Engineering immune cells to migrate to, infiltrate, persist, and expand in solid tumors

Abstract

The present disclosure provides engineered immune cells modified to overexpress tumor sensing enhancer proteins, thereby providing the engineered immune cells with an enhanced ability to migrate to and / or infiltrate tumors. In some examples, the tumor sensing enhancer proteins are tumor sensing receptors that specifically bind to non-chemokine receptors. The disclosure also provides methods including the provided engineered immune cells.

Description

PATENT Attorney Docket No.079445-1483820-014510PC Client Ref. No. S24-049 ENGINEERING IMMUNE CELLS TO MIGRATE TO, INFILTRATE, PERSIST, AND EXPAND IN SOLID TUMORS CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No. 63 / 631,049 filed April 8, 2024, the full disclosure of which is incorporated by reference in its entirety for all purposes. BACKGROUND

[0002] Breast cancer (BC) is the leading cause of cancer death in women worldwide with an estimated 2.3 million new cases and more than 685,000 deaths reported in 2020. Currently available BC treatment options involve a combination of surgery, radiotherapy, chemotherapy, endocrine, and targeted therapies, depending on disease subtype, hormone receptor status, genetic features, and other considerations. In the realm of immunotherapies, PD1 immune checkpoint blockade (ICB) is effective in a subset of BC patients, particularly those with relatively high levels of tumor infiltrating lymphocytes and Triple Negative BC (TNBC). However, even in this pre-selected patient population, ICB leads to only 6% improvement in overall survival at 18 months. In addition to ICB, an increasing number of clinical trials are evaluating BC vaccines, bispecific antibodies, oncolytic viruses, and adoptive cell therapies, including tumor infiltrating lymphocytes, T cell receptor (TCR) engineering, and different types of chimeric antigen receptor (CAR) T cells.

[0003] Alongside progress in T cell-based therapies, Natural Killer (NK) cells play an important role in immune surveillance during tumor initiation and metastatic progression. NK cells can recognize cancer cells in an antigen-independent manner through stress-induced proteins and loss of MHC class I, thus targeting malignant cells that are often missed by T cells. NK cells can also recognize cancer cells in an antigen-dependent manner via antibody dependent cellular cytotoxicity, thus synergizing with humoral immune responses and targeted therapies that are based on monoclonal antibodies. Clinical studies are under way to evaluate 79556009V.1NK cell-based therapies, including infusion of NK cell lines, expanded memory-like NK cells, and CAR NK cells.

[0004] Despite this progress and extensive research, key barriers remain. First, anti-tumor immune responses depend on the ability of effector T / NK cells and other immune cells to migrate to and infiltrate the tumor. Lack of response to ICB and the limited success of cell therapies in BC and other solid tumors has been attributed to low levels of tumor infiltrating lymphocytes. Second, while CAR T cells have been very effective in blood cancers (e.g., where most or all B cells are eliminated due to CD19 expression), BC associated antigens show variable expression in the cancer cells and are also expressed by other cell types. Third, antigen- dependent strategies are limited in the treatment of tumors with a low mutational load or inactivation of antigen presentation. These challenges underscore the need to identify new ways for effective immune “cell delivery” and the need for new mechanisms to unleash diverse anti- tumor immune responses. The present disclosure addresses these needs and provides associated and other advantages. BRIEF SUMMARY

[0005] This summary provides a high-level overview of various aspects of the disclosure and introduces some of the concepts that are described and illustrated in the present document and the accompanying figures. The summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used in isolation to determine the scope of the claimed subject matter. Covered embodiments of the disclosure are defined by the claims, not this summary. The subject matter should be understood by reference to appropriate portions of the entire specification, any or all figures, and each claim. Some of the exemplary embodiments of the present disclosure are discussed below.

[0006] In one aspect, the disclosure provides an engineered immune cell genetically modified to overexpress a tumor sensing enhancer protein. The tumor sensing enhancer protein includes a tumor sensing receptor that binds to a non-chemokine ligand, a tumor sensing protease, a tumor sensing transcription factor, a tumor sensing protein phosphatase, a tumor sensing ion channel protein, or a subunit or fragment thereof. The engineered immune cell exhibits increased migration to or infiltration of a tumor as compared to migration to or infiltration of the tumor by a corresponding immune cell not overexpressing the tumor sensing enhancer protein. 79556009V.1

[0007] In another aspect, the disclosure provides a method for producing an engineered immune cell. The method includes providing an immune cell. The method further includes introducing into the immune cell a polynucleotide encoding a tumor sensing enhancer protein, thereby producing the engineered immune cell. The tumor sensing enhancer protein includes a tumor sensing receptor that binds to a non-chemokine ligand, a tumor sensing protease, a tumor sensing transcription factor, a tumor sensing protein phosphatase, a tumor sensing ion channel protein, or a subunit or fragment thereof. The engineered immune cell exhibits increased migration to or infiltration of a tumor as compared to migration to or infiltration of the tumor by a corresponding immune cell not overexpressing the tumor sensing enhancer protein.

[0008] In another aspect, the disclosure provides another method for producing an engineered immune cell. The method includes providing an immune cell that includes a gene encoding a tumor sensing enhancer protein. The method further includes introducing into the immune cell a guide RNA targeting the gene, thereby producing the engineered immune cell. The tumor sensing enhancer protein includes a tumor sensing receptor that binds to a non- chemokine ligand, a tumor sensing protease, a tumor sensing transcription factor, a tumor sensing protein phosphatase, a tumor sensing ion channel protein, or a subunit or fragment thereof. The engineered immune cell exhibits increased migration to or infiltration of a tumor as compared to migration to or infiltration of the tumor by a corresponding immune cell not overexpressing the tumor sensing enhancer protein.

[0009] In another aspect, the disclosure provides a method for treating a cancer in a subject. The method includes engineering an immune cell of the subject to produce an engineered immune cell that overexpresses a tumor sensing enhancer protein, The tumor sensing enhancer protein includes a tumor sensing receptor that binds to a non-chemokine ligand, a tumor sensing protease, a tumor sensing transcription factor, a tumor sensing protein phosphatase, a tumor sensing ion channel protein, or a subunit or fragment thereof. The engineered immune cell exhibits increased migration to or infiltration of a tumor of the subject as compared to migration to or infiltration of the tumor by a corresponding immune cell not overexpressing the tumor sensing enhancer protein.

[0010] In another aspect, the disclosure provides another method for treating a cancer in a subject. The method includes administering to the subject a therapeutically effective amount of a population of any of the engineered immune cells disclosed herein. 79556009V.1

[0011] In another aspect, the disclosure provides a method for determining a personalized treatment for a cancer in a subject, the method includes administering to the subject two or more populations of engineered immune cells. For each population of the two or more populations, the engineered immune cells of the population are genetically engineered to overexpress a combination of one or more tumor sensing enhancer proteins. Each of the one or more tumor sensing enhancer proteins independently comprises a tumor sensing receptor that binds to a non-chemokine ligand, a tumor sensing protease, a tumor sensing transcription factor, a tumor sensing protein phosphatase, a tumor sensing ion channel protein, or a subunit or fragment thereof. The combination overexpressed by the engineered immune cells of each population is different from the combination overexpressed by the engineered immune cells of each other population. The method further includes analyzing a biological sample obtained from the subject subsequent to the administering. The method further includes determining, based on the analyzing, which one or more populations of the two or more populations of engineered immune cells exhibits a level of increased migration to, or infiltration of, a tumor of the subject, where the level is greater than a threshold. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG.1 presents a schematic illustration of an immune cell engineered to express one or more tumor sensing receptors (TSRs) in accordance with a provided embodiment.

[0013] FIG. 2 presents a schematic illustration of an in vivo CRISPR activation screen in NK-92 cells used to identify TSRs.

[0014] FIG. 3 presents a graph plotting results from the screen of FIG.2 as the significance (y-axis) and log-transformed fold-change (x-axis) of each target gene (dot) in the tumor compared to lung samples based on MAGeCK. There were no target genes showing a significant depletion in the tumor compared to lung samples.

[0015] FIG. 4 presents a graph (top) plotting robust rank aggregation (RRA) scores of all target genes matching the analysis shown in FIG. 3. FIG. 4 also presents the log-transformed fold change (bottom) of sgRNAs in tumor versus lung samples shown for sgRNAs targeting indicated genes.

[0016] FIG. 5 presents a graph plotting differential expression of top hits in tumor versus blood samples in breast cancer patients based on scRNA-Seq data. 79556009V.1

[0017] FIG. 6 presents schematic illustration of PRESTO-tango reporter cell lines where GPCR conformational changes in response to a ligand result in luciferase transcription for bioluminescence readouts.

[0018] FIG.7 presents a series of graphs with data showing that PRESTO-tango reporter cell lines generated for CXCR2, C5AR1, GPR34, GPR84, and GPR183 confirm IL8, C5a, LysoPS,6-OAU, and 7 ,25-OHC as the respective GPCR agonists (n = 3). One-way analysis of variance(ANOVA) was performed with Dunnett’s multiple comparisons< 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05).

[0019] FIG.8 presents a series of graphs with data showing GPCR-driven migration of NK- 92 cells to the respective ligand (n = 4 or 5). Data are representative of two independent experiments and presented as the mean ± s.e.m. One-way analysis of variance (ANOVA) was performed with Dunnett’s multiple comparisons< 0.0001).

[0020] FIG. 9 presents a schematic illustration of an experimental scheme for in vitro CRISPR activation screens in NK-92 cells.

[0021] FIG. 10 presents a graph plotting results from the in vitro chemotaxis screen of FIG. 9 as the significance (y-axis) and log-transformed fold-change (x-axis) of each target gene (dot) in the screen. Positive and negative values denote enrichment and depletion, respectively, in the NK cell population that migrated to the breast cancer supernatant. NS: Not significant.

[0022] FIG. 11 presents a graph plotting results from the breast cancer spheroids screen of FIG.9 as the significance (y-axis) and log-transformed fold-change (x-axis) of each target gene (dot) in the screen. Positive and negative values denote enrichment and depletion, respectively, in the NK cell population that populated the breast cancer spheroids. NS: Not significant.

[0023] FIG. 12 presents schematic illustrations of a perturb-seq workflow and UMAP of perturb-seq data, where each dot corresponds to an NK-92 cell colored by the sgRNA target gene.

[0024] FIG. 13 presents a graph plotting gene set enrichment analysis data for genes significantly differentially expressed in GPR183A+vs. NK-92 cells with NTC sgRNAs (i.e., control NK cells).

[0025] FIG.14 presents a heatmap of GPR183 differentially expressed genes in NK-92 cells: Each column corresponds to an NK-92 cell with either GPR183 or NTC sgRNAs, as indicated 79556009V.1by the topmost horizontal bar; each row corresponds to the topmost differentially expressed genes in the GPR183A+vs. controls NK-92 cells.

[0026] FIG. 15 presents bar plots showing data for cytotoxicity (left) and IFN secretion(right) in GPR183A+cells vs. control NK-92 cells (n = 3). Cytotoxicity to MDA-MB-231 cells was evaluated after 48 hours of coculture with the NK-92 cells, at varying effector-to-target(E:T). IFN secretion was evaluated at 1:1 E:T. Data are representative of two independentexperiments and presented as the mean ± s.e.m. Two-tailed unpaired Students’ t-test (***P < 0.001).

[0027] FIG. 16 presents bar plots depicting flow cytometry measures of IFN proteinexpression and CD107a levels on the NK-92 cell surface obtained upon stimulation across biological replicates (n = 3). Data are representative of two independent experiments and presented as the mean ± s.e.m. Two-tailed unpaired Students’ t-test< 0.0001).

[0028] FIG. 17 presents a series of graphs plotting flow cytometry measures of IFN proteinexpression and CD107a levels on the NK-92 cell surface in GPR183A+vs. control NK cells at baseline and upon stimulation with PMA / ionomycin.

[0029] FIG. 18 presents graphs with data quantifying proliferation of GPR183A+cells and control NK-92 cells via decay of cell-trace dye. Left, histogram plot. Right, relative mean fluorescence intensity (MFI) quantification (n = 3).

[0030] FIG.19 presents a bar plot with data demonstrating that GPR183A+NK-92 cells show a significant increase in migration to MDA-MB-231 cell conditioned media compared to control NK-92 cells based on transwell migration assay with flow cytometry cell count readouts (n = 10 for NTC and n = 13 for GPR183A+). MDA CM: MDA-MB-231 conditioned media. Three independent experiments were pooled. Data are presented as the mean ± s.e.m. Data analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons.

[0031] FIG. 20 presents a graph with data demonstrating that GPR183A+NK-92 cells show a significant increase in migration to tumor lysates compared to control NK-92 cells in competitive transwell migration assays with flow cytometry cell count readouts (n = 4 pergroup). 100 nM 7 ,25-OHC was used as a positive control in competitive migration assay.Data are representative of two independent experiments. Data analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons. 79556009V.1

[0032] FIG.21 presents a graph with data indicating overexpression of GPR183 on primary human NK cells via ORF.

[0033] FIG.22 presents a bar plot with data demonstrating that GPR183A+primary NK cellsshow a significant increase in migration to 7 ,25-OHC compared to control primary NK cellsbased on transwell migration assay with flow cytometry cell count readouts (n = 3 per group). Data are presented as the mean ± s.e.m. Data analysis was performed using one-way ANOVA with two-tailed unpaired Students’ t-test0.0001).

[0034] FIG.23 presents a graph with data indicating overexpression of GPR183 on primary human CD8 T cells via ORF.

[0035] FIG. 24 presents a graph with data demonstrating that GPR183A+primary T cellsshow a significant increase in migration to 7 ,25-OHC based on transwell migration with flowcytometry cell count readouts (n = 6 per group). Data are representative of two independent experiments. Data are presented as the mean ± s.e.m. Data analysis was performed using one- way ANOVA with Dunnett’s multiple comparisons0.0001).

[0036] FIG. 25 presents a graph with data demonstrating that GPR183A+primary T cells show a significant increase in migration to MDA-MB-231 cell conditioned media based on competitive transwell migration assays with flow cytometry cell count readouts (n = 5 per group). Data are representative of two independent experiments. Data analysis was performed using one-way ANOVA with two-tailed unpaired Students’ t-test (****P < 0.0001).

[0037] FIG. 26 presents a graph with data demonstrating that GPR183A+primary T cells show a significant increase in migration to tumor lysates based on competitive transwellmigration assays with flow cytometry cell count readouts (n = 5 per group). 100 nM 7 ,25-OHC was used as a positive control in competitive migration assay. Data are representative of two independent experiments. Data analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons.

[0038] FIG.27 presents graphs with data demonstrating that GPR183A+NK-92 cells show a significant increase in infiltration to breast cancer tumors in vivo (n = 5 per group), as seen based on representative flow cytometry plots (left), percentage (middle), and number of NK cells in the tumor (right). Data are presented as the mean ± s.e.m. Data analysis was performed using one-way ANOVA with two-tailed unpaired Students’ t-test.

[0039] FIG.28 presents schematic illustration of a design for a disease outcome study. 79556009V.1

[0040] FIG. 29 presents graphs plotting growth curve data from the study of FIG. 28 for MDA-MD-231 breast cancer tumors in NSG mice treated with cell-free media (n = 3 mice), control NK-92 (n = 6 mice) or GPR183A+(n = 6 mice) NK-92 cells. Data are shown as the mean ± s.e.m. (upper left) and individual growth curves per mouse; statistical significance was evaluated using a two-way ANOVA with Sidak correction for multiple hypotheses testing (****P < 0.0001).

[0041] FIG.30 presents a schematic illustration of an EpCAM CAR construct.

[0042] FIG. 31 presents graphs plotting growth curve data from the study of FIG. 28 for MDA-MD-231 breast cancer tumors in NSG mice treated with cell-free media (n = 3 mice), anti-EpCAM-CAR-NK-92 (n = 6 mice), or GPR183A+- anti-EpCAM-CAR-NK-92 (n = 6 mice). Data are shown as the mean ± s.e.m. (upper left) and individual growth curves per mouse; statistical significance was evaluated using a two-way ANOVA with Sidak correction for multiple hypotheses testing (****P < 0.0001).

[0043] FIG.32 presents a schematic illustration of G-protein coupled receptors in the surface of an engineered NK cell.

[0044] FIG. 33 presents a graph plotting RRA scores of target genes matching selection criteria in a CRISPR activation screen for infiltration to ovarian cancer tumors. DETAILED DESCRIPTION I. INTRODUCTION

[0045] The present disclosure generally relates to engineered immune cells (e.g., engineered T and Natural Killer (NK) cells) that are designed to express “tumor sensing enhancer proteins” (e.g., “tumor sensing receptors”) providing the engineered immune cells with an enhanced ability to migrate to, infiltrate, and target tumors (e.g., breast cancer (BC) tumors or other solid cancer tumors). When the tumor sensing enhancer proteins of the engineered immune cells are tumor sensing receptors, these receptors are proteins that surprisingly enhance the tumor sensing characteristics of the immune cells by responding to metabolites and other non- chemokine ligands. This mechanism is substantially different from that of chemokine receptors and integrins previously recognized as central regulators of T and NK (T / NK) cell migration to tumors, and used in comparative engineering strategies. Because the tumor sensing receptors described herein respond to a different set of bioactive molecules, including lipids, fatty acids, 79556009V.1and cholesterol derivatives, the materials and methods provided by the present disclosure significantly diverge from those of existing approaches, and advantageously offer a new mechanism to recruit engineered immune cells to the tumor.

[0046] As described in the Examples provided herein, CRISPR activation screens have been used to identify tumor sensing enhancer proteins and to demonstrate that their activation in NK cells significantly and repeatedly increases (> 8-fold) NK cell migration to BC. Results have shown, for example, that tumor sensing enhancer proteins specifically mark BC infiltrating immune cells in patients, and are mostly expressed by myeloid cells. Without being bound by a particular theory, it is therefore believed that tumor sensing enhancer proteins form an endogenous recruitment mechanism that is primarily most dominant in other (non-T / NK) immune cells but can be (epi)genetically or transiently activated in T / NK cells to advantageously elicit T / NK recruitment, infiltration, and expansion in the tumor. II. DEFINITIONS

[0047] Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs. In addition, any method or material similar or equivalent to a method or material described herein can be used in the practice of the present disclosure. For purposes of the present disclosure, the following terms are defined.

[0048] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a phospholipid” optionally includes a combination of two or more phospholipids, and the like.

