Improved engineered immune cell therapies

Abstract

The invention relates to an immune cell presenting enhanced migration and / or tumor cell killing while expressing a receptor recognizing a target antigen and having been in contact with an inhibitor of LAIR-1 resulting in the expression and / or function of LAIR-1 being reduced or eliminated. Furthermore, a method of manufacturing the immune cell is described, the method comprising contacting an immune cell extracted from a patient suffering from a tumor with an inhibitor of LAIR- 1 for a sufficient amount of time and under suitable conditions to reduce or eliminate the expression and / or function of LAIR-1. Finally, a method of treating a subject in need thereof, the method comprising administering the immune cell of the invention for a sufficient amount of time and under suitable conditions to treat the subject.

Description

[0001] Improved engineered immune cell therapies

[0002] Field of the invention

[0003] The invention pertains to the field of cancer immunotherapy, in particular to the field of modifying (engineered) immune or immune modulating cells for obtaining improved (tumor cell) killing and immune cell migration in immunosuppressive (tumor) or inflamed microenvironments.

[0004] Background

[0005] Adoptive T cell therapies (ACT) have risen, overthe past few decades, as one of the most promising novel treatments against cancers, such as leukemia’s or other hematological malignancies. Despite recent successes of immunotherapies in on-going clinical trials targeting several types of blood cancers, and the approval of CART therapies by the Food and Drug Administration (FDA), it is difficult to predict heterogenous responses of patients and the relatively high chance for relapse after treatment.

[0006] The ACT most extensively studied and clinically tested are chimeric antigen receptor (CAR) T cells, which target highly specific antigens expressed in tumor cells. However, the high dependency on the surface expression of these hyper specific antigens provides a window for the tumor to become resistant to the therapy via antigen escape, increasing relapse in patients. Hence, novel ACTs are being studied, such as ap T cells engineered to express a defined Vy9V62 TCR (TEGs) and novel dual targeting TEGs. yQTCRs recognize elevated levels of phosphoantigens (pAg) generated through the mevalonate pathway, allowing for MHC independent activation.

[0007] Despite considerable efforts put into improving these therapies, the major roadblock that all T cell immunotherapies face, the tumor microenvironment (TME), it’s still largely unknown and misunderstood. The complexity of its cellular composition, the non-cellular factors involved, and the cross-talk with tumor cells all create an adverse hostile environment which hinders anti-tumor responses and support tumor growth. So far, the role of MSCs in the TME has been recognized as having an effect on promoting tumor growth and possessing immunosuppressive features which further allow the tumor to escape immune surveillance. MSCs contribute substantially to the formation and remodeling of the extracellular matrix (ECM) of the TME, supporting tumor growth and metastasis. To do so, MSC secrete components of the fiber network, such as collagens, as well as metalloproteinase and lysyl oxidases which are enzymes involved in the remodeling of the morphology, thickness and stiffness of the matrix. In recent years, the contribution of the extracellular matrix (ECM) in this interplay has become increasingly clear. Tumor-associated ECM is distinct from healthy tissue, and plays a key role in promoting tumor progression and limiting therapy responses. Collagen is the major component of the ECM, and is involved in supporting tumor growth, initiating metastases, and excluding therapeutics and immune cells from the tumor nests. Collagen fibre alignment and density are key regulators of immune cell infiltration into the tumor site.

[0008] Immune cells express multiple receptors that interact with collagen and modulate immune functions, such as mobility and activation. LAIR-1 is an immune inhibitory collagen receptor expressed on almost all cells of the immune system. Upon activation, LAIR-1 can recruits downstream effector molecules, including SHP-1 and Csk and inhibit immune effector function, such as cytokine production and cell differentiation. Multiple studies support that LAIR-1 :collagen interaction is involved in tumor development and progression. For instance, increased tumoral collagen and LAIR-1 mRNA expression is associated with worse overall survival and circulating collagen fragments produced in cancer can inhibit T cell function through LAIR-1 , potentially contributing to systemic immune suppression. Is has been demonstrated that disrupting the LAIR-1 :collagen pathway in humanized mouse tumor models leads to increased immune cell numbers at the tumor site and inhibition of tumor growth. Currently, three clinical trials targeting LAIR-1 in cancer are ongoing (clinicaltrials.gov identifiers: NCT05572684, NCT04408599, NCT05311618). Although many studies have explored LAIR-1 as a therapeutic target in cancer, the exact role of LAIR-1 in the anti-tumor response remains to be fully elucidated.

[0009] Some of the biggest hurdles for cellular therapy in solid tumors are poor migration to the tumor, the physical barriers to infiltration, and the active suppression by the tumor microenvironment. The current challenge is to develop strategies to enhance the recruitment of endogenous or adoptively transferred lymphocytes to solid tumors and achieve optimal anti-tumor responses while decreasing off target effects.

[0010] Summary of the invention

[0011] In one aspect, there is provided an immune cell expressing a receptor recognizing a target antigen, wherein the expression and / or function of LAIR-1 has been reduced or eliminated.

[0012] In one aspect there is provided, a method of manufacturing the immune cell as described herein, the method comprising contacting an immune cell extracted from a patient suffering from a tumor with an inhibitor of LAIR-1 for a sufficient amount of time and under suitable conditions to reduce or eliminate the expression and / or function of LAIR-1 . In one aspect there is provided, an immune cell isolated from a patient suffering from a disease associated with expression of a tumor antigen, wherein the expression and / or function of LAIR-1 in said immune cell has been reduced or eliminated ex vivo.

[0013] In one aspect there is provided, an engineered immune cell characterized by a T cell expressing a chimeric antigen receptor (CAR) recognizing a cancer-associated target antigen, wherein said engineered T cell has been modified as such that the expression and / or function of LAIR-1 has been reduced or eliminated.

[0014] In one aspect there is provided, an ap T cell engineered to express a defined yQTCR (TEGs), or a dual targeting TEG, wherein the TEG cell presents enhanced migration and tumor cell killing and comprises an intracellular signaling domain or fragment that is functional in the absence of LAIR-1 , and wherein said engineered T cell has been modified as such that the expression and / or function of LAIR-1 has been reduced or eliminated.

[0015] In one aspect there is provided, a method of treating a subject in need thereof, the method comprising administering an immune cell as described herein for a sufficient amount of time and under suitable conditions to treat the subject.

[0016] In one aspect there is provided, an immune cell as described herein for use in treating a subject in need thereof.

[0017] In one aspect there is provided, the use of an immune cell as described herein in the manufacture of a medicament for treating a subject in need.

[0018] In one aspect there is provided, a kit comprising:

[0019] (a) an immune cell expressing a receptor recognizing a target antigen, wherein the expression and / or function of LAIR-1 has been reduced or eliminated; and

[0020] (b) instructions for administering the immune cell to a subject in need thereof.

[0021] Definitions

[0022] Various terms relating to the methods, compositions, formulations, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.

[0023] Methods of carrying out the conventional techniques used in methods of the invention will be evident to the skilled worker. The practice of conventional techniques in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing and related fields are well-known to those of skill in the art and are discussed, for example, in the following literature references: Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 and periodic updates; and the series Methods in Enzymology, Academic Press, San Diego.

[0024] “A,” “an,” and “the”, these singular form terms include plural referents unless the content clearly dictates otherwise. The indefinite article "a" or "an" thus usually means "at least one". Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

[0025] “About” and “approximately”, these terms, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1 %, and still more preferably ±0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods. Additionally, amounts, ratios, and other numerical values are sometimes presented herein in a range format. It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, and sub-ranges such as about 10 to about 50, about 20 to about 100, and so forth.

[0026] “And / or” refers to a situation wherein one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[0027] “Comprising”: this term is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components. “Exemplary", this terms means "serving as an example, instance, or illustration," and should not be construed as excluding other configurations disclosed herein.

[0028] The term "Chimeric Antigen Receptor" or "CAR" may refer to a set of polypeptides, which when in an immune effector cell, provides the cell with specificity for a target cell, typically a cancer cell, and with intracellular signal generation. A CAR may comprise at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as "an intracellular signaling domain") comprising a functional signaling domain derived from a primary signaling domain and / or costimulatory domain as defined below. The set of polypeptides may be contiguous with each other. In some embodiments, the set of polypeptides include a dimerization switch that, upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., can couple an antigen binding domain to an intracellular signaling domain. The CAR may comprise a target- specific binding element otherwise referred to as an antigen binding domain. The choice of moiety depends upon the type and number of ligands that define the surface of a target cell. For example, the antigen binding domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Thus examples of cell surface markers that may act as ligands for the antigen moiety domain in the CAR include those associated with viral, bacterial and parasitic infections, autoimmune disease and cancer cells The CAR can be engineered to target a tumor antigen of interest by way of engineering a desired antigen binding domain that specifically binds to an antigen on a tumor cell.

[0029] “Tumor antigen” or “hyperproliferative disorder antigen” or “antigen associated with a hyperproliferative disorder,” refers to antigens that are common to specific hyperproliferative disorders such as cancer. The antigens discussed herein are merely included by way of example. The list is not intended to be exclusive and further examples will be readily apparent to those with skill in the art. Tumor antigens are proteins that are produced by tumor cells that elicit an immune response, particularly T cell mediated immune responses. The selection of the antigen binding domain of the invention will depend on the particular type of cancer to be treated. Tumor antigens are well known in the art and include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), b-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1 , MN-CA IX, human telomerase reverse transcriptase, RU1 , RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, pro state- specific antigen (PSA), PAP, NY-ESO-1 , FAGE-la, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate- carcinoma tumor antigen- 1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, folate receptor (FRa) GD2, MUC-1 , BTNs, and mesothelin. The term "antibody," as used herein, may refer to a protein, or polypeptide sequence derived from an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be polyclonal or monoclonal, multiple or single chain, or intact immunoglobulins, and may be derived from natural sources or from recombinant sources.

[0030] The term "antibody fragment" may refer to at least one portion of an antibody that retains the ability to specifically interact with an epitope of an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CHI domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, multi- specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody. An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv.

[0031] The term “immune cell” as referred to herein includes cells that are of haematopoietic origin and that play a role in the immune response. Immune cells include lymphocytes, such as B cells and T cells; natural killer (NK) cells; tumor-infiltrating lymphocytes (TIL); myeloid cells, such as monocytes, macrophages, dendritic cells, eosinophils, mast cells, basophils, and granulocytes; or T cells engineered with CAR or cancer-antigen specific T cell receptors (TCR). In one embodiment, the immune cell is an immune effector cell. The term “immune effector cell,” as that term is used herein, refers to a cell that is involved in an immune response, e.g., in the promotion of an immune effector response. Examples of immune effector cells include T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid- derived phagocytes.

[0032] “Immune effector function or immune effector response,” as that term is used herein, refers to function or response, e.g., of an immune effector cell, that enhances or promotes an immune attack of a target cell. For example, an immune effector function or response refers a property of a T or NK cell that promotes killing or the inhibition of growth or proliferation of a target cell. In the case of a T cell, primary stimulation and co-stimulation are examples of immune effector function or response.

[0033] “Immune modulating cell” as referred to herein is any cell that impacts function of an immune cell, such as for example stromal cells in healthy and diseased tissue, epithelial cells or a tumor cell. The terms positive or negative are to be understood as being relative, i.e. positive cells have a much higher expression level as compared to cells being negative. Cells being negative in this sense may thus still have an expression level which may be detected.

[0034] The term “stimulation,” refers to a primary response induced by binding of a stimulatory molecule (e.g., a TCR / CD3 complex or CAR) with its cognate ligand (or tumor antigen in the case of a CAR) thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR / CD3 complex or signal transduction via the appropriate NK receptor or signaling domains of the CAR. Stimulation can mediate altered expression of certain molecules.

[0035] The term “CRISPR” refers to a caspase-based endonuclease comprising a caspase, such as Cas9, and a guide RNA that directs DNA cleavage of the caspase by hybridizing to a recognition site in the genomic DNA and is an example for all gene or base editing techniques.

[0036] The term "vector" or "expression construct" may refer to a nucleic acid molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for expression of the operably linked coding sequence (e.g. an insert sequence that codes for a product) in a particular cell. An expression vector construct may comprise sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vector constructs include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0037] The “vector” may also be a “transfer vector” which refers to a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “transfer vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to further include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno- associated virus vectors, retroviral vectors, lentiviral vectors, and the like. In some embodiments, the CAR sequences are delivered into cells using a retroviral or lentiviral vector. CAR-expressing retroviral and lentiviral vectors can be delivered into different types of eukaryotic cells as well as into tissues and whole organisms using transduced cells as carriers or cell-free local or systemic delivery of encapsulated, bound or naked vectors. The method used can be for any purpose where stable expression is required or sufficient.

[0038] The term "expression" may refer to the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0039] The term “encode" or “encoding” includes reference to nucleotides and / or amino acids that correspond to other nucleotides or amino acids in the transcriptional and / or translational sense.

