WO2025082603A1 - Engineered immune cells overexpressing cd74 molecule - Google Patents

Abstract

The invention relates to engineered immune cells overexpressing CD74 molecule which have improved properties.

Description

ENGINEERED IMMUNE CELLS OVEREXPRESSING CD74 MOLECULE

FIELD OF THE INVENTION

The invention pertains to the field of immunotherapy, in particular adoptive cell therapy of cancer. The invention relates to immune cells genetically engineered to overexpress CD74 molecule, which have improved properties.

BACKGROUND OF THE INVENTION

At present, cell-based therapies are in the forefront of cancer treatment. Some current therapies aim at genetically modifying human T cells to increase their tumor recognition and migration by introducing receptors for tumor antigens, as specific T cell receptors (TCR) or chimeric antigen receptors (CAR), and tumor homing-chemokine receptors. Despite such innovations, T cell persistence and anti-tumoral activity are usually hampered by the inefficiency of adoptively transferred cells to survive, reach, and accumulate in the hostile microenvironment of solid tumors. Therefore, there is a need for improved protocols for cell-based therapies.

SUMMARY OF THE INVENTION

Using transcriptomic datasets of CD4+ T conventional and regulatory T cells (Tregs) cells from human blood and tumors, the inventors have observed that a considerable extent of tumor Tregs overexpress CD74. CD74 has been described as chaperone for MHC-II, as a receptor for the macrophage migration inhibition factor (MIF) and as an intracellular mediator in different cell types. CD74 has already been targeted by Milatuzumab, a humanized IgGlk anti-CD74 blocking monoclonal antibody, designed to target B cells in lymphoma. However, its role in regulatory T cell (Treg) biology remains unknown. Indeed, CD74 manipulation to regulate T-cell retention in diseased tissue such as tumor has not yet been explored.

The inventors showed that, compared to wild type Tregs, CD74-deficient ones (obtained by CRISPR-Cas9 genetic knock-out of CD74 in human primary Tregs) accumulate less in the tumor and present perturbed suppressive function only in the tumor. Hence, CD74 upregulation seems to play a key role for Treg accumulation and fitness in tumors. Based on these findings, they have envisioned the engineering of anti-tumoral T cell types, such as CD4+, CD8+, NK or other immune cells to express increased levels of CD74, as a potential mean to augment their accumulation in the tumor and their anti-tumoral fitness. Additionally, increasing CD74 expression could be used to enhance Treg infiltration in inflamed tissues, for example in the case of autoimmune diseases.

CD74 overexpression was achieved using different forms of CD74 molecule: the full-length protein, and a truncated CD74 isoform with a shorter length of the intracellular domain correlating with a higher stabilization at the membrane (CD74dell-23). To this end, lentiviral CD74 gene transduction was used to achieve upregulated CD74 expression in primary human CD3+ T cells in vitro. Using a humanized mouse transplanted with human tumor, the inventors showed that CD3+ T cells overexpressing CD74 exhibit a higher capacity to infiltrate the tumor tissue and a higher anti-tumor activity than the mock transduced control cells. These results indicate that the manipulation of CD74 to induce its overexpression in anti-tumoral T- cell types represent a novel mean to increase their anti-tumor activity.

A new cell therapy approach is thus proposed where immune cells are genetically engineered to overexpress CD74 molecule, in order to increase their preferential retention in the diseased tissue and their capacity to control diseases such as cancer and auto-immune diseases. In particular, effector immune cells, in particular T cells, are engineered to increase their preferential retention in tumor and their anti-tumor activity; regulatory immune cells, in particular Tregs, are engineered to increase their preferential retention in inflamed tissues and their immunosuppressive activity.

Therefore, the invention relates to an engineered immune cell which is genetically engineered to overexpress CD74 molecule.

In some embodiments, the engineered immune cell is a T cell, NK cell, B cell, macrophage, or dendritic cell. In particular embodiments, the T cell is an effector T cell. In particular embodiments, the T cell is a regulatory T cell.

In particular embodiments, the engineered immune cell comprises a transgene encoding recombinant CD74 molecule of SEQ ID NO: 1 or a truncated form thereof. In some more particular embodiments, said truncated from comprises an N-terminal deletion of at least 12 amino acids in the cytoplasmic domain of CD74, preferably a N-terminal deletion of 23 amino acids. In some embodiments, the engineered immune cell, further expresses a genetically engineered antigen receptor, in particular a chimeric antigen receptor or a T cell receptor. In particular embodiments, the engineered immune cell is a CAR-T or TCR-T cell.

In some embodiments, the engineered immune cell, is isolated from a subject.

The invention also relates to an engineered immune cell according to the present disclosure for use as a medicament. In some embodiments, the engineered immune cell is for use in adoptive cell therapy of a subject. In some particular embodiments, the engineered immune cell is autologous. In some particular embodiments, the engineered immune cell is allogenic. In some particular embodiments, the engineered immune cell is for use in the treatment of inflammatory or autoimmune diseases, wherein said immune cell is a regulatory T cell. In some particular embodiments, the engineered immune cell is for use in the treatment of cancer, wherein said immune cell is an effector T cell, in particular a CAR-T or TCR-T cell.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to engineered immune cells overexpressing CD74 molecule which have a higher capacity to accumulate in diseased tissue. In the case of cancer, the invention relates to immune cells overexpressing CD74 molecule which have a higher anti-tumor activity. In the case of autoimmune disease, transplantation or inflammation, the invention relates to regulatory T cells overexpressing CD74 molecule which have a higher suppressive activity. The invention encompasses the engineered immune cells, the derived composition and their use in adoptive cell therapy, in particular of cancer, inflammatory and autoimmune diseases, transplantation, more particularly adoptive cell therapy of cancer.

Engineered immune cells

Immune cells

As used herein the term “immune cell” (or cell) refer to cells that are of hematopoietic origin and play a role in the immune response. Immune cell includes cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells. Immune cells include lymphocytes, such as B cells and T cells, natural killer cells and innate lymphoid cells (ILCs); myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes. Immune cells for engineering according to the invention include immune cell precursors and progeny thereof. Immune cell progeny includes effector and regulatory immune cells. Immune cells as used herein, refer to isolated immune cells or isolated population of immune cells. The population of immune cells as used herein may be a whole population of immune cells or may comprise one or more subsets or subpopulations of immune cells. The subpopulations of immune cells as used herein include those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. The cells for engineering according to the invention are typically eukaryotic cells, such as mammalian cells, preferably human cells. The cells may be primary cells, cells derived from stem cells or cell lines. Typically, the cells are primary cells, such as those obtained from or derived from a subject. Primary cells may be isolated directly from a subject and/or isolated from a subject and frozen. With reference to the subject to be treated, the cells of the invention may be allogeneic (or allogenic) and/or autologous.

In some embodiments, the immune cells are lymphocytes, monocytes, macrophages or dendritic cells. Lymphocytes include, B cells, T cells, NK cells and innate lymphoid cells (ILCs). In some particular embodiments, the lymphocytes are T cells, B cells and/or NK cells.

In some particular embodiments, the immune cells are T cells. As used herein, the term “T cell” includes cells bearing a T cell receptor (TCR). T cells include, whole T cell population, CD4+ cells, CD8+ cells, mixture of CD4+ and CD8+ cells and subpopulations thereof. Among the sub-types and subpopulations of T cells and/or of CD4+ and/or of CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, inflammatory T-lymphocytes, tumor-infiltrating lymphocytes (TILs), immature T cells, mature T cells, cytotoxic T cells, conventional T cells (Tconv), mucosa-associated invariant T (MAIT) cells, regulatory T (Treg) cells, follicular regulatory T cell, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH 17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells. In some particular embodiments, T cells are chosen from whole T cell population, CD4+ T cells, CD8+ T cells, mixture of CD4+ T cells and CD8+ T cells. In some preferred embodiments, T cells are Tregs. Tregs are a subset of CD4+ T cells that are Foxp3+. In some preferred embodiments, T cells exclude Tregs and comprise CD8+ T cells and/or Tconvs. Tconvs are a subset of CD4+ T cells that are Foxp3-. CD8+ T cells and Tconvs are effector T cells.

As used herein effector immune cells refer to immune cells other than Tregs.

In some embodiments, one or more of the T cell populations is enriched for, or depleted of, cells that are positive for or express high levels of one or more particular markers, such as surface markers, or that are negative for or express relatively low levels of one or more markers.

In some embodiments, such markers are those that are absent or expressed at relatively low levels on certain populations of T cells (such as non-memory cells) but are present or expressed at relatively higher levels on certain other populations of T cells (such as memory cells). In some particular embodiments, T cells are enriched in less differentiated T cell subsets that have greater proliferative capacity, such as naive T cells, stem cell memory T (TSCM) cells and central memory T (TCM) cells.

Cells according to the invention may also be immune cell progenitors, such as lymphoid progenitors and more preferably T cell progenitors. T cell progenitors typically express a set of consensus markers including CD44, CD117, CD135, and Sca-1 but see also Petrie HT, Kincade PW. Many roads, one destination for T cell progenitors. The Journal of Experimental Medicine. 2005;202(l): 11-13; Delacher et al., Immunity. 2020 Feb 18;52(2):295-312.el l. doi: 10.1016/j.immuni.2019.12.002. Epub 2020 Jan 7. PMID: 31924477; Pai et al., Cancer Cell, 2023, Apr 10;41(4):776-790.e7, doi: 10.1016/j.ccell.2023.03.009. Epub 2023 Mar 30.