[0049] As used herein, the terms “about” and “approximately,” when used to modify an amount specified in a numeric value or range, indicate that the numeric value as well as reasonable deviations from the value known to the skilled person in the art, for example ± 20%, ± 10%, or ± 5%, are within the intended meaning of the recited value.

[0050] As used herein, the term “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0051] As used herein, the terms “including,” “comprising,” “having,” “containing,” and variations thereof, are inclusive and open-ended and do not exclude additional, unrecited elements or method steps beyond those explicitly recited. As used herein, the phrase 79556009V.1“consisting of” is closed and excludes any element, step, or ingredient not explicitly specified. As used herein, the phrase “consisting essentially of” limits the scope of the described feature to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the disclosed feature.

[0052] The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0053] The terms “first,” “second,” “third,” and the like when used herein with reference to elements or properties, are simply to more clearly distinguish or identify multiple elements or properties, and are not intended to indicate an order or other serial or numerical limitation, or to require that each of the multiple elements or properties are present.

[0054] As used herein, the term “tumor sensing enhancer protein” refers to a protein, or a subunit or a fragment thereof, that, when expressed by a cell, enhances the ability of the cell to migrate to or infiltrate a tumor, e.g., a tumor cell of a tumor. Relatedly, the term “tumor sensing,” when used as a modifier in relation to a protein, indicates that the protein enhances the ability of a host cell to migrate to or infiltrate a tumor when the protein is expressed by the host cell. For example, a “tumor sensing receptor” is a receptor of a cell, where the receptor has a specific binding affinity for a ligand, and where binding of the ligand by the receptor enhances the ability of the cell to migrate to or infiltrate the tumor.

[0055] As used herein, the terms “protein, “peptide,” and “polypeptide” refer to polymers comprised of covalently linked natural or chemically modified amino acid residues.

[0056] As used herein, the terms “polynucleotide” and “nucleic acid molecule” refer to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. A nucleic acid sequence may comprise combinations of deoxyribonucleic acids and ribonucleic acids. Such deoxyribonucleic acids and ribonucleic acids include both naturally occurring molecules and synthetic analogs. The polynucleotides of the disclosure also encompass all forms of sequences including, but not limited to, single-stranded forms, double- stranded forms, hairpins, stem-and-loop structures, circular structures, and the like. 79556009V.1

[0057] As used herein, the term “variant,” when used in the context of polypeptides described herein, refers to polypeptides having a high degree of structural similarity to one another, with structural differences resulting from differences in polynucleotides encoding the polypeptide variants. Polypeptide variants may have amino acid sequences that are at least 80% similar to one another (% identity), e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similar to one another (% identity). Polypeptide variants may have the same biological functions as one another. For example, where polypeptide variants are enzymes, the polypeptide variants may each catalyze the same reaction. Alternatively, a variant of an original polypeptide may be specifically configured or selected to lack the biological activity or function of the original polypeptide.

[0058] As used herein, the term “recombinant” when used with reference to a protein refers to a protein prepared via genetic engineering. A recombinant cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, e.g., recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. A recombinant nucleic acid is a nucleic acid originally formed in vitro, in general, by the manipulation of nucleic acid, e.g., using polymerases and endonucleases, in a form not normally found in nature. A recombinant protein is made using recombinant techniques such as through the expression of a recombinant nucleic acid or DNA molecule as depicted above.

[0059] As used herein, the term “introducing,” in the context of a polynucleotide described herein, refers to presenting a nucleic acid sequence to a host cell in such a manner that the sequence gains access to the interior of the cell. Methods for introducing nucleic acid sequences into cells are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods. “Stable transformation” is intended to mean that the nucleotide construct introduced into a host cell integrates into the genome of the host cell and / or is capable of being inherited by the progeny of the transformed host cell. “Transient transformation” is intended to mean that a polynucleotide is introduced into the host cell and does not integrate into the genome and / or is not inherited by the progeny of the transformed host cell. 79556009V.1

[0060] As used herein, the term “gene” refers to a segment of DNA encoding a polypeptide chain. A gene may include regions preceding (i.e., leader regions) and following (i.e., trailer regions) a coding region, as well as intervening sequences (i.e., introns) between individual coding segments (i.e., exons).

[0061] As used herein, the term “metabolite” refers to a chemical entity produced by one or more enzymatic or non-enzymatic reactions as a result of exposure of an organism to a chemical substance.

[0062] As used herein, the term “fatty acid” refers to a carboxylic acid having an aliphatic tail, typically from 4 to 30 carbon atoms long. Fatty acids can be saturated, mono-unsaturated, or poly-unsaturated. Fatty acids include branched fatty acids such as iso-fatty acids.

[0063] As used herein, the term “lipid” refers to a chemical entity having a hydrophilic moiety covalently attached to one or more hydrophobic moieties. Lipid molecules can include, for example, fats, waxes, steroids, cholesterol, fat-soluble vitamins, monoglycerides, diglycerides, phospholipids, sphingolipids, glycolipids, cationic or anionic lipids, derivatized lipids, and the like.

[0064] As used herein, the term “NK cell” refers to any natural killer cell, including natural killer cells of various types, subtypes, lineages and sources. In one aspect, NK cells includeany non-B-cell and non-T-cell cytotoxic lymphocyte. NK cells encompass any CD56+, CD3immune cells, including CD56brightNK cells, CD56dimNK cells, tissue resident NK cells, and adaptive NK cells. In some embodiments, CD56brightNK cells include NK cells expressing any of CCR7, CSF2, CXCR3, IL2RB, KLRC1, and / or SELL. In some embodiments, CD56dimNK cells include cells expressing any of CX3CR1, CXCR1, ITGB2, KLGR1, and / or PRF1.

[0065] As used herein, the terms “treat,” “treating,” and “treatment” refer to a procedure resulting in any indicia of success in the elimination or amelioration of an injury, pathology, condition, or symptom (e.g., pain), including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the symptom, injury, pathology or condition more tolerable to the patient; decreasing the frequency or duration of the symptom or condition; or, in some situations, preventing the onset of one or more symptoms. The treatment or amelioration of symptoms can be based on any objective or subjective parameter; including, e.g., the result of a physical examination or laboratory test. 79556009V.1

[0066] As used herein, the term “subject” refers to a vertebrate, and preferably to a mammal. Mammalian subjects for which the provided composition is suitable include, but are not limited to, mice, rats, simians, humans, farm animals, sport animals, and pets. In some embodiments, the subject is human. In some embodiments, the subject is male. In some embodiments, the subject is female. In some embodiments, the subject is an adult. In some embodiments, the subject is an adolescent. In some embodiments, the subject is a child. In some embodiments, the subject is above 10 years of age, e.g., above 20 years of age, above 30 years of age, above 40 years of age, above 50 years of age, above 60 years of age, above 70 years of age, or above 80 years of age. In some embodiments, the subject is less than 80 years of age, e.g., less than 70 years of age, less than 60 years of age, less than 50 years of age, less than 40 years of age, less than 30 years of age, less than 20 years of age, or less than 10 years of age.

[0067] As used herein, the term “administering” refers to oral administration, administration as a suppository, topical contact, parenteral, intravenous, intraperitoneal, intramuscular, intralesional, intranasal or subcutaneous administration, intrathecal administration, or the implantation of a slow-release device e.g., a mini-osmotic pump, to the subject.

[0068] As used herein, the term “therapeutically effective amount” refers to an amount or dose that produces therapeutic effects for which it is administered. The exact amount or dose will depend on the purpose of the treatment, and will be readily ascertainable by one skilled in the art using known techniques.

[0069] As used herein, the term “biological sample” refers to any sample that is taken from a subject, e.g., a human or other animal. The biological sample can include or consist of a bodily fluid, such as blood, plasma, serum, urine, vaginal fluid, fluid from a hydrocele (e.g., of the testis), vaginal flushing fluids, pleural fluid, ascitic fluid, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchoalveolar lavage fluid, discharge fluid from the nipple, aspiration fluid from different parts of the body (e.g., thyroid, breast), intraocular fluids (e.g., the aqueous humor), amniotic fluid, etc. Stool samples can also be used as biological samples.

[0070] As used herein, the terms “threshold” and “cutoff” refer to a number, e.g., a predetermined number, used in an operation for classifying or categorizing a result. For example, a threshold value may be a value above or below which a particular classification applies. A cutoff or threshold may be “a reference value” or derived from a reference value that is representative of a particular classification or discriminates between two or more classifications. A cutoff may be predetermined with or without reference to the characteristics 79556009V.1of the sample or the subject. For example, cutoffs may be chosen based on the age or sex of the tested subject. A cutoff may be chosen after and based on output of the test data. As another example, reference subjects with known classifications of one or more conditions and measured characteristic values can be used to determine reference levels to discriminate between the different conditions and / or classifications of a condition (e.g., whether the subject has the condition). A reference value can be selected as representative of one classification (e.g., a mean) or a value that is between two clusters of the metrics (e.g., chosen to obtain a desired sensitivity and specificity). As another example, a reference value can be determined based on statistical simulations of samples. A particular value for a cutoff, threshold, reference, etc. can be determined based on a desired accuracy (e.g., a sensitivity and specificity). III. ENGINEERED IMMUNE CELLS

[0071] In one aspect, the present disclosure provides various engineered immune cells. The immune cells are generally modified to overexpress one or more tumor sensing enhancer proteins. The overexpression of these particular proteins provides the engineered immune cells with surprising improvements in their ability to migrate to or infiltrate tumors in a subject. For example, the engineered immune cells can exhibit an increased migration to a tumor that is at least about 1.3-fold greater than, at least about 2-fold greater than, at least about 3-fold greater than, at least about 5-fold greater than, at least about 8-fold greater than, at least about 15-fold greater than or at least about 30-fold greater than the migration to the tumor by a corresponding immune cell not overexpressing the one or more tumor sensing enhancer proteins. Additionally or alternatively, the engineered immune cells can exhibit an increased infiltration of a tumor that is at least about 1.3-fold greater than, at least about 2-fold greater than, at least about 3- fold greater than, at least about 5-fold greater than, at least about 10-fold greater than, at least about 15-fold greater than, or at least about 30-fold greater than the infiltration of the tumor by a corresponding immune cell not overexpressing the one or more tumor sensing enhancer proteins.

[0072] An advantage of the provided engineered immune cells is that their enhanced ability to migrate to and / or infiltrate tumors can allow them to more effectively treat cells of a solid tumor. For example, the engineered immune cells can exhibit an improved ability to migrate to and / or infiltrate cells of a renal cell carcinoma, nasopharyngeal carcinoma, glioblastoma, melanoma, lung carcinoma, cervix carcinoma, breast carcinoma, ovarian carcinoma, mesothelioma, or a metastatic cancer. In some embodiments, the engineered immune cells 79556009V.1exhibit improved migration to or infiltration of breast cancer tumors. In some embodiments, the engineered immune cells exhibit improved migration to or infiltration of tubo-ovarian cancer tumors.

[0073] The provided engineered immune cells can also have an improved ability to migrate to and / or infiltrate primary sites of liquid tumors or metastatic sites embedded in tissue and associated with liquid tumors. For example, the engineered immune cells can exhibit an improved ability to migrate to and / or infiltrate tumors of a blood cancer such as a lymphoma or leukemia. In some embodiments, the leukemia is acute myelogenous leukemia (AML), B- cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL or B-CLL), hairy cell leukemia, B-cell prolymphocytic leukemia, Non-Hodgkin lymphoma or Non- Hodgkin’s lymphoma, Hodgkin lymphoma or Hodgkin’s lymphoma, multiple myeloma (MM), or T-cell leukemia. In some embodiments, the non-Hodgkin lymphoma is diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, follicle center lymphoma, mantle cell lymphoma, Burkitt lymphoma (BL), or Waldenström macroglobulinemia. In some embodiments, the T- cell leukemia is anaplastic large cell lymphoma, peripheral T cell lymphoma, cutaneous T cell lymphoma, extranodal natural killer (NK) / T-cell lymphoma, Epstein-Barr virus associated T cell lymphoma, or T-cell acute lymphoblastic leukemia.

[0074] In some examples, the immune cell does not express (e.g., detectably express) the tumor sensing enhancer protein prior to being engineered, and the engineering results in the immune cell overexpressing the tumor sensing enhancer protein by enabling the immune cell to express some amount of the tumor sensing enhancer protein. In other examples, the immune cell does express (e.g., at a relatively low level) the tumor sensing enhancer protein prior to being engineered, and the engineering results in the immune cell overexpressing the tumor sensing enhancer protein by enabling the immune cell to express the tumor sensing enhancer protein at a relatively higher level. The engineered immune cell can overexpress one tumor sensing enhancer protein, two tumor sensing enhancer proteins, three tumor sensing enhancer proteins, four tumor sensing enhancer proteins, five tumor sensing enhancer proteins, six tumor sensing enhancer proteins, seven tumor sensing enhancer proteins, eight tumor sensing enhancer proteins, nine tumor sensing enhancer proteins, ten tumor sensing enhancer proteins, or more than ten tumor sensing enhancer proteins.

[0075] In some examples, at least one of the tumor sensing enhancer proteins overexpressed by the provided engineered immune cell is a tumor sensing receptor or a subunit or fragment 79556009V.1thereof. In some examples, each of the tumor sensing enhancer proteins overexpressed by the provided engineered immune cell is a tumor sensing receptor or a subunit or fragment thereof. The tumor sensing receptors generally specifically bind to a chemical entity that is not a chemokine. In this way, the tumor sensing receptors disclosed herein beneficially expand the repertoire of available receptors that can be used to modify immune cells for enhancing their effectiveness in, for example, cancer treatment or prevention.

[0076] In some examples, at least one of the tumor sensing receptors of a provided engineered immune cell specifically binds to a ligand that is a metabolite. In some examples, each of the tumor sensing receptors specifically binds to a ligand that is a metabolite. In some examples, at least one of the tumor sensing receptors of an engineered immune cell specifically binds to a ligand that is a fatty acid. In some examples, each of the tumor sensing receptors specifically binds to a ligand that is a fatty acid. In some examples, at least one of the tumor sensing receptors of an engineered immune cell specifically binds to a ligand that is a lipid. In some examples, each of the tumor sensing receptors specifically binds to a ligand that is a lipid. In some examples, at least one of the tumor sensing receptors of an engineered immune cell specifically binds to a ligand that is a product of a cancer cell, e.g., a chemical entity primarily or exclusively produced within a subject organism by a cancer cell. In some examples, each of the tumor sensing receptors specifically binds to a ligand that is a product of a cancer cell.

[0077] The tumor sensing receptors of a provided engineered immune cell can include or consist of one or more G protein coupled-receptors or subunits or fragments thereof (FIGS. 1 and 32). For example, the tumor sensing receptors can include or consist of GPR84. GPR84 can include, for example, the protein of UniProt protein accession number Q9NQS5. GPR84 is known for its role in phagocytosis, inflammation, and metabolic sensing, and induces cell migration to its agonists (e.g., medium chain fatty acids). In some examples, the tumor sensing receptor includes or consists of GPR18. GPR18 can include, for example, the protein of UniProt protein accession number Q14330. GPR18 initiates directional microglial migrationin the central nervous system and is required for the development of both CD8 intestinalintraepithelial lymphocytes and CD8 KLRG1+effector-memory T cells. GPR18 is alsorequired for sperm motility, demonstrating its role across diverse cell types. The tumor sensing receptors can include or consist of GPR34. GPR34 can include, for example, the protein of UniProt protein accession number Q9UPC5. GPR34 is highly expressed in microglia and plays a protective role against pathogen infection in the central nervous system. Its activation via lysophosphatidylserine (LysoPS) allows type 3 innate lymphoid cells (ILC3) to sense damage- 79556009V.1induced cell death in neutrophils and drive tissue repair. Without being bound by a particular theory, it is believed that GPR34 and other tumor sensing receptors allow engineered immune cells (e.g., engineered T / NK cells) to sense apoptotic cancer cells. Interestingly, T cells release LysoPS upon chemokine exposure and activate LysoPS synthesis in response to TCR stimulation, leading to an ability for GPR34 and other tumor sensing receptors to allow engineered T / NK cells to coordinate cell migration. The tumor sensing receptors can include or consist of GPR183. GPR183 can include, for example, the protein of UniProt protein accession number P32249. GPR183, is a key mediator of immune cell localization and movement within lymph nodes as is activated by oxysterols, such that GPR183-deficient B cells fail to migrate to the outer follicle. The tumor sensing receptors can include or consist of C5AR1. C5AR1 can include, for example, the protein of UniProt protein accession number P21730. C5AR1 is a receptor of complement component 5a (C5a; a chemoattractant of phagocytic and antigen presenting cells) and thus binds a ligand that is a protein rather than a metabolite. Other G protein coupled-receptors that can be overexpressed in an immune cell to yield an engineered immune cell as disclosed herein include, for example, formyl peptide receptor 3 (FPR3) (e.g., the protein of UniProt protein accession number P25089), FPR2 (e.g. UniProt protein accession number P25090), lysophosphatidic acid receptor 2 (LPAR2) (e.g., the protein of UniProt protein accession number Q9HBW0), GPR124 (e.g., the protein of UniProt protein accession number Q96PE1), GPR132 (e.g., the protein of UniProt protein accession number Q9UNW8), GPR137 (e.g., the protein of UniProt protein accession number Q96N19), GPR171 (e.g., the protein of UniProt protein accession number O14626), hydroxycarboxylic acid receptor 3 (HCAR3) (e.g., the protein of UniProt protein accession number P49019), platelet activating factor receptor (PTAFR) (e.g., the protein of UniProt protein accession number P25105), sphingosine-1-phosphate receptor 1 (S1PR1) (e.g., the protein of UniProt protein accession number P21453), S1PR4 (e.g., the protein of UniProt protein accession number O95977), and any combination thereof

[0078] In some examples, the tumor sensing receptors of the provided engineered immune cell include or consist of non-chemokine receptors other than G protein-coupled receptors. Other receptors that can be overexpressed in an immune cell to yield an engineered immune cell as disclosed herein include, for example, ephrin type-A receptor 6 (EPHA6) (e.g., the protein of UniProt protein accession number Q9UF33), inducible T-cell costimulator (ICOS) (e.g., the protein of UniProt protein accession number Q(Y6W8), syndecan binding protein (SDCBP), signaling lymphocytic activation molecule family member 1 (SLAMF1), talin-1 79556009V.1(TLN1), cytotoxic and regulatory T cell molecule (CRTAM), integrin-alpha L (ITGAL), cluster of differentiation 6 (CD6), and any combination thereof.

