WO2023023585A1

WO2023023585A1 - Genetically engineered tumor-specific effector cells for synthesis of enzymes

Info

Publication number: WO2023023585A1
Application number: PCT/US2022/075123
Authority: WO
Inventors: Parijat Bhatnagar; Harikrishnan RADHAKRISHNAN
Original assignee: Sri International
Priority date: 2021-08-18
Filing date: 2022-08-18
Publication date: 2023-02-23

Abstract

An example genetically engineered effector cell comprises an exogenous polynucleotide sequence that includes a receptor element, an actuator element, and an effector element. The receptor element encodes a chimeric antigen receptor (CAR) including an extracellular antigen binding domain operably linked to a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain recognizes an antigen on a surface of a tumor cell. The actuator element encodes a transcription factor binding site that upregulates synthesis of an enzyme configured to reduce a tumor-associated extracellular matrix (ECM) associated with the tumor cell. The effector element encodes the enzyme, wherein in response to the extracellular antigen binding domain of the CAR binding to the antigen of tumor cell, the genetically engineered effector cell is configured to activate and, to synthesize and secrete the enzyme to a tumor microenvironment (TME) associated with the tumor cell.

Description

GENETICALLY ENGINEERED TUMOR-SPECIFIC EFFECTOR CELLS FOR

SYNTHESIS OF ENZYMES

GOVERNMENT RIGHTS

[0001] This invention was made with Government support under grant numbers R33CA247739 and R21CA236640 both awarded by the National Cancer Institute of the National Institutes of Health, and under grant number DP2EB024245 awarded by the National Institutes of Biomedical Imaging and Bioengineering of the National Institutes of Health. The Government has certain rights in this invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0002] Incorporated by reference in its entirety is a computer-readable nucleotide sequence listing, an ASCII text file which is 187 kb in size, submitted concurrently herewith, and identified as follows: “S1647134111_SequenceListing” and created on August 8, 2022.

BACKGROUND

[0003] Cancer is a group of diseases involving abnormal cell growth, and which can invade or spread to other parts of the body. Example cancers include breast cancer, ovarian cancer, lung cancer, prostate cancer, colorectal cancer, skin cancer, cervical cancer, leukemia, and brain cancer, among others. Different types of cancers occur at different ages, with the rates of cancer generally increasing with age. Cancer can manifest in a variety of different organisms, including humans and other animals. Causes of cancers include genetics, and environment or lifestyle causes, such as smoking, diet and obesity, infections, radiation, and pollution. Different cancers cause a variety of symptoms, and ultimately death.

[0004] Cancer cells can form tumors, which are lumps of tissue that can be cancerous or noncancerous, e.g., benign. Cancerous tumors may spread or invade other tissues and can travel to other parts of the organism to form new tumors, sometimes referred to as “metastasis”. The ability of the cancer cells to metastasize, and for the cancer to progress, may be impacted by the cells forming tumors, sometimes referred to as “tumor cells”, and the tumor cell microenvironment (TME) including a tumor-associated extracellular matrix (ECM). The ECM is a non-cellular meshwork of crosslinked macromolecules including collagens, proteoglycans, and glycoproteins that form a molecular scaffold which maintains tissue structure and also provides various biological signals to modulate cellular function. Solid tumors consist both of the tumor cells and the ECM. As the ECM is dense, tumor cells may become refractory to treatment.

SUMMARY

[0005] The present invention is directed to overcoming the above-mentioned challenges and others related to tumors, such as involving a genetically engineered effector cell line which can activate in situ to cause synthesis of a human or non-human therapeutic protein (effector) against the extracellular matrix (ECM) of the tumor cells.

[0006] Various aspects of the present disclosure are directed to a genetically engineered effector cell comprising an exogenous polynucleotide sequence that includes, in operative association: a receptor element that encodes a chimeric antigen receptor (CAR) including an extracellular antigen binding domain operably linked to a transmembrane domain and an intracellular signaling domain, wherein the extracellular antigen binding domain recognizes an antigen on a surface of a tumor cell; an actuator element that encodes a transcription factor binding site that upregulates synthesis of an enzyme in response to the extracellular antigen binding domain of the CAR binding to the antigen of the tumor cell, the enzyme configured to reduce a tumor-associated ECM associated with the tumor cell; and an effector element that encodes the enzyme wherein, in response to the extracellular antigen binding domain of the CAR binding to the antigen of tumor cell, the genetically engineered effector cell is configured to activate and, to synthesize and secrete the enzyme to a tumor microenvironment (TME) associated with the tumor cell.

[0007] In some aspects, the genetically engineered effector cell is configured to synthesize and secrete an amount of the enzyme as a function of an amount of the tumor cell present in a sample or in situ.

[0008] In some aspects, the amount of the enzyme is proportional to the amount of tumor cell present in situ.

[0009] In some aspects, the effector element further encodes a signal peptide upstream of the enzyme, the signal peptide being non-native to the enzyme.

[0010] In some aspects, the enzyme is a modified form of a wild-type enzyme. [0011] In some aspects, the enzyme encoded by the effector element includes an active protein and a sub-portion of a pro-peptide of the wild-type enzyme.

[0012] In some aspects, the enzyme encoded by the effector element includes a removed native signal peptide, and the effector element further includes a signal peptide that is non-native to the enzyme.

[0013] In some aspects, the activation of the genetically engineered effector cell regulates stimulation of cytokines and causes the secretion of the enzyme to the TME. [0014] In some aspects, the intracellular signaling domain includes at least one of an intracellular signaling portion of a CD28, an intracellular signaling portion of a 4- IBB, and an intracellular signaling portion of a CD3 zeta.

[0015] In some aspects, the transcription factor binding site is selected from the group consisting of a nuclear factor of activated cell (NF AT) response element, a serum response element (SRE), and a cyclic AMP response element (CRE).

[0016] Various aspects of the present disclosure are directed to a population of genetically engineered effector cells, each of the genetically engineered effector cells of the population comprising an exogenous polynucleotide sequence that includes an actuator element, an effector element, and a receptor element, wherein: the receptor element encodes a CAR including an extracellular antigen binding domain operably linked to a transmembrane domain and an intracellular signaling domain, wherein the extracellular antigen binding domain recognizes an antigen on a surface of a tumor cell; the actuator element encodes a transcription factor binding site that upregulates synthesis of an enzyme in response to the extracellular antigen binding domain of the CAR binding to the antigen of the tumor cell, the enzyme configured to reduce a tumor- associated ECM associated with the tumor cell; and the effector element encodes the enzyme. Wherein, in response to the extracellular antigen binding domain of the CAR binding to the antigen of the tumor cell, the population of genetically engineered effector cells being configured to activate and, in response, to synthesize and secrete a calibrated amount of the enzyme based on a presence of the tumor cell and, in response, the enzyme reduces the tumor-associated ECM.