The cells for engineering according to the invention are generally isolated from a sample, notably a biological sample, e.g., obtained from or derived from a subject. Typically, the subject needs a cell therapy (adoptive cell therapy) and/or will receive the cell therapy. The subject is preferably a mammal, notably a human. In some embodiments of the invention, the subject has a cancer, is at risk of having a cancer, or is in remission of a cancer. The samples include tissues, fluids, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (for example transduction with viral vector), washing, and/or incubation. Therefore, the biological sample can be a sample obtained directly from a biological source or a sample that is processed. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), cord-blood, leukocytes, bone marrow, thymus, tissue biopsy, tissue from a site of infection, ascites, pleural effusion, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, and/or cells derived therefrom. Samples include, in the context of cell therapy (typically adoptive cell therapy) samples from autologous and allogeneic sources. Preferably, the sample from which the cells are derived or isolated is blood or a blood-derived sample or is or, is derived from, an apheresis or leukapheresis product.

Immune cells can be extracted from blood or derived from stem cells. The stem cells can be adult stem cells, embryonic stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells. Representative human stem cells are CD34⁺ cells.

Isolation of the cells includes one or more preparation and/or non-affinity-based cell separation steps according to well-known techniques in the field. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components.

Immune cells are isolated and cultured using standard methods that are well-known in the art. For example, immune cells may be isolated by leukapheresis, usually using differential centrifugation technique to separate leukocytes from whole blood. Specific sub-population of immune cells, in particular T cells, can be further isolated by positive or negative selection techniques, for example using flow cytometry assisted cell sorting or magnetic activated cell separation. For example, immune cells, in particular T cells, can be isolated using a combination of antibodies directed to surface markers unique to the positively selected cells, e.g., by incubation with antibody-conjugated beads for a time sufficient for positive selection of the desired immune cells. Alternatively, enrichment of immune cells, in particular T cells, can be performed by negative selection using a combination of antibodies directed to surface markers unique to the negatively selected cells. Non-limiting examples of specific cell-surface markers are well-known in the art and include: CD34 for hematopoietic stem cells; CD45 for hematopoietic cells; CD3 for T cells; CD56 for NK cells; CD19 for B cells; CD14 and CD16 for monocytes and macrophages; CDllc for dendritic cells; CD4, CD8 and further CD25 and/or CD 127 for T cell subpopulations; Tregs may be defined as CD45+ CD4+ CD25^hl CD127¹⁰ cells and Tconvs as CD45+ CD4+ CD25¹⁰ CD127^lo/hl.

In allogeneic immune cell therapy, immune cells are collected from healthy donors, rather than the patient. Typically, these are HLA matched to reduce the likelihood of graft vs. host disease. Alternatively, universal ‘off the shelf’ products that may not require HLA matching comprise modifications designed to reduce graft vs. host disease, such as disruption or removal of the TCRa0 receptor. See Graham et al., Cells. 2018 Oct; 7(10): 155 for a review. Because a single gene encodes the alpha chain (TRAC) rather than the two genes encoding the beta chain, the TRAC locus is a typical target for removing or disrupting TCRa[l receptor expression. Alternatively, inhibitors of TCRa[l signalling may be expressed, e.g. truncated forms of CD3^ can act as a TCR inhibitory molecule. Disruption or removal of HLA class I molecules has also been employed. For example, Torikai et al., Blood. 2013;122:1341-1349 used ZFNs to knock out the HLA-A locus, while Ren et al., Clin. Cancer Res. 2017;23:2255- 2266 knocked out Beta-2 microglobulin (B2M), which is required for HLA class I expression. Ren et al. simultaneously knocked out TCRa[l, B2M and the immune-checkpoint PD1.

The isolated immune cells, in particular T cells, are cultured in appropriate culture conditions that are well-known in the art. The culture may comprise one or more steps of incubation, stimulation, activation, and/or expansion of the immune cells. Generally, the immune cells are activated and expanded to be utilized in the adoptive cell therapy. The immune cells as herein disclosed can be expanded in vivo or ex vivo. The immune cells, in particular T-cells can be activated and expanded generally using methods known in the art. Generally, the T-cells are expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a co -stimulatory molecule on the surface of the T cells. In some embodiments, the stimulating agents include cytokines such as IL-2, IL-7 and/or IL- 15, for example, an IL-2 concentration of at least 10 units/m/L. In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. Such agents can include antibodies, such as those specific for a TCR component and/or costimulatory receptor, e.g., anti-CD3, anti-CD28, for example, bound to solid support such as a bead, and/or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti- CD3 and/or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml). Expansion of the immune cells, in particular T cells may be obtained by increasing the concentration of IL-2 (300 UI/mL) in the culture medium.

In some embodiments, the immune cells, in particular T cells, are expanded by adding to the culture-initiating composition feeder cells; and incubating the culture for a time sufficient to expand the numbers of immune cells, in particular T cells. In some embodiments, the feeder cells are non-dividing peripheral blood mononuclear cells (PBMC) or EBV-transformed lymphoblastoid cells (LCL), preferably irradiated. In some aspects, the feeder cells are added to culture medium prior to the addition of the populations of immune cells, in particular T cells.

In some embodiments, antigen-specific T cells, such as antigen- specific CD4+ and/or CD8+ T cells, are obtained by stimulating naive or antigen specific T lymphocytes with antigen. For example, antigen- specific T cell lines or clones can be generated to cytomegalovirus antigens by isolating T cells from infected subjects and stimulating the cells in vitro with the same antigen.

In some embodiments, the cell preparation includes steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and/or engineering. Any of a variety of known freezing solutions and parameters in some aspects may be used.

Engineered, immune cells

The immune cells according to the invention are genetically engineered (or modified) to overexpress CD74 molecule. Overexpression is typically obtained by standard gene genetic engineering techniques where nucleic acid is introduced into a cell to overexpress a gene such as CD74 that is already expressed in the cell. The nucleic acid may be a transgene or a system for targeted activation of the endogenous gene. Systems for targeted gene activation in a cell are well-known in the art and include for example nuclease-dead Cas proteins fused to transcriptional or epigenetic modulators (review in Chavez et al., Nature reviews nephrology, 2023, 19, 9-22).

In particular embodiments, the cell comprises a transgene encoding CD74 molecule, and the engineered cell expresses high level of recombinant CD74 molecule, which together with expression of endogenous CD74 molecule result in an engineered immune cell overexpressing CD74 molecule. The term “CD74” (CD74 antigen or molecule, HLA Class II Histocompatibility Antigen Gamma Chain, Major Histocompatibility Complex, Class II Invariant Chain, or invariant chain (li)) refers to a protein encoded by the CD74 gene in a mammalian genome. CD74 is a homotrimeric single-pass type II membrane protein which functions as a chaperone for MHC-II molecule, a receptor for MIF or a regulator for transcription factors (such as NFkb or Bcl-xl). Representative examples of CD74 include without limitation, human (NCBI Gene ID: 972), mouse (NCBI Gene ID: 16149) and other functional orthologs thereof.

CD74 comprises at least 5 isoforms in humans which are produced by alternative splicing and/or alternative initiation. They present different stability in the membrane and are exposed to different posttranscriptional modifications.

Human CD74 canonical sequence (long isoform) has the 296 amino acid sequence UniProtKB/Swiss-Prot P04233-1 or SEQ ID NO: 1. Isoform p35 (short isoform) lacks residues 209-272 of SEQ ID NO: 1 and corresponds to the 232 amino acid sequence UniProtKB/Swiss-Prot P04233-2. Isoform 3 lacks residues 161 to 296 of SEQ ID NO: 1 and has mutation at positions 148-160 (replacement of NADPLKVYPPLKG with SHWNWRTRLLGWV) and corresponds to the 160 amino acid sequence UniProtKB/Swiss- Prot P04233-3. Isoform p41 lacks residues 1-16 of SEQ ID NO: 1 and corresponds to the 280 amino acid sequence UniProtKB/Swiss-Prot P04233-4. Isoform p33 lacks both residues 1-16 and 209-272 of SEQ ID NO: 1 and corresponds to the 216 amino acid sequence UniProtKB/Swiss-Prot P04233-5.

Based on structure prediction and confirmation by crystallographic studies CD74 protein may be divided in 3 domains from its N-terminus to C-terminus: a Cytoplasmic domain (positions 1 to 46); a Transmembrane domain (positions 47 to 72) and an Extracellular domain (positions 73 to 296). The above-indicated positions of the different structural domains of CD74 protein may vary by few amino acids within the isoforms without altering the overall structure and activity of CD74 protein. Therefore, the invention encompasses domains which differ from the indicated positions by the addition or deletion of up to 5 consecutive amino acids at one or both ends of the domain.

In the following description, the amino acid residues are designated by the standard one letter amino acid code and the indicated positions are determined by alignment with SEQ ID NO: 1. One skilled in the art can easily determine the positions in another CD74 protein, by alignment with the reference sequence using appropriate software available in the art such as BLAST, CLUSTALW and others.

“a”, “an”, and “the” include plural referents, unless the context clearly indicates otherwise. As such, the term “a” (or “an”), “one or more” or “at least one” can be used interchangeably herein; unless specified otherwise, “or” means “and/or”.

