3-Fc), incorporating the CAR19 structure, a F2A domain, the previously characterized TIM-3-Fc structure and a T2A peptide followed by GFP (Figure 4A). The transduction rates of the CAR19-TIM-3-Fc bicistronic construct into activated primary T cells were significantly lower than that of the single CAR19 construct (13.5±2.2% vs 54.5±3.6%) (Figure 4B). We first confirmed that CAR19-TIM-3-Fc-T cells secreted TIM-3-Fc (5.3±0.4 ng / mL) (Figure 4C). The proliferation rates of untransduced cells, CAR19- and CAR19-TIM-3-Fc-T cells were very comparable (Figure 4D). Additionally, we determined the ratio of CD4 / CD8 and its distribution among naive, central memory, effector memory (EM) and terminally differentiated EM T-cell subsets in untransduced, CAR19- and CAR19-TIM-3-Fc-T cells and found no differences (Figure 4E, F). Similarly, no differences were observed in the expression of the ICRs TIM-3, LAG-3, and PD-1 in transduced T cells between CAR19- and CAR19-TIM-3-Fc-T cells, except for a significant decrease of LAG-3 expression in CAR19-TIM-3-Fc CD8+T cells (Figure 4G). Notably, a significantly higher percentage of T cells expressing the early T-cell activation marker CD69 was found in transduced (GFP+) CAR19-TIM-3-Fc-T cells than in CAR19-T cells, in both CD8+and CD4+subsets (Figure 4H).

[0363] We next evaluated the cytotoxic function of CAR19-TIM-3-Fc T cells in vitro. Four independent primary B-ALL cells expressing galectin-9 were used for cytotoxicity assays at an E: T ratio of 1:8 for 48h (Figure 41). CAR-T cells were identified as 7AAD-CD3+GFP+and live target cells as 7AAD-CD3-(Figure 4J). Compared to CAR19-T cells alone, CAR19-TIM-3-Fc-T cells had a more potent cytotoxic effect, with significantly lower absolute numbers of live blasts and a significantly higher numbers of GFP+T cells and total T cells, as well as increased IFN• release (Figure 4K). This confirms that CAR19-TIM-3-Fc-T cells exhibit greater in vitro B-ALL cell killing and T-cell expansion.

[0364] We next assessed in vivo, using a B-ALL PDX stress model, the enhanced impact of CAR19-TIM-3-Fc-T cells observed in vitro on cytotoxic activity and T-cell expansion. Four days after PDX injection, 1.5×106of either CAR19- or CAR19-TIM-3-Fc-T cells (12% transduction efficiency; 175.000 transduced cells) were administered (Figure 5A). No significant weight loss was observed in CAR19- and CAR19-TIM-3-Fc-treated mice during the follow-up period (Figure 5B).

[0365] At endpoint, soluble TIM-3-Fc was detected in plasma in only those animals treated with CAR19-TIM-3-Fc-T cells (24.4±4.4 ng / mL), confirming in vivo secretion of TIM-3-Fc by bicistronic T cells (Figure 5C). Analysis of the anti-leukemic effects revealed that the number of blasts in PB, spleen and BM was significantly lower in animals treated with CAR19-TIM-3-Fc-T cells than in animals treated with CAR19-T cells (Figure 5D). Furthermore, the total number of circulating GFP+T cells was higher in animals treated with CAR19-TIM-3-Fc-T cells (Figure 5E). Thus, CAR19-TIM-3-Fc-T cells have enhanced CAR19-T cell activity in vivo.

[0366] CAR19-TIM-3-Fc-T-cell treatment promotes the in vivo expansion of transduced and bystander adaptive and innate effector and memory T cells