[0017] In some aspects, the enzyme is a functional fragment of a wild-type enzyme. [0018] In some aspects, the enzyme encoded by the effector element includes an active protein and a sub-portion of a pro-peptide of the wild-type enzyme, the sub-portion including a different amino acid overhang than is present by natural post-translational modifications. [0019] In some aspects, the enzyme is configured to degrade and break down the tumor- associated ECM, as formed in a TME associated with the tumor cell, to treat or prevent a cancer infection.

[0020] In some aspects, the calibrated amount of the enzyme is a function of an amount of the tumor cell present in a plurality of cells or in a sample.

[0021] Various aspects of the present disclosure are directed to a method comprising contacting a plurality of cells with a volume of a genetically engineered effector cell, wherein the genetically engineered effector cell comprises a polynucleotide sequence that includes: a receptor element that encodes a CAR including an extracellular antigen binding domain operably linked to a transmembrane domain and an intracellular signaling domain, wherein the extracellular antigen binding domain recognizes an antigen on a surface of a tumor cell from the plurality of cells; an actuator element that encodes a transcription factor binding site; and an effector element that encodes an enzyme configured to reduce a tumor-associated ECM associated with the tumor cell. In response to contacting the plurality of cells with the volume of the genetically engineered effector cell and a presence of the tumor cell within the plurality of cells, the method includes causing binding of the extracellular antigen binding domain to the antigen of the tumor cell. And, in response to the extracellular antigen binding domain of the CAR binding to the antigen of the tumor cell, the method includes initiating expression and synthesis of the enzyme by the actuator element, secreting the enzyme by a signal peptide, and reducing the tumor-associated ECM by the enzyme.

[0022] In some aspects, the method further includes detecting expression of the enzyme, wherein detectable expression of the enzyme indicates the presence of the tumor cell. [0023] In some aspects, the method further includes, in response to the extracellular antigen binding domain of the CAR binding to the antigen of the tumor cell, activating the genetically engineered effector cell and, in response, synthesizing and secreting a calibrated amount of the enzyme based on the presence of the tumor cell.

[0024] In some aspects, the calibrated amount of the enzyme is proportional to an amount of the tumor cell present within the plurality of cells.

[0025] In some aspects, the method further includes stimulating cytokines by the genetically engineered effector cell. BRIEF DESCRIPTION OF THE DRAWINGS

Various example examples can be more completely understood in consideration of the following detailed description in connection with the accompanying drawings, in which:

[0026] FIG. 1 illustrates an example of a genetically engineered effector cell specific to a tumor cell, in accordance with the present disclosure.

[0027] FIG. 2 illustrates an example of a genetically engineered effector cell and a sequence of events triggered when in a diseased environment, in accordance with the present disclosure.

[0028] FIG. 3 illustrates an example population of genetically engineered effector cells in a diseased environment, in accordance with the present disclosure.

[0029] FIG. 4 illustrates an example method of contacting a plurality of cells with a volume of a genetically engineered effector cell, in accordance with the present disclosure.

[0030] FIGs. 5A-5D illustrate example expression constructs associated with genetically engineered effector cells, in accordance with the present disclosure.

[0031] FIGs. 6A-6F illustrate plots characterizing genetically engineered effector cells producing enzymes as a function of time, target, and cell number, in accordance with the present disclosure.

[0032] FIGs. 7A-7B illustrate plots characterizing expression of enzymes by genetically engineered effector cell responsive to stimulation by Phorbol 12-myristate 13 -acetate/ lonomycin (PMA/Io) as a function of cell number, in accordance with the present disclosure.

[0033] FIGs. 8A-8F illustrate plots characterizing genetically engineered effector cell function, in accordance with the present disclosure.

[0034] FIGs. 9A-9D illustrate plots characterizing different types of genetically engineered effector cells as compared to control cells, in accordance with the present disclosure.

[0035] FIGs. 10A-10E illustrate plots characterizing therapeutic activity of example genetically engineered effector cells, in accordance with the present disclosure.

[0036] FIGs. 11A-11C illustrate plots characterizing cytolytic activity of example genetically engineered effector cells, in accordance with the present disclosure. [0037] FIGs. 12A-12B illustrate plots characterizing example genetically engineered tumor cells which are modified to express human folate receptor alpha (FRa) tumor antigens, in accordance with the present disclosure.

[0038] FIGs. 13A-13B illustrate plots characterizing response of genetically engineered effector cells to genetically engineered tumor cells, in accordance with the present disclosure.

[0039] FIGs. 14A-14D illustrate plots characterizing cytolytic activity of example genetically engineered effector cells in response to the genetically engineered tumor cells, in accordance with the present disclosure.

[0040] FIGs. 15A-15C illustrate plots characterizing different enzymes expressed by example genetically engineered effector cells, in accordance with the present disclosure.

DETAILED DESCRIPTION

[0041] In the following detailed description, reference is made to the accompanying drawings which form a part hereof, and in which is shown by way of illustration specific examples in which the disclosure can be practiced. It is to be understood that other examples can be utilized, and various changes may be made without departing from the scope of the disclosure. The following detailed description, therefore, is not to be taken in a limiting sense, and the scope of the disclosure is defined by the appended claims. It is to be understood that features of the various examples described herein may be combined, in part or whole, with each other, unless specifically noted otherwise.