The term “functional ortholog” as used herein, refers to a gene from another species which encodes a protein having substantially the same activity than that of the initial gene. It is understood that polymorphisms or variants with different sequences exist in genomes from various mammalian species. The term CD74 protein according to the invention thus encompasses all mammalian variants of CD74 including the various isoforms of CD74, and polypeptides comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 1 that have CD74 activity (e.g. functional variants). CD74 activity according to the present disclosure relates in particular to the capacity of CD74 overexpressing immune cell to preferentially accumulate in the diseased tissue. CD74 activity can be assessed for example by evaluating the percentage of tumor infiltrating immune cells (in particular CD3+ T cells, or CD4+ and CD8+ subsets) expressing CD74 in mice bearing tumor injected with immune cells engineered with CD74 transgene and immune cells engineered with control transgene (encoding non-related protein), as disclosed in the examples. The percentage of tumor-infiltrating immune cells expressing CD74 is significantly increased in mice bearing tumor injected with the engineered immune cells according to the invention. In addition, tumor-growth is significantly decreased in mice bearing tumor injected with the engineered immune cells according to the invention.

As used herein, “a higher capacity to infiltrate diseased tissue”, with respect to the engineered immune cells according to the invention, refers to a higher capacity to accumulate, or a higher retention capacity in the diseased tissue. The accumulation of the engineered immune cells according to the invention in the diseased tissue such as tumor is selective since the engineered immune cells accumulate preferentially in the diseased tissue compared to other tissues. This accumulation results in an increased activity, in particular higher anti-tumor activity of the engineered immune effector cells according to the invention or higher immunosuppressive activity of the engineered immune regulatory cells according to the invention.

As used herein, the term “functional” with respect to a CD74 protein, including variants and fragments thereof refers to a CD74 protein having CD74 activity as disclosed herein.

As used herein, the term “variant” refers to a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the native sequence; preferably having at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the native sequence. The term variant includes fragments.

As used herein, the term “fragment” with respect to a CD74 protein or molecule, refers to a polypeptide having a sequence of at least 20 consecutive amino acids from a CD74 protein sequence. A CD74 fragment is also named truncated form of CD74.

The term “variant” refers to a functional variant having the activity of the native sequence. The activity of a variant may be assessed using methods well-known by the skilled person such as those disclosed in the examples.

The percent amino acid sequence or nucleotide sequence identity is defined as the percent of amino acid residues or nucleotides in a Compared Sequence that are identical to the Reference Sequence after aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity and not considering any conservative substitutions for amino acid sequences as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways known to a person of skill in the art, for instance using publicly available computer software such as the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program, or any of sequence comparison algorithms such as BLAST (Altschul et al., J. Mol. Biol., 1990, 215, 403-), FASTAor CLUSTALW. When using such software, the default parameters, are preferably used. The BLASTP program uses as default a word length (W) of 3 and an expectation (E) of 10.

In some embodiments, the term "variant" refers to a polypeptide having an amino acid sequence that differs from a native sequence by the substitution, insertion and/or deletion of less than 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10 or 5 amino acids. In a preferred embodiment, the variant differs from the native sequence by one or more conservative substitutions, preferably by less than 50, 40, 30, 25, 20, 15, 10 or 5 conservative substitutions. Conservative substitutions are substitutions of one amino acid with another having similar chemical or physical properties (size, charge or polarity), which substitution generally does not adversely affect the biochemical, biophysical and/or biological properties of the protein. Examples of conservative substitutions may be within the following groups : Group 1 -small aliphatic, non-polar or slightly polar residues (A, S, T, P, G); Group 2-polar, negatively charged residues and their amides (D, N, E, Q); Group 3-polar, positively charged residues (H, R, K); Group 4-large aliphatic, nonpolar residues (M, L, I, V, C); and Group 5-large, aromatic residues (F, Y, W); or within the groups of basic amino acids (R, K, H), acidic amino acids (D, E), polar amino acids (Q, N), hydrophobic amino acids (M, L, I, V), aromatic amino acids (F, W, Y), and small amino acids (G, A, S, T).

The term "expressing a polynucleotide" means when a polynucleotide is transcribed to mRNA and the mRNA is translated to a polypeptide. The term "overexpress" refers to any amount greater than an expression level exhibited by a reference standard (typically the corresponding non genetically modified cell or the corresponding cell genetically modified with a transgene encoding a non-related protein). The terms “upregulated expression”, “increased expression”, "overexpress," "overexpressing," "overexpressed" and "overexpression" in the present invention refer an expression of a gene product or a polypeptide at a level greater than the expression of the same gene product or polypeptide prior to a genetic alteration of the host cell or in a comparable host which has not been genetically altered at defined conditions. By “increased expression”, or “overexpression” it is meant herein that the expression is increased by at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5-fold or more as compared to normal levels in the corresponding cell control." Typically, CD74 can be co-expressed with a protein of interest (such as a GFP marker or a chimeric antigen protein). Thus, CD74 transgene expression in modified cells can be detected by way of measuring GFP or any other protein of interest such as the CAR. Alternatively, anti-CD74 antibodies can be used to detect expression of CD74 or fragment thereof.

In some embodiments, the cell comprises a transgene (e.g. heterologous gene) encoding CD74 protein. Preferably, the CD74 protein is human CD74.

As used herein, CD74 protein or molecule refers to wild-type CD74 protein including its various isoforms from various mammalian species and variants thereof which differ from the native sequence by amino acid mutations in particular substitutions, as well as fragments thereof. In some particular embodiments, the transgene encodes full-length CD74 protein (canonical CD74 protein sequence). Preferably, the full-length CD74 protein is human full- length CD74 (SEQ ID NO: 1).

In some particular embodiments, the transgene encodes a CD74 protein fragment comprising an N-terminal deletion of at least 12 amino acids in the cytoplasmic domain of CD74, preferably a N-terminal deletion from 12 to up to 46 amino acids; more preferably a deletion of 23 amino acids. It was previously observed that CD74 isoforms with a shorter length of the CD74 intracellular domain correlates with higher stabilization at the plasma membrane (cellsurface) by slowing down their endocytosis (Bakke et al., Cell, 1990, 63, 707-716). Immune cells expressing membrane-stabilized CD74 accumulate preferentially in diseased tissue.

In some particular embodiments, the cell comprises a transgene encoding a CD74 protein fragment consisting of the cytoplasmic domain or the cytoplasmic and transmembrane domains of CD74. In some more particular embodiments, the CD74 protein fragment consists of positions 1 to 42 or 1 to 82 of SEQ ID NO: 1. It was previously described that CD74 cytoplasmic domain acts as a transcription factor (Matza et al., Immunity, 2002 Nov;17(5):549-60. doi: 10.1016/sl074-7613(02)00455-7). Thus, it is expected that it is capable per se to increase cell survival in the diseased tissue, in particular tumor.

In some embodiments, the engineered immune cell, further expresses at least one recombinant molecule of interest (e.g. comprises another transgene of interest), in particular to improve the efficacy of the cellular-therapy, such as by promoting viability, persistence, infiltration and/or function of transferred cells or improving their therapeutic efficacy for treating a disease. Non- limiting examples of molecules of interest or genes including such molecules include : a genetically engineered antigen receptor, in particular a chimeric antigen receptor or a T cell receptor; inflammatory mediators such as cytokines, soluble immune-regulatory receptors and/or ligands to deliver into the tumour microenvironment; genes to provide a genetic marker for selection and/or evaluation of the cells, such as to assess in vivo survival or localization; genes to improve safety, for example, by making the cell susceptible to negative selection in vivo as described by Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992).

In addition, or as an alternative to the combination with checkpoint blockade, the immune cell of the present disclosure may also be genetically modified to render them resistant to immune- checkpoints using gene-editing technologies including but not limited to TALEN and CRISPR-Cas such as CRISPR-Cas9. Such methods are known in the art, see e.g. US20140120622. Gene editing technologies may be used to prevent the expression of immune checkpoints expressed by T cells including but not limited to PD-1, Lag-3, Tim-3, TIGIT, BTLA CTLA-4 and combinations of these. The immune cell as discussed here may be modified by any of these methods.

In some particular embodiments, the immune cell further comprises at least one genetically engineered antigen receptor. The genetically engineered antigen receptor binds an antigen of interest for the treatment of a disease. In some embodiments, the antigen is expressed in a cancer cell and/or is a universal tumor antigen. In some embodiments, the antigen is a pathogen-specific antigen, such as viral, bacterial and/or parasitic antigen.

Among the antigen receptors as per the invention are genetically engineered T cell receptors (TCRs) and components thereof, as well as functional non-TCR antigen receptors, such as chimeric antigen receptors (CAR). Non-limiting exemplary antigen receptors, including CARs and recombinant TCRs, as well as methods for engineering and introducing the receptors into cells, include those described, for example, in international patent application publication numbers W0200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, W02013/071154, W02013/123061 U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S. Patent Nos.: 6,451,995, 7,446,190, 8,252,592, , 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patent application number EP2537416, and/or those described by Sadelain et al., Cancer Discov. 2013 April; 3(4): 388- 398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75.