[0367] To further characterize the in v / vo-expanded T cell populations, spleens and BM were harvested from B-ALL xenografted mice four weeks after treatment with CAR19-T cells (n=5) and CAR19-TIM-3-Fc-T cells (n=5). Spleen and BM single-cell suspensions were analyzed by spectral flow cytometry using a 24-color T-cell module of the Euroflow-lmmune monitoring antibody panel3334(Figure 6A). The UMAP representation of 1,917,663 T cells, including both transduced (GFP+) and non-transduced / bystander (GFP-), were identified in spleen and BM from all mice and the major T-cell subsets, innate T cells, and functional and maturation-associated T cell subsets were classified and quantified (Figure 6B). CAR19-TIM-3-Fc-treated mice exhibited a notable expansion of multiple transduced and bystander T cell subpopulations in both spleen and BM compared to those treated with CAR19 alone, suggesting a greater polyclonal T cell response elicited by CAR19-TIM-3-Fc compared to CAR19 alone (Figure 6C). We thoroughly characterized the distinct T-cell populations that underwent a preferential significant expansion in each organ. In the spleens of animals treated with CAR19-TIM-3-Fc-T cells, the GFP+T-cell populations that showed the most pronounced expansion were effector • • T cells, consistent with their established cytotoxic capacity and their ability to migrate to lymphoid tissues3536(Figure 6C). Within the GFP-T-cell fraction in the spleen, there was a predominance of innate effector CD8+and CD4+mucosal-associated invariant T cells (MAIT), together with the CD8+and CD4+memory T cells. In T cells obtained from the BM of mice treated with CAR19-TIM-3-Fc-T cells, there was an overexpansion of GFP+CD4CD8-and CD8+EM T cells and GFP CD4CD8-, CD4+CD8+memory T cells and effector GFP CD4+ MAIT cells, all strong cytotoxic cells.36-39Finally, among all the T-cell populations significantly expanded in CAR19-TIM-3-Fc-treated mice, there were four GFP-T cell populations shared between spleens and BM. These include cytotoxic memory CD4+CD8+T cells and innate effector CD4+MAIT cells, which may be attributable to the bystander effect of the secreted TIM3-Fc decoy (Figure 6D). This multimodal identification of transduced and bystander T cells expanded in vivo provides insights into the diversity of T-cell responses following adoptive immunotherapy with CAR19-TIM-3-Fc-T cells.

[0368] CONCLUSIONS AND DISCUSSION CAR-T-cell therapy has emerged as an effective therapeutic approach for patients with R / R B-cell malignancies and multiple myeloma. Indeed, CD19-CAR-T cells have revolutionized the treatment of R / R B-ALL, but unfortunately, >50% of patients relapse at one year.6In this context, the design of the CAR constructs, the target of choice, the antigen density, the affinity and avidity of the antibody, the tumor burden and TME, and other factors that confer immune resistance to tumor cells, are key determinants of clinical response and durability.9’1041CAR-T-cell persistence after infusion is highly desirable; lower T-cell persistence correlates with worse overall survival in B-cell malignancies.42A number of emerging strategies hold promise for improving CAR-T-cell function and persistence. These include optimizing the design of CAR constructs, increasing the proportion of memory T cells, altering the TME through cytokine signaling, and overcoming IC inhibition with IC blockers.9’10’41’4344Here, we extensively characterized the protein expression of several ICRs and their ligands during B-ALL progression. Specifically, we investigated the expression of ICRs on T cells and IC ligands on B-ALL blasts and MSCs, which may play important role in the course of the disease.131445

[0369] The TIM-3-galectin9 axis was the only IC pathway that was consistently upregulated in the three cell types analyzed in our cohort of B-ALL samples, and it was also upregulated in the CAR19-T cell / B-ALL co-cultures. The TIM-3-galectin-9 axis has been studied in autoimmune diseases, infections and various cancer types,46-52but its role in cancer is still controversial. The effect of the TIM-3-galectin-9 axis in the context of CAR-T cells also remains unexplored. Our data show that galectin-9 clearly impairs CAR19-T cell cytotoxicity by increasing CAR-T cell apoptosis and decreasing T-cell proliferation.

[0370] We have developed an innovative therapeutic strategy based on an engineered TIM-3-Fc decoy delivered either by primary T cells co-administered with CAR19-T cells or by the CAR19-T cells themselves through a bicistronic all-in-one CAR19-TIM-3-Fc construct. Our data indicate that the presence of soluble TIM-3 increases CAR19-T cell persistence through autocrine and paracrine mechanisms. Importantly, the enhanced T-cell expansion improved the cytotoxicity effect of the CAR19 construct, especially the “all in one” CAR19-TIM-3-Fc construct, which was more active than CAR19-T cells alone, conferring a therapeutic advantage in vitro and in vivo. Because CAR19-T cells target CD19+blasts, the TIM-3-Fc decoy secreted locally to the site of disease by CAR19-TIM-3-Fc-T cells is likely to enhance the anti-leukemic effect compared to TIM-3 decoy secreted by other non-CAR T cells.