[0079] In some examples, at least one of the tumor sensing enhancer proteins overexpressed by the provided engineered immune cell is a metalloprotease or a subunit or fragment thereof. In some examples, each of the tumor sensing enhancer proteins overexpressed by the provided engineered immune cell is a metalloprotease or a subunit or fragment thereof. Metalloproteases that can be overexpressed in an immune cell to yield an engineered immune cell as disclosed herein include, for example, a disintegrin and metalloproteinase domain-containing protein 8 (ADAM8) (e.g., the protein of UniProt protein accession number P7832), ADAM9 (e.g., the protein of UniProt protein accession number Q13443), bone morphogenetic protein 1 (BMP1) (e.g., the protein of UniProt protein accession number P13497), carboxypeptidase D (CPD) (e.g., the protein of UniProt protein accession number O75976), matrix metallopeptidase 11 (MMP11) (e.g., the protein of UniProt protein accession number P24347), glutaminyl-peptide cyclotransferase (QPCT) (e.g., the protein of UniProt protein accession number Q16769), and any combination thereof..

[0080] In some examples, at least one of the tumor sensing enhancer proteins overexpressed by the provided engineered immune cell is a serine protease or a subunit or fragment thereof. In some examples, each of the tumor sensing enhancer proteins overexpressed by the provided engineered immune cell is a serine protease or a subunit or fragment thereof. Serine proteases that can be overexpressed in an immune cell to yield an engineered immune cell as disclosed herein include, for example, fibroblast activation protein-alpha (FAP), serine protease 23 (PRSS23) (e.g., the protein of UniProt protein accession number O95084), high-temperature requirement serine protease 2 (HTRA2) (e.g., the protein of UniProt protein accession number O43464), and any combination thereof.

[0081] In some examples, at least one of the tumor sensing enhancer proteins overexpressed by the provided engineered immune cell is a transcription factor or a subunit or fragment thereof. In some examples, each of the tumor sensing enhancer proteins overexpressed by the provided engineered immune cell is a transcription factor or a subunit or fragment thereof. Transcription factors that can be overexpressed in an immune cell to yield an engineered immune cell as disclosed herein include, for example, hypoxia-inducible factor 1-alpha (HIF1A) (e.g., the protein of UniProt protein accession number Q16665), interferon related 79556009V.1developmental regulator 1 (IFRD1) (e.g., the protein of UniProt protein accession number O00458), and a combination thereof.

[0082] In some examples, at least one of the tumor sensing enhancer proteins overexpressed by the provided engineered immune cell is a protein phosphatase or a subunit or fragment thereof. In some examples, each of the tumor sensing enhancer proteins overexpressed by the provided engineered immune cell is a protein phosphatase or a subunit or fragment thereof. Protein phosphatases that can be overexpressed in an immune cell to yield an engineered immune cell as disclosed herein include, for example, protein phosphatase 1 catalytic subunit beta (PPP1CB) (e.g., the protein of UniProt protein accession number P62140).

[0083] In some examples, at least one of the tumor sensing enhancer proteins overexpressed by the provided engineered immune cell is an ion channel protein or a subunit or fragment thereof. In some examples, each of the tumor sensing enhancer proteins overexpressed by the provided engineered immune cell is an ion channel protein or a subunit or fragment thereof. Ion channel proteins that can be overexpressed in an immune cell to yield an engineered immune cell as disclosed herein include, for example, cation channel sperm associated 2 (CATSPER2) (e.g., the protein of UniProt protein accession number Q96P56).

[0084] In a primary implementation, the foregoing tumor sensing enhancer proteins are human forms of the recited tumor sensing enhancer proteins. The disclosure also provides non- human orthologs, paralogs, and homologs, as well as hybrid, artificial and engineered forms of the recited tumor sensing enhancer proteins. The provided engineered immune cells can additionally or alternatively include variants or mutants of any of the proteins, protein subunits, or protein fragments described in this Section. For example, an engineered immune cell can overexpress a protein having an amino acid sequence that is at least about 80%, at least about 82%, at least about 84%, at least about 86%, at least about 88%, at least about 90%, at least about 92%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to an amino acid sequence of a wild-type tumor sensing enhancer protein described herein.

[0085] The provided engineered immune cell can generally be a modified version of any type of immune cell, engineered to overexpress the one or more tumor sensing enhancer proteins. For example, the engineered immune cell can be a T cell (including a primary T cell, a naïve T cell, a CD4 T cell, a CD8 T cell, a stimulated T cell, a cultured T cell, an immortalized T cell, a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a natural killer T 79556009V.1cell, an alpha / beta T cell, or a gamma / delta T cell), a B cell, a natural killer (NK) cell, a mast cell, a macrophage, a dendritic cell, a monocyte, an induced pluripotent stem cell, a hematopoietic stem cell, a myeloid progenitor cell, a lymphoid progenitor cell, or a myeloid- derived phagocytes. In particular examples, the immune cell is an NK cell, a T cell, a dendritic cell, a monocyte, or a macrophage. In some embodiments, the immune cell is an NK cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a dendritic cell. In some embodiments, the immune cell is a monocyte. In some embodiments, the immune cell is a macrophage.

[0086] In some examples, the immune cell is an autologous immune cell, i.e., an immune cell originally isolated from the subject (e.g., patient) who will receive the engineered immune cell. In other examples, the immune cell is an allogeneic cell, i.e., an immune cell isolated from a subject who is different from the patient who will receive the engineered immune cells. In some examples, the immune cell is an immune cell that expresses a chimeric antigen receptor (CAR). In some examples, the immune cell is an immune cell that expresses an engineered T- cell receptor (TCR).

[0087] In some examples, to enable the engineered immune cell to overexpress the one or more tumor sensing enhancer proteins, the engineered immune cell can be modified to include a CRISPR activation system effecting overexpression of the tumor sensing enhancer protein. Additionally or alternatively, the engineered immune cell can be modified to include an exogenous gene (e.g., an exogenous open reading frame, or ORF) that encodes the tumor sensing enhancer protein. IV. METHODS

[0088] In other aspects, the disclosure provides several methods for making and / or using the engineered immune cells described in Section III. The methods disclosed herein benefit from the improved properties of these provided materials, e.g., advantageously enhanced tumor sensing properties. These improved properties include, for example, a greater ability of the engineered immune cells to migrate to and / or infiltrate tumors, including solid tumors such as breast cancer or tubo-ovarian cancer. A. Producing an Engineered Immune Cell

[0089] Some methods provided by the disclosure are useful for producing an engineered immune cell, where the engineered immune cell is any of those described in Section III. In20 79556009V.1some examples, the engineered immune cell is produced by modifying an immune cell in vitro. The method can involve, for example, obtaining the immune cell from a subject (e.g., a human patient) in a biological sample from the subject. In other examples, the engineered immune cell is produced by modifying an immune cell in vivo, e.g., modifying the cell while the cell is within the subject.

[0090] In some examples, the provided method for producing an engineered immune cell includes a step of introducing a polynucleotide into the immune cell, where the polynucleotide encodes a tumor sensing enhancer protein (e.g., the polynucleotide can include or consist of an open reading frame or gene). In some embodiments, the polynucleotide encoding the tumor sensing enhancer protein includes DNA. In some embodiments, the polynucleotide encoding the tumor sensing protein includes RNA. The tumor sensing enhancer protein can be any of those describe in Section III. For example, the tumor sensing enhancer protein include or consist of a tumor sensing receptor that binds to a non-chemokine ligand, a tumor sensing protease, a tumor sensing transcription factor, a tumor sensing protein phosphatase, a tumor sensing ion channel protein, or a subunit or fragment thereof.

[0091] In some examples, the provided method for producing an engineered immune cell includes a step of introducing a polynucleotide into the immune cell, where the polynucleotide is a guide RNA (gRNA or sgRNA). The guide RNA can be one that targets a gene of the immune cell, where the gene encodes a tumor sensing enhancer protein (e.g., any of those described in Section III). In some embodiments, the gene targeted by the guide RNA is an endogenous gene of the immune cell. In other embodiments, the gene targeted by the guide RNA is an exogenous gene introduced to the immune cell prior to or concurrently with the introduction of the guide RNA to the immune cell. The immune cell can further include a CRISPR activation system that the guide RNA is a component of, such that the CRISPR activation system effects overexpression of the tumor sensing enhancer protein.

[0092] In some examples, the polynucleotide (e.g., the open reading frame or guide RNA) is combined with a delivery vehicle, e.g., a plasmid or a lipid or polymer vector (including exosomes, liposomes, and lipid nanoparticles (LNP)), that encapsulates, binds to, or otherwise is combined with the polynucleotide. The delivery vehicle can improve the delivery efficiency, and thus, the functional efficacy, of the polynucleotide in the engineered immune cell.

[0093] Accordingly, provided in this disclosure and included among its embodiments are nucleic acid constructs that include the polynucleotide. Some embodiments of the nucleic acid 79556009V.1constructs are purified nucleic acid molecules encoding tumor sensing enhancer proteins. For example, a nucleic acid construct can be an engineered (recombinant) DNA nucleic acid sequence comprising a promoter operably linked to a nucleic acid encoding a tumor sensing enhancer protein. A nucleic acid sequence is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. A promoter is a region or a sequence located upstream and / or downstream from the start of transcription that is involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A promoter is generally a nucleic acid sequence or sequences that function when in a relatively fixed location in regard to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements. A promoter included in nucleic acid constructs according to provided embodiments can be a eukaryotic or a prokaryotic promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a constitutive promoter. A promoter included in a nucleic acid construct according to the provided embodiments is capable of directing or driving expression of nucleic acid sequence encoding a tumor sensing enhancer protein in a host cell or host organism of interest. For preparing nucleic acid constructs according to the provided embodiments, nucleic acids may be manipulated, so as to provide for the nucleic acid sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the nucleic acid fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous nucleic acid sequences, removal of restriction sites, etc. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, and resubstitutions, such as transitions and transversions, may be involved.

[0094] A provided polynucleotide can be included in an expression cassette for expression of a tumor sensing enhancer protein encoded by the polynucleotide in a host cell or an organism of interest. In some embodiments, a provided polynucleotide can be codon-optimized forexpression in a host cell or an organism of interest. An expression cassette can include 5 and / or3 regulatory sequences operably linked to the polynucleotide encoding a tumor sensingenhancer protein according to a provided embodiment. An expression cassette can also include nucleic acid sequences encoding other polypeptides or proteins. An expression cassette can include a plurality of restriction sites and / or recombination sites for insertion of various nucleic acid sequences into the expression cassette and / or for insertion of the expression cassette into other nucleic acids, such as vectors. An expression cassette can include various regulatory 79556009V.1regions or sequences, such as, but are not limited to, transcriptional initiation start sites, operators, activators, enhancers, other regulatory elements, ribosomal binding sites, initiation codons, termination signals, and the like. Exemplary regulatory sequences included in the expression cassettes are promoters, transcriptional regulatory regions, and / or translational termination regions, which may be endogenous or heterologous to the host cell or host organism, or to each other. In this context, “heterologous” means a nucleic acid sequence that does not originate in the host cell or host organism, or is substantially modified from its form occurring in the host cell or host organism. An expression cassette can also include one or more selectable marker genes for the selection of host cells containing the expression cassette. Marker genes include, but are not limited to, genes conferring antibiotic resistance, such as those conferring hygromycin resistance, ampicillin resistance, gentamicin resistance, and / or neomycin resistance, to name a few. Additional selectable markers are known and any can beused. An exemplary expression cassette can include, in the 5 to 3 direction, a transcriptionaland translational initiation region (including a promoter), a polynucleotide sequence encoding a tumor sensing enhancer protein described in the present disclosure, and transcriptional and translational termination regions functional in the host cell or host organism of interest.

[0095] Also included among the provided embodiments are vectors including polynucleotides or nucleic acid constructs. Such vectors can include necessary functional elements that direct and regulate transcription of the nucleic acid sequences included in the vector. These functional elements include, but are not limited to, a promoter, regions upstream or downstream of the promoter, such as enhancers that may regulate the transcriptional activity of the promoter, an origin of replication, appropriate restriction sites to facilitate cloning of inserts adjacent to the promoter, antibiotic resistance genes or other markers that can serve to select for cells containing the vector or the vector containing the insert, RNA splice junctions, a transcription termination region, or any other region that may serve to facilitate the expression of the inserted gene or hybrid. The vector, for example, can be a plasmid.

[0096] Expression vectors according to provided embodiments can also include polynucleotides described in the present disclosure under the control of an inducible promoter such as the tetracycline inducible promoter or a glucocorticoid inducible promoter. The provided polynucleotides can also be under the control of a tissue-specific promoter to promote expression of the polynucleotide in specific cells, tissues, or organs. Any regulatable promoter, such as a metallothionein promoter, a heat-shock promoter, and other regulatable promoters is23 79556009V.1also contemplated. Furthermore, a Cre-loxP inducible system can also be used, as well as a Flp recombinase inducible promoter system.

[0097] In some embodiments, a polynucleotide encoding a provided tumor sensing enhancer protein is incorporated into a viral vector for delivery into a host cell or host organism. Accordingly, the vectors according to provided embodiments include viral vectors that transport the polynucleotides encoding tumor sensing enhancer proteins described in the present disclosure into cells without degradation and include a promoter yielding expression of the polynucleotides in the cells into which it is delivered. Suitable viral vectors include adenovirus vectors, adeno-associated viral (AAV) vectors, herpes viral vectors, retroviral vectors, poxviral vectors, or lentiviral vectors. Methods of constructing and using such vectors are well known. Typically, viral vectors contain, nonstructural early genes, structural late genes, an RNA polymerase III transcript, inverted terminal repeats necessary for replication and encapsidation, and promoters to control the transcription and replication of the viral genome. When engineered as vectors, viruses typically have one or more of the early genes removed and a gene or gene / promoter cassette is inserted into the viral genome in place of the removed viral DNA. The necessary functions of the removed early genes are typically supplied by cell lines that have been engineered to express the gene products of the early genes in trans.

[0098] For example, recombinant viruses in the pox family of viruses can be used as vectors for delivering the provided polynucleotides into a host cell or host organism. These include vaccinia viruses and avian poxviruses, such as the fowlpox and canarypox viruses. Methods for producing recombinant pox viruses are known. Representative examples of recombinant pox viruses include ALVAC, TROVAC, and NYVAC. In another example, adenovirus vectors can be used for delivering the provided polynucleotides into a host cell or host organism. In one more example, adeno-associated virus (AAV) vector systems can be used for delivering the provided polynucleotide into a host cell or host organism. In one more example, retroviral vectors can be used for delivering the provided polynucleotide into a host cell or host organism. Examples of retroviral vectors include, but are not limited to, vectors based on Murine Maloney Leukemia virus (MMLV), and retroviruses that express the desirable properties of MMLV as a vector. In yet another example, molecular conjugate vectors, such as the adenovirus chimeric vectors can be used for delivering a provided polynucleotide into a host cell or host organism. Vectors derived from the members of the Alphavirus genus, such as, but not limited to, Sindbis, Semliki Forest, and Venezuelan Equine Encephalitis viruses, can also be used for delivering a provided polynucleotide into a host cell or host organism.24 79556009V.1

[0099] In some examples, the polynucleotide is delivered to the immune cell via a virus or a viral vector. Examples of viruses and associated viral vectors (e.g., plasmids) include lentivirus, adeno-associated virus (AAV), adenovirus (e.g., a human adenovirus vector such as huAd5, huAd46; a chimpanzee adenovirus vector such as ChAdOx1, ChAd3; a rhesus macaque adenovirus vector such as RhAd54), flavivirus (e.g., a yellow fever (YF) virus vector), herpes simplex virus (HSV), measles virus, Newcastle disease virus (NDV), poxvirus (e.g., a vaccinia virus vector or a variola virus (VV) vector), retrovirus, or vesicular stomatitis virus (VSV). In some embodiments, the method includes a step of introducing the polynucleotide to the immune cell via viral transduction with a lentivirus.

[0100] In some embodiments, a polynucleotide encoding a provided tumor sensing enhancer protein may be incorporated into a transposon vector, which can be used as a part of a part of a transposon vector system to integrate a polynucleotide encoding a tumor sensing enhancer protein into transcriptionally active sites of the genome of a host cell. Transposon vector systems are discussed, for example, in Wei et al., 2022. Accordingly, the provided vectors include transposon vectors that transport the polynucleotides encoding the provided tumor sensing enhancer proteins into cells. A non-limiting example of a transposon vector system is a system that includes LEAP-IN TRANSPOSASE®available from ATUM (Newark, California) and its accompanying suite of transposon vectors. To use the above transposon vector system, transposase mRNA is co-transfected with the transposon vector encoding a protein of interest (for example, a provided tumor sensing enhancer protein) into a cell. After the mRNA is translated in the cell, the transposase protein transiently acts to integrate the transposon into the cell genome. The transposase mRNA is degraded through the normal cellular RNA turnover pathways. Pools of cells with the stably integrated DNA encoding the protein of interest can then be recovered, and individual cells can be cloned, thereby creating stable cell lines encoding the protein of interest, such as a provided tumor sensing enhancer protein.

[0101] A polynucleotide, a nucleic acid construct, or a vector according to provided embodiments can be transferred or introduced into the host cell by well-known methods, which vary depending on the type of the host cell. The “introducing” and the related terms or phrases used in the context of introducing a polynucleotide, a nucleic acid construct, or a vector into a cell refers to the translocation of the nucleic acid sequence from outside a cell to inside the cell. Various methods of such translocation are contemplated, including but not limited to, electroporation, nanoparticle delivery, viral delivery, contact with nanowires or nanotubes, 79556009V.1receptor mediated internalization, translocation via cell penetrating peptides, liposome mediated translocation, DEAE dextran, lipofectamine, calcium phosphate or any method now known or identified in the future for introduction of polynucleotides into cellular hosts. A targeted nuclease system (e.g., an RNA-guided nuclease (CRISPR-Cas9), a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), or a megaTAL (MT) can also be used to introduce a polynucleotide into a cell.

[0102] The present disclosure also provides methods and compositions for introducing and integrating a polynucleotide encoding a tumor sensing enhancer protein at a selected locus. In some embodiments, the selected locus is a safe harbor locus. Examples of safe harbor loci known to exist within mammalian cells include, but are not limited to, the CCR5 locus, the AAVS1 locus, the ROSA26 locus, and the CLYBL locus. B. Preventing or Treating a Cancer

[0103] Some methods provided by the disclosure are useful for preventing or treating a cancer of a subject. These methods generally include providing an engineered immune cell to the subject, either by administering the engineered immune cell, or a population of engineered immune cells, to the subject, or by modifying an immune cell of the subject in vivo, e.g., while the immune cell is within the subject. The engineered immune cell can be any of those described in Section III, or any of those prepared according to the method describe in Section IV.A. In some embodiments, the treating of the cancer includes decreasing or eliminating one or more signs or symptoms of the cancer.