[0040] The term "nucleic acid" includes a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. The terms “nucleic acid”, “nucleic acid molecule”, “nucleic acid sequence” and “polynucleotide” are used interchangeably herein unless the context indicates otherwise. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, siRNAs, shRNAs, RNAi agents, and primers. A polynucleotide can be modified or substituted at one or more base, sugar and / or phosphate, with any of various modifications or substitutions described herein or known in the art. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this invention that is a polynucleotide encompasses both the double-stranded form and each of two complementary single- stranded forms known or predicted to make up the double-stranded form. In some contexts, the terms “nucleic acid” or “polynucleotide” and the like encompass any material which conveys genetic information or performs a function of a nucleic acid or polynucleotide (e.g., it can be translated into a protein or act as an RNAi agent), even if such material is not strictly composed of nucleotides (which consist of a sugar, base and phosphate); such genetic material may comprise, as nonlimiting examples, peptide nucleic acid (PNA), locked nucleic acid (LNA), morpholino nucleotide, threose nucleic acid (TNA), glycol nucleic acid (GNA), arabinose nucleic acid (ANA), 2'-fl uoroarabinose nucleic acid (FANA), cyclohexene nucleic acid (CeNA), anhydrohexitol nucleic acid (HNA), and / or unlocked nucleic acid (UNA). The terms "protein" and "polypeptide" are used interchangeably and may refer to any polymer of amino acids (dipeptide or greater) linked through peptide bonds or modified peptide bonds. Polypeptides of less than about 10-20 amino acid residues are commonly referred to as "peptides." The polypeptides of the invention may comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a polypeptide by the cell in which the polypeptide is produced, and will vary with the type of cell. Polypeptides are defined herein, in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.

[0041] The terms “reduced” or “eliminated” or “disruption” or "disrupted" are used interchangeably herein to refer to any genetic modification that decreases or eliminates expression and / or the functional activity of the nucleic acid or an expression product thereof. For example, disruption of a gene includes within its scope any genetic modification that decreases or eliminates expression of the gene and / or the functional activity of a corresponding gene product ( e.g., mRNA and / or protein). Genetic modifications include complete or partial inactivation, suppression, deletion, interruption, blockage, or down- regulation of a nucleic acid (e.g., a gene). Illustrative genetic modifications include, but are not limited to, gene knockout, inactivation, mutation (e.g., insertion, deletion, point, or frameshift mutations that disrupt the expression or activity of the gene product), or use of inhibitory nucleic acids (e.g., inhibitory RNAs such as sense or antisense RNAs, molecules that mediate RNA interference such as siRNA, shRNA, miRNA; etc.), inhibitory polypeptides (e.g., antibodies, polypeptide-binding partners, dominant negative polypeptides, enzymes etc.) or any other molecule that inhibits the activity of the LAIR-1 gene or level or functional activity of an expression product of the LAIR-1 gene.

[0042] By “pharmaceutically acceptable carrier” is meant a solid or liquid filler, diluent or encapsulating substance that can be safely used in topical or systemic administration to an animal, preferably a mammal, including humans. Representative pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington’s Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient(s), its use in the pharmaceutical compositions is contemplated. The term “overexpressed” tumor antigen or “overexpression” of a tumor antigen is intended to indicate an abnormal level of expression of a tumor antigen in a cell from a disease area like a solid tumor within a specific tissue or organ of the patient relative to the level of expression in a normal cell from that tissue or organ. Patients having solid tumors or a haematological malignancy characterized by overexpression of the tumor antigen can be determined by standard assays known in the art.

[0043] The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized in part by unregulated cell growth. As used herein, the term “cancer” refers to non-metastatic and metastatic cancers, including early stage and late stage cancers. The term “precancerous” refers to a condition or a growth that typically precedes or develops into a cancer. By “non-metastatic” is meant a cancer that is benign or that remains at the primary site and has not penetrated into the lymphatic or blood vessel system or to tissues other than the primary site. Generally, a non-metastatic cancer is any cancer that is a Stage 0, 1 , or II cancer, and occasionally a Stage III cancer. By “early stage cancer” is meant a cancer that is not invasive or metastatic or is classified as a Stage 0, I, or II cancer. The term “late stage cancer” generally refers to a Stage III or Stage IV cancer, but can also refer to a Stage II cancer or a sub-stage of a Stage II cancer. One skilled in the art will appreciate that the classification of a Stage II cancer as either an early stage cancer or a late stage cancer depends on the particular type of cancer.

[0044] The term "cancer" includes but is not limited to, breast cancer, large intestinal cancer, lung cancer, small cell lung cancer, gastric (stomach) cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine sarcoma, ovarian cancer, rectal or colorectal cancer, anal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vulval cancer, squamous cell carcinoma, vaginal carcinoma, Hodgkin’s disease, non-Hodgkin’s lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue tumor, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, glioma, astrocytoma, glioblastoma multiforme, primary CNS lymphoma, bone marrow tumor, brain stem nerve gliomas, pituitary adenoma, uveal melanoma (also known as intraocular melanoma), testicular cancer, oral cancer, pharyngeal cancer or a combination thereof. Cancers that may be treated include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors. The cancers may comprise non-solid tumors (such as hematological tumors, for example, leukemias and lymphomas) or may comprise solid tumors. Types of cancers to be treated with the CARs, TCRs, or any other type of ligand-binding engineered receptors of the invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0045] The term “administering” refers to contacting, applying, injecting, transfusing or providing a composition of the present invention to a subject.

[0046] The term “treating" as used herein may refer to (1) preventing or delaying the appearance of one or more symptoms of the disorder; (2) inhibiting the development of the disorder or one or more symptoms of the disorder; (3) relieving the disorder, i.e., causing regression of the disorder or at least one or more symptoms of the disorder; and / or (4) causing a decrease in the severity of one or more symptoms of the disorder.

[0047] The term “subject” as used throughout the specification is to be understood to mean a human or may be a domestic or companion animal. While it is particularly contemplated that the methods of the invention are for treatment of humans, they are also applicable to veterinary treatments, including treatment of companion animals such as dogs and cats, and domestic animals such as horses, cattle and sheep, or zoo animals such as primates, felids, canids, bovids, and ungulates. The “subject” may include a person, a patient or individual, and may be of any age or gender.

[0048] Detailed description

[0049] The inventors surprisingly found that in addition to direct inhibition of tumor cell killing, the LAIR- 1 :collagen interaction also plays a role in suppressing immune cell migration. The inventors used in vitro and in vivo mouse models as well as in vitro human 3D tumor models to demonstrate the role of LAIR-1 in T cell migration and tumor cell killing in collagen-dense environments. With this knowledge, the invention herein solves the current problem of immunotherapy failure in collagen- rich and immunosuppressive microenvironments. In one method of the invention this is achieved by generating engineered immune cells expressing a receptor recognizing a target antigen wherein the expression and / or function of LAIR-1 has been reduced or eliminated. Therefore, in one aspect, there is provided an immune cell expressing a receptor recognizing a target antigen, wherein the expression and / or function of LAIR-1 has been reduced or eliminated. In some embodiments, the immune cell is selected from the group of an isolated non-modified immune cell, an engineered immune cell or an immune modulating cell. In some embodiments, the immune cell presents enhanced migration and / or tumor cell killing. LAIR-1

[0050] LAIR-1 may function as an inhibitory receptor on immune cells that binds to collagens. The interaction between LAIR-1 and collagen may suppress immune cell activation and migration. By reducing or eliminating LAIR-1 expression or function, immune cells may exhibit enhanced migration through collagen-rich environments such as the tumor stroma. Additionally, disrupting LAIR-1 signaling may increase the cytotoxic potential of immune cells against tumor targets. The inventors have demonstrated that genetic deletion or antibody-mediated blockade of LAIR-1 can improve T cell infiltration into tumors and augment anti-tumor responses in preclinical models. Therefore, targeting LAIR-1 may represent a promising strategy to enhance the efficacy of adoptive cell therapies and other immunotherapeutic approaches, particularly in solid tumors with abundant collagen deposition. In some embodiments, the immune cell comprises or has been in contact with an inhibitor of LAIR-1 that is either a blocking antibody or a nucleic acid sequence capable of downregulating or eliminating gene expression or modifying the function of LAIR-1 , wherein the nucleic acid capable of downregulating the gene expression of LAIR-1 is selected from the group consisting of an antisense RNA, antagomir RNA, siRNA, shRNA, a CRISPR system, a zinc finger nuclease system, a transcription activator-like effector based nuclease (TALEN) system, or interfering with binding or function through any base editing technique.

[0051] Methods for reducing or eliminating LAIR-1 expression or function in immune cells may be implemented using techniques known to those skilled in the art. Such methods may include, but are not limited to, genetic engineering approaches like CRISPR-Cas9 gene editing, RNA interference using siRNA or shRNA, or antibody-mediated blockade of LAIR-1. The skilled person may select an appropriate method based on factors such as the specific immune cell type, the desired level of LAIR-1 inhibition, and the intended therapeutic application. For genetic modification approaches, standard transfection or transduction protocols may be employed to introduce nucleic acids targeting LAIR-1 into isolated immune cells. In some cases, viral vectors like lentiviruses or adeno- associated viruses may be used to efficiently deliver gene editing components. For antibody-based approaches, the skilled person may optimize antibody concentrations and treatment durations to achieve effective LAIR-1 blockade while maintaining cell viability. Quality control measures such as flow cytometry, Western blotting, or functional assays may be implemented to confirm successful reduction of LAIR-1 expression or activity. The modified immune cells may then be expanded ex vivo using established cell culture protocols before administration to patients. Throughout the manufacturing process, good manufacturing practices (GMP) guidelines may be followed to ensure product quality and safety for clinical applications. In one aspect there is provided, a method of manufacturing the immune cell as described herein, the method comprising contacting an immune cell extracted from a patient suffering from a tumor with an inhibitor of LAIR-1 for a sufficient amount of time and under suitable conditions to reduce or eliminate the expression and / or function of LAIR-1 .The immune cell may be incubated with a LAIR- 1 blocking antibody for a period of time sufficient to achieve effective LAIR-1 blockade. The optimal incubation time may vary depending on factors such as the specific antibody used, the concentration of the antibody, and the type of immune cell being treated. Typically, incubation times may range from about 30 minutes to 24 hours. In some cases, shorter incubation times of 1-2 hours may be sufficient, while in other instances, longer incubation periods of 4-8 hours or overnight may be preferred to ensure maximal LAIR-1 blockade. The skilled person may determine the appropriate incubation time through routine optimization experiments, assessing LAIR-1 blockade efficiency and cell viability at various time points. Flow cytometry or functional assays may be used to confirm successful LAIR-1 blockade following antibody treatment.

[0052] In some embodiments, the inhibitor of LAIR-1 is selected from the group of a nucleic acid sequence capable of downregulating gene expression of LAIR-1 , a CRISPR system or another gene or base editing system interfering with function or expression.

[0053] The skilled person may adapt CRISPR techniques to reduce or eliminate LAIR-1 expression in immune cells using various approaches. In some cases, single guide RNAs (sgRNAs) targeting the LAIR-1 gene locus may be designed using publicly available tools and databases to identify optimal target sequences. Multiple sgRNAs may be tested to determine the most effective option for LAIR- 1 knockout. The selected sgRNA may then be combined with Cas9 or other CRISPR-associated nucleases, which may be delivered to immune cells as ribonucleoprotein complexes or encoded by viral vectors.

[0054] For more precise modifications, base editing or prime editing techniques may be employed to introduce specific mutations in the LAIR-1 gene without inducing double-strand breaks. These approaches may allow for subtle alterations that disrupt LAIR-1 function while minimizing off-target effects. The skilled person may optimize delivery methods, such as electroporation or viral transduction, to achieve efficient editing in primary immune cells.

[0055] To confirm successful CRISPR-mediated modification of LAIR-1 , various validation assays may be performed. These may include genomic PCR and sequencing to verify the intended genetic changes, as well as flow cytometry or Western blotting to assess LAIR-1 protein levels. Functional assays examining immune cell migration and tumor cell killing capacity may also be conducted to evaluate the phenotypic effects of LAIR-1 modification.

[0056] In some instances, the skilled person may implement inducible CRISPR systems to allow for temporal control over LAIR-1 editing. This approach may enable the study of LAIR-1 function at different stages of immune cell activation or tumor progression. Additionally, multiplexed CRISPR strategies may be used to simultaneously target LAIR-1 and other genes of interest, potentially enhancing the overall anti-tumor efficacy of engineered immune cells.

[0057] Immune Cell

[0058] In some embodiments, the immune cell is selected from the group of a T cell, from its subtypes ap T cell or y6 T cell, or a NK cell, ap T cells are a major subset of T lymphocytes that express T cell receptors (TCRs) composed of a and p chains. These cells play a central role in adaptive immunity and may recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, ap T cells may include CD4+ helper T cells and CD8+ cytotoxic T cells, which have distinct functions in coordinating immune responses and directly killing infected or malignant cells, respectively. y6 T cells represent a smaller population of T cells that express TCRs made of y and 6 chains. Unlike ap T cells, y6 T cells may recognize antigens in a non-MHC-restricted manner. These cells may have properties of both innate and adaptive immunity and may be involved in tissue homeostasis, infection control, and tumor surveillance. y6 T cells may recognize stress- induced ligands and metabolites produced by tumor cells or pathogens.

[0059] Natural killer (NK) cells are innate lymphoid cells that may play a crucial role in the early defense against viruses and tumors. NK cells may not require prior sensitization to exert their cytotoxic functions. They may recognize stressed, infected, or transformed cells through a balance of activating and inhibitory receptors. NK cells may also produce cytokines and chemokines that help shape adaptive immune responses. In the context of engineered immune cell therapies, each of these cell types may offer unique advantages, ap T cells may provide antigen-specific responses and memory formation. y6 T cells may offer broader recognition of tumor antigens and potential for allogeneic therapies. NK cells may provide rapid, innate-like responses against tumors and may be less susceptible to some tumor immune evasion mechanisms.