The transgene encoding CD74 molecule and optional transgene(s) encoding the molecule(s) of interest are usually included in a nucleic acid construct comprising a nucleotide sequence encoding the molecule. The nucleic acid construct may comprise or consist of DNA, RNA or a synthetic or semi-synthetic nucleic acid which is expressible in the individual’s target cells (immune cells). The sequence encoding the molecule may be codon-optimized for expression in the individual’s immune cells, preferably a human individual. Appropriate softwares for codon optimization in the desired individual are well-known in the art and publicly available.

The nucleic acid construct comprises an expression cassette wherein the coding sequence is operably linked to appropriate regulatory sequences for expression of a transgene in the individual’s target cells (immune cells). Such sequences which are well-known in the art include in particular a promoter, and further regulatory sequences capable of further controlling the expression of a transgene, such as without limitation, enhancer; terminator; intron; silencer, in particular tissue-specific silencer such as miRNAs; and post-translational regulatory element.

The promoter may be a tissue-specific, ubiquitous, constitutive or inducible promoter that is functional in the individual’s target cells. Examples of constitutive promoters which can be used in the present invention include without limitation: phosphoglycerate kinase promoter (PGK); elongation factor- 1 alpha (EF-1 alpha) promoter including the short form of said promoter (EFS); dihydrofolate reductase promoter; [l-actin promoter; viral promoters such as cytomegalovirus (CMV) immediate early enhancer and promoter, cytomegalovirus enhancer/chicken beta actin (CAG) promoter, SV40 early promoter and retroviral 5’ and 3’ LTR promoters including hybrid LTR promoters. Preferred ubiquitous promoter is EF-1 alpha promoter. Examples of inducible promoters which can be used in the present invention include Tetracycline-regulated promoters. The promoters are advantageously human promoters, i.e., promoters from human cells or human viruses. Such promoters are well-known in the art and their sequences are available in public sequence data base. Moreover, transgene expression levels can be improved by the inclusion, in the transgene expression cassette, of post-transcriptional regulatory elements such as the Woodchuck hepatitis virus (WHV) post-transcriptional regulatory element (WPRE), able to increase transcript levels and/or stability.

In some embodiments, the CD74 molecule including truncated form thereof is co-expressed with another molecule of interest as disclosed herein, in particular an engineered antigen receptor as disclosed herein . In order to assess the expression of a CD74 polypeptide, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected with the expression vector. In particular embodiments, the nucleic acid construct comprises separate expression cassettes for the CD74 molecule and the other protein(s) of interest. In other particular embodiments, the nucleic acid construct comprises a unique expression cassette wherein the coding sequences for the CD74 protein and the other protein of interest are separated by an Internal Ribosome Entry Site (IRES) or a viral 2A peptide.

The immune cells are genetically modified using standard nucleic acid delivery agents or systems suitable for the delivery and expression of nucleic acid into individual’s immune effector cells, in particular suitable for cellular therapy. The invention may use chemical, biological or physical delivery methods or a combination thereof. Such vectors that are well- known in the art include viral and non-viral vectors, wherein said vectors may be integrative or non-integrative; replicative or non- replicative.

Non-viral vector includes the various (non-viral) agents which are commonly used to either introduce or maintain nucleic acid into individual’s cells. Agents which are used to introduce nucleic acid into individual’s cells by various means include in particular polymer-based, particle-based, lipid-based, peptide -based delivery vehicles or combinations thereof, such as with no limitations cationic polymer, dendrimer, micelle, liposome, exosome, microparticle and nanoparticle including lipid nanoparticle (LNP); and cell penetrating peptides (CPP). CPP are in particular cationic peptides such as poly-L-Lysine (PLL), oligo-arginine, Tat peptides, Penetratin or Transportan peptides and derivatives thereof such as for example Pip. Agents which are used to maintain nucleic acid into individual’s cells (either integrated into chromosome(s) or else in extrachromosomal form) include in particular naked nucleic acid vectors such as plasmids, transposons and mini-circles, and gene-editing and RNA-editing systems. Transposon includes in particular the hyperactive Sleeping Beauty (SB100X) transposon system (Mates et al. 2009). Gene-editing and RNA-editing systems may use any site-specific endonuclease such as Cas nuclease, TALEN, meganuclease, zinc finger nuclease and the like. Gene-editing includes in particular targeted knock-in of the transgene. Nucleic acid may also be introduced into individual’s cells by physical means such as particle bombardment, micro injection, electroporation, and the like. In addition, these approaches can advantageously be combined to introduce and maintain the nucleic acid of the invention into individual’s cells.

Viral vectors are by nature capable of penetrating into cells and delivering nucleic acid(s) of interest into cells, according to a process named as viral transduction. As used herein, the term “viral vector” refers to a non-replicating, non-pathogenic virus engineered for the delivery of genetic material into cells. In viral vectors, viral genes essential for replication and virulence are replaced with an expression cassette for the transgene of interest. Thus, the viral vector genome comprises the transgene expression cassette flanked by the viral sequences required for viral vector production. As used herein, the term “recombinant virus” refers to a virus, in particular a viral vector, produced by standard recombinant DNA technology techniques that are known in the art. As used herein, the term “virus particle” or “viral particle” is intended to mean the extracellular form of a non-pathogenic virus, in particular a viral vector, composed of genetic material made from either DNA or RNA surrounded by a protein coat, called the capsid, and in some cases an envelope derived from portions of host cell membranes and including viral glycoproteins. As used herein, a viral vector refers to a viral vector particle.

A first type of vector for delivering the nucleic acid (nucleic acid construct) of the invention is a viral vector, in particular suitable for gene therapy of individual’s immune cells. In particular, the viral vector may be derived from a non-pathogenic parvovirus such as adeno- associated virus (AAV), a retrovirus such as a gammaretrovirus, spumavirus and lentivirus, and an adenovirus. The viral vector is preferably an integrating vector such as AAV or lentivirus vector, preferably lentivirus vector. The vector comprises the viral sequences required for viral vector production such as the lentiviral LTR sequences or the AAV ITR sequences flanking the expression cassette. Lentivirus vector may be pseudotyped with another envelope glycoprotein, preferably another viral envelope glycoprotein such as VSV- G. Pseudoptyped lentiviral vector improves the transduction of individual’s immune cells. Lentiviral vector is advantageously a self-inactivating (SIN) lentiviral vector (3^rd generation) as originally described in Zufferey et al. (J. Virol., 1998, 72, 9873-9880).

Another type of vector for delivering the nucleic acid (nucleic acid construct) of the invention is a particle or vesicle, macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, in particular lipid-based micro- or nano- vesicle or particle such as liposome or lipid nanoparticle (LNP). In more particular embodiments, the nucleic acid is RNA and the vector is a particle or vesicle as described above.

The present invention also relates to a method of producing a modified or engineered immune cell, comprising a step of overexpressing CD74 molecule in the cell by any appropriate mean, preferably by introducing a transgene encoding recombinant CD74 molecule or truncated form thereof as disclosed herein.

Preferably, the method for obtaining cells according to the invention further comprises a step of expressing in the cell a genetically engineered antigen receptor that specifically binds to a target antigen, such as a CAR or TCR, preferably by introducing a transgene encoding the genetically engineered antigen receptor into said immune cells.

In some embodiments, gene transfer is accomplished by first stimulating immune cell growth, proliferation, and/or activation, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical applications. The immune cells may be engineered in the presence of kinase inhibitors to generate CAR T cells with a less differentiated phenotype so as to improve engraftment, in vivo proliferation and effector activity of these cells.

Therapeutic applications

The engineered immune cells according to the present disclosure are useful, in particular in adoptive cell therapy, for treating various diseases such as cancer, inflammatory, infectious and immune diseases, transplantation; in particular cancer, in a subject in need thereof. The various engineered immune effector cells as disclosed herein which have a higher capacity to infiltrate the tumor tissue and a higher anti-tumor activity are useful in the treatment of cancer and infectious diseases.

Engineered Treg are predicted to have a higher capacity to infiltrate inflamed tissues and a higher suppressive activity in inflamed tissues. Therefore, engineered Treg useful in the treatment of various inflammatory and immune diseases. Inflammatory diseases include various acute or chronic inflammatory diseases, autoinflammatory diseases and inflammation secondary to viral infection.

The term "treating" or "treatment", as used herein, is defined as the application or administration of cells as per the invention or of a composition comprising the cells to a patient in need thereof with the purpose to reverse, alleviate, inhibit the progress of, or prevent the disorder or condition to which such term applies, or reverse, alleviate, inhibit the progress of, or prevent one or more symptoms of the disorder or condition to which such term applies. As used herein, the terms “treatment” or “treat” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patients at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and include suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment.

"Treating cancer" includes, without limitation, reducing the number of cancer cells or the size of a tumor in the patient, reducing progression of a cancer to a more aggressive form (i.e. maintaining the cancer in a form that is susceptible to a therapeutic agent), reducing proliferation of cancer cells or reducing the speed of tumor growth, killing of cancer cells, reducing metastasis of cancer cells or reducing the likelihood of recurrence of a cancer in a subject. Treating a subject as used herein refers to any type of treatment that imparts a benefit to a subject afflicted with cancer or at risk of developing cancer or facing a cancer recurrence. Treatment includes improvement in the condition of the subject (e.g., in one or more symptoms), delay in the progression of the disease, delay in the onset of symptoms, slowing the progression of symptoms and others.