[0104] In some examples, the method further includes engineering an immune cell, e.g., to produce the engineered cell that is then administered to the subject. The engineering of the immune cell can be performed using any of the methods described in Section III. For example, the engineering of the immune cell can include obtaining a biological sample from the subject. The engineering of the immune cell can further include partially or completely purifying or isolating the immune cell from the biological sample. The engineering of the immune cell can further include introducing a polynucleotide to the immune cell, where the polynucleotide can be one encoding a tumor sensing enhancer protein, or the polynucleotide can be a guide RNA targeting a gene that encodes a tumor sensing enhancer protein. In other examples, the engineering of the immune cell as part of the method of preventing or treating cancer can include introducing a polynucleotide to the subject, where the polynucleotide can be one encoding a tumor sensing enhancer protein, or the polynucleotide can be a guide RNA targeting 79556009V.1a gene that encodes a tumor sensing enhancer protein, and where introducing the polynucleotide to the subject modifies an immune cell of the subject in vivo.

[0105] Non-limiting examples of cancers that can be treated with the provided methods include Acanthoma, Acinic cell carcinoma, Acoustic neuroma, Acral lentiginous melanoma, Acrospiroma, Acute eosinophilic leukemia, Acute lymphoblastic leukemia, Acute megakaryoblastic leukemia, Acute monocytic leukemia, Acute myeloblastic leukemia with maturation, Acute myeloid dendritic cell leukemia, Acute myeloid leukemia, Acute promyelocytic leukemia, Adamantinoma, Adenocarcinoma, Adenoid cystic carcinoma, Adenoma, Adenomatoid odontogenic tumor, Adrenocortical carcinoma, Adult T-cell leukemia, Aggressive NK-cell leukemia, AIDS-Related Cancers, AIDS-related lymphoma, Alveolar soft part sarcoma, Ameloblastic fibroma, Anal cancer, Anaplastic large cell lymphoma, Anaplastic thyroid cancer, Angioimmunoblastic T-cell lymphoma, Angiomyolipoma, Angiosarcoma, Appendix cancer, Astrocytoma, Atypical teratoid rhabdoid tumor, Basal cell carcinoma, Basal-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, Biliary tract cancer, Bladder cancer, Blastoma, Bone Cancer, Bone tumor, Brain Stem Glioma, Brain Tumor, Breast Cancer, Brenner tumor, Bronchial Tumor, Bronchioloalveolar carcinoma, Brown tumor, Burkitt's lymphoma, Cancer of Unknown Primary Site, Carcinoid Tumor, Carcinoma, Carcinoma in situ, Carcinoma of the penis, Carcinoma of Unknown Primary Site, Carcinosarcoma, Castleman's Disease, Central Nervous System Embryonal Tumor, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Cholangiocarcinoma, Chondroma, Chondrosarcoma, Chordoma, Choriocarcinoma, Choroid plexus papilloma, Chronic Lymphocytic Leukemia, Chronic monocytic leukemia, Chronic myelogenous leukemia, Chronic Myeloproliferative Disorder, Chronic neutrophilic leukemia, Clear-cell tumor, Colon Cancer, Colorectal cancer, Craniopharyngioma, Cutaneous T-cell lymphoma, Degos disease, Dermatofibrosarcoma protuberans, Dermoid cyst, Desmoplastic small round cell tumor, Diffuse large B cell lymphoma, Dysembryoplastic neuroepithelial tumor, Embryonal carcinoma, Endodermal sinus tumor, Endometrial cancer, Endometrial Uterine Cancer, Endometrioid tumor, Enteropathy-associated T-cell lymphoma, Ependymoblastoma, Ependymoma, Epithelioid sarcoma, Erythroleukemia, Esophageal cancer, Esthesioneuroblastoma, Ewing Family of Tumor, Ewing Family Sarcoma, Ewing's sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Extramammary Paget's disease, Fallopian tube cancer, Fetus in fetu, Fibroma, Fibrosarcoma, Follicular lymphoma, Follicular thyroid cancer, Gallbladder Cancer,27 79556009V.1Gallbladder cancer, Ganglioglioma, Ganglioneuroma, Gastric Cancer, Gastric lymphoma, Gastrointestinal cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor, Gastrointestinal stromal tumor, Germ cell tumor, Germinoma, Gestational choriocarcinoma, Gestational Trophoblastic Tumor, Giant cell tumor of bone, Glioblastoma multiforme, Glioma, Gliomatosis cerebri, Glomus tumor, Glucagonoma, Gonadoblastoma, Granulosa cell tumor, Hairy Cell Leukemia, Hairy cell leukemia, Head and Neck Cancer, Head and neck cancer, Heart cancer, Hemangioblastoma, Hemangiopericytoma, Hemangiosarcoma, Hematological malignancy, Hepatocellular carcinoma, Hepatosplenic T-cell lymphoma, Hereditary breast- ovarian cancer syndrome, Hodgkin Lymphoma, Hodgkin's lymphoma, Hypopharyngeal Cancer, Hypothalamic Glioma, Inflammatory breast cancer, Intraocular Melanoma, Islet cell carcinoma, Islet Cell Tumor, Juvenile myelomonocytic leukemia, Kaposi Sarcoma, Kaposi's sarcoma, Kidney Cancer, Klatskin tumor, Krukenberg tumor, Laryngeal Cancer, Laryngeal cancer, Lentigo maligna melanoma, Leukemia, Leukemia, Lip and Oral Cavity Cancer, Liposarcoma, Lung cancer, Luteoma, Lymphangioma, Lymphangiosarcoma, Lymphoepithelioma, Lymphoid leukemia, Lymphoma, Macroglobulinemia, Malignant Fibrous Histiocytoma, Malignant fibrous histiocytoma, Malignant Fibrous Histiocytoma of Bone, Malignant Glioma, Malignant Mesothelioma, Malignant peripheral nerve sheath tumor, Malignant rhabdoid tumor, Malignant triton tumor, MALT lymphoma, Mantle cell lymphoma, Mast cell leukemia, Mediastinal germ cell tumor, Mediastinal tumor, Medullary thyroid cancer, Medulloblastoma, Medulloblastoma, Medulloepithelioma, Melanoma, Melanoma, Meningioma, Merkel Cell Carcinoma, Mesothelioma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Metastatic urothelial carcinoma, Mixed Mullerian tumor, Monocytic leukemia, Mouth Cancer, Mucinous tumor, Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma, Multiple myeloma, Mycosis Fungoides, Mycosis fungoides, Myelodysplastic Disease, Myelodysplastic Syndromes, Myeloid leukemia, Myeloid sarcoma, Myeloproliferative Disease, Myxoma, Nasal Cavity Cancer, Nasopharyngeal Cancer, Nasopharyngeal carcinoma, Neoplasm, Neurinoma, Neuroblastoma, Neuroblastoma, Neurofibroma, Neuroma, Nodular melanoma, Non-Hodgkin Lymphoma, Non-Hodgkin lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Ocular oncology, Oligoastrocytoma, Oligodendroglioma, Oncocytoma, Optic nerve sheath meningioma, Oral Cancer, Oral cancer, Oropharyngeal Cancer, Osteosarcoma, Osteosarcoma, Ovarian Cancer, Ovarian cancer, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Paget's disease of the breast, Pancoast tumor, Pancreatic Cancer, Pancreatic cancer, Papillary thyroid cancer, Papillomatosis, Paraganglioma, Paranasal Sinus28 79556009V.1Cancer, Parathyroid Cancer, Penile Cancer, Perivascular epithelioid cell tumor, Pharyngeal Cancer, Pheochromocytoma, Pineal Parenchymal Tumor of Intermediate Differentiation, Pineoblastoma, Pituicytoma, Pituitary adenoma, Pituitary tumor, Plasma Cell Neoplasm, Pleuropulmonary blastoma, Polyembryoma, Precursor T-lymphoblastic lymphoma, Primary central nervous system lymphoma, Primary effusion lymphoma, Primary Hepatocellular Cancer, Primary Liver Cancer, Primary peritoneal cancer, Primitive neuroectodermal tumor, Prostate cancer, Pseudomyxoma peritonei, Rectal Cancer, Renal cell carcinoma, Respiratory Tract Carcinoma Involving the NUT Gene on Chromosome 15, Retinoblastoma, Rhabdomyoma, Rhabdomyosarcoma, Richter's transformation, Sacrococcygeal teratoma, Salivary Gland Cancer, Sarcoma, Schwannomatosis, Sebaceous gland carcinoma, Secondary neoplasm, Seminoma, Serous tumor, Sertoli-Leydig cell tumor, Sex cord-stromal tumor, Sezary Syndrome, Signet ring cell carcinoma, Skin Cancer, Small blue round cell tumor, Small cell carcinoma, Small Cell Lung Cancer, Small cell lymphoma, Small intestine cancer, Soft tissue sarcoma, Somatostatinoma, Soot wart, Spinal Cord Tumor, Spinal tumor, Splenic marginal zone lymphoma, Squamous cell carcinoma, Stomach cancer, Superficial spreading melanoma, Supratentorial Primitive Neuroectodermal Tumor, Surface epithelial-stromal tumor, Synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocyte leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, Teratoma, Terminal lymphatic cancer, Testicular cancer, Thecoma, Throat Cancer, Thymic Carcinoma, Thymoma, Thyroid cancer, Transitional Cell Cancer of Renal Pelvis and Ureter, Transitional cell carcinoma, Urachal cancer, Urethral cancer, Urogenital neoplasm, Uterine sarcoma, Uveal melanoma, Vaginal Cancer, Verner Morrison syndrome, Verrucous carcinoma, Visual Pathway Glioma, Vulvar Cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor, and combinations thereof.

[0106] The treated or prevented cancerous tumor can be a solid cancerous tumor or a liquid cancerous tumor. The solid cancerous tumor can be, for example, breast cancer tumor or a tubo- ovarian cancer tumor. A tumor treated with the methods disclosed herein can result in stabilized tumor growth (e.g., one or more tumors do not increase more than 1%, 5%, 10%, 15%, or 20% in size, and / or do not metastasize). In some embodiments, a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks. In some embodiments, a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months. In some embodiments, a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years. In some embodiments, the size of a tumor or the number of tumor cells is reduced by at least about 5%, 79556009V.110%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. In some embodiments, the tumor is completely eliminated, or reduced below a level of detection. In some embodiments, a subject remains tumor free (e.g. in remission) for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks following treatment. In some embodiments, a subject remains tumor free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months following treatment. In some embodiments, a subject remains tumor free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years after treatment.

[0107] One skilled in the art will also appreciate that the provided engineered immune cells can be co-administered with other therapeutic agents for the treatment of cancer. Suitable anti- cancer agents for combination therapy include, without limitation, cytotoxins and agents such as antimetabolites, alkylating agents, anthracyclines, antibiotics, antimitotic agents, procarbazine, hydroxyurea, asparaginase, corticosteroids, interferons, radiopharmaceuticals,peptides with anti-tumor activity such as TNF- , pharmaceutically acceptable salts thereof;derivatives thereof, prodrugs thereof, and combinations thereof. For example, the provided engineered immune cells can be administered to a patient before, during, or after administration of an anti-cancer agent or combination of anti-cancer agents either before, during, or after chemotherapy.

[0108] In some embodiments, treatment with the provided engineered immune cells results in stable disease, partial remission or complete remission in the subject (e.g., the methods described herein comprise administering to the subject a dose of the provided engineered immune cells that kills or otherwise slows the growth or progression of cancer cells and leads to stable disease or to partial or complete remission of the cancer in the subject). In some embodiments, treatment with the provided engineered immune cells results in a reduction in metastases of the cancer in the subject (e.g., the methods described herein comprise administering to the subject a dose of the provided engineered immune cells that reduces metastases of the cancer in the subject). In some embodiments, treatment with the provided engineered immune cells results in a reduction in volume, size, or growth of a tumor in the subject (e.g., the methods described herein comprise administering to the subject a dose of the provided engineered immune cells that reduces the volume, size, or growth of a tumor in the subject). In some embodiments, treatment with the provided engineered immune cells results in an increased responsiveness of the cancer to a subsequently administered anti-cancer agent (e.g., the methods described herein comprise administering to the subject a dose of the provided 79556009V.1engineered immune cells that increases responsiveness of the cancer to a subsequently administered anti-cancer agent).

[0109] In some embodiments, the provided method further includes obtaining a test sample from the subject. The test sample can include, for example, a blood sample, a tissue sample, a urine sample, a saliva sample, a cerebrospinal fluid sample, or a combination thereof. In some embodiments, the provided method further includes determining the level of one or more biomarkers in the obtained test sample. Determining the presence or level of biomarkers(s) can be used to, as non-limiting examples, determine response to treatment or to select an appropriate composition for the prevention or treatment of the disease.

[0110] In some embodiments, the provided method further includes comparing the determined level of the one of more biomarkers in the obtained test sample to the level of the one or more biomarkers in a reference sample. The reference sample can be obtained, for example, from the subject, with the reference sample being obtained prior to the obtaining of the test sample, e.g., prior to the administering to the subject of the therapeutically effective amount of the provided materials. In this way, the reference sample can provide information about baseline levels of the biomarkers in the sample before the treatment, and the test sample can provide information about levels of the biomarkers after the treatment.

[0111] Alternatively, the reference sample can be obtained, for example, from a different subject, e.g., a subject in which the treatment is not provided according to the provided methods. In this way, the reference sample can provide information about baseline levels of the biomarkers without treatment, and the test sample can provide information about levels of the biomarkers with treatment. The reference sample can also be obtained, for example, from a population of subjects, e.g., subjects in which the treatment is not provided according to the provided method. In this way, the reference sample can provide population-averaged information about baseline levels of the biomarkers without treatment, and the test sample can provide information about levels of the biomarkers with treatment.

[0112] The reference sample can also be obtained from an individual or a population of individuals after treatment is provided according to the provided methods, and can serve as, for example, a positive control sample. In some embodiments, the reference sample is obtained from normal tissue. In some embodiments, the reference sample is obtained from abnormal tissue. 79556009V.1

[0113] Depending on the biomarker, an increase or decrease relative to a normal control or reference sample can be indicative of the presence of a disease, or response to treatment for a disease. In some embodiments, an increased level of a biomarker in a test sample, and hence the presence of a disease, e.g., a cancer, increased risk of the disease, or response to treatment is determined when the biomarker levels are at least, 1.1-fold, e.g., at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, or at least 20-fold higher in comparison to a control. In other embodiments, a decreased level of a biomarker in the test sample, and hence the presence of the disease, increased risk of the disease, or response to treatment is determined when the biomarker levels are at least 1.1-fold, e.g., at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, or at least 20-fold lower in comparison to a control.

[0114] The biomarker levels can be detected using any method known in the art, including the use of antibodies specific for the biomarkers. Exemplary methods include, without limitation, polymerase chain reaction (PCR), Western Blot, dot blot, ELISA, radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, FACS analysis, electrochemiluminescence, and multiplex bead assays, e.g., using Luminex or fluorescent microbeads. In some instances, nucleic acid sequencing is employed. In some embodiments, the detection of levels of biomarkers such as metabolites can include use of mass spectrometry (e.g., liquid chromatography linked to mass spectrometry (LC-MS)) and / or receptor (e.g., GPCR) reporting, for example as described in Example 3.

[0115] In certain embodiments, the presence of decreased or increased levels of one or more biomarkers is indicated by a detectable signal, e.g., a blot, fluorescence, chemiluminescence, color, or radioactivity in an immunoassay or PCR reaction, e.g., quantitative PCR. This detectable signal can be compared to the signal from a reference sample or to a threshold value. 79556009V.1

[0116] In some embodiments, the results of the biomarker level determinations are recorded in a tangible medium. For example, the results of diagnostic assays, e.g., the observation of the presence or decreased or increased presence of one or more biomarkers, and the diagnosis of whether or not there is an increased risk or the presence of a disease, e.g., a cancer, or whether or not a subject is responding to treatment can be recorded, for example, on paper or on electronic media, e.g., audio tape, a computer disk, a CD-ROM, or a flash drive.

[0117] In some embodiments, the provided method further includes the step of providing to the subject a diagnosis and / or the results of treatment. C. Determining a Personalized Cancer Treatment

[0118] Some methods provided by the disclosure are useful for determining a personalized cancer treatment for a subject. These methods generally include administering a plurality of (i.e., two or more) populations of engineered immune cells to the subject. Each of the administered populations of cells differs from the other, such that the ability of each different population to enhance the ability of immune cells to migrate to and / or infiltrate tumors can be compared to one another or to a desired threshold metric, thereby determining which one or more of the populations is suitable for use in the personalized cancer treatment. Accordingly, while the cells of each population of the plurality of populations of engineered immune cells are engineered to overexpress a combination of one or more tumor sensing enhancer proteins, the overexpressed combination in the cells of one population is different from the overexpressed combination in the cells of each other population.

[0119] The provided method for determining a personalized cancer treatment can further include a step of obtaining a biological sample from the subject following the administration of the plurality of populations of engineered immune cells. An amount of time between the administration step and the step of obtaining the biological sample can be selected to be sufficient for the engineered immune cells to cause a measurable effect on tumors of the subject. The method can further include analyzing the biological sample, and based on the analyzing, determining which one or more of the plurality of administered populations of immune cells exhibits a sufficiently high level of increase migration to, or infiltration of tumors of the subject. In some embodiments, a level is considered sufficiently high when the level is greater than or equal to a threshold. The threshold can be a predetermined value, e.g., a value based on a desired performance. Alternatively, the threshold can be a value determined based 79556009V.1on the performance of each of, or at least a subset of, the administered populations of engineered immune cells. V. EXEMPLARY EMBODIMENTS

[0120] The following embodiments are contemplated. All combinations of features and embodiments are contemplated.

[0121] Embodiment 1: An engineered immune cell genetically modified to overexpress a tumor sensing enhancer protein, the tumor sensing enhancer protein comprising a tumor sensing receptor that binds to a non-chemokine ligand, a tumor sensing protease, a tumor sensing transcription factor, a tumor sensing protein phosphatase, a tumor sensing ion channel protein, or a subunit or fragment thereof, wherein the engineered immune cell exhibits increased migration to or infiltration of a tumor as compared to migration to or infiltration of the tumor by a corresponding immune cell not overexpressing the tumor sensing enhancer protein.