[0060] In some embodiments, the method as described herein, comprises the ex vivo or in vivo modification of a T cell or NK cell extracted from a patient suffering from a tumor, wherein the tumor microenvironment of said tumor is characterized by an immunosuppressive and collagen-rich environment and / or increased presence of immunosuppressive mesenchymal stem cells (MSC). In some embodiments, the immune cell may be an autologous cell derived from the patient to be treated. In other embodiments, the immune cell may be an allogeneic cell from a healthy donor. The immune cell may be isolated from peripheral blood, bone marrow, umbilical cord blood, or other tissues. In some cases, the immune cell may be expanded ex vivo prior to genetic modification to reduce or eliminate LAIR-1 expression or function. The immune cell may be further modified to express additional receptors or molecules that enhance its therapeutic potential. For example, the immune cell may be engineered to express chemokine receptors that promote migration to tumor sites, cytokines that enhance persistence and function in the tumor microenvironment, or costimulatory molecules that amplify activation signals. In some embodiments, the immune cell may be a naive T cell, memory T cell, or effector T cell. The specific subset of T cells used may depend on the intended therapeutic application and may influence the cell's capacity for expansion, persistence, and effector function following adoptive transfer. For NK cells, both conventional NK cells and adaptive NK cells may be utilized. In some cases, NK cells may be further modified to express chimeric antigen receptors (CARs) or other targeting moieties in addition to having reduced LAIR-1 expression or function. The immune cell may also be selected or modified based on its metabolic profile to enhance survival and function in the nutrient-poor tumor microenvironment. For instance, cells with increased mitochondrial fitness or the ability to utilize alternative fuel sources may be preferentially selected or engineered. In some embodiments, the immune cell may be genetically modified to be resistant to immunosuppressive factors present in the tumor microenvironment, such as TGF-p or adenosine, in addition to having reduced LAIR-1 expression or function. This multi-pronged approach may further enhance the cell's anti-tumor efficacy.

[0061] In one aspect there is provided, an immune cell isolated from a patient suffering from a disease associated with expression of a tumor antigen, wherein the expression and / or function of LAIR-1 in said immune cell has been reduced or eliminated ex vivo.

[0062] In one aspect there is provided, an engineered immune cell characterized by a T cell expressing a chimeric antigen receptor (CAR) recognizing a cancer-associated target antigen, wherein said engineered T cell has been modified as such that the expression and / or function of LAIR-1 has been reduced or eliminated. In a preferred embodiment, in the engineered or natural immune cell or engineered or natural T cell of the invention expressing a receptor recognizing a target antigen the expression of LAIR-1 , has been reduced.

[0063] In an embodiment the engineered T cell is an ap T cell engineered to express a defined yQTCR (TEGs), or a dual targeting TEG. apT cells may be defined with respect to function as T lymphocytes that express an apTCR, which recognize peptides bound to MHC molecules (major histocompatibility complex), which are expressed on the surface of various cells. MHC molecules present peptides derived from the proteins of a cell. When for example a cell is infected with a virus, the MHC will present viral peptides, and the interaction between the apTCR on the T cell and the MHC-complex on the target cell (i.e. the virus infected cell) activates specific types of T-cells which initiate and immune responses to eliminate the infected cell. Hence, apT cells may be functionally defined as being cells capable of recognizing peptides bound to MHC molecules. apT cells may be selected from peripheral blood for example via the CD3 antigen, as the large majority of T cells have the apTCR. apT cells may also be selected with an antibody specific for the apTCR. From such selected cells, the nucleic acid (or amino acid) sequence corresponding to the aT-cell receptor chain and the pT-cell receptor chain may be determined by sequencing. Hence, apT-cells may also be defined as being cells comprising a nucleic acid (or amino acid) sequence corresponding to the aT-cell receptor chain and / or the pT-cell receptor chain. yQT-cells may be functionally defined in that they are specifically and rapidly activated by e.g. a set of non-peptidic phosphorylated isoprenoid precursors, collectively named phosphoantigens which modulate members of the butyrophilin (BTN) family (e.g. BTN2 and BTN3) or stress signals medicated by non classical HLA molecules like CD1 . Phosphoantigens are produced by virtually all living cells, though the levels are usually very low in healthy cells, and increased in transformed I malignant cells or cells infected with e.g. mycobacterium tuberculosis, which deliver a derivate of phosphoantigens and modulate the spatial expression and conformation of BTN2 and BTN3. The most common phosphoantigen found in human cells (including cancer cells) is isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP). Activation of yQT-cells comprises clonal expansion, cytoxic activity and expression and release of cytokines. yQT-cells are also defined by expression of the y6T cell receptor. For example, cells may be selected using an antibody specific for the y6T cell receptor such as described below. From such selected cells, the nucleic acid (or amino acid sequence) sequence corresponding to the yT-cell receptor chain and / or the 6T-cell receptor chain may be determined by sequencing, preferably as carried out in the experimental part. Hence, yQT-cells may also be defined as being cells naturally comprising a nucleic acid (or amino acid) sequence corresponding to a yT-cell receptor chain and / or a 6T-cell receptor chain. The person skilled in the art is well capable of selecting and / or identifying immune cell populations characterized by expression of an antigen or receptor on the surface of the immune cell such as described throughout herein. It is understood that with regard to expression on the surface of immune cells, such as CD3, CD4, CD8, apTCR, and yQTCR, this is typically done in a population of cells of which a portion of cells have a much higher level of expression of the antigen when compared to cells having a lower level of expression. The immune cells used for the engineering of the immune cells of the invention hereunder may be T-cells from primary cells, for example from a subject, such as described in the examples for a human subject. Engineering can occur in vitro or in vivo. The engineered T-cells may be ap or y6 T-cells derived from a human subject. Alternatively, the T-cells may be T cell lines, such as SupT- 1 , Jurkat, or Raji cells or any other widely available cell line. Any cell type, being a primary cell or any other cell line will suffice, as long as the cell population, or a substantial part thereof, expresses the T-cell receptor, i.e. such as being positive for the apT- cell or the yQTCR receptor in a FACS sorting, such a cell population may be contemplated. Also, any cell or cell population may be contemplated that, when provided with a yQTCR according to the invention is capable of forming a functional TCR complex and exerting e.g. a functional cytoxic response and / or cytokine production. The cell that is provided may also be a progenitor cell, preferably a blood progenitor cell such as a thymocyte or a blood stem cell, which after it has been provided with the right stimuli can develop into T cells. Preferably, T cells provided express or are able to express a y6 TCR. T cells may have been transduced to express a y6 TCR or already express a yTCR and have been transduced to express a 6TCR(or respectively already express a QTCRand have been transduced to express a yTCR), comprising the nucleic acid sequences encoding the sequence obtained in step a). All theoretical combinations of a y with a 6 chain of the TCR is encompassed. In an embodiment of the invention, the yQTCP is y962TCR. In another embodiment the yQTCR is y561TCR. In another embodiment, the yQTCR is y865TCR. In another embodiment, the yQTCR is y465TCR. In another embodiment, the yQTCP is y261TCR. In another embodiment the yQTCR is y861TCR, nevertheless in principle all combination of VyVQTCR are possible.

[0064] In one aspect there is provided, an ap T cell engineered to express a defined yQTCR (TEGs), or a dual targeting TEG, wherein the TEG cell presents enhanced migration and tumor cell killing and comprises an intracellular signaling domain or fragment that is functional in the absence of LAIR-1 , and wherein said engineered T cell has been modified as such that the expression and / or function of LAIR-1 has been reduced or eliminated. TEGs, or T cells engineered to express gamma delta T cell receptors, are ap T cells that have been genetically modified to express a defined y6 T cell receptor (TCR). This engineering approach combines the advantages of ap T cells, such as their capacity for expansion and memory formation, with the unique properties of y6 T cells, including their ability to recognize tumor antigens in a non-MHC-restricted manner.

[0065] Dual targeting TEGs a are engineered to express two distinct receptor systems: 1 . A defined y6 TCR, which may recognize stress-induced ligands or metabolites commonly produced by tumor cells; 2. An additional targeting moiety, such as a chimeric antigen receptor (CAR) or a second TCR, which may recognize a different tumor-associated antigen. This dual targeting approach may enhance the specificity and efficacy of the engineered T cells. By recognizing multiple tumor- associated antigens simultaneously, dual targeting TEGs may reduce the risk of tumor escape through antigen loss and may provide a more robust anti-tumor response. The combination of different recognition mechanisms (e.g., TCR and CAR) may also allow these cells to overcome some of the limitations associated with single-targeting strategies.

[0066] In some cases, the dual targeting approach may be designed to improve tumor infiltration, persistence within the tumor microenvironment, or activation of other immune cell types. The specific combination of targeting moieties may be tailored to the characteristics of particular tumor types or to address specific challenges in the tumor microenvironment.

[0067] Tumour Micro Environment

[0068] The inventors identified two major causes of an immunosuppressive TME that lead to failure of most immunotherapies: the presence of immunosuppresive mesenchymal stem cells (sMSC) and an increased presence of collagen. Regarding the presence of sMSC, the inventors built an experimental and analysis pipeline that integrates single-cell functional data from a multicellular 3D BM model into single-cell RNA sequencing analysis. In this system the inventors distinguished immunosuppressive (s-MSC) from permissive MSC (p-MSC). These MSC donor-intrinsic phenotypes differ in their capacity to support or suppress engineered T cell mediated tumor cell killing. Permissive MSCs (p-MSCs) display dynamic transcriptomic profiles that adapt to the surrounding cellular environment, particularly in the presence of tumor and immune cells, while the transcriptomic heterogeneity of immunosuppressive MSCs (s-MSCs) is more limited. Furthermore, the inventors show that suppressive versus permissive MSCs differ in their collagen gene expression profile and suppress the migratory and cytolytic capacity of genetically engineered T cells, which is dependent on the inhibitory collagen receptor LAIR-1 on T cells. The tumor microenvironment (TME) may play a crucial role in cancer progression and response to therapy. It consists of various cellular and non-cellular components that interact with tumor cells and influence their behaviour. The TME may be highly heterogeneous and dynamic, evolving as the tumor progresses. It may create an immunosuppressive environment that hampers anti-tumor immune responses and limits the efficacy of immunotherapies. Factors such as increased collagen deposition and the presence of immunosuppressive mesenchymal stem cells (sMSCs) may contribute to this suppressive milieu. Understanding and targeting the TME may be essential for developing more effective cancer treatments and improving patient outcomes.

[0069] In one embodiment the one or more tumors to be treated with the immune cell described herein, is first characterized. In one embodiment the tumor is characterized by having an immunosuppressive and collagen-rich microenvironment and / or increased presence of immunosuppressive mesenchymal stem cells (MSC). In one embodiment the tumor is characterized by the immunomodulatory properties of the MSCs in the tumor. In one preferred embodiment the MScs are characterized by the expression of one or more immunomodulatory genes. In one preferred embodiment the MScs are characterized by the expression of one or more immunomodulatory genes from Table 1 . The tumor microenvironment (TME) as described herein refers to the complex interplay of many different cell types, including tumor cells, immune cells, and stromal cells. In recent years, the contribution of the extracellular matrix (ECM) in this interplay has become increasingly clear. Tumor-associated ECM is distinct from healthy tissue, and plays a key role in promoting tumor progression and limiting therapy responses. Collagen is the major component of the ECM, and is involved in supporting tumor growth, initiating metastases, and excluding therapeutics and immune cells from the tumor nests. Collagen fibre alignment and density are key regulators of immune cell infiltration into the tumor site. The dominant component of the ECM is fibrillar collagen, which comprises approximately 30 % of its total protein. Tumors to be treated with the invention hereunder are preferably, but are not limited to, gastric cancer, hepatocellular carcinoma (HCC), lung cancer, and pancreatic cancer.

[0070] Bone marrow-mesenchymal stem cells (BM-MSCs) as described herein comprise a very heterogeneous population of cells with self-renewal capacity and the ability to differentiate to multiple lineages: osteocytes, adipocytes and chondrocytes. In the tumor microenvironment, MSCs promote growth of tumor cells and possess immunosuppressive features which further allow the tumor to escape immune surveillance. The term MSCs as described herein refers also to MSCs from any other source like umbilical cord blood derived MSC. MSCs contribute substantially to the formation and remodeling of the extracellular matrix (ECM) of the TME, supporting tumor growth and metastasis as well as to inhibit overshooting immune responses in the context of auto-immune diseases like GVHD. To do so, MSC secrete components of the fiber network, such as collagens, as well as metalloproteinase and lysyl oxidases which are enzymes involved in the remodeling of the morphology, thickness and stiffness of the matrix.

[0071] Using a 3D co-culture model the inventors reveal donor-intrinsic differences in the capacity of MSC to suppress anti-tumor T cell activity in the bone marrow niche through extracellular matrix remodeling, possibly explaining patient-specific responses to engineered T cell therapy. Besides observing the diversity in MSC phenotypes in the TME the inventors have further identified and characterized the specific phenotype of immunosuppressive MSCs in the bone marrow niche (s- MSCs) responsible for immunotherapy failure in such tumors, which could lead to further stratification of patients before immunotherapy and is considered to be a specific embodiment of the invention. Therefore, in one embodiment, a subject is first stratified prior to treatment with the immune cell as described herein. In one embodiment, the subject is stratified using the phenotype of immunosuppressive MSCs in the bone marrow niche.