[0042] Tumors caused by cancer can be treated with a variety of antitumor agents, with the current standard care for solid tumors being chemotherapy. While different types of cancers are unique in terms of presentation, the dense ECM tissue is common among solid tumors of many types of cancers. The ECM has unique physical and chemical properties that can synergistically blunt the antitumor response of chemo and immune therapies by limiting the influx of antitumor agents to the tumor cells and/or reducing the efflux of tumor metabolite to create an immunosuppressive environment that supports tumor cell growth. The ECM can assist in creating a dynamic and living TME that can require increasing dosages of antitumor agents for a response, which can impact healthy tissue and cells. For example, excess chemotherapy damages healthy tissues and the therapy may be stopped when a host can no longer tolerate it or may cause cachexia or death of the host. [0043] Various examples are directed to a genetically engineered effector cell that reduces the tumor-associated ECM, which can assist with normalizing the TME and can reduce treatment resistance and immunosuppression in tumors. The genetically engineered effector cell is targeted to a tumor cell, with binding to the tumor cell activating generation of an enzyme that can reduce the ECM. As used herein, reducing the ECM includes and/or refers to degrading and/or breaking down the ECM as formed and/or as forming. In some examples, reducing the ECM includes degrading the ECM or otherwise reducing ECM formed and/or as forming or remodeling. Because the activation is responsive to binding to the tumor cell, the enzyme is generated only in the presence of tumor cells, which may reduce the impact of the enzyme on healthy or normal tissue as compared to generally or globally treating the host with the enzyme. Further, as the ECM is common to many types of cancer, the genetically engineered effector cell can be designed to bind to specific types of tumor cells, and/or for treating many different types of cancer. In some examples, the genetically engineered effector cell can be used to provide immunotherapies, which offers the potential for complete transmission with reduced side effects.

[0044] Examples of the present disclosure include cells and cell lines that are genetically engineered with CARs to specifically detect (e.g., bind) antigens expressed on the surface of tumor cells. In response to the CAR binding to the antigen of the target tumor cell, an enzyme is expressed. For example, the genetically engineered effector cell can autonomously synthesize calibrated amounts of the enzyme directly in the TME after engaging the antigen-presenting tumor cells. The expression of the enzyme can be directly proportional to the number of tumor cells present, such that the amount of enzyme present is proportional to the disease burden and the enzymes are synthesized in the TME. The enzymes synthesized can be used to treat solid tumors by reducing, such as degrading, the ECM as formed and/or as forming, which may allow for other antitumor agents to more easily infiltrate the TME as compared to a fully functioning and/or formed ECM. The antitumor agents can be part of the innate immune system or can be administered drugs or other treatments. As the enzymes are synthesized in the TME and in response to binding to the tumor cells, the methodology mitigates the undesired systemic effect of directly infusing such enzymes, and a degraded or otherwise reduced ECM can more easily be infiltrated by the innate immune system and other antitumor agents. Efflux of the tumor metabolites from the TME can reduce the immunosuppressive effects and can allow for the efflux of the tumor metabolites to reduce the immunosuppressive nature of the TME.

[0045] In some examples, the cell can be engineered to express genetic elements including transmembrane receptor(s) that autonomously regulate the intracellular transcriptional machinery. The genetic elements of the cell can be modular and/or a cell can include multiple genetic elements to yield an engineered effector cell having the capacity to serve as a vector for a variety of in vitro, ex vivo, and in vivo applications. Such cells can be modular in that parts can be conserved, and parts can be changed for different applications. The genetically engineered effectors cells can be used for therapeutics and treatment methods that self-regulate the therapeutic response upon stimulation by the disease tumor cells and that are applicable to a variety of cancers that evade the immune system or involve its malfunction. In other examples, the genetically engineered effector cells can be used in diagnostics, such as diagnosing a stage of cancer or assessing the progress of treatment.

[0046] Various examples demonstrate the successful implementation of the artificial cell-signaling pathway in a cell line. In some experimental examples, the cell line was transformed into a vector for engaging antigen-presenting tumor cells and to trigger the synthesis of calibrated amounts of proteins in situ, herein sometimes referred to as “effector proteins”. In some examples, the genetically engineered effector cells having specificity towards the folate receptor alpha (FRa) are engineered by encoding for an effector protein of collagenase-2, sometimes referred to as “MMP8”. However, examples are not so limited and can include other tumor antigens and/or other enzymes. [0047] As used herein, a “genetically engineered effector cell” includes and/or refers to a cell that is genetically engineered or modified to comprise a (i) receptor element, (ii) actuator element, and (iii) effector element, each of which can be modular. The terms “modular” and “modularity” include and/or refer to the versatility associated with recombinant sequence domains and the resulting recombinant polypeptides when assembled in various combinations for introduction into an engineered effector cell. The genetically engineered effector cells can be modified for different functionalities by changing portions of the effector element and/or receptor element to develop cell with the different functionalities and for different implementations, such as therapeutics, diagnostics, and/or reporters, as further described herein.

[0048] As used herein, "receptor element" includes and/or refers to a polynucleotide sequence encoding a transmembrane receptor, such as a CAR, capable of a specific interaction with a target cell, such as a tumor cell, a genetically engineered tumor cell that expresses an antigen specific to a type of cancer, and/or other molecules. Depending on the particular application, the receptor element can be reprogrammed by exchanging the single chain variable fragment (scFv) portion of the CAR for an extracellular antigen binding domain specific for a different cancer-associated antigen or general for an antibody or other molecule (e.g., cytokines, chemokines, proteins).

[0049] As used herein, “actuator element" includes and/or refers to a polynucleotide sequence encoding a transcription factor binding site that initiates transcription and translation events downstream of a triggering signal (e.g., binding of the receptor element to a target antigen). In general, the underlying molecular mechanism of the actuator element is based on the intracellular calcium [Ca2+]i dynamics, a mechanism used by cells to regulate their functions. Exemplary response elements include, without limitation, NF AT ("nuclear factor of activated T cells") response element (NFAT-RE), serum response element (SRE), and cyclic AMP response element (CRE).

[0050] As used herein, "effector element" includes and/or refers to a polynucleotide sequence encoding an effector protein, and in some instances, an effector protein operably linked to a signal peptide. Example effector proteins include an enzyme, a detectable reporter protein, and an antitumor agent, among others. The polynucleotide sequence encoding the effector protein can be, for example, a sequence derived from a human gene, a sequence derived from a gene of a non-human species, a recombinant sequence, a sequence encoding a detectable reporter molecule, a sequence encoding a detectable imaging molecule, a sequence encoding a therapeutic molecule, and the like. [0051] The genetically engineered effector cell into which the receptor element, the actuator element, and the effector element are introduced can be any cell type including human cells or non-human cells (e.g., mammal, reptiles, plants, among others). In this manner, the genetically modified cellular "source" of the modular elements provides a cellular chassis or frame providing, among other things, transcriptional and translational machinery for expression and presentation of the receptor element, the actuator element, and the effector element. In some examples, the effector cells can be from a source (e.g., a first human), modified, and administered to an organism that is different than the source (e.g., the host which is a second human). In other examples, the cells can be from the source (e.g., a first human), modified, and administered back to the source (e.g., the source is the host). [0052] As used herein, the TME includes and/or refers to an environment that includes an ECM (e.g., is rich in ECM) associated with a tumor cell. For example, the TME can be an environment including and around a tumor, which includes tumor cells, surrounding blood vessels, immune cells, and the ECM. As previously described, the ECM is a non-cellular meshwork of crosslinked macromolecules including collagens, proteoglycans, and glycoproteins that form a molecular scaffold which maintains tissue structure and provides biological signals to modulate cellular function. Solid tumors include both of the tumor cells that form an abnormal mass of tissue and the ECM.