The therapeutic uses according to the present disclosure comprise adoptive T cell therapy which may be combined with another therapy. Adoptive cell therapy (ACT) also called adoptive T cell therapy, adoptive cell transfer, cellular adoptive immunotherapy or T-cell transfer therapy is a type of immunotherapy in which immune cells, in particular T cells, are administered to a patient to help the immune system fight diseases such as cancer, infectious diseases and others. The immune cells used in adoptive cell therapy may be autologous or allogenic immune cells. Allogenic refers to histocompatible (HLA-compatible) cells. Immune cells for Adoptive cell therapy (ACT) may be engineered to recognize an antigen of interest for therapy (redirected T cell immunotherapy, CAR T cell therapy). The immune cells for adoptive T cell therapy may be delivered to the individual in need thereof by any appropriate mean such as for example by intravenous injection (infusion or perfusion), or injection in the tissue of interest (implantation).

As used herein, the term “cancer” refers to any member of a class of diseases or disorders characterized by uncontrolled division of cells and the ability of these cells to invade other tissues, either by direct growth into adjacent tissue through invasion or by implantation into distant sites by metastasis. Metastasis is defined as the stage in which cancer cells are transported through the bloodstream or lymphatic system. The term cancer according to the present invention also comprises cancer metastases and relapse of cancer. Cancers are classified by the type of cell that the tumor resembles and, therefore, the tissue presumed to be the origin of the tumor. For example, carcinomas are malignant tumors derived from epithelial cells. This group represents the most common cancers, including the common forms of breast, prostate, lung, and colon cancer. Lymphomas and leukemias include malignant tumors derived from blood and bone marrow cells. Sarcomas are malignant tumors derived from connective tissue or mesenchymal cells. Mesotheliomas are tumors derived from the mesothelial cells lining the peritoneum and the pleura. Gliomas are tumors derived from glia, the most common type of brain cell. Germinomas are tumors derived from germ cells, normally found in the testicle and ovary. Choriocarcinomas are malignant tumors derived from the placenta. As used herein, “cancer” refers to any cancer type including solid and liquid tumors. As used herein, the term “cancer” refers to any cancer that may affect any one of the following tissues or organs: breast; liver; kidney; heart, mediastinum, pleura; floor of mouth; lip; salivary glands; tongue; gums; oral cavity; palate; tonsil; larynx; trachea; bronchus, lung; pharynx, hypopharynx, oropharynx, nasopharynx; esophagus; digestive organs such as stomach, intrahepatic bile ducts, biliary tract, pancreas, small intestine, colon; rectum; urinary organs such as bladder, gallbladder, ureter; rectosigmoid junction; anus, anal canal; skin; bone; joints, articular cartilage of limbs; eye and adnexa; brain; peripheral nerves, autonomic nervous system; spinal cord, cranial nerves, meninges; and various parts of the central nervous system; connective, subcutaneous and other soft tissues; retroperitoneum, peritoneum; adrenal gland; thyroid gland; endocrine glands and related structures; female genital organs such as ovary, uterus, cervix uteri; corpus uteri, vagina, vulva; male genital organs such as penis, testis and prostate gland; hematopoietic and reticuloendothelial systems; blood; lymph nodes; thymus.

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

In some embodiments the cancer is selected from the group comprising: non-small cell lung cancer (NSCLC); breast, skin, ovarian, kidney and head and neck cancers; and rhabdoid tumors; preferably non-small cell lung cancer (NSCLC) and breast cancer.

As used herein infectious diseases refers to any disease caused by a pathogenic agent or microorganism such as virus, bacteria, fungi, parasite and the like.

Inflammatory and immune diseases include in particular acute or chronic inflammatory diseases, autoinflammatory diseases, miscarriage, allergic diseases, autoimmune or infectious diseases, graft- versus-host disease and graft-rejection, and tissue repair (wound healing).

Non-limiting examples of autoimmune diseases include: type 1 diabetes, rheumatoid arthritis, psoriasis and psoriatic arthritis, multiple sclerosis, Systemic lupus erythematosus (lupus), Inflammatory bowel disease such as Crohn’s disease and ulcerative colitis, Addison’s disease, Grave’s disease, Sjogren’s disease, alopecia areata, autoimmune thyroid disease such as Hashimoto’s thyroiditis, myasthenia gravis, vasculitis including HCV-related vasculitis and systemic vasculitis, uveitis, myositis, pernicious anemia, celiac disease, Guillain-Barre Syndrome, chronic inflammatory demyelinating polyneuropathy, scleroderma, hemolytic anemia, glomerulonephritis, autoimmune encephalitis, fibromyalgia, aplastic anemia, and others. Non-limiting examples of inflammatory and allergic diseases include: neuro- degenerative disorders such as Parkinson disease, chronic infections such as parasitic infection or disease like Trypanosoma cruzi infection, allergy such as asthma, atherosclerosis, chronic nephropathy, and others. The disease maybe allograft rejection including transplant-rejection, graft-versus-host disease (GVHD) and spontaneous abortion.

The subject of the invention (i.e. patient) is a mammal, typically a primate, such as a human. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects.

In some embodiments of the invention, said subject has a cancer, is at risk of having a cancer, or is in remission of a cancer.

In some embodiments of the invention, said subject is suffering from or is at risk of an inflammatory, infectious or immune disease as disclosed herein.

In particular, the invention relates to a pharmaceutical composition comprising a cell according to the present disclosure as an active component.

In particular embodiments, the cell is not a Treg; particularly the cell is a T cell, B cell, NK cell, monocyte, macrophage or dendritic cell as disclosed herein; more particularly a cell comprising an engineered antigen receptor as disclosed herein, including CAR-T, CAR- macrophage, CAR-DC, CAR-monocyte, CAR-NK, TCR-T and others. In more particular embodiments, the cell is an effector T cell such as Tconv or CD8+ T cell as disclosed herein, preferably comprising an engineered antigen receptor such as CAR-T or TCR-T cell.

In other particular embodiments, the cell is a Treg as disclosed herein.

In particular, the invention relates to a pharmaceutical composition comprising a cell according to the present disclosure, as an active component.

In the various embodiments of the present invention, the pharmaceutical composition comprises a therapeutically effective amount of the cell according to the disclosure. In the context of the invention a therapeutically effective amount refers to a dose sufficient for reversing, alleviating or inhibiting the progress of the disorder or condition to which such term applies, or reversing, alleviating or inhibiting the progress of one or more symptoms of the disorder or condition to which such term applies. The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve, or at least partially achieve, the desired effect. The effective dose is determined and adjusted depending on factors such as the composition used, the route of administration, the physical characteristics of the individual under consideration such as sex, age and weight, concurrent medication, and other factors, that those skilled in the medical arts will recognize. The effective dose can be determined by standard clinical techniques. In addition, in vivo and/or in vitro assays may optionally be employed to help predict optimal dosage ranges.

In the various embodiments of the present invention, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and/or vehicle. In some aspects, the choice of carrier in the pharmaceutical composition is determined in part by the particular engineered CAR or TCR, vector, or cells expressing the CAR or TCR, as well as by the particular method used to administer the vector or host cells expressing the CAR. Accordingly, there are a variety of suitable formulations. The pharmaceutical vehicles and carriers are those appropriate to the planned route of administration, which are well known in the art. The pharmaceutical composition is formulated for administration by a number of routes, including but not limited to parenteral and local. The pharmaceutical composition of the present invention is generally administered according to known procedures, at dosages and for periods of time effective to induce a therapeutic effect in the patient. The pharmaceutical composition may be administered by any convenient route, such as in a non-limiting manner by injection, perfusion or implantation. The administration can be systemic, local or systemic combined with local. Systemic administration is preferably intravascular such as intravenous (IV) or intraarterial; intraperitoneal (IP) or else. In some preferred embodiments, the administration is parenteral, preferably intravascular such as intravenous (IV) or intraarterial. The parenteral administration is advantageously by injection or perfusion.

In some embodiments, said pharmaceutical composition is for adoptive cell therapy, in particular adoptive cell therapy of cancer. The pharmaceutical composition may also comprise an additional therapeutic agent, in particular an agent useful for the treatment of a disease according to the present disclosure. The additional therapeutic agent is preferably an antigen specific of the disease such as a cancer antigen, or an anticancer, anti-infectious or immunomodulatory agent.

Another aspect of the invention relates to the cell or pharmaceutical composition according to the present disclosure as a medicament, in particular for use in the treatment of a disease according to the present disclosure; preferably for use in adoptive cell therapy of the disease.

Methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided cells and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg et al; US Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.

In some embodiments, the cell therapy, e.g., adoptive cell therapy, is carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.

In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., is carried out by allogeneic transfer, in which the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject, of the same species. The first and second subjects are genetically similar, in particular they are histocompatible (e.g., HLA compatible); more particularly, the second subject expresses the same HLA class or supertype as the first subject.

The present invention also relates to a method of treatment of a disease as disclosed herein and notably an adoptive cell therapy, preferably an adoptive cell therapy of cancer, comprising administering an effective amount of a cell or composition as disclosed herein to a subject in need thereof. The invention provides also a method for treatment, notably adoptive cell therapy, of a patient in need thereof, comprising: providing at least one immune cell from a subject; engineering said immune cell to obtain an immune cell overexpressing CD74 according to the present disclosure; and administering said engineered immune cell, in particular in the form of a pharmaceutical composition, to a subject in need thereof.