[0072] The inventors identified from healthy individuals s-MSC and p-MSC which differ in transcriptomic heterogeneity, transcriptomic plasticity, and function. While s-MSC have been rigid in terms of transcriptomic heterogeneity, plasticity and were mainly immune suppressive, p-MSC showed more transcriptomic plasticity not only themselves in the presence of cancer cells and engineered immune cells but were also able to induce transcriptomic plasticity in CD8+ T cells and associated with an improved killing of leukemic blasts. P-MSC created a milieu with more pro-inflammatory cytokine profiles. In addition, s-MSC and p-MSC rearranged the extracellular matrix differently with varying composition of collagens. This allowed to interfere in the presence of s-MSC with either collagen polymerization or blocking collagen binding via LAIR1 which increased both killing and infiltration capacity of mainly cancer specific CD8+ T cells.

[0073] The inventors have identify that the differential MSC phenotypical profiles found are intrinsic to different individuals as distinct phenotypes grew out from different donors. This observation is in line with the observation that indeed MSC from different donors have different abilities to be immune suppressive when used for the treatment of, for example, acute graft versus host disease. The inventors demonstrate that gene expression differences between s- and p-MSCs are responsible for the immunomodulatory features. While s-MSC have been stable in transcriptomic heterogeneity, p-MSC further changed profiles once T cells and cancer cells were added. However, functional consequences of s- and p-MSC have been maintained regardless of the cancer type present in the environment implying that this observation is not only relevant for the more extensively studied AML, but also other hematological cancer cell types like MM. Therefore, the immune cell as described herein has a wide range of suitability.

[0074] Transcriptomic heterogeneity in the infused engineered T cell product has been most recently described as major predictor for clinical outcome for various hematological malignancies. The inventors described that transcriptomic heterogeneity correlates with T cell behaviour against solid cancers can be further modulated by different types of co-stimulation against liquid and solid cancers. The current data set implies that such 3D models might be too simplistic as simply the addition of MSC even in the absence of cancer cells further alters transcriptomic heterogeneity of engineered immune cells. Most importantly intrinsic abilities of MSC and the preference in s- or p- MSC has further major impact on shaping transcriptomic heterogeneity and most likely needs to be taken into consideration in individualized therapies. The inventors demonstrated that the migration capacity of T cells to the cancer microenvironment was also strongly hindered by s-MSCs, while p- MSC induced pro-inflammatory genes in T cells. Therefore, in one embodiment, the subject’s cancer microenvironment is assessed before and / or after to treatment with the immune cell as described herein.

[0075] An important part of the invention is that s- and p-MSCs are able to remodel the extracellular matrix via expressing key proteins involved in matrix stiffening in line also with other reports. Furthermore, different types of collagens are observed in either s- or p-MSC, which play an important role in the differential phenotype between MSCs. As a further part of the invention, the inventors showed that s-MSCs preferentially express type I, III and VI collagens, while p-MSC express type IV and XII collagens. Type I and III collagens are fibril-forming collagens that when highly expressed, lead to a dense fibrillary network which stiffness the ECM, a known hallmark of cancer. Type I and III collagens have been also reported to be high affinity ligands for inhibitory receptor LAIR-1 and to inhibit the effector cytotoxic activity of CD8+ T cells. In an embodiment of the invention, CD8+ TEGs expressed at the cell surface higher levels of LAIR-1 , and thus, benefited the most from LAIR-1 blocking treatment in the suppressive MSC environment. This effect is due to a higher infiltration capacity of mainly CD8+ TEGs into the cancer microenvironment. Altogether, the inventors describe varying preferences for s- or p-MSC in different individuals, which associated most likely with different cell matrix already in healthy tissues. Intrinsic differences between s- and p-MSC derived from different individuals drive for many individuals a stronger suppressive phenotype through the modulation of the collagen composition. Once cancer develops cell matrix produced due to the presence of s-MSC can be detrimental for cancer immune surveillance and cancer immune therapies. Characterizing the individual preferences for MSC in patients' prior therapies will allow to further individualize immune therapies. Therefore, in one embodiment, the subject’s preferences for MSC is characterized prior to treatment with the immune cell as described herein.

[0076] Treatment & Kits

[0077] In one aspect there is provided, a method of treating a subject in need thereof, the method comprising administering an immune cell as described herein for a sufficient amount of time and under suitable conditions to treat the subject.

[0078] The present invention provides engineered immune cells and methods for treating cancer, autoimmune diseases, or infection, among other diseases. In one embodiment, the invention provides an engineered immune cell (e.g., T cell) engineered to express a CAR wherein the cell exhibits an antitumor property or an anti-pathogen property. The cells can also be engineered so that they (i) express various therapeutic / beneficial factors (e.g., one or more different CARs, pro- function factors, and graft sustaining factors) and (ii) lack the expression of various harmful factors (e.g., rejection factors, host targeting factors, and infection-risk factor) as described herein.

[0079] In one embodiment a method of treating a subject in need thereof is described. In a preferred embodiment the subject has a tumor. The definition of tumor as used herein includes, but is not limited to, a disease associated with expression of a tumor antigen as described herein or condition associated with cells which express a tumor antigen as described herein including, e.g., proliferative diseases such as a cancer or malignancy or a precancerous condition such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia; or a noncancer related indication associated with cells which express a tumor antigen as described herein. In one embodiment, a tumor associated with expression of a tumor antigen as described herein is a hematological cancer. In one embodiment, a tumor associated with expression of a tumor antigen as described herein is a solid cancer. Further diseases associated with expression of a tumor antigen described herein include, but not limited to, e.g., atypical and / or non-classical cancers, malignancies, precancerous conditions or proliferative diseases associated with expression of a tumor antigen as described herein. Non-cancer related indications associated with expression of a tumor antigen as described herein include, but are not limited to, e.g., autoimmune disease, (e.g., lupus), inflammatory disorders (allergy and asthma) and transplantation. In some embodiments, the tumor antigen expressing cells express, or at any time expressed, mRNA encoding the tumor antigen. In an embodiment, the tumor antigen-expressing cells produce the tumor antigen protein (e.g., wild- type or mutant), and the tumor antigen protein may be present at normal levels or reduced levels. In an embodiment, the tumor antigen-expressing cells produced detectable levels of a tumor antigen protein at one point, and subsequently produced substantially no detectable tumor antigen protein. In an embodiment, the tumor antigen-expressing cells overexpresses the tumor antigen protein.

[0080] In some embodiments, the immune cells may be administered to the subject through various routes. Intravenous administration may be used to deliver the cells systemically. Alternatively, intratumoral injection may allow for direct delivery of the cells to the tumor site. In some cases, the immune cells may be administered via intraperitoneal injection, particularly for tumors located in the abdominal cavity. Subcutaneous administration may be employed for certain types of solid tumors.The immune cells may also be delivered using implantable devices or scaffolds that allow for controlled release over time. In some instances, the cells may be encapsulated in biodegradable materials to protect them during administration and promote sustained delivery. Intradermal or transdermal administration methods may be explored for specific tumor types or to enhance migration to lymph nodes. In certain embodiments, the immune cells may be administered in combination with other therapeutic agents or treatment modalities. For example, the cells may be co-administered with checkpoint inhibitors, cytokines, or small molecule drugs that enhance their function or persistence. The timing and sequencing of cell administration relative to other treatments may be optimized to maximize therapeutic efficacy.

[0081] The dosing regimen may vary depending on factors such as the type and stage of cancer, the patient's overall health status, and the specific characteristics of the engineered immune cells. Multiple doses may be administered over time to maintain therapeutic levels of the cells. In some cases, lymphodepletion or other preconditioning regimens may be employed prior to cell administration to enhance engraftment and expansion of the transferred cells. In some embodiments, the dosage of administered immune cells may range from about 1 x 10A5 to 1 x 10A9 cells per kilogram of body weight. The specific dose may be adjusted based on factors such as the patient's condition, tumor burden, and response to previous treatments. For example, an initial dose of 1 x 10A6 cells / kg may be administered, followed by subsequent doses that may be increased or decreased based on the patient's response and tolerability. In certain cases, the immune cells may be administered in multiple infusions over a period of time. For instance, patients may receive 3-5 infusions of cells, spaced 2-4 weeks apart. This approach may help maintain therapeutic levels of the engineered cells in the body and promote a sustained anti-tumor response.

[0082] The concentration of cells in each infusion may also be optimized. In some embodiments, the cells may be suspended in a suitable carrier solution at a concentration of about 1 x 10A6 to 1 x 10A8 cells / mL. The infusion rate may be adjusted to deliver the cells over a period of 30-60 minutes, which may help minimize potential infusion-related reactions. For intratumoral administration, lower cell numbers may be used, typically ranging from 1 x 10A5 to 1 x 10A7 cells per injection site. The frequency of intratumoral injections may vary depending on the tumor type and location, with some protocols utilizing weekly injections for a defined treatment course.

[0083] In cases where the immune cells are delivered using controlled-release devices or scaffolds, the dosing may be designed to provide a sustained release of cells over days to weeks. The total number of cells loaded into such devices may range from 1 x 10A7 to 1 x 10A9, with the release kinetics tailored to maintain therapeutic cell levels at the target site. It is important to note that optimal dosing regimens may vary between different types of engineered immune cells and tumor types. Therefore, dose-escalation studies and careful monitoring of patient responses may be necessary to determine the most effective and safe dosing strategy for each specific application. In some embodiments, the subject has a tumor, wherein the tumor microenvironment of said tumor is characterized by an immunosuppressive collagen-rich environment and / or increased presence of immunosuppressive mesenchymal stem cells (MSC). The skilled person may assess an immunosuppressive collagen-rich environment and / or increased presence of immunosuppressive mesenchymal stem cells (MSC) through various methods and techniques. In some cases, histological analysis of tumor biopsies may be performed to evaluate collagen content and architecture. Masson's trichrome staining or picrosirius red staining may be used to visualize and quantify collagen fibers. Second harmonic generation microscopy may provide detailed imaging of collagen structure and organization within the tumor microenvironment. Immunohistochemistry or immunofluorescence techniques may be employed to detect specific collagen types, such as collagen I, III, or IV, as well as markers associated with immunosuppressive MSCs. These may include CD73, CD90, CD105, and others. The spatial distribution and density of these markers may be analyzed to assess the presence and extent of immunosuppressive elements. In some instances, flow cytometry analysis of dissociated tumor samples may be used to quantify the proportion of MSCs and characterize their phenotype. Markers such as CD29, CD44, CD73, CD90, and CD105, in combination with the absence of hematopoietic markers, may be used to identify MSCs. Additional markers like PD-L1 , IDO, or IL-10 may indicate an immunosuppressive phenotype.

[0084] Gene expression profiling of tumor samples, using techniques such as RNA sequencing or quantitative PCR, may reveal upregulation of collagen-related genes and genes associated with immunosuppressive functions in MSCs. This may include analysis of extracellular matrix components, immunomodulatory factors, and cytokines. Proteomic analysis of tumor tissue or interstitial fluid may be performed to detect elevated levels of collagen proteins and soluble factors produced by immunosuppressive MSCs. Mass spectrometry-based approaches may provide comprehensive profiling of the tumor microenvironment composition. Functional assays may be conducted to assess the immunosuppressive capacity of isolated MSCs from tumor samples. These may include T cell proliferation assays, cytokine production assays, or assessment of regulatory T cell induction in the presence of tumor-derived MSCs. In some approaches, imaging techniques such as magnetic resonance imaging (MRI) with specific contrast agents or positron emission tomography (PET) with radiolabeled tracers may be used to non-invasively assess collagen content or MSC presence in tumors. The skilled person may also consider analyzing circulating biomarkers in patient blood samples. Elevated levels of certain collagen fragments or MSC-derived factors in serum or plasma may correlate with an immunosuppressive tumor microenvironment. Comparative analysis with normal tissue or less aggressive tumors may help establish thresholds for what constitutes "increased" collagen or MSC presence. The skilled person may need to consider tissuespecific and tumor type-specific variations when making these assessments. In some cases, a combination of these approaches may be necessary to comprehensively characterize the immunosuppressive nature of the tumor microenvironment. The specific methods chosen may depend on the availability of tumor samples, technical capabilities, and the particular research or clinical context.

[0085] In one aspect there is provided, an immune cell as described herein for use in treating a subject in need thereof.

[0086] In one aspect there is provided, a method of enhancing the efficacy of adoptive cell therapy, comprising: administering to a subject an immune cell as described herein. In some embodiments, the method further comprising administering one or more additional therapeutic agents selected from the group consisting of checkpoint inhibitors, cytokines, and small molecule drugs.

[0087] In one aspect there is provided, the use of an immune cell as described herein in the manufacture of a medicament for treating a subject in need.

[0088] In one aspect there is provided, a kit comprising:

[0089] (a) an immune cell expressing a receptor recognizing a target antigen, wherein the expression and / or function of LAIR-1 has been reduced or eliminated; and

[0090] (b) instructions for administering the immune cell to a subject in need thereof.

[0091] In some embodiments, the kit may further comprise one or more of:

[0092] (c) a pharmaceutically acceptable carrier;

[0093] (d) a cryopreservation medium;

[0094] (e) reagents for assessing viability or function of the immune cells;

[0095] (f) additional therapeutic agents for combination therapy;

[0096] (g) devices or materials for administration of the immune cells.

[0097] In some cases, the kit may include multiple doses of the immune cells, which may be stored separately. The kit may also contain materials for ex vivo manipulation or expansion of the immune cells prior to administration. In certain embodiments, the kit may include components for genetic modification of the immune cells, such as viral vectors or CRISPR reagents for reducing LAIR-1 expression. The instructions may provide guidance on dosing, administration routes, and monitoring of potential side effects.

[0098] The invention is summarized in the following embodiments: Embodiment 1. An immune cell expressing a receptor recognizing a target antigen, wherein the expression and / or function of LAIR-1 has been reduced or eliminated.