[0053] Turning now to the figures, FIG. 1 illustrates an example of a genetically engineered effector cell specific to a tumor cell, in accordance with the present disclosure. The genetically engineered effector cell 100, herein generally referred to as “an effector cell", can be modular in that genetic elements 102, 106, 110 can be adjusted for different target tumor cells and to synthesize different enzymes or other effector proteins.

[0054] The effector cell 100 comprises an exogenous polynucleotide sequence 101 that includes, in operative association, a receptor element 102, an actuator element 106, and an effector element 110. A variety of different types of cells can be genetically modified to form the effector cell 100. Example cells include an immune cell (e.g., a T-cell, a natural killer cell), a pluripotent stem cell, a multipotent stem cell, an epithelial cell, or a K562 cell. The cell modified to generate the effector cell 100 can include a living cell from an organism, e.g., a basic membrane-bound unit that contains structural and functional elements.

[0055] In some examples, the exogenous polynucleotide sequence 101 is selected from SEQ ID NOs: 1, 16, 23, and 28. In some examples, the exogenous polynucleotide sequence 101 includes SEQ ID NOs: 2, 17, 24, or 29. In some examples, the exogenous polynucleotide sequence 101 includes SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 28, or SEQ ID NO: 29. Examples are not so limited and the exogenous polynucleotide sequence 101 can include other sequences, such as a sequence with at least 80 percent (%), 85%, 90%, 95%, or 99% sequence identity to one of the sequences set forth in SEQ ID NOs: 1, 2, 16, 17, 23, 24, 28, and 29, among other sequences.

[0056] The receptor element 102 encodes a CAR 104. A CAR is sometimes called a “chimeric receptor”, a “T-body”, or a “chimeric immune receptor (CAR).” As used herein, a CAR includes and/or refers to an artificially constructed hybrid protein or polypeptide including extracellular antigen binding domain(s) 103 of an antibody (e.g., scFv, VHH) operably linked to a transmembrane domain 105 and at least one intracellular signally domain 107. For example, the CAR 104 includes an extracellular antigen binding domain 103 operably linked to the transmembrane domain 105 and the intracellular signaling domain 107. The CAR 104 can be designed to identify an antigen on a surface of a tumor cell, such as a target tumor cell of a host or a genetically modified target tumor cell. The CAR 104 can mobilize internal Ca+2 stores for intracellular Ca+2 release in response to antigen binding. For example, the extracellular antigen binding domain 103 of the CAR 104 can recognize an antigen on a surface of a tumor cell, such as diseased tumor cells of a host.

[0057] The antigen can include a tumor-specific antigen, which are present only on tumor cells, or a tumor-associated antigen, which are present on tumor cells and other cells, such as normal cells. Example antigens of a tumor cell includes FRa, Mucin 16 (Ca-125), Mucin 1 (MUC-1), Epithelial tumor antigen (ETA), Epidermal Growth Factor Receptor (EGFR) antigen, Mesothelin (MSLN), Cancer/testis antigen 1 (CTAG1B), Melanoma-associated antigen 1 (MAGE-A1), MAGE- A3 alpha protein (AFP) antigen, carcinoembryonic antigen (CEA), Mucin 16 (MUC-16), sometimes referred to as CA- 125, Claudin 18.2, human epidermal receptor (Her)-2, prostate-specific membrane antigen (PSMA), tumor protein D52 (TPD52), New York esophageal squamous cell carcinoma (NY-ESO-1), cyclin-dependent kinase (CDK-4), -catenin, Caspase-8, glycoprotein (Gp) 100, melanoma antigen recognized by T-cells (MART-1), Tyrosinase, prostate specific antigen (PSA), prostate acid phosphatase (PAP), RAS and K-RaS antigens, BRAF antigen, p53, Wilms tumor 1 (WT1) antigen, 5T4 onofetal antigen, Glypican-3 (GPC-3), insulin-like growth factor-II messenger RNA (mRNA)-binding protein-3 (IMP-3), Human Chorionic Gonadotropin //-Subunit (hCG P), Thomsen- Friedenreich antigen, Stage-specific embryonic antigen 1 (SSEA-1), gangliosides, such as ganglioside fucosyl-GMl(FucGM), MYC antigen, osteopontin (OPN), murine double-minute 2 oncoprotein (MDM2), survivin, Epstein-Barr virus (EBV) antigen, and Carcinogenembyronic antigen (CEA), among others and combinations thereof.

[0058] In some examples, the extracellular antigen binding domain 103 includes SEQ ID NO: 5. In other examples, the extracellular antigen binding domain 103 includes any of SEQ ID NOs: 5, 6, 7, and 8, and/or combinations thereof. Examples are not so limited and the extracellular antigen binding domain 103 can include other sequences, such as a sequence with at least 80%, 85%, 90%, 95%, or 99% sequence identity to one of the sequences set forth in SEQ ID NOs: 5, 6, 7, and/or 8, among other sequences. [0059] As used herein, the extracellular antigen binding domain 103 includes and/or refers to a polynucleotide sequence that is complementary to a target, such as an antigen of the tumor cell. The extracellular antigen binding domain 103 can bind to the surface antigen of the tumor cell, as described above.

[0060] The transmembrane domain 105 includes and/or refers to a polynucleotide sequence encoding a transmembrane segment of a transmembrane protein, e.g., a type of membrane protein that spans the membrane of a cell, such as the membrane of the effector cell 100. The transmembrane domain 105 can be derived from a natural polypeptide, or can be artificially designed. A transmembrane domain 105 derived from a natural polypeptide can be obtained from any membrane-binding or transmembrane protein. For example, a transmembrane domain of a T-cell receptor a or [3 chain, a CD3^ chain, CD28, CD3E, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or a GITR can be used.