The immune cell is prepared as disclosed herein; the immune cell may be obtained from a subject sample or derived from a subject sample as disclosed herein. This includes in particular isolating immune cells from a subject sample or deriving immune cells from stem cells derived from a subject sample.

In particular embodiments of the method, the cell is autologous. In particular embodiments of the method, the cell is allogenic. In particular embodiments of the method, the cell is not a Treg; particularly the cell is a T cell, B cell, NK cell, monocyte, macrophage or dendritic cell as disclosed herein. In more particular embodiments, the cell is an effector T cell such as Tconv or CD8+ T cell as disclosed herein. In other particular embodiments, the cell is a Treg as disclosed herein. In particular embodiments, the cell is further modified to express one or more genetically modified antigen receptor(s) as disclosed herein before administration to the subject. In particular embodiments of the method, the cell is expanded before administration to the subject. In particular embodiments, the method is adoptive cell therapy of cancer. In particular embodiments of the method, said subject in need thereof has a cancer, is at risk of having a cancer, or is in remission of a cancer.

The present invention also relates to the use of a cell or composition according to the present disclosure in the manufacture of a medicament for treatment of a disease as disclosed herein in a subject in need thereof, in particular for treatment of cancer, inflammatory and immune diseases, particularly for treatment of cancer.

The present invention also relates to the use of a cell or composition according to the present disclosure for the treatment of a disease as disclosed herein, in a subject in need thereof, in particular for treatment of cancer, inflammatory and immune diseases, particularly for treatment of cancer. The present invention also relates to a pharmaceutical composition for treatment of a disease as disclosed herein, comprising a cell according to the disclosure as active component.

The present invention also relates to a pharmaceutical composition comprising a cell according to the disclosure for treating of a disease as disclosed herein.

The cell or pharmaceutical composition of the invention may be used in combination with another therapy, wherein the combined therapies may be simultaneous, separate or sequential.

The additional therapy is in particular an anticancer, anti-infectious therapy and/or immunotherapy. Anti-infectious therapy includes the use of the known antibacterial, antiviral, antiparasitic, antifungal agents currently used for treating infectious diseases. Anticancer therapy includes chemotherapy, targeted therapy, radiotherapy, surgery, and immunotherapy. Immunotherapy includes but is not limited to immune checkpoint modulators (i.e. inhibitors and/or agonists) such as immune checkpoint inhibitors and co-stimulatory antibodies; monoclonal antibodies; and cancer vaccines.

Preferably, administration of cell in an adoptive cell therapy according to the invention is combined with administration of immune checkpoint modulators, notably checkpoint inhibitors. Checkpoint inhibitors include, but are not limited to, PD-1 inhibitors, PD-L1 inhibitors, Lag-3 inhibitors, Tim-3 inhibitors, TIGIT inhibitors, BTLA inhibitors, V-domain Ig suppressor of T-cell activation (VISTA) inhibitors and CTLA-4 inhibitors, IDO inhibitors for example. Co-stimulatory antibodies deliver positive signals through immune -regulatory receptors including but not limited to ICOS, CD 137, CD27 OX-40 and GITR. Most preferably, the immune checkpoint modulators comprise a PD-1 inhibitor (such as anti-PD- 1), a PDL1 inhibitor (such as antiPDLl) and/or a CTLA4 inhibitor.

Administration of at least one cell according to the invention to a subject in need thereof may be combined with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the cells are co -administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cell populations are administered prior to the one or more additional therapeutic agents. In some embodiments, the cell populations are administered after to the one or more additional therapeutic agents. The various embodiments of the present disclosure can be combined with each other and the present disclosure encompasses the various combinations of embodiments of the present disclosure.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques which are within the skill of the art. Such techniques are explained fully in the literature.

The invention will now be exemplified with the following examples, which are not limitative, with reference to the attached drawings in which:

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. CD74 promotes d accumulation of Tregs specifically in the tumor. MDA- MB231 tumor cells were engrafted subcutaneously in the flank of immunodeficient NSG mice. 10 days later (when tumors are palpable), HLA.A2+ PBMCs were injected intravenously alone or together with WT, or CD74KO in vitro expanded HLA.A2- Tregs (expTregs), a group of mice was not injected as a control. A. Individual tumor-growth curves for each group of mice injected with one representative donor of expTregs. B. Ratio of absolute numbers of expTregs (HLA.A2-)/PBMCs (HLA.A2+) in the spleen and tumors 6 days after cell injection. Statistical analyses were performed using an unpaired t-test with pVal < *:0.01; **:0.001; ***:0.0001; ****:0.00001.

Figure 2. CD74 KO Tregs show defective accumulation in the tumor tissue.

MDA-MB23 1 tumor cells were engrafted subcutaneously in the flank of immunodeficient NSG mice, and 14 days later, PBMCs, CFSE⁺ WT expTregs and CTV⁺ CD74KO expTregs were injected intravenously. Six days later, spleens, livers and tumors were analyzed by FACS. Shown is the ratio of numbers of CD74KO versus WT cells among expTregs (gated as live huCD45+CD4+ HLA-A2- cells) in spleen, liver, and tumor. Statistical analyses were performed using a paired t-test with pVal: ** <0.001.

Figure 3. CD74 overexpressing Tregs show enhanced accumulation in the tumor tissue.

MDA-MB231 tumor cells were engrafted subcutaneously in the flank of immunodeficient NSG mice. 14 days later, HLA.A2+ PBMCs were injected intravenously together with WT or CD74OE expanded HLA.A2- Tregs. 6 days after T-cell injection, spleen, liver and tumor were analyzed by FACS. Shown is the proportion of cells expressing surface CD74 (gated as live huCD45+CD4+ HLA-A2- cells) among expTregs, in the different tissues.

Figure 4: Generation of CD74-overexpressing T cells.

A-B. Lentivirial vectors containing the encoding genes for (A) CD74 WT GFP and CD74 del 1-23 GFP; and (B) the reporters for control of transduction coding for mCherry GFP or for the non-related gene: TCR (for HPV16 E7). These vectors were packaged in lentiviral particles and used to transduce in vitro-cultured activated CD3+ T cells derived from human PBMCs.

Figure 5: Phenotyping of in vitro CD74 WT and CD74 dell-23-transduced-T cells cultures. CD3+T cells transduced with the vectors coding for CD74 WT GFP, CD74 del 1-23 GFP, or for HPV16 E7 TCR (control) as described in Figure 4, were in vitro expanded for 8- 21 days. The efficiency of transduction, verified before injection to the mice, was of 41% for CD74 WT GFP and of 31% for CD74 del 1-23 GFP (determined as GFP+ cells among total live CD3+ cells); and 80% for HPV16 E7 TCR (determined as TCR+ cells among total live CD3+ cells). Shown is the Mean Florescence Intensity (MFI) of surface CD74 expression on total CD3+ cells determined by FACS.

Figure 6: In vivo anti-tumoral effect of CD74-expressing T cells. NSG mice were s.c. inoculated with MDA-MB231 tumor cells, and when tumors were palpable, mice were cotransferred with PBMC (HLA. A2+) and CD3+ T cells (HLA. A2-) transduced with CD74 WT, CD74 dell-23, or HPV16 E7 TCR. A) Monitoring of tumor size post T cell injection (day 0 to day 6). Tumor volume is shown in the upper panel and the % of increase in tumor volume is shown in the lower panel. B) At day 6 after transfer, cells were collected from liver, spleen and tumor and analyzed by FACS, and the % of surface CD74 expression was determined on total live CD3+, CD4+, and CD8+ T cells: Liver: on total CD3+ (TCR: 0.41%; CD74 WT: 0.46%; CD74 dell-23: 0.78%); on CD4+ (TCR: 0.36%; CD74 WT: 0.39%; CD74 dell-23: 0.72%); on CD8+ (TCR: 0.53%; CD74 WT: 0.58%; CD74 dell-23: 0.82%). Spleen: on total CD3+ (TCR: 0.99%; CD74 WT: 1.26%; CD74 dell-23: 1.48%); on CD4+ (TCR: 0.23%; CD74 WT: 0.61%; CD74 dell-23: 0.92%); on CD8+ (TCR: 0.18%; CD74 WT: 0.32%; CD74 dell-23: 0.54%). Tumor: on total CD3+ (TCR: 12.5%; CD74 WT: 23.6%; CD74 dell-23: 19.1%); on CD4+ (TCR: 13.1%; CD74 WT: 23.3%; CD74 dell-23: 19.4%); on CD8+ (TCR: 9.79%; CD74 WT: 24.2%; CD74 dell-23: 17.5%). Variation of CD74+ expression in these cell types are represented as fold increase with respect to control TCR condition (n=l).