[0099] Embodiment 2. The immune cell from embodiment 1 , wherein the immune cell is selected from the group of an isolated non-modified immune cell, an engineered immune cell or an immune modulating cell.

[0100] Embodiment 3. The immune cell from embodiment 1 or 2, wherein the immune cell presents enhanced migration and / or tumor cell killing.

[0101] Embodiment 4. The immune cell of any of embodiments 1 to 3, wherein the immune cell comprises or has been in contact with an inhibitor of LAIR-1 that is either a blocking antibody or a nucleic acid sequence capable of downregulating or eliminating gene expression or modifying the function of LAIR-1 , wherein the nucleic acid capable of downregulating the gene expression of LAIR-1 is selected from the group consisting of an antisense RNA, antagomir RNA, siRNA, shRNA, a CRISPR system, a zinc finger nuclease system, a transcription activator-like effector based nuclease (TALEN) system, or interfering with binding or function through any base editing technique .

[0102] Embodiment 5. The immune cell of any of embodiments 1 to 4, wherein the immune cell is selected from the group of a T cell, from its subtypes ap T cell or y6 T cell, or a NK cell.

[0103] Embodiment 6. A method of manufacturing the immune cell of any of embodiments 1 to 5, the method comprising contacting an immune cell extracted from a patient suffering from a tumor with an inhibitor of LAIR-1 for a sufficient amount of time and under suitable conditions to reduce or eliminate the expression and / or function of LAIR-1 .

[0104] Embodiment 7. The method of embodiment 6, wherein the inhibitor of LAIR-1 is selected from the group of a nucleic acid sequence capable of downregulating gene expression of LAIR-1 , a CRISPR system or another gene or base editing system interfering with function or expression.

[0105] Embodiment 8. The method of embodiment 6 or 7, wherein the method comprises the ex vivo or in vivo modification of a T cell or NK cell extracted from a patient suffering from a tumor, wherein the tumor microenvironment of said tumor is characterized by an immunosuppressive and collagen- rich environment and / or increased presence of immunosuppressive mesenchymal stem cells (MSC). Embodiment 9. An immune cell isolated from a patient suffering from a disease associated with expression of a tumor antigen, wherein the expression and / or function of LAIR-1 in said immune cell has been reduced or eliminated ex vivo.

[0106] Embodiment 10. An engineered immune cell characterized by a T cell expressing a chimeric antigen receptor (CAR) recognizing a cancer-associated target antigen, wherein said engineered T cell has been modified as such that the expression and / or function of LAIR-1 has been reduced or eliminated.

[0107] Embodiment 11. An ap T cell engineered to express a defined yQTCR (TEGs), or a dual targeting TEG, wherein the TEG cell presents enhanced migration and tumor cell killing and comprises an intracellular signaling domain or fragment that is functional in the absence of LAIR-1 , and wherein said engineered T cell has been modified as such that the expression and / or function of LAIR-1 has been reduced or eliminated.

[0108] Embodiment 12. A method of treating a subject in need thereof, the method comprising administering an immune cell according to any of embodiments 1 to 5 for a sufficient amount of time and under suitable conditions to treat the subject.

[0109] Embodiment 13. The method of embodiment 12, wherein the subject has a tumor, wherein the tumor microenvironment of said tumor is characterized by an immunosuppressive collagen-rich environment and / or increased presence of immunosuppressive mesenchymal stem cells (MSC).

[0110] Embodiment 14. An immune cell according to any one of embodiments 1 to 5 for use in treating a subject in need thereof.

[0111] Embodiment 15. Use of an immune cell according to any one of embodiments 1 to 5 in the manufacture of a medicament for treating a subject in need.

[0112] Embodiment 16. A method to identify the effectiveness of an immunotherapy and additional potential modulations in relation to the tumor microenvironment or an auto-immune disease like GVHD, the method comprising the steps of: a. Determining the immunomodulatory effects of mesenchymal stem cells (MSCs) in a 3D bone marrow model or defined gene profile b. Categorizing the MSCs into immunosuppressive MSCs (s-MSC) or immunopermissive MSCs (p-MSC) based on their ability to influence T cell-specific tumor killing or induce tolerance c. Conducting single-cell RNA sequencing in the MSCs groups d. Identifying genes selected from Table 1 associated with the immunomodulatory effects of the MSC groups e. Modify the expression of one or several gene candidates from (d) to boost or inhibit T cellkilling f. Selecting MSC for infusion into patients in order to dampen or activate the immune system g. Selecting patients most suitable for immunotherapies or suitable for LAIR interference when using immune modulators like immune cell engager including but not limited to bispecific molecules. h. Selecting patients for in vivo modulation or gene editing of LAIR expression.

[0113] Embodiment 17. The method of embodiment 16 wherein the mesenchymal stem cells are from a human donor.

[0114] Embodiment 18. The method of embodiment 16 wherein the 3D bone marrow model comprises a. Co-culturing the MSCs with T cells and tumor cells b. Embedding the tumor cells and MSCs in a hydrogel mimicking the extracellular matrix

[0115] Embodiment 19. The method of embodiment 16 wherein the gene candidate selected in (d) is LAIR- 1

[0116] Features of any of the examples or embodiments outlined above may be combined to create additional examples or embodiments without losing the intended effect. It should be understood that the description of an embodiment or example provided above is by way of example only, and various modifications could be made by one skilled in the art. Furthermore, one skilled in the art will recognise that numerous further modifications and combinations of various aspects are possible. Accordingly, the described aspects are intended to encompass all such alterations, modifications, and variations that fall within the scope of the appended claims.

[0117] Figure legends

[0118] Figure 1. 3D bone marrow model reveals donor-dependent MSC immunomodulation that affects migration, persistence, and tumor-killing capacity of T-cells.

[0119] (A) Schematic depicting the components and timeline of 3D in vitro model designed to mimic the bone marrow TME (designed with Biorender). (B) Killing of RPMI-8226 cells by TEG001 relative to TEG-LM1 control on day 6 when co-cultured with MSCs from 5 different healthy donors. (C) Total migrated TEG001 cells in bottom compartment of Transwell system on day 6 when co-cultured with RPMI-8226 and immunosuppressive (MSC06) or permissive (MSC18) stromal cells. (D) Killing of patient-derived AML cells (donors 01 , 02, 03) and AML cell lines by TEG001 on day 6 when co- cultured with MSC06 or MSC18 (normalized to TEG-LM1 control). (E) Killing of RPMI-8226 by BCMA CAR T-cells on day 6 when co-cultured with MSC06 or MSC18 (normalized to TEG-LM1 control). (F)Total MSC cell counts on day 6 after co-culture with RPMI-8226 and TEG001 (normalized to TEG-LM1 control). (G) Relative amount of T cells normalized to TEG-LM1 control in a co-culture with MSC donor 6 and 18 in matrigel after 2 days.

[0120] Figure 2. Immunosuppressive MSCs display a more rigid transcriptomic profile at steady state with enriched transcription of cell division genes compared to the more variable profile of permissive MSCs.

[0121] (A) Principal component analysis (PCA) plot of RNAseq transcriptomic analysis done on immunosuppressive MSCs (donors 3, 6, 8, 9) and permissive MSCs (donors 10, 14, 16) cultured at steady state. The immunomodulation of these donors was previously characterized using the 3D BM model. (B) Gene set enrichment analysis (GSEA) plot depicting the statistically significant biological pathways for the immunosuppressive and permissive MSCs (left). Unsupervised clustering of the MSCs based on relevant genes from marked pathways supports the phenotypic split between the immunosuppressive and permissive MSCs (right). (F) GO-term analysis of MSC clusters.

[0122] Figure 3. Single-cell analysis reveals uniformity in the transcriptome of suppressive MSCs independent of TEG or tumor cell presence, while the transcriptomic activity of permissive MSCs is more susceptible to its environment. (A) Process of single cell sequencing of the 3D co-cultures of the bone marrow niche (B) UMAP plot of unsupervised clustering of the combined dataset, annotated by experiment (cell type composition). (C) GSEA plot of a subset of HALLMARK genesets over the pseudobulk profiles of MSC subsets per experiment. (D) UMAP plot displaying the unsupervised clustering of MSC subsets from all experiment. (E) Cluster composition of the MSC population per experiments (%). The colors correspond to those of the clusters in (D).

[0123] Figure 4. Permissive MSCs allow for a transcriptomic shift primarily in the TEG population, while suppressive MSCs constrain TEG to a distinct, transcriptomic profile independent of tumor presence.

[0124] (A) CD4 I CD8 composition of the TEG subset from each experiment (%), according to our type prediction. (B) UMAP plot depicting the unsupervised cluster analysis of CD4+ TEGs. (C) Cluster composition of the CD4+ TEG population per experiments (%). The colors correspond to those of the clusters in (B). (D) Density dot plot of the percentage of cells from CD4+ TEG clusters (from B) that express a given gene ('percent expressed') and the scaled average expression of canonical marker representing the marked terms. (E) UMAP plot depicting the unsupervised cluster analysis of CD8+ TEGs. (F) Cluster composition of the CD8+ TEG population per experiments (%). The colors correspond to those of the clusters in (E). (G) Density dot plot of the percentage of cells from CD8+ TEG clusters (from B) that express a given gene ('percent expressed') and the scaled average expression of canonical marker representing the marked terms. (H) UMAP plot displaying the unsupervised clustering of TEG subsets from all experiment. (I) CD4 and CD8A expression of the predicted CD4 and CD8 TEG subsets. Grey color mark lack of expression. (J) UMAP plot displaying the predicted CD4 and CD8 types of TEG cells from all experiment. (K) CD4 I CD8 composition of the TEG clusters in (H) (%), according to our type prediction. (L) GO-term analysis per CD4 TEG clusters. M) GO-term analysis per CD8 TEG clusters.

[0125] Figure 5. MSCs represent an important cell-cell communication hub in the model marrow model

[0126] (A) Comparison of numbers of interaction within the complete suppressive and permissive experiments. (B) Number of detected channels per experiment. (C) List of detected channel groups, marking their representation per experiment. (D) List of incoming channel groups, per cell type (source) per experiment. (E) List of outgoing channel groups, per cell type (receiver) per experiment. (F) Scaled expression of main detected collagen genes, per MSC-clusters (from Figure 3D). (G) Scaled expression of main detected collagen genes, in MSC cells per experiment (from Figure 3B). (H) Scaled expression of LAIR-1 and COL3A1 , per cell types per experiment. (I) LAIR-1 expression and mean fluorescence intensity (MFI) of CD4+ and CD8+ TEGs compared to unstained cells.

[0127] Figure 6. Interference of collagen folding in MSCs and LAIR1 -mediated binding to collagen in TEG001 both increase tumor killing in the suppressive microenvironment, while blocking BAG6-NKp30 interactions between MSCs and TEG001 ameliorates tumor killing and infiltration by TEG001 in both the suppressive and permissive microenvironments. (A) TEG001 -specific killing of RPMI-8226 and (B) TEG001 infiltration into bottom compartment of 3D model on day 6 after b subunit of prolyl 4-hydroxylase (P4HB) gene was knocked out in MSC06 and MSC18 on day 0. (C) TEG001 -specific killing of RPMI-8226 and (D) TEG001 infiltration on day 6 after antibody blocking of LAIR1 on TEG001 (+ isotype control) prior to T-cell administration on day 4. (E) Total amount of migrated CD4+ and CD8+ TEG001 and TEG-LM1 , treated with LAIR Isotype and LAIR1 blocking antibody, in the bottom compartment of the transwell system of an in vitro 3D RPMI-8226 model made with MSC18 and MSC06.

[0128] Figure 7. LAIR-1 is a key regulator of T cell migration. A. Wild type and LAIR-1 -CD3 NFAT- GFP reporter cells were used to determine if Matrigel at different densities is capable of inducing LAIR-1 signalling. (A). Data from 3-4 independently performed experiments is summarised. (B). Lair1+,+and Lairt7OT-1 cells were stained with separate dyes, embedded in Matrigel and migration was tracked using live cell immunofluorescence imaging. Tracks were calculated using Imaris software and are depicted for one representative experiment out of 4 experiments (C). Quantification of data as in B. Data is aggregated from four separate experiments. Both Lair1+,+and Lairt1' OT-1 cells were stained with calcein AM and CellTracker Orange CMTMR within the same experiment. As dyes did not influence migration (Figure 1 G), measurements from both dyes were combined. D. RNA sequencing was performed on naive CD8+T cells from Lair1+,+and Lairt1' spleens. Differential expression analysis was done with DESeq2. Significantly differentially expressed genes are depicted in orange, non-significant genes are depicted in blue. (E). Data corresponding to Figure 1 B, where data of Lair1+,+or Lairt1' OT-1 T cells was combined to determine the effect of staining cells with either calcein AM or CellTracker Orange CMTMR on migration. We confirmed that the use of different dyes did not affect migration through Matrigel. (F). As in A., but migration of Lair1+,+and Lairt1' OT-1 cells is determined separately to show that differences between Lair1+,+and Lairt1' OT-1 T cells are mostly maintained, independent of the dye used. (G). Lair1+,+and Lairt1' OT-1 cells were activated using SIINFEKL peptides for 3 days, and expression of LAIR-1 and chemokine receptors CCR5 and CCR7 was determined by flow cytometry.