[0061] The intracellular signaling domain 107 includes and/or refers to a polynucleotide sequence encoding any oligopeptide or polypeptide that function as a domain and transmit a signal to cause activation or inhibition of a biological process in a cell. Example intracellular signaling domains include an intracellular signaling portion of a CD28, an intercellular signaling portion of a 4- IBB, and an intracellular signal portion of a CD3-zeta. In some examples, the intracellular signaling domain 107 includes the intracellular signaling portion of CD28, the intercellular signaling portion of 4-1BB, and the intracellular signal portion of CD3-zeta. In some examples, the intracellular signaling domain 107 includes the intracellular signaling portion of (i) CD3-zeta, (ii) CD3-zeta and CD28, (iii) CD3-zeta and 4-1BB, or (iv) CD3-zeta, CD28, and 4-1BB. In some examples, the intracellular signaling domain 107 includes the CD28 transmembrane portion that is upstream of the intracellular signaling portion of 4- IBB (e.g., 4-1BB cytoplasmic domain) which is optionally upstream of the intracellular signal portion of CD3-zeta (e.g., CD3-zeta cytoplasmic domain). However, examples are not so limited and can include other types and combinations of intracellular signaling domains in combination with the intracellular signal portion of CD3-zeta. For example, the intracellular signaling domain 107 can include encode any molecule that can transmit a signal into a cell when the extracellular antigen binding domain 103 present within the same molecule binds to (interacts with) the antigen. [0062] Generally, the extracellular antigen binding domain 103 of a CAR 104 has specificity for a particular antigen expressed on the surface of a target cell of interest, e.g., a tumor cell. As described above, the extracellular antigen binding domain 103 is capable of binding to an antigen and includes any oligopeptide or polypeptide that can bind to the antigen. Example extracellular antigen binding domain 103 include an antigen-binding domain of an antibody and a ligand-binding domain of a receptor. The extracellular antigen binding domain 103 binds to and interacts with the antigen present on a cell surface of the tumor cell, and thereby imparts specificity to an effector cell 100 expressing the CAR 104. In some examples, the receptor element 102 encodes a CAR 104 including an extracellular antigen binding domain 103 having specificity for the antigen associated with tumor cells, such as but not limited to the FRa antigen.

[0063] The actuator element 106 encodes a transcription factor binding site 108. The transcription factor binding site 108 includes and/or refers to a binding site for a protein that upregulates synthesis of an enzyme 112 in response to the extracellular antigen binding domain 103 of the CAR 104 binding to the antigen of the tumor cell. The transcription factor binding site 108 can bind to transcription factors as triggered by [Ca2+], which release in response to the antigen binding. In some examples, the transcription factor binding site 108 is selected from a NF AT response element (NFAT- RE), SRE, and a CRE. The actuator element 106 can thereby include a sequence for binding the factors triggered by [Ca2+], and can trigger amplified synthesis of the enzyme 112 in response to [Ca2+]i rise.

[0064] In some examples, the actuator element 106 encodes a NF AT transcription factor binding site for a transcription factor protein. In some examples, the actuator element 106 encodes a set of NF AT transcription factor binding sites, such as at least two transcription factor binding sites, three transcription factor binding sites, or six transcription factor binding sites (e.g., six NFAT-RE), among other amounts. NF AT transcription factor family consists of five members NFATcl, NFATc2, NFATc3, NFATc4, and NFAT5. See Sharma S et al. (2011) PNAS, 108(28); Hogan PG et al. (2010) Ann Rev Immunol, 28; Rao A, Hogan PG (2009) Immunol Rev, 231(1); Rao A (2009) Nat Immunol, 10(1), M. R. Muller and A. Rao, Nature Reviews Immunology, 2010, 10, 645-656; M. Oh-Hora and A. Rao, Curr. Opin. Immunol., 2008, 20, 250-258. Crabtree & Olson EN (Apr 2002), Cell 109 Suppl (2): S67-79, which are each hereby incorporated herein in their entireties for their specific and general teachings. NFATcl through NFATc4 are regulated by calcium signaling. Calcium signaling is critical to NF AT activation because calmodulin, a calcium sensor protein, activates the serine/threonine phosphatase calcineurin. The underlying molecular mechanism is based on intracellular Ca+2 ([Ca2+]i) dynamics (as further shown by FIG. 2). The [Ca2+]i dynamics are common to many cell types, and the approach is broadly applicable. The [Ca2+]i rise from CAR-mediated stimulation of cells leads to dephosphorylation of the nuclear factor of an activated effector cell 100 (through Ca+2/calmodulin-dependent serine phosphatase calcineurin), which is then translocated to the nucleus and interacts with the NFAT-RE to upregulate expression of enzyme 112. In parallel, the NFAT-RE also performs the function of inducing Interleukin-2 in the activated effector cell 100 that regulates clonal expansion proportional to the disease burden.

[0065] The effector element 110 encodes the enzyme 112, and in some examples, encodes the enzyme 112 operably linked to a signal peptide 114. As further illustrated herein, in some examples, the signal peptide 114 is upstream of the enzyme 112. The signal peptide 114 can be non-native to the enzyme 112. For example, the enzyme 112 can be unable to secrete into the extracellular environment without the addition of the signal peptide 114. However, examples are not so limited and in some examples, the enzyme 112 includes a native signal peptide. For example, the enzyme 112 can (natively) include the signal peptide 114. In other examples, the native signal peptide of the enzyme 112 can be removed and a non-native signal peptide 114 can be added.

[0066] As used herein, the terms “secretor”, “secretory peptide and “signal peptide” are used interchangeable and include and/or refer to a peptide that assists or directs the synthesized enzyme 112 into the extracellular environment (e.g., assists with translocating the effector element 110). The signal peptide 114 can be operably linked or fused to the enzyme 112 for release into the extracellular environment. In this manner, the signal peptide 114 can direct movement of the enzyme 112 outside of the effector cell 100. A signal peptide 114 is particularly advantageous when included in the effector cell 100 expressing an enzyme 112 that is unable to and/or minimally able to translocate natively, where the enzyme 112 can remain inside the effector cell 100 in the absence of the signal peptide 114 and/or can translocate at a rate below a threshold.