C) Experiment similar to B, but with n=3 mice per group and using mCherry encoding lentivector as mock-transduction control (instead of HPV16 E7 TCR). At day 6 after transfer, cells were collected from liver, spleen and tumor and analyzed by FACS, and the % of surface CD74 expression was determined on total live CD3+, CD4+, and CD8+ T cells. Mean values of % of surface CD74 expression were as follows: Liver: on CD3+ (mCherry: 1.35%; CD74 WT: 0.98%; CD74 dell-23: 1.13%); on CD4+ (mCherry: 1,11%; CD74 WT: 0,87%; CD74 dell-23: 1.03%); on CD8+ (mCherry: 1.77%; CD74 WT: 1.21%; CD74 dell-23: 1.26%). Spleen: on CD3+ (mCherry: 1.58%; CD74 WT: 1.44%; CD74 dell-23: 1.86%); on CD4+ (mCherry: 0.92%; CD74 WT: 1.21%; CD74 dell-23: 1.90%); on CD8+ (mCherry: 0.78%; CD74 WT: 0.90%; CD74 dell-23: 1.49%). Tumor: on CD3+ (mCherry: 8.93%; CD74 WT: 16.88%; CD74 dell-23: 27.03%); on CD4+ (mCherry: 10.61%; CD74 WT: 20.16%; CD74 dell-23: 31.83%); on CD8+ (mCherry: 13.32%; CD74 WT: 23.5%; CD74 dell-23: 35.4%). Variation of CD74+ expression in these cell types were represented as fold increase with respect to control mCherry condition (n=3). *p<0.05, two-tailed t test.

EXAMPLES

Example 1: CD74 specifically stabilizes the suppressive phenotype of Tregs within tumors and is specifically required for retention of Tregs within tumors

Material and Methods

Mouse studies

NSG mice (^O'D.C -Prkdc^sad Il2r^^mIWjlISz], JAX#005557) are bred in Curie Institute SPF animal facility. Female mice used in the experiments were age-matched and euthanized by cervical dislocation. Human endpoints were used for tumor-bearing mice as maximal ethical size of tumors subcutaneously grafted of 2 cm3, more than 20% of weight loss, signs of altered mobility-eating ability and cachexia. Transfer of human cells into immunodeficient host

After 7 days of adaptation, NSG mice were injected intravenously with 100pL of PBS containing the different cell mixes. Total PBMCs were stained to evaluate the percentage of CD3+ cells. The three different cell suspensions were made of 5.10⁶ CD3+ cells/50pL with 1- 50pL of PBS; 2- 50pL containing 5.10⁶ WT Tregs; 3- 5.10⁶ CD74KO Tregs. Mice were assessed twice a week for the GvHD signs: weight loss, skin integrity, posture, fur texture, activity and diarrhea. For survival study, mice were monitored 5 times a week, and unhealthy mice, according to ethical grade, were euthanized and counted as nonsurvivors. 3 mice from each group were euthanized 14 days after cell injection and liver and spleen were collected for FACS analysis. Livers and spleens were processed to obtain cell suspensions and a staining was performed to analyze the human infiltrating cells. In the case of livers, percoll was used to remove debris.

Generation of tumor model in humanized mice

After 7 days of adaptation, NSG mice were subcutaneously injected on the flank with 100pL of PBS containing 5.10⁶ MDA-MB231 cells (day 0). When tumors were palpable (between day 6 and 10), mice were injected intravenously with lOOpL of PBS containing the different cell mixes. Mice were assessed twice a week to measure tumor size. When tumors exceeded the ethical size, mice were euthanized. 3 mice from each group were euthanized 6 days after cell injection, and liver, spleen and tumor tissues were collected for FACS analysis. . For liver and tumor, a percoll was done to remove debris. Cells from spleens, livers and tumors are stained to evaluate the phenotype and repartition of Tregs.

Isolation of regulatory T cells

Blood buffy coats from healthy donors were collected at the Etablissement Frangais du sang (Paris, France). Peripheral blood mononuclear cells (PBMC) were obtained by gradient centrifugation using ficoll tubes filled with lymphoprep (STEMCELL -Cat#07851). CD25+ cells were isolated from total PBMCs by a positive selection with the CD25 Microbeads II kit (Miltenyi - Cat#l 30-092-983). Recovered cells were washed with PBS and stained with Live -Dead fixable Aqua, anti-CD4, anti-CD8, anti-CD25 and anti-CD127. Cells were resuspended at 20.10⁶cell/mL in PBS with EDTA (2mM) and FBS (0,5%). Cells were FACS sorted as Aqua-negative, CD4-positive, CD127-negative and CD25high-positive. The sorted Tregs were centrifugated and resuspended at 10⁶ cell/mL in X-vivo medium completed with IL-2 (300IU/mL, Novartis - Proleukine) and with soluble aCD3aCD28aCD2 (251jL/mL, STEMCELL - Cat#10970) for genome edition or with aCD3aCD28 beads (Ibead: Icell, THERMOFISHER - Cat#11132D) for expansion. 10,000 Tregs were stained with anti-Foxp3 antibody after fixation and permeabilization to evaluate the expression of Foxp3 on the sorted Tregs before expansion. The X-vivo 15 media (OZYME - Cat#BE02-060F) is completed with human serum (10%), b-mercaptoethanol (501jM), Pen/Strep (1%), non- essential amino acids (1 %). To expand sorted Tregs, fresh IL-2 was added every 3 days and cells were reactivated every week.

Genome edition with CRISPR-Cas9 technique

Seven days after sort Tregs were stimulated with IL-2 (300IU/mL) and soluble aCD3aCD28aCD2 (251jL/mL) 24 hours before genome edition by electroporation. CRISPR RNA (crRNA, CD74 guides: IDT - Cat#229055317 and -318 and control Cat#1072544) and trans-activating crRNAs (IDT - Cat#1072532) were mixed at an equimolar ratio and incubated 30 minutes in a 37°C- 5% CO2 incubator to form a single- guide RNA at lOOpM. HiFi-Cas9 protein (IDT - Cat#1081060) was mixed with sgRNA at a ratio lsgRNA:2Cas9 to form a CRISPR ribonucleoproteic complex (crRNP) at a concentration of 50pM. Cell suspension was done at 1.10⁶cells in 20pL of the supplemented P3 Primary Cell Nucleofector Solution (Lonza - Cat#V4XP-3032). 5pL of crRNP (50pM) was mixed to 20 pL of cell suspension and incubated at room temperature for 10 minutes, cells with crRNP were transfered into a 16-well Nucleocuvette (Lonza - Cat#V4XP-3032) and electroporated by EH100 pulse (Lonza -Cat#AAF-1003X). 5 days after electroporation, the efficacy of gene deletion was assessed by FACS and the electroporated Tregs were reactivated with IL-2 (300IU/mL) and aCD3aCD28 coated beads (Ibead: Icell) for further expansion. Retroviral transduction of human primary Tregs

Sorted Tregs were stimulated with IL-2 (300IU/mL) and aCD3aCD28 coated beads (lbead:lcell) 24 hours before transduction. Tregs were transferred to retronectin- coated plates and transduced by spinoculation with retrovirus. Retrovirus were designed by VectorBuilder with two promoters, the first one for the expression of the target sequence (or mCherry as a control) and the second one for EGFP. Seven days after, transduction efficiency was assessed by FACS, and Tregs were reactivated for further expansion.

Antibody staining and flow cytometry

Cells were stained with a viability marker in PBS during 15 minutes at 4°C. Following this step, they were stained with a mix of fluorochrome-conjugated antibodies against surface protein in staining buffer (PBS, 2mM EDTA, 0.5% BSA) during 20 minutes at 4°C. After two washes with staining buffer, cells were fixed in Fixation/Permeabilization kit during 30 minutes at 4°C (ThermoFisher scientific - Cat#15151976) and washed twice with Permeabilization Buffer (ThermoFisher - Cat# 12766048). Intracellular staining was done by diluting the fluorochrome -conjugated antibodies in the Permeabilization Buffer during 20 minutes at room temperature protected from light. Cells were washed twice with staining buffer and analyzed on a Fortessa flowcytometer (BD) or ZE5 flowcytometer (BioRad) and finally using FlowJo software (V. 10).

Results

The inventors investigated whether in the tumor tissue - where the surface upregulation of CD74 in Tregs was originally observed - the absence of CD74 impacts Treg function. For this, immunodeficient NSG mice were s.c. grafted with MDA-MB231 triple negative breast cancer cells. At day ten, when tumors were palpable, mice were left untreated (control) or were injected with fresh PBMCs alone or co -injected with in vitro expanded WT or CD74KO Tregs (expTregs). Fig.lA shows the individual tumor growth curves for one representative donor (donor 1). It was observed that tumors grew exponentially in control mice and that injection of PBMCs led to tumor rejection. Addition of the WT expTregs slightly delayed tumor elimination. In sharp contrast, CD74KO expTregs significantly accelerated the tumor rejection. Although some inter-donor variability existed, similar results were obtained with 3 additional donors, as well as when using frozen instead of fresh expTregs.

This set of experiments revealed that CD74 expression in Tregs is required for Tregs to maintain their suppressive function in the tumor. Furthermore, the enhanced tumor rejection observed with the transfer of CD74KO expTregs suggests that in the absence of CD74, Tregs no longer suppress antitumor immune responses but may further acquire anti-tumoral functions.