[0122] Embodiment 2: An embodiment of embodiment 1, wherein the engineered immune cell exhibits increased migration to the tumor as compared to migration to the tumor by the corresponding immune cell not overexpressing the tumor sensing enhancer protein.

[0123] Embodiment 3: An embodiment of embodiment 1 or 2, wherein the engineered immune cell exhibits increased infiltration of the tumor as compared to infiltration of the tumor by the corresponding immune cell not overexpressing the tumor sensing enhancer protein.

[0124] Embodiment 4: An embodiment of any one of embodiments 1-3, wherein the tumor sensing enhancer protein comprises the tumor sensing receptor or the subunit or fragment thereof.

[0125] Embodiment 5: An embodiment of embodiment 4, wherein the engineered immune cell exhibits increased migration to the tumor in a presence of the non-chemokine ligand, as compared to migration to the tumor by the corresponding immune cell not overexpressing the tumor sensing receptor.

[0126] Embodiment 6: An embodiment of embodiment 4 or 5, wherein the engineered immune cell exhibits increased infiltration of the tumor in a presence of the non-chemokine ligand, as compared to infiltration of the tumor by the corresponding immune cell not overexpressing the tumor sensing receptor. 79556009V.1

[0127] Embodiment 7: An embodiment of any one of embodiments 4-6, wherein the non- chemokine ligand comprises a metabolite.

[0128] Embodiment 8: An embodiment of any one of embodiments 4-7, wherein the non- chemokine ligand comprises a fatty acid, a lipid, or a derivative thereof.

[0129] Embodiment 9: An embodiment of any one of embodiments 4-8, wherein the tumor sensing receptor comprises a G protein-coupled receptor or a subunit or fragment thereof.

[0130] Embodiment 10: An embodiment of embodiments 9, wherein the tumor sensing receptor or the subunit or fragment thereof comprises G protein-coupled receptor 84 (GPR84), GPR18, GPR34, GPR183, complement component 5a receptor 1 (C5AR1), formyl peptide receptor 3 (FPR3), FPR2, lysophosphatidic acid receptor 2 (LPAR2), or a combination thereof.

[0131] Embodiment 11: An embodiment of embodiment 9 or 10, wherein the tumor sensing receptor or the subunit or fragment thereof comprises GPR124, GPR132, GPR137. GPR171, hydroxycarboxylic acid receptor 3 (HCAR3), platelet activating factor receptor (PTAFR), sphingosine-1-phosphate receptor 1 (S1PR1), S1PR4. or a combination thereof.

[0132] Embodiment 12: An embodiment of any one of embodiments 4-11, wherein the tumor sensing receptor or the subunit or fragment thereof comprises ephrin type-A receptor 6 (EPHA6), inducible T-cell costimulator (ICOS), syndecan binding protein (SDCBP), signaling lymphocytic activation molecule family member 1 (SLAMF1), talin-1 (TLN1), cytotoxic and regulatory T cell molecule (CRTAM), integrin-alpha L (ITGAL), cluster of differentiation 6 (CD6), or any combination thereof.

[0133] Embodiment 13: An embodiment of any one of embodiments 1-12, wherein the tumor sensing enhancer protein comprises the tumor sensing protease or the subunit or fragment thereof.

[0134] Embodiment 14: An embodiment of embodiment 13, wherein the tumor sensing protease comprises a metalloprotease or a subunit or fragment thereof.

[0135] Embodiment 15: An embodiment of embodiment 14, wherein the metalloprotease or the subunit or fragment thereof comprises a disintegrin and metalloproteinase domain- containing protein 8 (ADAM8), ADAM9, bone morphogenetic protein 1 (BMP1), carboxypeptidase D (CPD), matrix metallopeptidase 11 (MMP11), glutaminyl-peptide cyclotransferase (QPCT), or a combination thereof. 79556009V.1

[0136] Embodiment 16: An embodiment of any one of embodiments 13-15, wherein the tumor sensing protease comprises a serine protease or a subunit or fragment thereof.

[0137] Embodiment 17: An embodiment of any one of embodiments 16, wherein the serine protease or the subunit or fragment thereof comprises fibroblast activation protein-alpha (FAP), serine protease 23 (PRSS23), high-temperature requirement serine protease 2 (HTRA2), or a combination thereof.

[0138] Embodiment 18: An embodiment of any one of embodiments 1-17, wherein the tumor sensing enhancer protein comprises the tumor sensing transcription factor or the subunit or fragment thereof.

[0139] Embodiment 19: An embodiment of embodiment 18, wherein the tumor sensing transcription factor or the subunit or fragment thereof comprises hypoxia-inducible factor 1- alpha (HIF1A), interferon related developmental regulator 1 (IFRD1), or a combination thereof.

[0140] Embodiment 20: An embodiment of any one of embodiments 1-19, wherein the tumor sensing enhancer protein comprises the tumor sensing protein phosphatase or the subunit or fragment thereof.

[0141] Embodiment 21: An embodiment of embodiment 20, wherein the tumor sensing protein phosphatase or the subunit or fragment thereof comprises protein phosphatase 1 catalytic subunit beta (PPP1CB).

[0142] Embodiment 22: An embodiment of any one of embodiments 1-21, wherein the tumor sensing enhancer protein comprises the tumor sensing ion channel protein or the subunit or fragment thereof.

[0143] Embodiment 23: An embodiment of embodiment 22, wherein the tumor sensing ion channel protein or the subunit or fragment thereof comprises cation channel sperm associated 2 (CATSPER2).

[0144] Embodiment 24: An embodiment of any one of embodiments 1-23, wherein the tumor is a solid tumor.

[0145] Embodiment 25: An embodiment of embodiment 24, wherein the tumor comprises a breast cancer or a tubo-ovarian cancer. 79556009V.1

[0146] Embodiment 26: An embodiment of any one of embodiments 1-25, wherein the engineered immune cell is a natural killer (NK) cell, a T cell, a dendritic cell, a monocyte, or a macrophage.

[0147] Embodiment 27: An embodiment of any one of embodiments 1-26, wherein the engineered immune cell expresses a chimeric antigen receptor (CAR).

[0148] Embodiment 28: An embodiment of embodiment 27, wherein the CAR targets EpCAM or HER2

[0149] Embodiment 29: An embodiment of any one of embodiments 1-28, wherein the engineered immune cell expresses an engineered T-cell receptor (TCR).

[0150] Embodiment 30: An embodiment of any one of embodiments 1-28, wherein the engineered immune cell comprises a CRISPR activation system effecting overexpression of the tumor sensing enhancer protein or the subunit or fragment thereof by the engineered immune cell.

[0151] Embodiment 31: An embodiment of any one of embodiments 1-30, wherein the engineered immune cell comprises an exogenous gene encoding the tumor sensing enhancer protein or the subunit or fragment thereof.

[0152] Embodiment 32: An embodiment of any one of embodiments 1-31, wherein the engineered immune cell is genetically modified to overexpress two or more tumor sensing enhancer proteins.

[0153] Embodiment 33: A method for producing an engineered immune cell, the method comprising: providing an immune cell; and introducing into the immune cell a polynucleotide encoding a tumor sensing enhancer protein, thereby producing the engineered immune cell; wherein the tumor sensing enhancer protein comprises a tumor sensing receptor that binds to a non-chemokine ligand, a tumor sensing protease, a tumor sensing transcription factor, a tumor sensing protein phosphatase, a tumor sensing ion channel protein, or a subunit or fragment thereof; and wherein the engineered immune cell exhibits increased migration to or infiltration of a tumor as compared to migration to or infiltration of the tumor by a corresponding immune cell not overexpressing the tumor sensing enhancer protein.

[0154] Embodiment 34: An embodiment of embodiment 33, wherein introducing the polynucleotide comprises viral transduction of the polynucleotide into the immune cell. 79556009V.1

[0155] Embodiment 35: A method for producing an engineered immune cell, the method comprising: providing an immune cell comprising a gene encoding a tumor sensing enhancer protein; and introducing into the immune cell a guide RNA targeting the gene, thereby producing the engineered immune cell; wherein the tumor sensing enhancer protein comprises a tumor sensing receptor that binds to a non-chemokine ligand, a tumor sensing protease, a tumor sensing transcription factor, a tumor sensing protein phosphatase, a tumor sensing ion channel protein, or a subunit or fragment thereof; and wherein the engineered immune cell exhibits increased migration to or infiltration of a tumor as compared to migration to or infiltration of the tumor by a corresponding immune cell not overexpressing the tumor sensing enhancer protein.

[0156] Embodiment 36: An embodiment of embodiment 35, wherein the gene is an endogenous gene of the immune cell.

[0157] Embodiment 37: An embodiment of embodiment 35, wherein the gene is an exogenous gene, and wherein the method further comprises introducing the exogenous gene to the immune cell.

[0158] Embodiment 38: An embodiment of any one of embodiments 33-37, wherein the engineered immune cell exhibits increased migration to the tumor as compared to migration to the tumor by the corresponding immune cell not overexpressing the tumor sensing enhancer protein.

[0159] Embodiment 39: An embodiment of any one of embodiments 33-38, wherein the engineered immune cell exhibits increased infiltration of the tumor as compared to infiltration of the tumor by the corresponding immune cell not overexpressing the tumor sensing enhancer protein.

[0160] Embodiment 40: An embodiment of any one of embodiments 33-39, wherein the tumor sensing enhancer protein comprises the tumor sensing receptor or the subunit or fragment thereof.

[0161] Embodiment 41: An embodiment of embodiment 40, wherein the engineered immune cell exhibits increased migration to the tumor in a presence of the non-chemokine ligand, as compared to migration to the tumor by the corresponding immune cell not overexpressing the tumor sensing receptor. 79556009V.1

[0162] Embodiment 42: An embodiment of embodiment 40 or 41, wherein the engineered immune cell exhibits increased infiltration of the tumor in a presence of the non-chemokine ligand, as compared to infiltration of the tumor by the corresponding immune cell not overexpressing the tumor sensing receptor.

[0163] Embodiment 43: An embodiment of any one of embodiments 40-42, wherein the non- chemokine ligand comprises a metabolite.

[0164] Embodiment 44: An embodiment of any one of embodiments 40-43, wherein the non- chemokine ligand comprises a fatty acid, a lipid, or a derivative thereof.

[0165] Embodiment 45: An embodiment of any one of embodiments 40-44, wherein the tumor sensing receptor comprises a G protein-coupled receptor or a subunit or fragment thereof.

[0166] Embodiment 46: An embodiment of embodiment 45, wherein the tumor sensing receptor or the subunit or fragment thereof comprises GPR84, GPR18, GPR34, GPR183, C5AR1, FPR3, FPR2, LPAR2, or a combination thereof.

[0167] Embodiment 47: An embodiment of any one of embodiments 45 or 46, wherein the tumor sensing receptor or the subunit or fragment thereof comprises GPR124, GPR132, GPR137. GPR171, HCAR3, PTAFR, S1PR1, S1PR4. or a combination thereof.

[0168] Embodiment 48: An embodiment of any one of embodiments 40-47, wherein the tumor sensing receptor or the subunit or fragment thereof comprises EPHA6, ICOS, SDCBP, SLAMF1, TLN1, CRTAM, ITGAL, CD6, or any combination thereof.

[0169] Embodiment 49: An embodiment of any one of embodiments 33-48, wherein the tumor sensing enhancer protein comprises the tumor sensing protease or the subunit or fragment thereof.

[0170] Embodiment 50: An embodiment of embodiment 49, wherein the tumor sensing protease comprises a metalloprotease or a subunit or fragment thereof.

[0171] Embodiment 51: An embodiment of embodiment 50, wherein the metalloprotease or the subunit or fragment thereof comprises ADAM8, ADAM9, BMP1, CPD, MMP11, QPCT, or a combination thereof.

[0172] Embodiment 52: An embodiment of any one of embodiments 49-51, wherein the tumor sensing protease comprises a serine protease or the subunit or fragment thereof. 79556009V.1

[0173] Embodiment 53: An embodiment of embodiment 52, wherein the serine protease or the subunit or fragment thereof comprises FAP, PRSS23, HTRA2, or a combination thereof.

[0174] Embodiment 54: An embodiment of any one of embodiments 33-53, wherein the tumor sensing enhancer protein comprises the tumor sensing transcription factor or the subunit or fragment thereof.

[0175] Embodiment 55: An embodiment of embodiment 54, wherein the tumor sensing transcription factor or the subunit or fragment thereof comprises HIF1A, IFRD1, or a combination thereof.

[0176] Embodiment 56: An embodiment of any one of embodiments 33-55, wherein the tumor sensing enhancer protein comprises the tumor sensing protein phosphatase or the subunit or fragment thereof.

[0177] Embodiment 57: An embodiment of embodiment 56, wherein the tumor sensing protein phosphatase or the subunit or fragment thereof comprises PPP1CB.

[0178] Embodiment 58: An embodiment of any one of embodiments 33-57, wherein the tumor sensing enhancer protein comprises the tumor sensing ion channel protein or the subunit or fragment thereof.

[0179] Embodiment 59: An embodiment of embodiment 58, wherein the tumor sensing ion channel protein or the subunit or fragment thereof comprises CATSPER2.

[0180] Embodiment 60: An embodiment of any one of embodiments 33-59, wherein the tumor is a solid tumor.

[0181] Embodiment 61: An embodiment of embodiment 60, wherein the tumor comprises a breast cancer or a tubo-ovarian cancer.

[0182] Embodiment 62: An embodiment of any one of embodiments 33-61, wherein the engineered immune cell is an NK cell, a T cell, a dendritic cell, a monocyte, or a macrophage.

[0183] Embodiment 63: An embodiment of any one of embodiments 33-62, wherein the engineered immune cell expresses a CAR.

[0184] Embodiment 64: Am embodiment of embodiment 63, wherein the CAR targets EpCAM or HER2 79556009V.1

[0185] Embodiment 65: An embodiment of any one of embodiments 33-64, wherein the engineered immune cell expresses a TCR.

[0186] Embodiment 66: A method for treating a cancer in a subject, the method comprising: engineering an immune cell of the subject to produce an engineered immune cell that overexpresses a tumor sensing enhancer protein comprising a tumor sensing receptor that binds to a non-chemokine ligand, a tumor sensing protease, a tumor sensing transcription factor, a tumor sensing protein phosphatase, a tumor sensing ion channel protein, or a subunit or fragment thereof, wherein the engineered immune cell exhibits increased migration to or infiltration of a tumor of the subject as compared to migration to or infiltration of the tumor by a corresponding immune cell not overexpressing the tumor sensing enhancer protein.

[0187] Embodiment 67: An embodiment of embodiment 66, wherein the method further comprises: obtaining the immune cell from the subject prior to engineering the immune cell; and administering the engineered immune cell to the subject.

[0188] Embodiment 68: An embodiment of embodiment 67, wherein engineering the immune cell comprises introducing into the immune cell a polynucleotide encoding the tumor sensing enhancer protein.

[0189] Embodiment 69: An embodiment of embodiment 68, wherein introducing the polynucleotide comprises viral transduction of the polynucleotide into the immune cell.

[0190] Embodiment 70: An embodiment of any one of embodiments 67-69, wherein engineering the immune cell comprises introducing into the immune cell a guide RNA targeting a gene encoding the tumor sensing enhancer protein.

[0191] Embodiment 71: An embodiment of embodiment 66, wherein engineering the immune cell comprises administering to the subject a polynucleotide encoding the tumor sensing enhancer protein.

[0192] Embodiment 72: An embodiment of embodiment 66 or 70, wherein engineering the immune cell comprises administering to the subject a guide RNA targeting a gene encoding the tumor sensing enhancer protein.

[0193] Embodiment 73: An embodiment of any one of embodiments 66-72, wherein the engineered immune cell exhibits increased migration to the tumor as compared to migration to 79556009V.1the tumor by the corresponding immune cell not overexpressing the tumor sensing enhancer protein.

[0194] Embodiment 74: An embodiment of any one of embodiments 66-73, wherein the engineered immune cell exhibits increased infiltration of the tumor as compared to infiltration of the tumor by the corresponding immune cell not overexpressing the tumor sensing enhancer protein.

[0195] Embodiment 75: An embodiment of any one of embodiments 66-74, wherein the tumor sensing enhancer protein comprises the tumor sensing receptor or the subunit or fragment thereof.

[0196] Embodiment 76: An embodiment of embodiment 75, wherein the engineered immune cell exhibits increased migration to the tumor in a presence of the non-chemokine ligand, as compared to migration to the tumor by the corresponding immune cell not overexpressing the tumor sensing receptor.

[0197] Embodiment 77: An embodiment of embodiment 75 or 76, wherein the engineered immune cell exhibits increased infiltration of the tumor in a presence of the non-chemokine ligand, as compared to infiltration of the tumor by the corresponding immune cell not overexpressing the tumor sensing receptor

[0198] Embodiment 78: An embodiment of any one of embodiments 75-77, wherein the non- chemokine ligand comprises a metabolite.

[0199] Embodiment 79: An embodiment of any one of embodiments 75-78, wherein the non- chemokine ligand comprises a fatty acid, a lipid, or a derivative thereof.

[0200] Embodiment 80: An embodiment of any one of embodiments 75-79, wherein the tumor sensing receptor comprises a G protein-coupled receptor or a subunit or fragment thereof.

[0201] Embodiment 81: An embodiment of embodiment 80, wherein the tumor sensing receptor or the subunit or fragment thereof comprises GPR84, GPR18, GPR34, GPR183, C5AR1, FPR3, FPR2, LPAR2, or a combination thereof.

[0202] Embodiment 82: An embodiment of embodiment 80 or 81, wherein the tumor sensing receptor or the subunit or fragment thereof comprises GPR124, GPR132, GPR137. GPR171, HCAR3, PTAFR, S1PR1, S1PR4. or a combination thereof. 79556009V.1

[0203] Embodiment 83: An embodiment of any one of embodiments 75-82, wherein the tumor sensing receptor or the subunit or fragment thereof comprises EPHA6, ICOS, SDCBP, SLAMF1, TLN1, CRTAM, ITGAL, CD6, or any combination thereof.

[0204] Embodiment 84: An embodiment of any one of embodiments 66-83, wherein the tumor sensing enhancer protein comprises the tumor sensing protease or the subunit or fragment thereof.

[0205] Embodiment 85: An embodiment of embodiment 84, wherein the tumor sensing protease comprises a metalloprotease or a subunit or fragment thereof.