[0371] Decoys occur naturally in humans and are very useful for targeting receptors / ligands with redundant functions. Indeed, receptor-ligand promiscuity is a hallmark of the chemokine / cytokine network, but is also evident for ICRs, such as TIM-3, where the same receptor has multiple ligands that can bind to the same or distinct sites within the extracellular domain.57-59Several anti-TIM-3 mAbs have been clinically used in several tumors with limited therapeutic efficacy, and significant antitumor effects have only been shown when co-administered with anti-PD-1 or anti-PD-L1 mAbs.60-62In contrast to the TIM-3 decoy, available anti-TIM-3 antibodies bind to a specific site on the TIM-3 receptor, disrupting its binding to PtdSer and CEACAM1, but not to galectin-9, thereby compromising the inhibition of the TIM-3 inhibitory pathway and reducing their therapeutic effect.31 32In addition to mAbs, intrinsic IC silencing in CAR-T cells using CRISPR / Cas9 has shown efficacy.63-67Two independent studies showed that a TIM-3 knockout mouse model had impaired T-cell responses, supporting the idea that TIM-3 is essential for optimal T-cell function.7071Accordingly, using an IC blocker without altering the innate function of the ICR seems the most promising approach.

[0372] Spectral flow cytometry studies allowed us to comprehensively identify in vivo overexpanded T-cell populations in B-ALL-xenografted mice treated with CAR19-TIM3-Fc-T cells. We identified specific functional and maturation-associated subpopulations of both adaptive and innate transduced and bystander T cells overexpanded in mice treated with CAR19-TIM3-Fc-T cells. Among all the populations overexpanded in CAR19-TIM-3-Fc-treated mice, there is a predominance of innate effector and memory T cells, such as •• T cells, MAIT and double negative T cells. As these innate CAR19-T cells are likely to show exhausted immunophenotype / proliferation due to their strong cytotoxic activity,38’4072-75the local secretion of the TIM-3-Fc decoy may benefit the persistence of these T-cell subtypes.

[0373] In conclusion, our novel strategy using CAR19-TIM-3-Fc-T cells that continuously secrete a TIM-3 decoy in vivo improves CAR19-T-cell persistence and efficacy in pre-clinical B-ALL PDX models. Furthermore, T cells engineered to secrete the TIM-3-Fc decoy may be extended to different CAR-T cells or other malignancies involving the TIM-3-galectin-9 axis.

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Claims

CLAIMS1. A T cell secreting a soluble fusion protein consisting of SEQ ID NO: 3, 2 or 9.

2. The T cell according to claim 1, wherein the cell further expresses a targeting moiety capable of binding to a tumour associated antigen.

3. The T cell according to claim 2, wherein the targeting moiety capable of binding to a tumour associated antigen is a chimeric antigen receptor (CAR).

4. The T cell according to claim 3, wherein the CAR is a CAR against CD19.

5. A composition comprising a T cell as defined in any of claims 1 to 4, or a population of said T cells.

6. The T cell as defined in any one of claims 1 to 4, or the composition as defined in claim 5, for use in medicine or as a medicament.

7. The T cell or composition for use as defined in claim 6, for use in the treatment or prevention of cancer.

8. The T cell as defined in claim 4, or a composition comprising said T cell, for use in the treatment or prevention of CD19+ B-cell malignancies, preferably B-cell acute Lymphoid Leukemia.

9. A combination product comprising:- a fusion protein consisting of SEQ ID NO: 3, SEQ ID NO: 2, or SEQ ID NO: 9, or a T cell expressing said fusion protein, and- a chimeric antigen receptor (CAR), or a T cell expressing said CAR,for use in a method of treatment or prevention of cancer, wherein the use comprises administering the CAR and the fusion protein, or the respective cells expressing them, either simultaneously or sequentially.

10. The combination product for use according to claim 9, wherein the T cell expressing the fusion protein and the CAR is the same cell.

11. The combination product for use according to claim 9 or 10, wherein the CAR is a CAR against CD19.

12. The combination product for use according to claim 11, wherein the use is in the treatment or prevention of CD19+ B-cell malignancies, preferably B-cell acute Lymphoid Leukemia.

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