Generally, signal peptides are located at the N-terminus of nascent secreted proteins and characteristically have three domains: (1) a basic domain at the N-terminus, (2) a central hydrophobic core, and (3) a carboxy-terminal cleavage region. Any signal peptide can be used. For example, the signal peptide 114 can be the signal peptide of Interleukin-6, Interleukin-2, from an Interferon, such as the signal peptide from Interferon alpha-2a. [0067] In various examples, in response to the extracellular antigen binding domain 103 of the CAR 104 binding to the antigen of the tumor cell, the effector cell 100 is configured to activate, and to synthesize and secrete the enzyme 112 to a TME associated with the tumor cell. For example, the effector cell 100 can synthesize and secrete an amount of the enzyme 112 as a function of an amount of the tumor cell and/or an amount of other antigen-presenting cells in the environment (e.g., the extracellular environment), such as secreting an amount of the enzyme 112 in the TME that is proportional to the number of tumor cells present in a sample or in situ.

[0068] The enzyme 112 can include a variety of different types of enzymes configured to reduce a tumor-associated ECM. As previously described, reducing the ECM includes degrading and/or breaking down the ECM as formed and/or as forming. In some examples, the enzyme 112 degrades the tumor-associated ECM formed. In some examples, the enzyme 112 suppresses the tumor-associated ECM as forming. As used herein, an enzyme includes and/or refers to a protein or other substance that catalyzes or otherwise participates in the breakdown of another protein, such as degrading or breaking down the tumor-associated ECM. Example enzymes include collagenases, such as collagenase-2, collagenase-3, stromelysin- 1, and other metalloproteinases (e.g., MMP9), heparanase (HPE), matrilysin-2, hyaluronidase, bacterial Sialidase (b- Sialidase), elastase, trypsin3, gelatinase A, plasminogen, serine proteases, such as neutrophil elastase and cathepsin G, among other enzymes and proteases. In some examples, the enzyme 112 is a protease, such as collagenase-2 or a derivative thereof. In some examples, the enzyme 112 is a transferase, a hydrolase, and/or a lyase, among other types of enzymes that are configured to reduce a tumor-associated ECM.

[0069] In some examples, the enzyme 112 encoded by the effector element 110 can include a modified form of a wild-type enzyme. Said differently, the enzyme 112 encoded can be a genetically modified enzyme, such as a functional fragment of the wild-type enzyme configured to reduce the tumor-associated ECM. In some examples, the wild-type enzyme includes a signal peptide, a pro-peptide, and an active protein. The pro-peptide, sometimes referred to as a “pro-peptide portion”, includes an inactive portion (e.g., amino acid sequence) of the enzyme that is removed by post-translational modification, such as by breaking off a piece of enzyme or adding another molecule, to form an active protein (e.g., an activated enzymes). The active protein or active protein portion includes a portion of the enzyme that performs a function, e.g., breaks down proteins. The signal peptide transports the enzyme across the cellular membrane, and then the enzyme is activated by removing the pro-peptide portion such that the active protein remains with an overhang of amino acids from the pro-peptide. The active protein with the naturally occurring overhang of amino acids (e.g., occurs from natural post-translational modification) is sometimes herein referred to as “an activated enzyme”. As used herein, an amino acid overhang or overhang of amino acids includes and/or refers to a sub-portion (e.g., a number amino acids) of the pro-peptide from a wild-type enzyme. In physiological in vivo settings, activation can occur with the assistance of proteinases in the extracellular milieu. To prepare a therapeutic enzyme, the enzyme (such as collagenase-2) can be treated in vitro with proteinases, mercurial compounds, and oxidants. In some instances, activated enzymes can have a half-life below a threshold.

[0070] In some examples, the enzyme 112 encoded includes a functional fragment of the wild-type enzyme, such as the active protein portion with the sub-portion of the propeptide portion from the wild-type enzyme. As described above, the sub-portion of propeptide can include a different overhang of amino acids from the pro-peptide than is present by natural post-translational modification. The overhang of amino acids can be longer or shorter than the natural post-translational modification, and can result in a superactivated enzyme. A superactivated enzyme (e.g., a superactivated protease), as used herein, includes and/or refers to an enzyme that includes an active protein and an overhang of amino acids from the pro-peptide that is different than an activated enzyme, and which can exhibit greater activity than the activated enzyme. In some examples, the wild-type signal peptide is further removed and a non-naturally occurring signal peptide is added to the effector element 110 for transporting the enzyme 112 across the cell membrane to the extracellular space. The non-naturally occurring signal peptide can provide greater transporting efficiency than the wild-type signal peptide.

[0071] In some examples, the enzyme 112 can be encoded by and/or include SEQ ID NOs: 4, 20, 26, and/or 30. As such, in some examples, the effector element 110 can include one or more of SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 26, and SEQ ID NO: 30. In some examples, the effector element 110 can encode a modified enzyme (e.g., a modified protease) and a signal peptide that is non-natural to the enzyme. In such examples, the enzyme 112 can be encoded by and/or include SEQ ID NOs: 20 or 26 and the signal peptide 114 can be encoded by SEQ ID NO: 19. In various examples, the full effector element 110 can include any of SEQ ID NOs: 4, 18, 19, 20, 25, 26, and 30, as well as combinations thereof. However, examples are not so limited and the effector element 110, the enzyme 112, and/or the signal peptide 114 can include other sequences, such as a sequence with at least 80%, 85%, 90%, 95%, or 99% sequence identity to one or more of the sequences set forth in SEQ ID NOs: 4, 18, 19, 20, 25, 26, and 30, among other sequences.

[0072] In some examples, the effector cell 100 can stimulate production of other therapeutic proteins in the host and/or can otherwise trigger the natural immune system by recruiting immune cells. As the enzyme 112 can reduce the ECM as formed and/or as forming, the stimulated therapeutic proteins and/or recruited immune cells can have greater infiltration and/or impact on tumor growth and progression as compared to a fully intact ECM in the TME, such as inhibiting growth and/or killing tumor cells. For example, the activation of the effector cell 100 can regulate stimulation of cytokines and/or other proteins in the host. In such examples, the activation of the effector cell 100 regulates stimulation of cytokines and causes the secretion of the enzyme 112 to the TME. In addition and as described above, the release of cytokines stimulates other immune cells. For example, the NFAT-RE of the effector cell 100 can induce Interleukin-2, which can enhance functionality of immune cells, such as T-cells, dendritic cells, macrophages, natural killer cells, and/or B-cells, which are recruited by the effector cell 100 and which can infiltrate and/or impact tumor growth and progression more easily due to the enzyme 112 reducing the tumor-associated ECM.