[0206] Embodiment 86: An embodiment of embodiment 85, wherein the metalloprotease or the subunit or fragment thereof comprises ADAM8, ADAM9, BMP1, CPD, MMP11, QPCT, or a combination thereof.

[0207] Embodiment 87: An embodiment of any one of embodiments 84-86, wherein the tumor sensing protease comprises a serine protease or a subunit or fragment thereof.

[0208] Embodiment 88: An embodiment of embodiment 87, wherein the serine protease or the subunit or fragment thereof comprises FAP, PRSS23, HTRA2, or a combination thereof.

[0209] Embodiment 89: An embodiment of any one of embodiments 66-88, wherein the tumor sensing enhancer protein comprises the tumor sensing transcription factor or the subunit or fragment thereof.

[0210] Embodiment 90: An embodiment of embodiment 89, wherein the tumor sensing transcription factor or the subunit or fragment thereof comprises HIF1A, IFRD1, or a combination thereof.

[0211] Embodiment 91: An embodiment of any one of embodiments 66-90, wherein the tumor sensing enhancer protein comprises the tumor sensing protein phosphatase or the subunit or fragment thereof.

[0212] Embodiment 92: An embodiment of embodiment 91, wherein the tumor sensing protein phosphatase or the subunit or fragment thereof comprises PPP1CB.

[0213] Embodiment 93: An embodiment of any one of embodiments 66-92, wherein the tumor sensing enhancer protein comprises the tumor sensing ion channel protein or the subunit or fragment thereof. 79556009V.1

[0214] Embodiment 94: An embodiment of embodiment 93, wherein the tumor sensing ion channel protein or the subunit or fragment thereof comprises CATSPER2.

[0215] Embodiment 95: An embodiment of any one of embodiments 66-94, wherein the tumor is a solid tumor.

[0216] Embodiment 96: An embodiment of embodiment 95, wherein the tumor comprises a breast cancer or a tubo-ovarian cancer.

[0217] Embodiment 97: An embodiment of any one of embodiments 66-96, wherein the engineered immune cell is an NK cell, a T cell, a dendritic cell, a monocyte, or a macrophage.

[0218] Embodiment 98: An embodiment of any one of embodiments 66-97, wherein the engineered immune cell expresses a CAR.

[0219] Embodiment 99: An embodiment of embodiment 98, wherein the CAR targets EpCAM or HER2.

[0220] Embodiment 100: An embodiment of any one of embodiments 66-99, wherein the engineered immune cell expresses a TCR.

[0221] Embodiment 101: A method for treating a cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a population of the engineered immune cell of any one of embodiments 1-32.

[0222] Embodiment 102: A method for determining a personalized treatment for a cancer in a subject, the method comprising: administering to the subject two or more populations of engineered immune cells, wherein: for each population of the two or more populations, the engineered immune cells of the population are engineered to overexpress a combination of one or more tumor sensing enhancer proteins that each independently comprise a tumor sensing receptor that binds to a non-chemokine ligand, a tumor sensing protease, a tumor sensing transcription factor, a tumor sensing protein phosphatase, a tumor sensing ion channel protein, or a subunit or fragment thereof; and the combination overexpressed by the engineered immune cells of each population is different from the combination overexpressed by the engineered immune cells of each other population; analyzing a biological sample obtained from the subject subsequent to the administering; and based on the analyzing, determining which one or more populations of the two or more populations of engineered immune cells exhibits a level of 79556009V.1increased migration to, or infiltration of, a tumor of the subject, wherein the level is greater than a threshold. EXAMPLES

[0223] The present disclosure will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes only, and are not intended to limit the disclosure in any manner. Example 1. Identification of metabolite-sensing GPCRs as enhancers of cell migration to breast cancer tumors.

[0224] To identify mechanisms to mobilize NK cells to solid tumors, in vivo CRISPR activation screens were designed to identify genes that significantly enhance NK cell ability to migrate to and populate breast cancer tumors in mice (FIG. 2). Screens were conducted in a clinically applicable human NK cell line (Nowakowska, P. et al., Cancer Immunol. Immunother.67, (2018): 25; Klingemann, H., Boissel, L. & Toneguzzo, F., Front. Immunol.7, (2016): 91) (NK-92) that has been widely used for chimeric antigen receptor (CAR) NK studies and in clinical trials (Boyiadzis, M. et al., Cytotherapy 19, (2017): 1225; Williams, B.A. et al., Oncotarget 8, (2017): 89256). To power the screens, we first identified genes that are more likely to drive recruitment to breast cancer tumors in patients were first identified by analyzing scRNA-seq data collected from 22 breast cancer patients with matched samples from tumor and peripheral blood (Zhang, Y. et al., Cancer Cell 39, (2021): 1578). For each immune cell type with sufficient representation in the data, genes were identified that were significantly overexpressed in the tumor compared to blood samples [Benjamini-Hochberg (BH) False Discovery Rate (FDR) < 0.05, log fold change > 0.1, MAST test (Finak, G. et al., Genome Biol. 16, (2015): 278)]. These analyses were not constrained only to NK cells because of a hypothesis that there may be genes that facilitate or drive the recruitment of other (non-NK) immune cells to solid tumors and can elicit similar effects in NK cells if expressed exogenously [e.g., via CRISPR activation or an Open Reading Frame (ORF)].

[0225] To test the candidate recruitment enhancing genes, a library of 1,070 CRISPR activation single guide RNAs (sgRNAs) was generated, where these sgRNAs target a total of 256 genes [4 sgRNAs per target gene, and 46 non-targeting control (NTC) sgRNAs], including both candidate genes identified in patients as well as additional genes with well-established roles in NK biology or immune chemoattraction and invasion, as proteases, adhesion 79556009V.1molecules, integrins, and chemokine receptors. Next, NK-92 cells were transduced to stably express CRISPR / dCas9-SAM (synergistic activation mediator) system (Konermann, S. et al., Nature 517, (2015): 583) and sgRNA-based gene overexpression at the protein level was confirmed via flow cytometry. Then, the CRISPR / dCas9-SAM NK-92 cells were transduced with the sgRNA library at a low multiplicity of infection (MOI < 0.3) to generate a pool of engineered NK cells, each carrying a single sgRNA to activate one of the 256 genes.

[0226] By intravenously transferring the pool of engineered NK cells to breast cancer tumor- bearing mice, in vivo screens were performed to identify engineered NK cells with superior ability to migrate to and infiltrate the tumors compared to control NK cells (transduced with NTC sgRNAs). The screens were performed in non-obese diabetic scid gamma (NSG) female mice engrafted with triple-negative breast cancer cells (MDA-MB-231), either subcutaneously or orthotopically in the left inguinal (4th) mammary fat pad and in transgenic NOG mice that express the human IL2 gene (hIL2-NOG) and are better at retaining NK cells. In all three models, tumor and lung samples were collected 24 hours after the NK cell adoptive transfer for genomic DNA (gDNA) extraction and sgRNA sequencing. As control samples, sequencing was performed for both the NK cell pool from which samples were drawn for in vivo transfer (denoted as the “pre-injection pool”), as well as the lung tissue samples. The lung was chosen as a control site because it has been reported to accumulate and sequester adoptively transferred tumor-reactive lymphocytes, both decreasing tumor control (Hong, Y. et al., Nat. Immunol.24, (2023): 1007; Kaur, K., Sanghu, J., Memarzadeh, S. & Jewett, A., Vaccines 12, (2024): 677; Skovgard, M.S. et al., Mol. Ther. Oncolytics 22, (2021): 355) and resulting in severe lung toxicities (Qin, D. et al., Oncoimmunol. 9, (2020): 1806009; Ahmed, E.N., Cutmore, L.C. & Marshall, J.F. Cancers 16, (2024): 3186; Morgan, R.A. et al., Mol. Ther.18, (2010): 843). All samples were processed and sequenced together and demultiplexed post-sequencing (FIG.2).

[0227] Animal-matched paired statistical data analyses was performed to identify gene activations that resulted in substantial, significant, and reproducible tumor enrichment, both compared to the lung and compared to the pre-injection NK cell pool (using MAGeCK [Li, W. et al., Genome Biol.16, (2015): 281; Li, W. et al., Genome Biol.15, (2014): 554]). Eight genes– GPR183, GPR84, GPR34, GPR18, LPAR2, FPR3, C5AR1, and CXCR2–were consistently ranked as the top hits (BH FDR < 1×10-7, Fisher test) in all three screens (FIGS.3 and 4). All the hits were initially identified as candidates based on the scRNA-seq data (Zhang, Y. et al., Cancer Cell 39, (2021): 1578) analyses, as they were significantly overexpressed in breast cancer tumors compared to blood samples in the cell types where they are endogenously 79556009V.1expressed. Similar hits were also identified in a corresponding screen for infiltration to ovarian cancer tumors (FIG.33). Based on this data C5AR1, FPR3, GPR34, and GPR84 are primarily expressed by myeloid and dendritic cells, GPR18 is mostly expressed by lymphocytes [e.g., T cells, B cells, and innate lymphoid cells (ILCs)], CXCR2 and LPAR2 show a relatively low expression across all immune cell types in this dataset, and GPR183 is expressed across a broader set of immune cell types (FIG.5).

[0228] Collectively, the screens repeatedly identified eight GPCRs as enhancers of NK-92 cell migration to breast cancer tumors in mice, with only one of them being a chemokine receptor. This prompted further investigation into the molecular mechanisms that mediate these effects. Example 2. Redirection of NK cells to migrate to chemoattracting metabolites and factors released from breast cancer cells.

[0229] According to the International Union of Basic and Clinical Pharmacology (IUPHAR) (Harding, S.D. et al., Nucleic Acids Res. 52, (2024): D1438) classification, all eight hits identified in the screens of Example 1 are GPCRs coupled with Gi / oprotein, a primary signal transducer for most conventional chemokine receptors. Yet, unlike chemokine receptors, five out of the eight hits—GPR183, GPR84, GPR34, GPR18, and LPAR2—have been reported to bind and respond to bioactive metabolites, including lipid, fatty acid, and cholesterol derivatives (Wang, X. et al., Immunity 54, (2021): 1123; McHugh, D. et al., BMC Neurosci. 11, (2010): 44; Wang, J. et al., J. Biol. Chem. 281, (2006): 34457; Liu, C. et al., Nature 475, (2011): 519).

[0230] Focusing on GPR183, GPR84, and GPR34, examinations were conducted to determine whether these GPCRs sense bioactive metabolites and redirect NK cell migration to these metabolites. First, using the PRESTO-tango system (Kroeze, W.K. et al., Nat. Struct. Mol. Biol.22, (2015): 362), reporter lines were generated for these three hits as well as C5AR1 and CXCR2 (top hits marking well-established complement- and chemokine-based chemotaxis that were used here as controls). Using the PRESTO-tango reporter lines, where the conformational change of the GPCR in response to its agonist ligand is converted to abioluminescence signal (FIG. 6), results confirmed that 7 ,25-dihydroxycholesterol (7 ,25-OHC), lysophosphatidylserine (LysoPS), 6-OAU, complement component 5a (C5a), and interleukin 8 (IL8) are the agonists of GPR183, GPR34, GPR84, C5AR1, and CXCR2, respectively (FIG. 7). Then, NK-92 lines were generated that constitutively express each one 79556009V.1of these hits via CRISPR activation or ORF. These syngeneic NK-92 lines were used to track and quantify migration to the different agonists, showing GPR183-, GPR34-, GPR84-, C5AR1-, and CXCR2- dependent migration to 7 ,25-OHC, LysoPS, medium 6-OAU, C5a, and IL8,respectively (FIG.8).

[0231] Thus, it was hypothesized that the identified hits surface a different form of immune cell recruitment to the tumor that is based on chemoattracting metabolites released from the cancer cells. To test this hypothesis and systematically identify genes that enhance NK cell chemoattraction to breast cancer cells, two additional in vitro screens were performed (FIG.9). The first in vitro screen was designed to identify perturbations that enhance chemotaxis to factors released from breast cancer cells (MCF7 and MDA-MB-231) using a transwell device (FIGS. 9 and 10). The second in vitro screen was conducted by coculturing the pool of genetically modified NK cells with breast cancer (MCF7) spheroids and selecting for the NK cells that migrated and penetrated to the spheroids within 6, 24, and 48 hours (FIGS.9 and 11). All eight hits identified in the in vivo screen were among the top 10 hits in both the chemotaxis and spheroid in vitro screens (FIGS.10 and 11). The chemotaxis screen identified several other hits, including the chemokine receptor CXCR3, the integrins ITGAD, ITGAL, ITGAX, and ITGAM, which are known for their role in lymphocyte adhesion and transmigration across blood vessels, and EPHA1, which is known for its role in NK cell migration (Darling, T.K. & Lamb, T.J., Front. Immunol. 10, (2019): 1473). The only other hits identified in the spheroid screen were ICOS and ITGAL.

[0232] Collectively, these findings demonstrate that the GPCRs identified as enhancers of NK cell migration to breast cancer tumors in mice are enhancing NK cell ability to sense and migrate to chemoattracting metabolites and soluble factors released by breast cancer cells. Example 3. Alteration of the NK cell transcriptome and effector functions with GPR183.

[0233] Tumor metabolism has often been reported to be immunosuppressive, with a recent study demonstrating that LysoPS prevents type 1 ILCs (ILC1s) activation via GPR34 (Yan, J. et al., Nat. Immunol. 25, (2024): 2057). Thus, while the screens of Example 1 selected for perturbations that enhance NK cell migration to breast cancer tumors, there is no guarantee that these perturbations will not interfere with NK cell effector functions, surfacing the need to determine the impact of the hits on NK cell state and functionality. As GPCR signaling results in transcriptional regulation, additional experiments were conducted to examine if and how the hits impact the NK cell transcriptome by conducting a Perturb-seq (Dixit, A. et al., Cell 167, 79556009V.1(2016): 1853; Replogle, J.M. et al., Nat. Biotechnol. 38, (2020): 954) screen, where scRNA- seq was combined with matched sgRNA detection at the single cell level in NK-92 cells 159 (FIG. 12). The Perturb-seq data confirmed on-target gene activation and demonstrated that GPR183, C5AR1, and CXCR2 CRISPR activation result in a substantial impact on the NK-92 cell transcriptome, with hundreds of differentially expressed genes in comparison to the control NK-92 cells (transduced with NTC sgRNAs; FIGS.13 and 14).

[0234] NK cells with GPR183 CRISPR activation (denoted as GPR183A+) overexpressed genes involved in NK cell cytotoxicity (e.g., FASLG encoding Fas ligand, GZMB encoding granzyme B, PRF1 encoding for perforin, ICAM1, IFNG, NFAT5, SH2D1B, and SH3BP2) and chemokines (CCL18, CCL3, CCL3L1, CCL4, CCL4L2, CCL5; FIGS. 13 and 14), suggesting that GPR183 CRISPR activation may also support NK cell cytotoxicity and ability to recruit other immune cells to the tumor, respectively. Aligned with GPR183 ligand being a form ofoxidized cholesterol (7 ,25-OHC), the genes downregulated in GPR183A+ cells were enrichedfor cholesterol and sterol biosynthesis genes (P < 1×10-10, e.g., ACLY, CNBP, CYP51A1, DHCR7, FDFT1, FDPS, HMGCS1, IDI1, INSIG1, LSS, MSMO1, MVD, SQLE, SERBP1, and TM7SF2), as well as oxidative phosphorylation, chromatin organization and other gene sets (FIGS.13 and 14).

[0235] These transcriptional readouts indicated that GPR183 does not disrupt and may enhance NK cell cytotoxicity and effector functions, as we turned to examine via functional readouts. Aligned with the transcriptional data, GPR183A+NK cells show a more specific and efficacious response to stimuli compared to control NK cells. GPR183A+NK cells exhibitedincreased IFN secretion and cytotoxicity in coculture with MDA-MB-231 breast cancer cells(FIG. 15). Upon PMA / ionomycin stimulation, GPR183A+NK cells also show a significant increase in degranulation, as indicated by the surface expression of CD107a—a membrane-bound molecule commonly used as a proxy for cytotoxic degranulation—and IFN production(FIGS. 16 and 17). Interestingly, MDA-MB-231 cells express the genes encoding oxysteroland 7 ,25-OHC synthesizing enzymes and overexpress them in response to IFN and TNF,indicating a potential positive regulation across GPR183A+NK and cancer cells. Importantly,GPR183A+ cells show minimal degranulation and IFN production at baseline (FIG. 17) andproliferate at similar rates to those of control NK cells, as quantified via cell-trace dye assays over 4 days (FIG. 18). These features are particularly important due to the potential risks associated with cell therapies, including cytokine release syndrome (Morris, E.C., Neelapu, S.S., Giavridis, T. & Sadelain, M., Nat. Rev. Immunol. 22, (2022): 85) and malignant 79556009V.1transformation of the transferred immune cells (Verdun, N. & Marks, P., N. Engl. J. Med.390, (2024): 584).

[0236] Given these findings, further studies focused on GPR183 to examine whether metabolite-sensing GPCRs can make engineered lymphocytes spatially targeted and more efficacious in vivo. Example 4. Enhancement of directional migration of NK and CD8 T cells to breast cancer via GPR183 activation.

[0237] Given the biochemical and biophysical properties of GPR183, its association with tumor infiltration in multiple cell types, including CD8 T cells (FIG.5), and the high degree of transcriptional similarity between NK cells and CD8 T cells, additional experiments were conducted to investigate GPR183-mediated directional migration in NK-92, as well as human primary NK cells and human primary CD8 T cells. In vitro, GPR183A+NK-92 showed a significant increase in cell migration to MDA-MB-231 supernatant and breast cancer tumor lysate compared to control NK-92 cells (FIGS. 19 and 20). To test GPR183 in primary NK cells, primary NK cells from human peripheral blood mononuclear cells (PBMCs) were isolated and expanded using bead-based isolation and a feeder-cell free expansion method (Huang, R.-S., Lai, M.-C., Shih, H.-A. & Lin, S., J. Exp. Med.218, (2021): e20201529), which resulted in highly enriched CD56+CD3- NK cells at > 92%. Then the primary NK cells were transduced using spin-infection with high titer with either GPR183 ORF or control lentivirus (FIG. 21). As observed in NK-92 cells, GPR183A+primary NK cells exhibited significantlyincreased migration to 7 ,25-OHC compared to control NK cells from the same donor, whichwere transduced with the control lentivirus (FIG.22). To test GPR183 in primary CD8 T cells, CD8 T cells were isolated from human PBMCs and transduced with either GPR183 ORF orcontrol lentivirus (FIG. 23). GPR183A+ primary T cells displayed increased migration to 7 ,25-OHC (FIG. 24), MDA-MB-231 supernatant (FIG. 25), and breast cancer tumor lysate (FIG. 26) compared to control CD8 T cells from the same donor, which were transduced with the control lentivirus. Focusing on NK-92 for in vivo testing, GPR183A+NK-92 cells intravenously transferred into MDA-MB-231 xenograft-bearing mice were found to be significantly more abundant in the tumors compared to control NK-92 cells (FIG.27). 79556009V.1Example 5. Improvement to breast cancer tumor control resulting from GPR183 activation in NK-92 and CAR NK-92.