[0129] Figure 8. Phospho-proteomic profiling identifies enriched migration pathways in LairT' OTI T cells compared to Lair1+ / +in a collagen-rich 3D environment (A). Schematic overview of the experimental protocol. (B). Volcano plot representing the differentially expressed phospho-peptides between Lair and Lairl+ / +OT1 T cells. C. Pathway enrichment analysis of upregulated genes in Lairt ' OT1 T cells (p<0.1). (D). Dendogram highlighting the candidate kinases predicted to target the top 50 phosphopeptides in Lairt7and Lair1+ / +OT1 T cells. Node size is proportional to the number of the queried phosphosites targeted by the kinases. Major kinase families are annotated in the dendogram which include: TK (Tyrosine Kinases), TKL (Tyrosine Kinase-Like), STE (Sterile kinases; homologs of the yeast STE7, STE11 and STE20 kinases), CK1 (Casein Kinase 1), AGC (comprising Protein kinase A / PKA, PKG and PKC kinase sub-families), CAMK (Calcium / Calmodulin-dependent kinases) and CMGC (comprising cyclin-dependent kinase (CDK), mitogen- activated protein kinase (MAPK), glycogen synthase kinase (GSK) and CDC-like kinase (CLK)). (E). Phospho-peptides identified in Lairl Lair1+ / +OT1 T cells and Matrigel alone. (F). Phosphopeptides identified per protein. (G). Dendogram highlighting the candidate kinases predicted to target the top 50 unique phosphopeptides in Lair1+ / +OT1 T cells. (H). Dendogram highlighting the candidate kinases predicted to target the top 50 unique phosphopeptides in Lairt7OT1 T cells. Node size is proportional to the number of the queried phosphosites targeted by the kinases. Major kinase families are annotated in the dendogram which include: TK (Tyrosine Kinases), TKL (Tyrosine Kinase-Like), STE (Sterile kinases; homologs of the yeast STE7, STE11 and STE20 kinases), CK1 (Casein Kinase 1), AGC (comprising Protein kinase A / PKA, PKG and PKC kinase sub-families), CAMK (Calcium / Calmodulin-dependent kinases) and CMGC (comprising cyclin- dependent kinase (CDK), mitogen-activated protein kinase (MAPK), glycogen synthase kinase (GSK) and CDC-like kinase (CLK)).

[0130] Figure 9. LAIR-1 impairs tumour cell killing by inhibiting T cell migration through Matrigel. (A). MC38-OVA-GFP cells were embedded in 50% Matrigel and Lair1+,+or Lairt1' OT-1 T cells were added on top of the Matrigel. Tumour cell killing was assessed by loss of GFP signal by live cell imaging. Data is aggregated from 2 experiments with similar measurements, out of 5 experiments performed. (B). MC38-OVA cells were allowed to form a monolayer for 3 days, after which Lair1+,+or Lairt1' OT-1 T cells were added in different effectortarget ratios. Tumour cell killing was assessed by loss of GFP signal by live cell imaging. Data is aggregated from 6 separate experiments. (C). MC38-OVA cells were allowed to form a spheroid in low attachment plates for 3 days, after which Lair1+,+or Lairt1' OT-1 T cells were added in different effectortarget ratios. Tumour cell killing was assessed by loss of GFP signal by live cell imaging. Data is aggregated from 3 experiments with similar measurements, out of 8 experiments performed. (D). MC38-OVA target cells were labelled with chromium-51 and activated Lair1+,+or Lairt1' OT-1 T cells were added in suspension. Cytotoxicity was determined after 4 or 24 hours by measuring chromium release in supernatant. Data is aggregated from 3 independent experiments. E. MC38-OVA and wild type OT-1 T cells were co-cultured at different effector:target ratios in the presence or absence of agonist anti-LAIR-1 antibodies. Tumour killing was determined by flow cytometry. (E). MC38-OVA-GFP cells were assessed for expression of collagen by immunofluorescence. Cells were stained with anti-collagen I antibody, followed by a secondary antibody. Data is representative of 2 experiments.

[0131] (F). Expression of collagen genes was determined using publicly available RNA sequencing data of MC38 cells (GSE93017)54. Only genes with normalized counts > 1 in both samples are depicted.

[0132] (G). The ability of MC38 cells to induce LAIR-1 signalling was determined by GFP response in wild type and LAIR-1 -CD3 NFAT-GFP reporter cells when cultured on top of a MC38 monolayer. (H). MC38-OVA cells were allowed to form a monolayer for 3 days, and treated with collagenase, after which Lair1+,+or Lairt1' OT-1 T cells were added in different effector:target ratios. Tumour cell killing was assessed by loss of GFP signal and by viability dye using flow cytometry. Data is aggregated from 3-4 separate experiments.

[0133] Figure 10. LAIR-1 -deficient T cells migrate more efficiently towards the tumour site in vivo. (A). Lair1+I+and Lairt1' OT-1 T cells were adoptively transferred at a 1 :1 ratio into MC38-OVA tumour bearing mice. After 12 hours, the contribution of Lair1+I+and Lairt1' OT-1 T cells in the tumour and spleen were determined using congenic markers on flow cytometry. Data is aggregated from 2 independent experiments with n = 7-10 mice each. (B). Mice were injected subcutaneously with 500,000 MC38-OVA cells and tumours were allowed to establish. On day 30, at an average tumour size per group of approximately 500 mm3, 3 million Lair1+,+or Lairt1' OT-1 T cells or PBS were adoptively transferred by intravenous injection. Afterwards, tumour growth was followed over time. Data is from one experiment with 6 mice per group. (C). Percentage of mice clearing tumours, i.e., reaching tumour size < 50 mm3corresponding to C. Three out of six mice in each group did not control tumour growth and reached HEP. (D). Schematic overview and gating strategy for experiment performed as in Figure 3A. (E). Transferred Lair1+,+and Lairt1' OT-1 T cells were measured by flow cytometry to confirm 1 :1 ratio.

[0134] Figure 11. LAIR-1 -deficiency or blockade increase TEG capacity to kill and migrate towards HT29 tumor cells in an in vitro collagen rich 3D tumor model. (A). Schematic overview.

[0135] (B). Percentage of LAIR1' ' and LAIR1+ / +TEG specific killing. (C). Percentage of LAIRt' and LAIR1+ +TEG infiltration. (D). Number of CD4 and CD8 infiltrating LAIR1' ' and LAIR1+ +TEGs.

[0136] (E). Percentage of TEG infiltration after LAIR-1 blocking antibodies or control. (F). Impact of LAIR- 1 deficiency on CD4 and CD8 TEG cells for tumor killing. (G). LAIR-1 expression on CD4 and CD8 TEGs. (H). LAIR 1 expression on CD4 and CD8 TEGs before and after LAIR-1 knockout generation by CRISPR

[0137] Figure 12. LAIR 1 depletion on TEGs rescues HT29 expressed collagen induced tumor cell killing impairment (A). Percentage of LA / RT / _and LAIR1+ +TEG specific killing. (B). Percentage of LAIR17' and LAIR1+ +TEG infiltration (C). Number of CD4 and CD8 infiltrating TEGs

[0138] Figure 13. Effects of LAIR-1 on early T-cell infiltration into a collagen matrix. (A) Analysis pipeline for T-cell infiltration. Either wild type or LAIR1 KO TEG001 cells were separated to CD4+ and CD8+ fractions and labelled separately with either Calcein AM or CellTrace Violet viability dyes [ThermoFischer). T cells were then placed on top of a BME matrix consisting of preconditioned media using HT29 cell line and imaged for 6.5h. The cell coordinates per timepoint are extracted, and the cell positions at t=0 are used to infer the topology of the BME surface. The cell depth distributions overtime are used to determine the rate and speed of cell infiltration. (B) Cell infiltration over time (C) Infiltration speed, (D) Infiltration rate and (E) infiltration at timepoint 6,5h. The experiment was done twice with 4 technical replicates. Bars represent the SD. * p<0.05; ** p<0.01 ; ns: non-significant. Figure 14. Figure 7. Knocking out LAIR1 expression in TEG-001 T-cells increases tumor killing and infiltration rate in the suppressive tumor microenvironment. (A) TEG001 -specific killing of RPMI-8226 and (B) TEG001 infiltration into bottom compartment of 3D model containing RPMI-8226 and s-MSC donor 06 on day 6 after the LAIR1 gene was knocked out in either TEG- 001 CD4+, CD8+ or both. (C) Total amount of migrated CD4+ and CD8+ TEG001 knocked out for LAIR1 in the bottom compartment of the transwell system of an in vitro 3D RPMI-8226 model made with s-MSC donor 06. (n>2)

[0139] Tables

[0140] Table 1. List of top 50 genes with the highest Iog2 fold-change (log2-FC) in MSC clusters. Table 2. List of top 50 genes with the highest log2-FC in MSCs from different experiments.

[0141] Table 3. List of top 50 genes with the highest log2-FC in TEG_CD4 clusters.

[0142] Table 4. List of top 50 genes with the highest log2-FC in TEG_CD8 clusters.

[0143] Table 5. Differentially expressed genes (bulk RNAseq, DEGs) between pMSC and ssMSC in a steady-state experiment, not co-cultured.

[0144] Examples

[0145] Example 1- Donor-dependent MSC immunomodulation affects T-cell efficacy and migration

[0146] To assess the immunomodulatory effects of MSC from different individuals on T-cell immunotherapies, we designed a multifaceted 3D model with tumor cells, MSC, endothelial cells and different types of immune effector cells to mimic the malignant bone marrow niche. (Figure 1A). When using TEGs as immune effector cells, killing efficacy differed in the presence of five randomly chosen MSC donors: When using MSC from donor 03, 06 and 09 TEGs were unable to kill RPMI- 8226 tumor cells in the presence of Pamidronate while when using MSCs from donor 13 and 18 TEGs were able to kill up to 60% of all tumor cells (Figure 1 B). Consequently, MSC donors were categorized accordingly into two primary groups based on their ability to influence T cell-specific tumor killing: immunosuppressive MSCs (s-MSC: donors 03, 06, and 09) and immunopermissive MSCs (p-MSC: donors 13 and 18). Migration ofTEGs into the bottom compartment of the Transwell system was likewise significantly diminished when s-MSC were utilized in comparison to p-MSC (Figure 1 C). Immunomodulatory effects by s-MSC on TEGs was again seen when using various primary pediatric leukemia samples and leukemic cell lines as tumor targets, indicating the immunoregulation of TEGs by MSCs is donor-specific and independent of the tumor target (Figure 1 D). Similarly, the immunosuppressive effect of s-MSC on T-cells compared to p-MSC was also observed when introducing BCMA CAR T-cells instead of TEGs to target RPMI-8226 (Figure 1 E). Importantly, s- and p-MSCs did not differ in their proliferation capacity in culture neither in the cocultures settings (Figure 1 F). Together, these findings suggest that MSCs have a donor-dependent effect on tumor targeting by T-cells independent of the leukemic model or T-cell therapy implemented in the model.

[0147] Example 2- Transcriptomic profiles of p-MSCs ands-MSCs reflect distinct programs

[0148] To enhance our understanding of how mesenchymal stem cells (MSCs) with different capacities to modulate T cell-mediated tumor cytotoxicity are regulated at the transcriptomic level, we conducted RNA sequencing (RNAseq) on a panel of s-MSCs and p-MSCs. Analysis of the transcriptomic data via principal component analysis demonstrated that s-MSCs displayed a more compact cluster in the space defined by the first two principal components, indicating a more homogenous gene expression profile relative to p-MSCs (Figure 2A). Gene Set Enrichment Analysis (GSEA) showed that, p-MSCs had higher enrichment of terms such as ‘leukocyte migration’, ‘chemotaxis’, ‘immune effector process’, suggesting their role in fostering an immune-tolerant milieu even in the absence of T-cells or tumor cells. Conversely, s-MSCs had increased enrichment of biological pathways like ‘cell division’, ‘metabolic processes’, and ‘protein production’ (Figure 2B). Despite the increased enrichment of cell division related genes in the transcriptomic profiles, we did not detect higher numbers of s-MSCs in co-cultures, as shown in Figure 1 F.

[0149] Example 3- Identification of Marker Genes (scRNAseq):

[0150] Differentially expressed genes were identified using the Seurat package’s FindAIIMarkers function, employing a Wilcoxon Rank Sum test as the default statistical method. This analysis was constrained to genes exhibiting a minimum of 0.25 Iog2 fold change in average gene expression within a cluster relative to the combined average expression across all other clusters. The investigation was limited to genes that serve as positive markers and are detected in at least 25% of cells in any of the two populations compared (parameters: only.pos = TRUE, min. pct = 0.25, logfc.threshold = 0.25). The resulting gene lists ('clusterDEG_' worksheets) were split corresponding to each cluster and ranked individually by fold change. The ‘topDEGs worksheets enumerate the top 50 genes with the highest fold changes. In case of the ‘experimentDEGJ worksheets, the transcriptomic profiles of MSC cells from different experimental conditions were contrasted with the same parameters as above.

[0151] Example 4- Identification of Marker Genes bulk (RNAseq):

[0152] Differentially expressed genes were identified using the DESeq2 package by contrasting samples with suppressive (n=4) and permissive (n=3) phenotypes. To minimize sex-related bias, genes located on chromosomes X and Y were excluded from the analysis. Furthermore, the study was restricted to 'protein coding' genes. The criteria for significant differential expression included a minimum Iog2 fold change of 3, an adjusted p-value of less than 0.05 (Wald test), and a baseMean of counts greater than 20 following normalization.