[0073] Different parts of the genetic elements 102, 106, 110 of the effector cell 100 can be modular and other parts can be conserved (e.g., may not change for different implementations). For example, in some examples, the intracellular signaling domain 107, the actuator element 106, and the optional signal peptide 114 are constant domains, and the extracellular antigen binding domain 103 and the enzyme 112 are variable domains. As an example, the extracellular antigen binding domain 103 can be changed for different cancer targets and/or the enzyme 112 can be changed to cause in situ synthesis of different proteins, while the intracellular signaling domain 107, the actuator element 106, and the signal peptide 114 remain the same for the different implementations. Keeping parts conserved can reduce production time. However, examples are not so limited, and any part of the effector cell 100 can be modified.

[0074] In some examples, the effector cell 100 can include multiple (e.g., two or more) of some or all of the genetic elements 102, 106, 110. For example, the effector cell 100 can include multiple receptor elements 102, multiple actuator elements 106, and/or multiple effector elements 110. In some examples, multiplicity takes the form of providing multiple genetically engineered effector cells (e.g., a plurality of cells) modified as described herein to a host to provide more than one therapeutic task for treating cancer and/or for other purposes.

[0075] In some examples, the effector element 110 can encode multiple effector proteins, such as at least two enzymes, an enzyme and a detectable reporter protein (as further defined below), or at least two detectable reporter proteins, among other types of effector protein combinations. For example, two effector proteins can be encoded linked together by a 2A linker peptide. A 2A linker peptide, as used herein, includes and/or refers to a peptide which induces ribosomal skipping during translation of a protein complex (e.g., encoding of two proteins or peptides linked by the 2 A linker peptide) in a cell, such that the protein complex is translated into two proteins that independently fold. Example 2A linker peptides include F2A, P2A, E2A, and T2A, among others. Such peptides are generally 18-22 amino acids long, and derived from viruses.

[0076] In some examples, the actuator element 106 is connected to and/or associated with the effector element 110. In some examples, the exogenous polynucleotide sequence 101 includes the actuator element 106 connected to the effector element 110 connected to the receptor element 102, which are all formed on a single plasmid vector. For example, the exogenous polynucleotide sequence 101 can include the actuator element 106 connected to and upstream from the effector element 110, and the effector element 110 connected to and upstream or downstream from the receptor element 102, wherein the signal peptide 114 is upstream from the enzyme 112. In other examples, the receptor element 102 can be on a different plasmid vector than the actuator element 106 and the effector element 110.

[0077] The effector cell 100 can be specific to different types of cancers and to specificantigens of cancers. For example, the effector cell can be specific to an antigen associated breast cancer, ovarian cancer, lung cancer, prostate cancer, colorectal cancer, skin cancer, cervical cancer, stomach cancer, pancreatic cancer, leukemia, or brain cancer, among others. As compared to prior approaches, the effector cell 100 can be used in immunotherapies, and can offer the potential for complete remission with reduced side effects. By targeting the dense fibrous ECM tissue growth that is common in many solid tumors, and in which the TME resides, drug resistance and immunosuppression can be reduced as compared to prior approaches. Increasing the therapeutic dosage in the systemic circulation may not improve efficacy and may cause cachexia and morbidity of the host. Further, the selective activation of the effector cell 100 responsive to binding to the tumor cell can reduced side effects to healthy tissue caused by the enzyme 112. In addition, the effector cell 100 can improve immune response to the tumor cells by actively recruiting immune cells and while reducing the tumor-associated ECM, resulting in greater infiltration and/or impact on tumor growth and progression by the immune cells as compared to a fully intact ECM in the TME. [0078] FIG. 2 illustrates an example of a genetically engineered effector cell and a sequence of events triggered when in a diseased environment, in accordance with the present disclosure. The genetically engineered effector cell 200, herein generally referred to as the “effector cell 200” for ease of reference, can be used as or act as a living vector to synthesize the enzyme 212 using the artificial cell-signaling pathway and/or to trigger a sequence of events 220. The effector cell 200 synthesizes the engineered enzyme 212 in situ upon interacting with the antigen-presenting target cell 225, as shown at 222.

[0079] As previously described, the effector cell 200 can comprise a polynucleotide sequence 201 including the receptor element encoding the extracellular antigen binding domain 203, transmembrane domain 205, and the intracellular signaling domain 207, the actuator element 206 encoding the transcription factor binding site (e.g., NF AT), and the effector element 210 encoding the enzyme 212 and, optionally, the signal peptide 214. The polynucleotide sequence 201 can comprise a single plasmid (e.g., a single construct including each of) comprising constant domains (e.g., the actuator element 206, the signal peptide 214, and portions of the receptor element, such as the transmembrane domain 205 and the intracellular signaling domain 207), and variable domains (e.g., the extracellular antigen binding domain 203 (labeled as the “sensor”) and enzyme 212) arranged in cis. In some examples, the polynucleotide sequence 201 can comprise multiple plasmids, such as a first plasmid comprising the actuator element 206 and the effector element 210, and a second plasmid comprising the receptor element.

[0080] The constant domains can be configured to provide functionality to the effector cell 200. The constant domains form part of the intracellular signaling pathway and include a transmembrane molecule (e.g., transmembrane domain 205) that mobilizes the calcium-dependent transcriptional machinery (e.g., actuator element 206) to upregulate the effector transgene (e.g., enzyme 212) fused to the signal peptide 214 that assists in transporting the effector transgene into the extracellular space 223. In various examples, the enzyme 212 can include a native signal peptide, which forms part of the enzyme 212, or a non-native signal peptide which is linked to the enzyme 212.

[0081] The variable domains can be responsible for the applicability of the effector cell 200 to different cancers, target cells, therapy, and/or other applications. For example, the variable domains can impart specificity to the effector cell 200 against particular cancer diseases. The variable domains can include a variable heavy-light (VH-VL) chain (e.g., the extracellular antigen binding domain 203, labeled as the “sensor”) to identify the antigen biomarker on the target cell (e.g., labeled “diseased cell”) independent of the peptide-major histocompatibility complex, and the effector transgene (e.g., enzyme 212). The variable domains are modular. For example, the extracellular antigen binding domain 203 can be exchanged or revised to reprogram the effector cell 200 to target biomarkers specific to different cell-based cancer diseases. As another example, the enzyme 212 can be exchanged or revised with different therapeutic transgenes, such as for neutralizing the pathology that activated the effector cell 200 and essentially creating an off-shelf living vector, which is enhanced further by the innate cytolytic activity of effector cell 200.