[0238] As insufficient recruitment and infiltration is a limiting factor of cell therapies in solid tumors, additional experiments were conducted to examine the efficacy of GPR183A+NK cells in controlling breast cancer tumors in vivo (FIG.28). First, intravenous transfer of GPR183A+NK-92 cells to NSG female mice bearing MDA-MB-231 xenografts significantly delayed tumor growth compared to both untreated mice (i.e., mice treated with cell-free media) and mice treated with control NK cells (i.e., NK-92 cells transduced with control lentivirus; FIG. 29), such that the same regimen of control NK-92 cell transfer had no impact on tumor growth. Second, to test whether GPR183 activation can enhance the in vivo efficacy of CAR NK cells, we cloned a CAR construct that targets EpCAM (epithelial cell adhesion molecule) on the surface of breast cancer cells (FIG.30) and co-introduced the construct (anti-human EpCAM- CAR-GFP) with GPR183-ORF-mCherry or empty-ORF-mCherry into NK-92 cells, resulting in antigen-specific cell cytotoxicity. Intravenous transfer of GPR183A+CAR-NK-92 showed significantly better control of MDA-MB-231 tumor growth compared to both the untreated group (i.e., treated with cell-free media) and the group treated with control (i.e., empty-ORF- mCherry) CAR-NK-92 cells, demonstrating the synergistic effect of CAR and GPR183 (FIG. 31). Together, these experiments demonstrate that engineering NK cells to express GPR183 provides a mechanism to enhance NK cell antitumor efficacy in vivo. Example 6. Production of TSR ligands by BC or T / NK cells.

[0239] TSRs are activated in primary T / NK cells using CRISPRa and ORFs expression constructs, and differential chemotaxis of TSR+vs. control cells, as well as the GPCR PRESTO-tango report system are used to test the following TSR ligand and ligand sources: (1) commercially available TSR ligands (medium chain free fatty acids (C9 to C14) for CPR84; N-arachidonyl glycine (NAGly) and Resolvin-D2 (derived from omega-3 fatty acids) forGPR18; Lysophosphatidylserine (LysoPS) for GPR34; oxysterols and 7 ,25-dihydroxycholesterol (7 ,25-OHC) for GPR183; and Complement component 5a (C5a) forC5Ar1) tested at various concentrations (2) different BC cell lines (MCF7, SKBR3, MDA- MB-231, and BT549) cultured in standard monoculture or following treatment with IFN / TNF (40 ng / μl) or apoptosis inducers (e.g., doxorubicin); (3) T / NK cells with and without chemokine stimulation (CXCL8, CXCL12, and CXCL16); and (4) T cells with and without CD3 / CD28 beads for TCR activation. In addition, TSR activation is combined with CXCR2, 79556009V.1CXCR6, or CXCR4 activation / KO in the same T / NK cells to test for synergy in enhancing CXCL8, CXCL16, or CXCL12 mediated chemotaxis, respectively. Examinations are conducted to determine if there is a significantly synergistic effect when activating a TSR together with a chemokine receptor in response to the respective chemokine via drugZ, mixed effects, and maximum likelihood estimation methods.

[0240] Primary CD8+T cells and primary NK cells are isolated from blood samples (buffy coat) of healthy donors and expanded. T cells are activated with CD3 / CD28 beads for expansion and transduced to express a TCR to target specific antigens (e.g., NY-ESO1, mesothelin, EGFR), which are expressed by the BC cells, either endogenously or via ORF lentivirus transduction. T / NK cells are transduced with lentivirus to stably express CRISPR / dCas9 SAM system, with the MPH activator helper complex. T / NK cells stably expressing dCas9-MPH are transduced with TSR sgRNAs to generate lines with a stable activation of one the TSRs or with non-targeting control (NTC) sgRNAs. In an alternative approach, T / NK cells are transduced with lentivirus to express one of the different ORF isoforms of the five TSRs or with a control ORF lentivirus. Both CRISPRa and ORF edited T / NK cells are sorted via fluorescence-activated cell sorting (FACS) with an antibody targeting the pertaining TSR to obtain a homogeneous cell population with confirmed TSR protein expression on the cell surface.

[0241] TSR+vs. control T / NK cells chemotaxis is tracked via devices that maintain a stable gradient for up to 48 hours and allow tracking of cell locomotion along the gradient (e.g., - Slide). Time-lapse video microscopy is conducted for 48 hours and analyzed to quantify chemotaxis and distinguish it from chemokinesis (i.e., random migration) using ImageJ and a Python / R package that is developed for this purpose. Cells that have migrated to the target site are collected after 6, 24, and 48 hours. These are analyzed via flow cytometry, microplate reader for cell counting, and NGS to quantify the relative abundance of cells with different perturbations compared to control cells. Boyden chamber is used for validation.

[0242] TSR+vs. control T / NK cell responses are also determined by measuring cAMPsynthesis inhibition (G i / o), mobilization of intracellular calcium (Ca2+), andRhoA / Rac1 / Cdc42 GTPases activation. HEK293 PRESTO-Tango reporter cells that were optimized are transduced with the PRESTO-Tango constructs of each of the TSRs to detect agonists via bioluminescence or fluorescence. Confirmed TSR ligands are measured in the different cell supernatant samples using liquid chromatography (LC) linked to mass 79556009V.1spectrometry (MS) (LC-MS). If migrating or activated T / NK cells secrete TSR ligands, then combined TSR and chemokine receptors activation will be synergistic and can be used to design cell migration circuits. If BC cells secrete TSR ligands, then direct testing of TSR signaling (e.g., using PRESTO-tango) in response to BC cells can form a biomarker for the efficacy of TSR-engineered T / NK cells. Example 7. TSR activation to increase T / NK cell migration, expansion, and function in a diverse set of 3D BC spheroids.

[0243] The dynamics of clonally labeled engineered T / NK cells migration, expansion, and destruction of BC spheroids is tracked via clonal labeling and live cell imaging. All experiments are done separately in T and NK cells. Clonal labeling is achieved using a highdiversity barcoding system. The barcode consists of a random 28-mer in the 5 untranslatedregion of an enhanced GFP trans-gene under control of a ubiquitous EF1a promoter. Edited T / NK cells with activation of one of the five TSRs, positive / negative control genes (e.g., CXCR2, PD-1, TIM-3), or control sgRNAs / ORFs are barcoded separately and allowed to double for 2 cycles. A subsample of the barcoded population of each sample is sequenced to determine the barcode-to-perturbation mapping before pooling per cell type. Clonally labeled T / NK cells are co-cultured with four different types of 3D BC spheroids: MCF7, SKBR3, MDA-MB-231, and BT549. BC spheroids are collected at different time points (6, 24, and 48 hours) and NGS is used to measure the number and size of each clone for each perturbation, both within and outside the spheroids at each time point. The NGS-based readouts are compared to live cell imaging that is used to track fluorescently labeled T / NK cells location and expansion within and outside the spheroid, and to track cytotoxicity via live / dead cell staining and spheroid destruction. Cytotoxicity is also tested in 2D co-cultures.

[0244] Clonal barcoding allows for the determination of TSR impact on migration and expansion based on the number and size of clones within and outside the spheroid, with further validations via live cell imaging. Quantifying of BC cell killing confirms that TSR activation does not disrupt effector cytotoxic functions. TSRs increase migration (number of clones) with some increase in expansion (clone size), leading to improvement in cytotoxicity in 3D co- cultures. Example 8. Control of GPR84 ligand synthesis or release by APMAP.

[0245] GPR84 activation has been shown to enhance macrophage phagocytosis, and cancer cells resist this enhanced phagocytosis via expression of a poorly characterized enzyme called 79556009V.1APMAP (adipocyte plasma membrane-associated protein). Given APMAP homology to the paraoxonase family of lipid hydrolases, APMAP may hydrolyze and / or prevent the accumulation of lipids that function as agonists of GPR84. Alternatively, because GPR84 is desensitized at high levels of its ligands, it is also possible that APMAP increases the release or production of the GPR84 ligands.

[0246] The combinatorial effects of GPR84 activation are tested in T / NK cells in conjunction with activation vs. KO of APMAP in BC cells via the chemotaxis, PRESTO-Tango, and spheroid models described above. Similarly, genes involved in the synthesis or degradation / deactivation of TSR ligands are probed via CRISPRa and KO in the BC or T / NK cells and studied in a similar manner. Example 9. TSR activation in T / NK cells to improve T / NK cell ability to migrate and expand in BC tumors.

[0247] Xenotransplantation of human BC lines (MCF7 and MDA-MB-231) is done via orthotopic inguinal (4th) mammary fat pad injection in a cohort of 6-8-week-old hIL-2 NOG female mice (PrkdcscidIl2rgtm1SugTg(CMV-IL2)4-2Jic / JicTac; hIL-2 NOG mice are immunocompromised but express human IL2, thus providing an improved model to study human T / NK cells in human tumor bearing mice). T cells are transduced to express a TCR for an antigen that the cancer cells express endogenously or via transduction (e.g., NY-ESO1, MSLN, EGFR). Syngeneic EO771-OVA+BC cells (i.e., EO771 cells that are transduced to express the ovalbumin antigen) are orthotopically engrafted to the inguinal mammary fat pad of immunocompetent CD45.1 C57BL / 6 female mice. OT1 CD45.2 CD8+T cells are modified to express one of the five TSR mouse orthologs and used in this immunocompetent syngeneic model. In both models, the mice are blindly split to two groups, one for T cell experiments, and another for NK experiments. Once tumors become palpable, mouse body weights and caliper measurements of tumor size are recorded every 3 days. Two pools of edited and clonally barcoded T / NK cells (one for each cell type) are generated as described above, such that each cell has an activation of one of the TSRs or a control sgRNA / ORF. The T or NK cell pool are injected in the mice intravenously (IV) after 2-3 weeks from tumor engraftment, or when the tumors reach a size of 100 mm3. One group of mice is euthanized 3 days following the T / NK cell injection, and another after 2 weeks. Tumor, spleen, lung, and blood samples are collected from each mouse and used for NGS and single cell readouts, as described below. 79556009V.1

[0248] Genomic DNA is extracted from each sample followed by 2-step PCR to amplify the barcodes. The samples are indexed and pooled to a single library for NGS. NGS readouts are used to determine for each TSR whether it significantly increased the number and size of engineered T / NK cell clones in BC tumors compared to those in other tissues and blood.

[0249] NGS data is demultiplexed, the number of reads and number of unique clonal barcodes representing each sgRNA / ORF is quantified to determine the relative number of cells carrying each perturbation in each sample and their clonal identity. MAGECK, casTLE, and mixed-effects statistical models that are developed for this purpose are used to quantify the statistical significance (BH FDR) and effect size of each of the five TSRs in increasing the number and size of engineered T / NK cell clones compared to control T / NK cells in the tumor. The T / NK cell pools before injection (t0) and non-tumor samples (spleen, lung, and blood) provide the null distributions. Experiments and analyses are done per cell type (T / NK) and per model (MCF7, MDA-MB-231, and EO771) and combined to summary statistics. Example 10. TSR activation in T / NK cells to increase the abundance of adopted cytotoxic effector T / NK cells in BC tumors.

[0250] The pool of clonally labeled engineered T / NK cells from the tumor, spleen, lung, and blood samples are sorted and sequenced, as well as the T / NK cells before injection via ECCITE-seq (Expanded CRISPR-compatible cellular indexing of transcriptomes and epitopes by sequencing) for multimodal single cell sequencing of transcriptome, surface protein,perturbation identifier (sgRNA / ORF direct capture), and clonal barcodes. Five prime (5 )droplet-based single cell sequencing (10x genomics) is used with direct capture of the sgRNA protospacer, clonal barcodes, and antibody barcodes. Surface proteins including the TSR proteins, are profiled via antibodies conjugated to oligonucleotide barcodes.

[0251] Data is processed using cellranger and Seurat pipelines and an R package (scTrack) that is developed for this purpose. Clonal barcodes are used to determine and control for the impact of “lineage” on cell states and examine if all the cells with a specific TSR activation show a consistent and generalizable “transcriptional reprogramming” (across and within clones). Differentially expressed genes and proteins are identified per tissue per TSR compared to the control cells using standard methods and multilevel hierarchical modeling with interaction terms to determine TSR-driven T / NK transcriptional reprogramming as a function of tissue type. Curated T and NK cell state signatures derived from pan-cancer and BC single cell studies are used to examine different cell states (e.g., exhausted, cytotoxic, memory, 79556009V.1cycling, etc.). Because GPR84, GPR34, and C5AR1 are mostly expressed by macrophages, it is also determined if macrophage-specific genes are up-regulated in TSR+ vs control T / NK cells. Example 11. TSR activation in T / NK cells to increases T / NK cell infiltration and spreading within the tumor.

[0252] To track the location and microenvironment of engineered T / NK cells within the tumor, in situ Perturb-Seq is applied for spatial transcriptomics with matching multiplexed sgRNA / ORF detection. Tumor tissues are preserved as FFPE (formalin-fixed paraffin- embedded), sectioned to 5 μm sections, and processed on the CosMx instrument with the customized in situ Perturb-CosMx probe panel (6,000 genes, 64 proteins, and 35 sgRNA / ORF barcodes). For comparison, in situ Perturb-Seq is also applied to T / NK-BC spheroids as described above using similar embedding and sectioning.

[0253] A computational pipeline is developed for end-to-end analysis of in situ Perturb-Seq data. In brief, cell segmentation is performed on raw immunofluorescent images of nuclear and cell membrane staining using a deep-learning based image processing algorithm, Mesmer (DeepCell pipeline). Each cell is defined based on its gene expression and microenvironment composition at a range of radiuses (10 μm, 30 μm, and 50 μm). Data is normalized, and annotated for cell types and subtypes via a recursive clustering-based cell type annotation procedure and a distributionally robust clustering method (Yeh et al., biorxiv 2023, under review). In situ data is then combined with matching ECCITE-seq data via co-embedding methods as RPCA (reciprocal principal component analysis) and scANVI (single-cell Annotation using Variational Inference). Lastly, differentially expressed genes and microenvironments are identified per TSR per cell type (i.e., T or NK cells) using an extension of the computational method DIALOGUE developed for identification of multicellular modules. Example 12. GPR84 activation to increases T / NK cell ability to control BC tumors and recruit other immune cells to the tumor.

[0254] Immunocompromised female mice (hIL-2 NOG) with human BC xenografts (MCF7 or MDA-MB-231) are split to five experimental groups for IV injection of either GPR84+CD8 T cells, GPR84+NK cells, NTC CD8 T cells, NTC NK cells, or no cell transfer. GPR84+denotes cells where GPR84 was activated. All T cells are transduced and confirmed to express a TCR matching a cancer cell antigen. Likewise, immunocompetent CD45.1 C57BL / 6 female 79556009V.1mice with engraftment of EO771-OVA+ cells are split to three groups, with IV injection of either CD45.2 OT1 / GPR84+cells, CD45.2 OT1 / NTC cells, or no cell transfer. Cancer cells are engrafted, and T / NK cells are transferred as described above. Mice are monitored for survival and tumor growth for up to 6 months from the time of tumor engraftment. Additionally, live T / NK cell tracking experiments are conducted with T / NK cells expressing luciferase to detect the cells in the mice via bioluminescence in vivo imaging. Imaging is done every 3 days. Five mice per group are euthanized 2 weeks after T / NK cell injections, and the tissues are used to construct a TMA for in situ Perturb-Seq profiling.

[0255] Paired t-tests are performed to compare the tumor growth in each time point across the different groups and at the endpoint. Survival analysis is done via standard log-rank and COX regression test, using Kaplan-Meier estimator to generate survival curves and to estimate median survival values. Spatial data processing and analysis is done as described above, with comparisons across different experimental groups. The composition and architecture of tumors is compared to determine if GPR84+T / NK cells are significantly better at populating the BC tumor and recruiting other endogenous effector immune cells compared to the control T / NK cells and no cell transfer. The endogenous response is tested primarily in the syngeneic model where endogenous adaptive immune responses are expected to be activated, as further described below. Example 13. GPR84 activation to enhance T / NK cell ability to unleash endogenous antitumor immune responses.

[0256] The increased recruitment of GPR84+T cells to the tumor can lead to secondary recruitment of antigen presenting cells and endogenous T cells, thus leading to wider elimination and protection against cancer cells even if these do not express the specific (neo)antigen (in this case, ovalbumin) that the transferred T cells target. This applies also to a setting where a CAR is used instead of a TCR, as some cancer cells may not express the CAR target.

[0257] CD45.1 C57BL / 6 female mice are engrafted with two different tumors in the two contralateral inguinal mammary fat pads. One tumor includes a pool of EO771-OVA+and EO771 cells (without ovalbumin expression), and the other tumor includes only EO771 cancer cells. The anti-tumor immune response or rejection of the ovalbumin negative tumor is unlikely to result from the transferred OT1 cells and thus is used to examine the endogenous immune response in the mice with transfer of CD45.2 OT1 / NTC, OT1 / GPR84+, and no OT1. 79556009V.1

[0258] Survival, tumor growth, and tumor tissue remodeling is analyzed and compared across the three groups, this time monitoring and analyzing two tumors per mouse. In addition, CD45.1 T cells are sorted from the tumors, spleen, lung, and blood for TCR sequencing to examine TCR clonal expansion and tumor reactivity in co-culture with EO771 cells.

[0259] Although the foregoing disclosure has been described in some detail by way of illustration and example for purpose of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications within the spirit and scope of the disclosure may be practiced, e.g., within the scope of the appended claims. It should also be understood that aspects of the disclosure and portions of various recited embodiments and features can be combined or interchanged either in whole or in part. In the foregoing descriptions of the various embodiments, those embodiments which refer to another embodiment may be appropriately combined with other embodiments as will be appreciated by one of skill in the art. Furthermore, those of ordinary skill in the art will appreciate that the foregoing description is by way of example only and is not intended to limit the disclosure. In addition, each reference provided herein is incorporated by reference in its entirety for all purposes to the same extent as if each reference was individually incorporated by reference. 79556009V.1

Claims

WHAT IS CLAIMED IS:

1. An engineered immune cell genetically modified to overexpress a tumor sensing enhancer protein, the tumor sensing enhancer protein comprising a tumor sensing receptor that binds to a non-chemokine ligand, a tumor sensing protease, a tumor sensing transcription factor, a tumor sensing protein phosphatase, a tumor sensing ion channel protein, or a subunit or fragment thereof, wherein the engineered immune cell exhibits increased migration to or infiltration of a tumor as compared to migration to or infiltration of the tumor by a corresponding immune cell not overexpressing the tumor sensing enhancer protein.