[0153] Example 5- s-MSC and p-MSC do not only have a different transcriptomic heterogeneity, but also different plasticity

[0154] To deepen our comprehension of the transcriptomic regulation in mesenchymal stem cells (MSCs) within a malignant bone marrow environment, particularly when these cells are in contact with tumor cells and T cells, we established co-cultures of either s-MSCs or p-MSCs with pediatric AML blasts (referred to as ‘cancer’ in our single cell RNA-sequencing (scRNA) experiments) embedded in Matrigel and TEGs (Figure 3A). We designed an experimental matrix that incorporated various cellular combinations to replicate incremental complexity levels of the bone marrow microenvironment. After co-culture, samples were processed per experiment into scRNA libraries and submitted for sequencing. The initial cell type separation per experiments was facilitated by single nucleotide polymorphism (SNP) profiles as the experimental components originated from distinct individuals. The separation was corroborated using uniform manifold approximation and projection (UMAP) alongside cell type annotation methodology, which divided the combined datasets into three major cluster groups corresponding to the expected cell types: cancer, TEGs, and MSCs, each identified by the expression of their canonical marker genes (Figure 3B). When we labelled the UMAP-plots according to the experiments cell phenotypes we noticed that s- and p-MSC types showed the highest levels of heterogeneity within cell types. Further, to investigate the interactions of MSCs with the components of our constructed bone marrow model, we examined the transcriptomic profiles of each element individually, but aggregated from all experimental runs.

[0155] Initiating our analysis, we conducted gene set enrichment analysis (GSEA) on the pseudobulk transcriptomes of MSC populations across our data collection, focusing on a selection of hallmark gene sets from molecular signature databases. This analysis reaffirmed the proliferative nature of s-MSCs and the pro-inflammatory milieu associated with p-MSCs, consistent across various experimental conditions (Figure 3C). To profile the cellular compositions of each dataset from our experimental conditions, we applied unsupervised clustering and identified eight distinct MSC clusters (Figure 3D).

[0156] After clustering we split the dataset per experiments to reveal the effect of conditions over the cluster compositions. In case of s-MSCs, clusterO (top differential markers: KRT19, TIMP3, CHI3L1) and cluster! (TOP2A, CENPF, MKI67) were predominant, with their proportions remaining stable irrespective of whether cancer cells or TEGs were present alone or in combination (Figure 3E, column 1-3). GO-term analysis of cluster 0 revealed terms related to ossification and organ development, which led us to investigate the published references of the leading marker genes (Figure 3F). Both KRT19 (keratin 19) as an intermediate filament molecule, the metalloprotease inhibitorTIMP3 (TIMP metalloprotease inhibitors) and the secreted glycoprotein CHI3L1 (chitinase- 3-like proteinl) have been associated with immunosuppression and exctracellular matrix, hinting at a potential role in fostering immunoresistance within the tumor microenvironment (TME). Cluster 1 represented the cells with proliferative signatures, mirroring the proliferative profiles of s-MSCs in the (bulk)RNAseq experiments.

[0157] Contrariwise, p-MSCs displayed a different profile as compared to s-MSC in the presence of TEGs or cancer cells (Figure 3E, column 4-6). Under conditions with exclusively TEGs or cancer cells clusters 2 and 6 prevailed. These clusters were characterized by differential gene expression associated with hypoxia and apoptosis (cluster2 (BNIP3, HMOX1 , LDHA)) and stress response pathways (clusters (PLCG2, PLK2, NFKBIZ)). In addition, in the presence of both TEGs and cancer cells, cluster 2 and 6 of p-MSCs showed in contrast to s-MSC a major shift towards cluster 3 and 4, reflecting an upregulation of genes implicated in extracellular matrix construction, (clusters (COL4A1 , POSTN, THBS1)), and genes involved in immune cell infiltration, like CRYAB (cluster4).- These findings suggest that the transcriptomic landscape of s-MSCs is not only different from p- MSCs but also comparatively stable, whereas p-MSCs exhibit an additional degree of plasticity in the presence of tumor and immune cells.

[0158] Example 6 - s- and p-MSC impact transcriptomic heterogeneity of TEGs differently

[0159] Since s- and p-MSCs affected tumor targeting of T cells differently, we aimed to understand whether the s- and p-MSC impact transcriptomic heterogeneity of TEGs. To this end, we dissected TEG transcriptomic data from all experiments and performed unsupervised clustering. First, we focused on the distinction of CD4+ and CD8+ TEGs. Given the absence of proteomic information for the single-cell transcriptomic profiles, we utilized a support vector machine (SVM) algorithm to predict the CD4 / 8 status of TEGs. This prediction was based on our earlier multiomic single-cell RNA sequencing experiment that provided us with profiles of FACS-sorted CD4+ and CD8+ TEG populations. Upon comparing the CD4 / CD8 distribution across different experimental TEG populations, we observed a greater proportion of CD8+ TEGs in the milieu containing p-MSCs (Figure 4A).

[0160] Firstly CD4+ and CD8+ TEGs cultured alone, had a different cluster composition when compared to transcriptomic composition of TEGs in the presence of MSC. For CD4+ TEGs (Figure 4B-C-D), a predominantly GZMA and GZMA-related cytotoxicity clusterl gave way to clusterO, characterized by GNLY-associated cytotoxicity along with genes implicated in 'response to external stimulation' and 'apoptosis' (Figure 4L). The newly appeared clusterO represented the majority of CD4+ TEG cells in all co-culture setups involving s-MSCs, in the presence and absence of tumor cells (Figure 4C and D). In contrast, the combination of p-MSCs with TEGs spawned an additional cluster (cluster4) indicative of T-cell activity and exhaustion, distinguished by CCL3 and CCL4 expression. This cluster disappeared when tumor cells were introduced and CD4+ TEGs in the presence s- MSC or p-MSC did not differ substantially, except for the lower representation of clusterO. In Figure 3 C and F we ordered the two most complete co-culturing experiments, with different MSC-types, next to each other and squared them to guide the visual comparison.

[0161] Similarly, CD8+ TEG populations (Figure 4E-F-G) showed clusters 1 and 2 as dominant clusters in the absence of any additional components of the model, associated with TCR-binding (cluster! (HLA-DRB1 / 5)), adhesion (cluster2 (CXCR6)). The clusters of the CD8 ‘TEG alone’ condition were replaced by a GNLY-marked cluster that (unlike in CD4s) additionally expressed genes related to oxidative phosphorylation (NDUFA1 , ATP5MD) (Figure 4M). Again, p-MSCs alone induced the emergence of a new cluster (cluster4) expressing CCL3 and CCL4. In contrast to CD4+ TEG in the presence of p-MSC, the CD8+ TEG cluster composition changed markedly with the addition of tumor cells when compared to comparable experimental conditions with s-MSC (Figure 4, marked with the square). The exclusively identified cluster in this scenario (clusters in p-MSC + CD8 TEG + cancer cells) was defined by terms such as ‘response to stress’ and ‘apoptosis’ (BTG1 , BCL2L11). We conclude, that both s-MSC and p-MSC changed transcriptomic heterogeneity of CD4+ and CD8+ TEGs, however only in the presence of p-MSC did CD8+ TEGs show additional plasticity in transcriptomic heterogeneity once they encountered tumors.

[0162] Example 7 - Immunopermissive environments display higher number of potential cell-to- cell interactions

[0163] While we established that s- and p-MSCs distinctly influence tumor-specific T cell functions within the 3D bone marrow niche, the question arose whether this modulation stemmed from specific receptor-ligand interactions. To explore this, we utilized the cell-chat algorithm, which predicts potential receptor-ligand interactions by calculating communication probabilities based on gene expression data, of a list of receptors and their ligands, and their overall representation throughout the compared groups.

[0164] Our analyses focused on comparing two separate experiments, each containing all three components (MSC + CD4 TEG + CD8 TEG + cancer cells; the CD4-CD8 annotations of the TEG cells were from our previous analysis), with the only variable being s- or p-MSC. As such, each half of the analysis contains cells that could potentially interact (unlike when combining the cells from all experiments). We identified communication between every compartment of the model and also pathways within the same compartment. The MSC > MSC communication represented a prominent part of the complete list of potentially active channels (Figure 5A). Overall, in permissive conditions we detected higher number of different channels (Figure 5B).

[0165] We next compared the information flow (defined by the sum of communication probabilitis among all pairs of cell groups in our analysis), for specific signaling pathways in suppressive versus permissive conditions. Certain pathway groups, such as VISFANTIN and NOTCH prominently increased their information flow when s-MSCs were present, while with p-MSCs, pathways like BAG, GALECTIN and MHC-I were more prominent (Figure 5C). To further dissect these findings, we categorized the channels into sending (outgoing, green) and receiving (incoming, blue) pathways and compared their heir prevalence across the three cell types under both conditions (Figure 5D, E). Both s- and p-MSCs were highly represented both as senders and as receivers, underscoring the importance of MSC > MSC signaling within our models. The lower halves if these plots show channels that were detected in permissive but not in suppressive conditions. The higher number of detected channels at both the outgoing and the incoming sides in the permissive conditions is mirrored again on the lower halves of Figure 5 D and E. The channels listed there are only detected on the permissive environments. The plasticity of CD8 TEGs that we described earlier in Figure 4F is now gaining an additional level of explanation as these cells did not only express higher number of ligand-receptor pairs compared to the CD4 TEGs, but also the number of their active channels were higher in the permissive environment (compared to the permissive). This groups of channels in Figure 5 D and E contained potential points of interventions, leading us to initiate validation and exploratory studies. However, first we followed up the COLLAGEN pathway, a channel that was highly represented in both conditions (Figure 5C), and is biologically relevant considering the MSCs and the extracellular matrix.

[0166] We analyzed the data according to the fibrillar and non-fibrillar classification of collagens, attributing per-cluster annotations to identify the primary sources of various collagen types (Figure 5F, as clusters form Figure 3D,E). Clusters showed high levels of multiple types of collagens which reflected on the associated GO-terms (es Supplemental Figure2), along with clusters 6 and 8. To draw comparative insights into the collagen production influenced by p- or s-MSCs under differing conditions, we combined the MSC profiles per each experiment (Figure 4G) and discerned that a fibrillar-type collagen signature (involving COL1A1 , COL1A2, COL3A1 , COL5A1) was more associated with s-MSCs, in contrast to the non-fibrillar types (COL4A1 , COL4A2, COL8A1) prevalent in the permissive conditions. This prompted us is the idea to investigate the COLLAGEN pathway through the lens of a receptor that was not present in the original cell-chat reference dataset but is described to bind to collagen; LAIR-1 [34-36], an immunoinhibitory receptor. Specifically, COL3A1 's link to adverse cancer prognosis potentially via LAIR-1 interaction garnered our attention.

[0167] Next, in a comparative analysis of LAIR-1 and COL3A1 expression across the entire dataset, grouping the cells per cell-type per experiment (Figure 5H) confirmed the MSCs as the primary source of collagen expression and both CD4 and CD8 TEGs as components that express the collagen-binding LAIR-1 receptors. We made several novel observations: firstly, not only TEGs, but also the cancer cells expressed LAIR-1 . Secondly, this expression was more prominent when the cancer cells were co-cultured with s-MSCs, compared to the p-MSC condition. Lastly, LAIR-1 was induced in TEGs when co-cultured with MSCs.

[0168] Example 8 - Interference with LAIR1 increases cancer killing in the presence of s-MSC

[0169] To validate that collagen structures in s-MSCs play a role in modulating anti-cancer CD8+ T cell functions via cell-cell interactions and thus play a role in the immunosuppression of T cell-mediated cancer killing, we investigated whether collagen binding by LAIR-1 impacts T cell function. LAIR-1 was higher in expression on CD8+ as compared to CD4+ TEGs (Figure 5I). Furthermore, we knocked out the protein disulphid isomeresase (P4HB), an enzyme responsible for collagen polymerization in cells, in both s-MSC and p-MSC using CRISPR Cas (MSCP4HB-KO). These s- MSCP4HB-KO were then seeded in 3D bone marrow niches containing RPMI-8226 and treated with CD4+ and CD8+ TEGs in 1 :1 ratio. Using S-MSCP4HB-KO instead of s-MSC resulted in significant increase in cancer killing by TEG cells p-MSC and p-MSCP4HB-KO did not affect cancer (Figure 6A). Migration capacity of TEGs in the 3D bone marrow niche was however not affected by S-MSCP4HB-KO (Figure 6B). To further investigate the mechanism how collagens produced by MSCs could modulate T cell function, we inhibited LAIR-1 , that binds to collagens by using a-LAIR- 1 blocking antibodies. We pretreated TEGs with either a-LAIR-1 or isotype control antibodies and used them as treatment in the 3D bone marrow niche. LAIR-1 blocking on TEGs rescued cancer killing only when s-MSC was present but not in the presence of p-MSC (Figure 6C), gain emphasizing the role of LAIR-1 in the context of s-MSC. Altogether our data demonstrate that collagen expression by s-MSCs play an important role in creating an immunosuppressive milieu in the malignant bone marrow impacting anti-cancer potential of cancer specific T cells.