Given that CD74KO-Tregs accumulated less in the tumor than WT Tregs (Fig.lB) and that CD74 can act as a receptor for chemokines produced by the tumor (Tkachev et al., Science Translational Medicine, 2021 , 13, 576) an experiment was designed to more accurately quantify the contribution of CD74 deficiency on Treg infiltration of the tumor. For this, tumor-bearing NSG mice were co-injected with a mix of PBMCs, and WT and CD74KO expTregs stained with different cell trace dyes and the amount of WT and CD74 KO expTregs present in different organs was quantified six days later. Similar absolute numbers and frequencies of WT and CD74KO expTregs were found in the spleen and the liver, whereas a significantly lower proportion of CD74KO expTregs accumulated within the tumor (Fig.2). Indeed, only 28.67% of CD74KO expTregs were found in the tumor as compared to 50.04% in the spleen and 45.38% in the liver. These results point to a non-redundant role for CD74 in favoring Treg migration and/or accumulation specifically in the tumor. To validate this observation, CD74-overexpressing Tregs (CD74OE Tregs) were generated by transducing FACS-sorted Tregs with an intracellular-truncated CD74 mutant previously described to stabilize CD74 membrane expression by slowing down its endocytosis (Bakke, Cell, 1990, 63, 707-716). Then, tumor-bearing NSG mice were co-injected with PBMCs plus WT or CD74OE expTregs and analyzed six days later. Mirroring the observation in human samples, a higher proportion of WT expTregs expressed CD74 at the surface in the tumor compared to the spleen. Additionally, the CD74OE expTregs showed the highest tumor accumulation, validating the role of CD74 in facilitating Treg accumulation in the tumor (Fig.3). Overall, these results indicate that CD74 plays a non-redundant and specific role in Treg recruitment and/or retention within tumors.

Example 2: CD74 over expression in anti-tumoral T cell types increases their tumor infiltration and antitumor activity

Material and Methods

Lentiviral gene transduction

CD74 overexpression was achieved by lentiviral gene transduction. For this, human PBMCs were purified from healthy donor blood by Ficoll, and the CD3+ T cell fraction was obtained by negative selection using the Human Pan T cell isolation kit from Miltenyi. Cells were seeded at a density of one million per ml in Xvivo complete media (Xvivo 15, 5% Human serum, 1% penicillin streptomycin) and activated with anti-CD3/CD28 activation beads overnight. Activated cells were transduced with lentiviruses encoding either wild type CD74 (CD74 WT) or a deletion mutant lacking the N terminal amino acid sequence 1-23, which increases its retention at the plasma membrane (CD74 dell -23). As control, a GFP-encoding gene under an independent promoter was introduced in each construction, serving as reporter of transduction efficacy. In addition, lentiviral constructions encoding a non-related T cell receptor (TCR) recognizing the HPV16 E7 protein or encoding the mCherry protein, were used as a mock-transduction control (Figure 4A-B). The multiplicity of infection (MOI) for transduction was set as 50 after performing a MOI dose test (not shown). This condition is consistent with gene addition studies (increment in the copy number of an endogenous gene). CD74 upregulation was measured by detecting surface CD74 expression by FACS. T cell cultures were maintained at 37 degrees in the incubator, addition of fresh media supplemented with IL-2 100 U/ml was performed every 3 days. Reactivation of cells was done every 7 days.

Mice model of human tumor

Humanized immunodeficient NSG (NOD scid gamma) mice were engrafted with human breast tumors. 5 million of MDA-MB231 breast tumor cells were injected subcutaneously in the flank . After two weeks, when tumors were palpable, mice were i. v. injected with a mixture of 5 million CD3+ T cells from the CD74 WT or CD74 dell -23 T cell cultures (both HLA.A2-) and 5 million of CD3+T cells from human PBMCs (HLA-A2+). After 6 days, mice were sacrificed, and tumor size was measured.

Results

At day 9 after transduction, around 80% TCR+ cells were observed in the control TCR- transduced CD3+ T culture, and 41% and 31% of GFP+ cells were detected in the CD74 WT and CD74 del 1-23 transduced T cell cultures, altogether confirming an adequate transduction efficiency. The global percentages of surface CD74 expressing cells in the total CD3+ T cell culture were 18,8%, 23,2% and 37% respectively. Next, transduced and untransduced populations within these cultures were compared (Figure 5). The mock transduced culture did not present considerable differences in the % of surface CD74 expressing cells in these populations (19,8% in TCR+ and 15% in TCR-), indicating that the transduction itself does not affect considerably the expression of endogenous CD74 (not shown). In contrast, upregulation of approximately 3 and 3,4-fold were observed in the case of CD74 WT and CD74 del 1-23 -transduced cultures (37,7% in GFP+/13% in GFP- for CD74 WT; 72,8% in GFP+/ 21,1% in GFP- for CD74 dell-23) (not shown). Additionally, MFI expression values for surface CD74 were higher in cells transduced with CD74WT than in control cells, and the highest value was observed in CD74 del 1-23 -transduced ones (Figure 5). Altogether, these results validate the feasibility of the in vitro upregulation of CD74 expression by the lentiviral transduction system, giving rise to T cells cultures with increased numbers of CD74- expressing cells and increased levels of CD74 expression. The global differences in CD74 expression for these CD3+ T cell cultures could be augmented by further optimizing the transduction conditions or performing sorting and expansion of transduced populations in each case (TCR+ and GFP+).

As next step, to evaluate the anti-tumor effect of these T cell cultures in vivo, humanized immunodeficient NSG mice were engrafted with human breast tumor cell line. Mice receiving CD74 WT and CD74 del 1-23 T cell cultures showed a lower tumor growth compared to the one receiving the mock-TCR-T cell culture. Interestingly, while in the latter the tumor size almost doubled, in the former two it remained almost unaltered (Figure 6A). Furthermore, analysis of the tumors revealed that in mice injected with CD74-transduced T cells, the tumor infiltrating CD3+ T cells (HLAA2-), as well as their CD4+ and CD8+ subsets, presented a higher percentage of surface CD74-expressing cells (almost double) than in tumors from mock TCR injected mice (average of 22% vs 11%) (Figure 6B). Of note, the increased accumulation of CD74+ T cells was selective to the tumor, and not observed in the spleen nor in the liver (not shown).

Overall, in this first study, CD74 WT or CD74 del 1-23 T-cell cultures conferred similar in vivo phenotypes. CD74 del 1-23 did not provide a higher percentage of CD74+ T cells in the tumor infiltrate nor a superior tumor control than CD74 WT, suggesting that in vivo the two versions of the CD74 molecule behave similarly.

In a second study, 3 mice per group were analysed, and the mCherry-mock transduced cells were used as control (Figure 6C). Similar to the previous experiment, mice injected with T cells transduced with CD74WT or CD74 dell -23 showed increased levels of T cells in the tumor, being the increase observed with the T cells transduced with the latter construct statistically significantly different.

Taken together, these data indicate that: i) CD74 WT or CD74 del 1-23 -transduced T cells, present a selective advantage to accumulate in the tumor, compared to the mock-transduced cells; and ii) the higher proportion of surface CD74+ cells in the tumor T cell compartment correlates with an improved tumor growth control. These results reinforce the concept that manipulating CD74 levels in T cell types represent a novel mean to increase their anti-tumor activity.

List of sequences disclosed in the present application

SEQ ID NO: 1 : Human CD74 protein

MHRRRSRSCREDQKPVMDDQRDLISNNEQLPMLGRRPGAPESKCSRGALYTGFS ILVTLLL AGQATTAYFLYQQQGRLDKLTVTSQNLQLENLRMKLPKPPKPVSKMRMATPLLMQALPMGA LPQGPMQNATKYGNMTEDHVMHLLQNADPLKVYPPLKGSFPENLRHLKNTMETIDWKVFES WMHHWLLFEMSRHSLEQKPTDAPPKVLTKCQEEVSHIPAVHPGSFRPKCDENGNYLPLQCY GS IGYCWCVFPNGTEVPNTRSRGHHNCSESLELEDPSSGLGVTKQDLGPVPM

Claims

I. An engineered immune cell which is genetically engineered to overexpress CD74 molecule.

2. The engineered immune cell according to claim 1 , which is a T cell, NK cell, B cell, macrophage, or dendritic cell.

3. The engineered immune cell according to claim 2, which is an effector T cell.

4. The engineered immune cell according to claim 2, which is a regulatory T cell.

5. The engineered immune cell according to any one of claims 1 to 4, which comprises a transgene encoding recombinant CD74 molecule of SEQ ID NO: 1 or a truncated form thereof.

6. The engineered immune cell according to claim 5, wherein said truncated form comprises an N-terminal deletion of at least 12 amino acids in the cytoplasmic domain of CD74, preferably a N-terminal deletion of 23 amino acids.

7. The engineered immune cell according to any one of claims 1 to 3 and 5 to 6, which further expresses a genetically engineered antigen receptor, in particular a chimeric antigen receptor or a T cell receptor.

8. The engineered immune cell according to claim 7, which is a CAR-T or TCR-T cell.

9. The engineered immune cell according to any one of claims 1 to 8, which is isolated from a human subject.

10. The engineered immune cell according to any one of claims 1 to 9 for use as a medicament.

II. The engineered immune cell according to claim 10, which is for use in adoptive cell therapy of a subject.

12. The engineered immune cell for use according to claim 11, which is autologous.

13. The engineered immune cell for use according to claim 11, which is allogenic.

14. The engineered immune cell for use according to any one of claims 10 to 13, which is for use in the treatment of cancer, wherein said immune cell is an effector T cell, in particular a CAR-T or TCR-T cell.

15. The engineered immune cell for use according to any one of claims 10 to 13, which is for use in the treatment of inflammatory or autoimmune diseases, wherein said immune cell is a regulatory T cell.