2. The engineered immune cell of claim 1, wherein the engineered immune cell exhibits increased migration to the tumor as compared to migration to the tumor by the corresponding immune cell not overexpressing the tumor sensing enhancer protein.

3. The engineered immune cell of claim 1, wherein the engineered immune cell exhibits increased infiltration of the tumor as compared to infiltration of the tumor by the corresponding immune cell not overexpressing the tumor sensing enhancer protein.

4. The engineered immune cell of claim 1, wherein the tumor sensing enhancer protein comprises the tumor sensing receptor or the subunit or fragment thereof.

5. The engineered immune cell of claim 4, wherein the engineered immune cell exhibits increased migration to the tumor in a presence of the non-chemokine ligand, as compared to migration to the tumor by the corresponding immune cell not overexpressing the tumor sensing receptor.

6. The engineered immune cell of claim 4, wherein the engineered immune cell exhibits increased infiltration of the tumor in a presence of the non-chemokine ligand, as compared to infiltration of the tumor by the corresponding immune cell not overexpressing the tumor sensing receptor.

7. The engineered immune cell of claim 4, wherein the non-chemokine ligand comprises a metabolite.

8. The engineered immune cell of claim 4, wherein the non-chemokine ligand comprises a fatty acid, a lipid, or a derivative thereof. 79556009V.

19. The engineered immune cell of claim 4, wherein the tumor sensing receptor comprises a G protein-coupled receptor or a subunit or fragment thereof.

10. The engineered immune cell of claim 9, wherein the tumor sensing receptor or the subunit or fragment thereof comprises G protein-coupled receptor 183 (GPR183), GPR84, GPR34, GPR18, lysophosphatidic acid receptor 2 (LPAR2), complement component 5a receptor 1 (C5AR1), formyl peptide receptor 3 (FPR3), FPR2, or a combination thereof.

11. The engineered immune cell of claim 9, wherein the tumor sensing receptor or the subunit or fragment thereof comprises GPR124, GPR132, GPR137. GPR171, hydroxycarboxylic acid receptor 3 (HCAR3), platelet activating factor receptor (PTAFR), sphingosine-1-phosphate receptor 1 (S1PR1), S1PR4. or a combination thereof.

12. The engineered immune cell of claim 4, wherein the tumor sensing receptor or the subunit or fragment thereof comprises ephrin type-A receptor 6 (EPHA6), inducible T-cell costimulator (ICOS), syndecan binding protein (SDCBP), signaling lymphocytic activation molecule family member 1 (SLAMF1), talin-1 (TLN1), cytotoxic and regulatory T cell molecule (CRTAM), integrin-alpha L (ITGAL), cluster of differentiation 6 (CD6), or any combination thereof.

13. The engineered immune cell of claim 1, wherein the tumor sensing enhancer protein comprises the tumor sensing protease or the subunit or fragment thereof.

14. The engineered immune cell of claim 13, wherein the tumor sensing protease comprises a metalloprotease or a subunit or fragment thereof.

15. The engineered immune cell of claim 14, wherein the metalloprotease or the subunit or fragment thereof comprises a disintegrin and metalloproteinase domain- containing protein 8 (ADAM8), ADAM9, bone morphogenetic protein 1 (BMP1), carboxypeptidase D (CPD), matrix metallopeptidase 11 (MMP11), glutaminyl-peptide cyclotransferase (QPCT), or a combination thereof.

16. The engineered immune cell of claim 13, wherein the tumor sensing protease comprises a serine protease or a subunit or fragment thereof. 79556009V.

117. The engineered immune cell of claim 16, wherein the serine protease or the subunit or fragment thereof comprises fibroblast activation protein-alpha (FAP), serine protease 23 (PRSS23), high-temperature requirement serine protease 2 (HTRA2), or a combination thereof.

18. The engineered immune cell of claim 1, wherein the tumor sensing enhancer protein comprises the tumor sensing transcription factor or the subunit or fragment thereof.

19. The engineered immune cell of claim 18, wherein the tumor sensing transcription factor or the subunit or fragment thereof comprises hypoxia-inducible factor 1- alpha (HIF1A), interferon related developmental regulator 1 (IFRD1), or a combination thereof.

20. The engineered immune cell of claim 1, wherein the tumor sensing enhancer protein comprises the tumor sensing protein phosphatase or the subunit or fragment thereof.

21. The engineered immune cell of claim 20, wherein the tumor sensing protein phosphatase or the subunit or fragment thereof comprises protein phosphatase 1 catalytic subunit beta (PPP1CB).

22. The engineered immune cell of claim 1, wherein the tumor sensing enhancer protein comprises the tumor sensing ion channel protein or the subunit or fragment thereof.

23. The engineered immune cell of claim 22, wherein the tumor sensing ion channel protein or the subunit or fragment thereof comprises cation channel sperm associated 2 (CATSPER2).

24. The engineered immune cell of claim 1, wherein the tumor is a solid tumor.

25. The engineered immune cell of claim 24, wherein the tumor comprises a breast cancer or a tubo-ovarian cancer. 79556009V.

126. The engineered immune cell of claim 1, wherein the engineered immune cell is a natural killer (NK) cell, a T cell, a dendritic cell, a monocyte, or a macrophage.

27. The engineered immune cell of claim 1, wherein the engineered immune cell expresses a chimeric antigen receptor (CAR).

28. The engineered immune cell of claim 27, wherein the CAR targets EpCAM or HER2.

29. The engineered immune cell of claim 1, wherein the engineered immune cell expresses an engineered T-cell receptor (TCR).

30. The engineered immune cell of claim 1, wherein the engineered immune cell comprises a CRISPR activation system effecting overexpression of the tumor sensing enhancer protein or the subunit or fragment thereof by the engineered immune cell.

31. The engineered immune cell of claim 1, wherein the engineered immune cell comprises an exogenous gene encoding the tumor sensing enhancer protein or the subunit or fragment thereof.

32. The engineered immune cell of claim 1, wherein the engineered immune cell is genetically modified to overexpress two or more tumor sensing enhancer proteins.

33. A method for producing an engineered immune cell, the method comprising: providing an immune cell; and introducing into the immune cell a polynucleotide encoding a tumor sensing enhancer protein, thereby producing the engineered immune cell; wherein the tumor sensing enhancer protein comprises a tumor sensing receptor that binds to a non-chemokine ligand, a tumor sensing protease, a tumor sensing transcription factor, a tumor sensing protein phosphatase, a tumor sensing ion channel protein, or a subunit or fragment thereof; and wherein the engineered immune cell exhibits increased migration to or infiltration of a tumor as compared to migration to or infiltration of the tumor by a corresponding immune cell not overexpressing the tumor sensing enhancer protein. 79556009V.

134. The method of claim 33, wherein introducing the polynucleotide comprises viral transduction of the polynucleotide into the immune cell.

35. A method for producing an engineered immune cell, the method comprising: providing an immune cell comprising a gene encoding a tumor sensing enhancer protein; and introducing into the immune cell a guide RNA targeting the gene, thereby producing the engineered immune cell; wherein the tumor sensing enhancer protein comprises a tumor sensing receptor that binds to a non-chemokine ligand, a tumor sensing protease, a tumor sensing transcription factor, a tumor sensing protein phosphatase, a tumor sensing ion channel protein, or a subunit or fragment thereof; and wherein the engineered immune cell exhibits increased migration to or infiltration of a tumor as compared to migration to or infiltration of the tumor by a corresponding immune cell not overexpressing the tumor sensing enhancer protein.

36. The method of claim 35, wherein the gene is an endogenous gene of the immune cell.

37. The method of claim 35, wherein the gene is an exogenous gene, and wherein the method further comprises introducing the exogenous gene to the immune cell.

38. The method of claim 33, wherein the engineered immune cell exhibits increased migration to the tumor as compared to migration to the tumor by the corresponding immune cell not overexpressing the tumor sensing enhancer protein.

39. The method of claim 33, wherein the engineered immune cell exhibits increased infiltration of the tumor as compared to infiltration of the tumor by the corresponding immune cell not overexpressing the tumor sensing enhancer protein.

40. The method of claim 33, wherein the tumor sensing enhancer protein comprises the tumor sensing receptor or the subunit or fragment thereof.

41. The method of claim 40, wherein the engineered immune cell exhibits increased migration to the tumor in a presence of the non-chemokine ligand, as compared to 79556009V.1migration to the tumor by the corresponding immune cell not overexpressing the tumor sensing receptor.

42. The method of claim 40, wherein the engineered immune cell exhibits increased infiltration of the tumor in a presence of the non-chemokine ligand, as compared to infiltration of the tumor by the corresponding immune cell not overexpressing the tumor sensing receptor.

43. The method of claim 40, wherein the non-chemokine ligand comprises a metabolite.

44. The method of claim 40, wherein the non-chemokine ligand comprises a fatty acid, a lipid, or a derivative thereof.

45. The method of claim 40, wherein the tumor sensing receptor comprises a G protein-coupled receptor or a subunit or fragment thereof.

46. The method of claim 45, wherein the tumor sensing receptor or the subunit or fragment thereof comprises GPR183, GPR84, GPR34, GPR18, LPAR2, C5AR1, FPR3, FPR2, or a combination thereof.

47. The method of claim 45, wherein the tumor sensing receptor or the subunit or fragment thereof comprises GPR124, GPR132, GPR137. GPR171, HCAR3, PTAFR, S1PR1, S1PR4. or a combination thereof.

48. The method of claim 40, wherein the tumor sensing receptor or the subunit or fragment thereof comprises EPHA6, ICOS, SDCBP, SLAMF1, TLN1, CRTAM, ITGAL, CD6, or any combination thereof.

49. The method of claim 33, wherein the tumor sensing enhancer protein comprises the tumor sensing protease or the subunit or fragment thereof.

50. The method of claim 49, wherein the tumor sensing protease comprises a metalloprotease or a subunit or fragment thereof.

51. The method of claim 50, wherein the metalloprotease or the subunit or fragment thereof comprises ADAM8, ADAM9, BMP1, CPD, MMP11, QPCT, or a combination thereof. 79556009V.

152. The method of claim 49, wherein the tumor sensing protease comprises a serine protease or the subunit or fragment thereof.

53. The method of claim 52, wherein the serine protease or the subunit or fragment thereof comprises FAP, PRSS23, HTRA2, or a combination thereof.

54. The method of claim 33, wherein the tumor sensing enhancer protein comprises the tumor sensing transcription factor or the subunit or fragment thereof.

55. The method of claim 54, wherein the tumor sensing transcription factor or the subunit or fragment thereof comprises HIF1A, IFRD1, or a combination thereof.

56. The method of claim 33, wherein the tumor sensing enhancer protein comprises the tumor sensing protein phosphatase or the subunit or fragment thereof.

57. The method of claim 56, wherein the tumor sensing protein phosphatase or the subunit or fragment thereof comprises PPP1CB.

58. The method of claim 33, wherein the tumor sensing enhancer protein comprises the tumor sensing ion channel protein or the subunit or fragment thereof.

59. The method of claim 58, wherein the tumor sensing ion channel protein or the subunit or fragment thereof comprises CATSPER2.

60. The method of claim 33, wherein the tumor is a solid tumor.

61. The method of claim 60, wherein the tumor comprises a breast cancer or a tubo-ovarian cancer.

62. The method of claim 33, wherein the engineered immune cell is an NK cell, a T cell, a dendritic cell, a monocyte, or a macrophage.

63. The method of claim 33, wherein the engineered immune cell expresses a CAR.

64. The method of claim 63, wherein the CAR targets EpCAM or HER2.

65. The method of claim 33, wherein the engineered immune cell expresses a TCR. 79556009V.

166. A method for treating a cancer in a subject, the method comprising: engineering an immune cell of the subject to produce an engineered immune cell that overexpresses a tumor sensing enhancer protein comprising a tumor sensing receptor that binds to a non-chemokine ligand, a tumor sensing protease, a tumor sensing transcription factor, a tumor sensing protein phosphatase, a tumor sensing ion channel protein, or a subunit or fragment thereof, wherein the engineered immune cell exhibits increased migration to or infiltration of a tumor of the subject as compared to migration to or infiltration of the tumor by a corresponding immune cell not overexpressing the tumor sensing enhancer protein.

67. The method of claim 66, wherein the method further comprises: obtaining the immune cell from the subject prior to engineering the immune cell; and administering the engineered immune cell to the subject.

68. The method of claim 67, wherein engineering the immune cell comprises introducing into the immune cell a polynucleotide encoding the tumor sensing enhancer protein.

69. The method of claim 68, wherein introducing the polynucleotide comprises viral transduction of the polynucleotide into the immune cell.

70. The method of claim 67, wherein engineering the immune cell comprises introducing into the immune cell a guide RNA targeting a gene encoding the tumor sensing enhancer protein.

71. The method of claim 66, wherein engineering the immune cell comprises administering to the subject a polynucleotide encoding the tumor sensing enhancer protein.

72. The method of claim 66, wherein engineering the immune cell comprises administering to the subject a guide RNA targeting a gene encoding the tumor sensing enhancer protein. 79556009V.

173. The method of claim 66, wherein the engineered immune cell exhibits increased migration to the tumor as compared to migration to the tumor by the corresponding immune cell not overexpressing the tumor sensing enhancer protein.

74. The method of claim 66, wherein the engineered immune cell exhibits increased infiltration of the tumor as compared to infiltration of the tumor by the corresponding immune cell not overexpressing the tumor sensing enhancer protein.

75. The method of claim 66, wherein the tumor sensing enhancer protein comprises the tumor sensing receptor or the subunit or fragment thereof.

76. The method of claim 75, wherein the engineered immune cell exhibits increased migration to the tumor in a presence of the non-chemokine ligand, as compared to migration to the tumor by the corresponding immune cell not overexpressing the tumor sensing receptor.

77. The method of claim 75, wherein the engineered immune cell exhibits increased infiltration of the tumor in a presence of the non-chemokine ligand, as compared to infiltration of the tumor by the corresponding immune cell not overexpressing the tumor sensing receptor.

78. The method of claim 75, wherein the non-chemokine ligand comprises a metabolite.

79. The method of claim 75, wherein the non-chemokine ligand comprises a fatty acid, a lipid, or a derivative thereof.

80. The method of claim 75, wherein the tumor sensing receptor comprises a G protein-coupled receptor or a subunit or fragment thereof.

81. The method of claim 80, wherein the tumor sensing receptor or the subunit or fragment thereof comprises GPR183, GPR84, GPR34, GPR18, LPAR2, C5AR1, FPR3, FPR2, or a combination thereof.

82. The method of claim 80, wherein the tumor sensing receptor or the subunit or fragment thereof comprises GPR124, GPR132, GPR137. GPR171, HCAR3, PTAFR, S1PR1, S1PR4. or a combination thereof. 79556009V.

183. The method of claim 75, wherein the tumor sensing receptor or the subunit or fragment thereof comprises EPHA6, ICOS, SDCBP, SLAMF1, TLN1, CRTAM, ITGAL, CD6, or any combination thereof.

84. The method of claim 66, wherein the tumor sensing enhancer protein comprises the tumor sensing protease or the subunit or fragment thereof.

85. The method of claim 84, wherein the tumor sensing protease comprises a metalloprotease or a subunit or fragment thereof.

86. The method of claim 85, wherein the metalloprotease or the subunit or fragment thereof comprises ADAM8, ADAM9, BMP1, CPD, MMP11, QPCT, or a combination thereof.

87. The method of claim 84, wherein the tumor sensing protease comprises a serine protease or a subunit or fragment thereof.

88. The method of claim 87, wherein the serine protease or the subunit or fragment thereof comprises FAP, PRSS23, HTRA2, or a combination thereof.

89. The method of claim 66, wherein the tumor sensing enhancer protein comprises the tumor sensing transcription factor or the subunit or fragment thereof.

90. The method of claim 89, wherein the tumor sensing transcription factor or the subunit or fragment thereof comprises HIF1A, IFRD1, or a combination thereof.

91. The method of claim 66, wherein the tumor sensing enhancer protein comprises the tumor sensing protein phosphatase or the subunit or fragment thereof.

92. The method of claim 91, wherein the tumor sensing protein phosphatase or the subunit or fragment thereof comprises PPP1CB.

93. The method of claim 66, wherein the tumor sensing enhancer protein comprises the tumor sensing ion channel protein or the subunit or fragment thereof.

94. The method of claim 93, wherein the tumor sensing ion channel protein or the subunit or fragment thereof comprises CATSPER2.

95. The method of claim 66, wherein the tumor is a solid tumor. 79556009V.

196. The method of claim 95, wherein the tumor comprises a breast cancer or a tubo-ovarian cancer.

97. The method of claim 66, wherein the engineered immune cell is an NK cell, a T cell, a dendritic cell, a monocyte, or a macrophage.

98. The method of claim 66, wherein the engineered immune cell expresses a CAR.

99. The method of claim 98, wherein the CAR targets EpCAM or HER2.

100. The method of claim 66, wherein the engineered immune cell expresses a TCR.

101. A method for treating a cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a population of the engineered immune cell of claim 1.

102. A method for determining a personalized treatment for a cancer in a subject, the method comprising: administering to the subject two or more populations of engineered immune cells, wherein: for each population of the two or more populations, the engineered immune cells of the population are engineered to overexpress a combination of one or more tumor sensing enhancer proteins that each independently comprise a tumor sensing receptor that binds to a non-chemokine ligand, a tumor sensing protease, a tumor sensing transcription factor, a tumor sensing protein phosphatase, a tumor sensing ion channel protein, or a subunit or fragment thereof; and the combination overexpressed by the engineered immune cells of each population is different from the combination overexpressed by the engineered immune cells of each other population; analyzing a biological sample obtained from the subject subsequent to the administering; and based on the analyzing, determining which one or more populations of the two or more populations of engineered immune cells exhibits a level of increased migration to, or infiltration of, a tumor of the subject, wherein the level is greater than a threshold. 79556009V.1

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