[0170] Example 9 - LAIR1 is a key regulator of in vitro T cell migration in a collagen-rich 3D environment We first determined if LAIR1 affects T cell migration using in vitro live imaging of 3D matrigel cultures. We used mouse LAIR-1 -CD3 NFAT-GFP reporter cells to determine if matrigel, of which collagen is a core component, can ligate LAIR-1 and induce productive signalling, as measured by GFP production. 3D culture in matrigel activated LAIR1-CD3£-transfected reporter cells, but not wild type reporter cells in a concentration-dependent manner (Figure 7A) indicating that matrigel is a functional LAIR-1 ligand. We next embedded Lair1+,+and Lairt7OT-1 T cells in matrigel, and observed that Lairt1' T cells moved faster and moved over longer distances than Lair1+,+T cells. Although genetic ablation of LAIR1 in mice did not result in intrinsic changes in gene expression of naive splenic CD8+T cells (Figure 7D), we did observe higher C-C chemokine receptor type 5 (CCR5), but not CCR7, expression in Lairt1' OT-1 T cells than in Lair1+,+T cells after OVA peptide stimulation (Figure 7C). Together, this shows that LAIR-1 suppresses T cell migration in collagen- dense matrices

[0171] Example 10 - Phospho-proteomic profiling identifies enriched migration pathways in Lairl 'AOT1 T cells compared to Lairl+ / +in a collagen-rich 3D environment

[0172] To study the role of LAIR-1 in T cell migration we allowed Lair1+ / +and Lairt ' OT1 T cells to migrate through high density matrigel for 30 min and performed phospho-proteomics by mass spectrometry to comprehensively profile the signal transduction events downstream of LAIR-1 (Figure 8). This effort enabled the quantification of 7657 phospho-peptides (Figure 8B), where 2656 were exclusively present in La / rt / OT1 cells and 911 were exclusively present in Lair1+ / +OT1 T cells. Additionally, we mainly detected 1 phosphosite per protein with some proteins having more than one. In agreement with an inhibitory role for LAIR-1 , 61 peptides were upregulated in Lairt7compared to Lair1+ / +while only 2 peptides were upregulated in the Lair1+ / +. By performing a pathway enrichment analysis, we observed that Lairt7OT1 cells had enriched signalling cascades related with actin cytoskeleton and RHOA GTPases compared to Lair1+ / +cells, known to be involved in cellular migration. We also observed enrichment of pathways related with metabolic and transcription regulation. Interestingly, by analysing the top 50 differently upregulated phosphopeptides in Lairt7and LAIR1+ / +OT1 T cells we identified motifs with enriched residues that resembled the consensus sequence for TKL and CMGC target sites respectively (Figure 8D). A similar analysis on the top 50 unique phospho-peptides in Lair1+ / +and LAIR1+ / +suggest activation of different kinases related with transcription regulation and cell survival / cell death. Overall, these findings show that Lairl -deficient OT1 T cells exhibit enhanced activation of signalling pathways associated with migration compared to Lairl -expressing OT1 T cells when placed in a 3D collagen- rich matrix.

[0173] Example 11 - LAIR-1 impairs in vitro tumor cell killing by inhibiting T cell migration

[0174] Next, we investigated if LAIR1 impacts tumor cell killing in collagen-dense environments. We cultured GFP-expressing MC38-OVA adenocarcinoma cells in matrigel, added SIINFEKL peptide- stimulated Lair1+ / +or Lairt1' OT-1 T cells on top of the matrigel and assessed antigen-specific tumor cell killing by loss of GFP expression by microscopy. We found that at lower E:T ratios Lairt ' OT- 1 T cells killed MC38-OVA cells more efficiently compared to Lair1+ / +OT-1 cells (Figure 9A). To investigate whether this was due to enhanced migration through the collagen matrix, and not solely due to enhanced cytotoxicity, we also performed killing assays in the absence of matrigel or other LAIR-1 ligands. We observed no differences in MC38-OVA tumor cell killing between activated Lair1+,+and Lairt1' OT-1 T cells in 2D monolayer cultures in the absence of collagen-rich matrices (Figure 9B). Importantly, although MC38 tumor cells do express collagen, they did not induce potent LAIR-1 signalling in LAIR-1 reporter cells. In line with this, removal of MC38-produced collagen by collagenase treatment did not impact T cell-mediated tumor cell killing. Next, we determined cytotoxicity in a MC38-OVA spheroid model to which we added activated OT-1 T cells in suspension. Again, there was no major difference in tumor cell killing between Lair1+,+and Lairt1' T cells, although Lairt1' OT-1 T cells did show improved killing at the highest E:T ratio (Figure 9C). Similarly, we found in a 4-hour chromium release assay that Lairt1' T cells killed MC38-OVA cells in suspension to a higher extent than Lair1+,+T cells (Figure 9D). Lastly, we co-cultured OT-1 T cells and MC38-OVA cells in 2D, in the presence or absence of LAIR-1 agonistic antibodies and found that antibody-induced LAIR-1 signalling did not reduce the cytotoxic ability of Lair1+,+or Lairt

[0175] T cells (Figure 9E). These findings suggest that LAIR-1 -deficiency promotes tumor cell killing by increasing migration through matrigel, and that LAIR-1 can limit killing capacity independent of migration in some MC38-OVA models, in line with previous findings.

[0176] Example 12 - LAIR-1 -deficient T cells migrate more efficiently towards the tumor site in vivo Next, we investigated whether LAIR-1 not only impacts cell movement within the tumor microenvironment, but also impacts migration towards tumor cells in vivo. To test this, we adoptively transferred SIINFEKL peptide-stimulated Lair1+,+CD45.1 and Lairt1' OT-1 CD45.2 T cells into MC38-OVA tumor-bearing CD45.1.2 recipient mice at a 1 :1 ratio and determined T cell infiltration in the resected MC38-OVA tumor after 12 hours. Using the CD45.1 and CD45.2 congenic markers, we detected significantly more infiltrating Lairt1' OT1 T cells in the tumor compared to Lair1+,+OT1 T cells, while we observed fewer Lairt1' T cells in the spleen compared to Lair1+,+(Figure 10A). Next, we transferred either single Lair1+,+or Lairt1' OT-1 T cells into separate MC38-OVA tumorbearing recipient mice and determined tumor growth over time. While control mice were not capable of controlling tumor outgrowth, 3 / 6 mice injected with either Lair1+,+or Lairt1' OT-1 cells cleared smaller tumors equally well (Figure 10B-C). Thus, although LAIR-1 limited migration of T cells towards the tumor site in vivo, in this experimental setting it did not affect tumor cell clearance. Example 13 - LAIR-1 blockade or deficiency in genetically engineered T cells leads to increased migration and killing of tumor cells in a 3D model

[0177] We have previously demonstrated that human apT cells engineered to express a defined yQTCR, so-called TEGs, show strong antitumor reactivity. However, migration and infiltration of these cells into tumor nests remain a problem in solid tumors. Both CD4 and CD8 in vitro expanded TEG highly express LAIR-1 . Here we tested if we could improve TEG migration towards tumors by blocking or depleting LAIR-1 in an in vitro 3D collagen rich tumor model. Interestingly, when imaging matrigel for the presence of TEGs cells by confocal microscopy we observed that a large fraction of Lair1+ / +TEGs remained at the surface of the gel compared to Lair1 / _TEGs which were able to infiltrate. Additionally, we observed that LAIR / _TEGs in migrated faster and for longer distances when embedded in matrigel compared to LAIR+ / +TEGs (Figure 11). Blocking LAIR-1 antibodies enhanced TEG killing and migration of HT29 cells compared to isotype control antibodies. A similar effect was observed when using Lair1' TEGs generated by CRISPR (Figure 11 B and 11 C). For all experiments a 1 :1 mix of CD4 and CD8 TEGs was used. However, CD4 TEGs migrated more efficiently than CD8 TEGs (Figure 11 D). We next questioned whether LAIR-1 on CD4+ or CD8+ cells was essential to suppress migration and tumor cell killing. As we reported previously, CD4 T help was required for CD8 T cell migration independently of LAIR1 expression (Figure 11 J). However, the increased killing capacity of LAIR1 deficient-CD8 TEGs was not influenced by the expression of LAIR1 on CD4 T cells (Figure 11). Overall, these data show that LAIR-1 blockade or genetic deletion in TEGs leads to increased T cell migration and killing of HT29 tumor cells in a 3D model.

[0178] Example 14 - LAIR-1 mediated inhibition of tumor killing is dependent on tumor-expressed collagen, while migration is suppressed by ECM collagens

[0179] HT29 cells express a high level of transmembrane collagen, which is capable of binding LAIR-1. In order to distinguish the differential contribution of tumoral expressed collagen versus ECM collagen we generated a prolyl 4-hydroxylase (P4HB) HT29 deficient tumor line. P4H enzymes are essential for the stability of the collagen triple helix and deficiency leads to reduced collagen expression. Indeed, P4HB HT29 deficient cells lost the capacity to bind LAIR-1. In 3D cultures, HT29 cells deficient in P4HB did not result in differences in TEG migration (Figure 12B), but did result in higher tumor cell killing (Figure 12A). In contrast, genetic deletion of LAIR-1 resulted in both enhanced T cell migration and tumor cell killing, which was not further enhanced by P4HB deficiency in the tumor cells. These data suggests that tumoral collagen directly mediates LAIR-1 mediated inhibition of killing, while inhibition of migration is suppressed by ECM collagens. Indeed, the direct impact of tumoral collagen on tumor cell killing was confirmed by our finding that HT29 cells directly inhibited TEG tumor killing in 2D cultures, which was abrogated by genetic deletion of P4HB in HT29 cells or LAIR-1 in T cells respectively. Thus, blocking LAIR-1 simultaneously release the inhibitory effect on T cell migration and T cell cytotoxicity by collagens expressed by tumor stroma as well as tumor cells respectively.

[0180] Example 15 - Effects of LAIR-1 on early T-cell infiltration into a collagen matrix. Either wild type or LAIR1 KO TEG001 cells were separated to CD4+ and CD8+ fractions and labelled separately with either Calcein AM or CellTrace Violet viability dyes (ThermoFischer). T cells were then placed on top of a BME matrix consisting of preconditioned media using HT29 cell line and imaged for 6.5h. The cell coordinates per timepoint are extracted, and the cell positions at t=0 are used to infer the topology of the BME surface. The cell depth distributions over time are used to determine the rate and speed of cell infiltration (Figure 13B-E).

[0181] Example 16 - CD4 and CD8 TEGs both benefit from LAIR1 KO, using a malignant bone marrow niche.

[0182] TEG001 -specific killing of RPMI-8226 and TEG001 infiltration into bottom compartment of 3D model containing RPMI-8226 and s-MSC donor 06 on day 6 after the LAIR1 gene was knocked out in either TEG-001 CD4+, CD8+ or both (Figure 14). Total amount of migrated CD4+ and CD8+ TEG001 knocked out for LAIR1 in the bottom compartment of the trans-well system of an in vitro 3D RPMI-8226 model made with s-MSC donor 06.

Claims

Claims1 . An immune cell expressing a receptor recognizing a target antigen, wherein the expression and / or function of LAIR-1 has been reduced or eliminated.

2. The immune cell from claim 1 , wherein the immune cell is selected from the group of an isolated non-modified immune cell, an engineered immune cell or an immune modulating cell.

3. The immune cell from claim 1 or 2, wherein the immune cell presents enhanced migration and / or tumor cell killing.

4. The immune cell of any of claims 1 to 3, wherein the immune cell comprises or has been in contact with an inhibitor of LAIR-1 that is either a blocking antibody or a nucleic acid sequence capable of downregulating or eliminating gene expression or modifying the function of LAIR-1 , wherein the nucleic acid capable of downregulating the gene expression of LAIR-1 is selected from the group consisting of an antisense RNA, antagomir RNA, siRNA, shRNA, a CRISPR system, a zinc finger nuclease system, a transcription activatorlike effector based nuclease (TALEN) system, or interfering with binding or function through any base editing technique .

5. The immune cell of any of claims 1 to 4, wherein the immune cell is selected from the group of a T cell, from its subtypes ap T cell or y6 T cell, or a NK cell.

6. A method of manufacturing the immune cell of any of claims 1 to 5, the method comprising contacting an immune cell extracted from a patient suffering from a tumor with an inhibitor of LAIR-1 for a sufficient amount of time and under suitable conditions to reduce or eliminate the expression and / or function of LAIR-1.

7. The method of claim 6, wherein the inhibitor of LAIR-1 is selected from the group of a nucleic acid sequence capable of downregulating gene expression of LAIR-1 , a CRISPR system or another gene or base editing system interfering with function or expression.

8. The method of claim 6 or 7, wherein the method comprises the ex vivo or in vivo modification of a T cell or NK cell extracted from a patient suffering from a tumor, wherein the tumor microenvironment of said tumor is characterized by an immunosuppressive and collagen-rich environment and / or increased presence of immunosuppressive mesenchymal stem cells (MSC).

9. An immune cell isolated from a patient suffering from a disease associated with expression of a tumor antigen, wherein the expression and / or function of LAIR-1 in said immune cell has been reduced or eliminated ex vivo.

10. An engineered immune cell characterized by a T cell expressing a chimeric antigen receptor (CAR) recognizing a cancer-associated target antigen, wherein said engineered T cell has been modified as such that the expression and / or function of LAIR-1 has been reduced or eliminated.11 . An ap T cell engineered to express a defined yQTCR (TEGs), or a dual targeting TEG, wherein the TEG cell presents enhanced migration and tumor cell killing and comprises an intracellular signaling domain or fragment that is functional in the absence of LAIR-1 , and wherein said engineered T cell has been modified as such that the expression and / or function of LAIR-1 has been reduced or eliminated.

12. A method of treating a subject in need thereof, the method comprising administering an immune cell according to any of claims 1 to 5 for a sufficient amount of time and under suitable conditions to treat the subject.

13. The method of claim 12, wherein the subject has a tumor, wherein the tumor microenvironment of said tumor is characterized by an immunosuppressive collagen-rich environment and / or increased presence of immunosuppressive mesenchymal stem cells (MSC).

14. An immune cell according to any one of claims 1 to 5 for use in treating a subject in need thereof.

15. Use of an immune cell according to any one of claims 1 to 5 in the manufacture of a medicament for treating a subject in need.

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