[0082] In some examples, the receptor element encodes a CAR. Characteristics of CARs include the ability to redirect cell specificity and reactivity toward a selected target in a non-major histocompatibility complex (MHC)-restricted manner, exploiting the antigenbinding properties of monoclonal antibodies. The non-MHC-restricted antigen recognition gives effector cells expressing CARs the ability to recognize antigens independent of antigen processing. Referring to FIG. 2, expression of a transmembrane CAR enables an effector cell 200 to sense and bind to the target antigen 227 expressed on the surface of target cell 225, such as a tumor cell. Binding of the CAR and target antigen 227 on the target cell 225 activates the effector cell 200, which triggers an activation cascade leading to the expression of the enzyme 212. For example, expression of the enzyme 212, or other effector proteins, is autonomously expressed as part of the effector cell 200 activation cascade in response to binding of the transmembrane receptor to the target antigen 227 presented on the target cell 225.

[0083] More particularly, the effector cell 200 expressing a CAR binds to a tumorspecific antigen via the CAR, and in response, a signal is transmitted into the effector cell 200 and the effector cell 200 is activated. The activation of the effector cell 200 expressing the CAR is varied depending on the kind of target cell and an intracellular domain of the CAR, and can be confirmed based on, for example, release of a cytokine, improvement of a cell proliferation rate, change in a cell surface molecule, or the like as an index. For example, release of a cytotoxic cytokine (e.g., tumor necrosis factor, a lymphotoxin, etc.) from the activated effector cell 200 causes destruction of the target cell 225 expressing the target antigen 227. In addition, release of a cytokine or change in a cell surface molecule stimulates other immune cells, for example, a B-cell, a dendritic cell, a natural killer cell, and a macrophage.

[0084] As shown by FIG. 2, an example sequence of events 220 triggered by or related to the effector cell 200 includes (1) the effector cell 200 actively migrates to the diseased environment, (2) the CAR on the effector cell 200 surface engages the target antigen 227 of the target cell 225 that comprises a tumor cell, (3) the effector cell activates, (4) upregulation of the enzyme 212 with the signal peptide 214 through the NF AT, (5) the signal peptide 214 is cleaved off and the enzyme 212 is transported into the extracellular space 223 that includes a TME and the enzyme 212 reduces the tumor- associated ECM, and (6) antigen stimulation regulates cytokines that modulate cell expansion in response to the disease burden.

[0085] FIG. 3 illustrates an example population of genetically engineered effector cells in a diseased environment, in accordance with the present disclosure. The population

331 can include a plurality of genetically engineered effector cells 300-1, 300-2, 300-3, 300-4, 300-5, 300-6, 300-N (herein generally referend to as “the effector cells 300” for ease of references). Each of the effector cells 300 can include at least substantially the same components and features as the effector cell 100 of FIG. 1, the details of which are not repeated for ease of reference.

[0086] In the example illustrated by FIG. 3, the environment is an extracellular space 330 that includes a presence of target tumor cell(s) 332, such that the extracellular space 330 can be referred to as a diseased environment or a TME. The population 331 of effector cells 300 can bind to the antigens of the target tumor cell(s) 332 via the extracellular antigen binding domain of the CAR. In response to the binding, the effector cells 300 can activate and, in response, synthesize and secrete a calibrated amount of the enzyme based on a presence of the target tumor cell(s) 332. The calibrated amount of the enzyme can be a function of an amount of the target tumor cell

332 present in a plurality of (host) cells, such as in an extracellular space 330 or in a sample. As previously described, the calibrated amount of the enzyme can be proportional to the amount of the target tumor cell 332. Although the extracellular space 330 of FIG. 3 illustrates effector cells 300 and the target tumor cells 332, the extracellular space 330 and the plurality of (host) cells can further include other normal and/or diseased cells, among other non-cellular components.

[0087] As described above, the target tumor cell(s) 332 include a tumor-associated ECM and the effector cells 300 can secrete an enzyme in response to binding to the target tumor cell(s) 332. The enzyme is configured to reduce the tumor-associated ECM to treat or prevent a cancer infection. For example, the enzyme, such as a protease, can degrade (e.g., breakdown) or otherwise reduce the ECM as formed and/or as forming. In some examples, the enzyme can suppress ECM formation or as forming.

[0088] In some examples, the enzyme is a functional fragment of a wild-type enzyme, e.g., a modified enzyme. For example, the effector elements can encode an active protein and sub-portion of the pro-peptide of a wild-type enzyme, with the sub-portion including a different amino acid overhang than is present by natural post-translational modifications. The functional fragment of the wild-type enzyme can include a superactivated enzyme, as previously described.

[0089] In some examples, different effector cells of the population 331 can encode different enzymes and/or can encode multiple enzymes or other effector proteins. For example, a first subset of the population 331 of effector cells 300 can include the effector element that encodes a first enzyme and a second subset of the population 331 of effector cells 300 can include the effector element that encodes a second enzyme. In other examples, each of the effector cells 300 or a sub-portion thereof can include effector elements that encode the first enzyme bound to the second enzyme by a 2A linker peptide.

[0090] FIG. 4 illustrates an example method of contacting a plurality of cells with a volume of a genetically engineered effector cell, in accordance with the present disclosure. The method 440 can be implemented using the effector cell 100 illustrated by FIG. 1, effector cell 200 of FIG. 2, and/or the population 331 of effector cells 300 illustrated by FIG. 3.

[0091] At 442, the method 440 includes contacting a plurality of cells with a volume of a genetically engineered effector cell. The cells can be contacted by contacting a sample with or administering the volume of the genetically engineered effector cell to a host, such as a patient. The genetically engineered effector cell can include at least some of substantially the same components and features as previously described by the effector cell 100 of FIG. 1, the details of which are not repeated for ease of reference. [0092] At 444, in response to contacting the plurality of cells with the genetically engineered effector cell and a presence of the tumor cell within the plurality of cells, the method 440 includes causing binding of the extracellular antigen binding domain to an antigen on a surface of the tumor cell. The plurality of cells, including the infected tumor cell, can include cells of a host (e.g., host cells and target host cells).

[0093] At 446, in response to the extracellular antigen binding domain of the CAR binding to the antigen of the tumor cell, the method 440 includes initiating expression and synthesis of (e.g., transcription and translation of) the enzyme by the actuator element, secreting the enzyme by the signal peptide, and reducing the tumor-associated ECM by the enzyme. As previously described, the enzyme can be a modified form of a wild-type enzyme, and/or the signal peptide can be native to the enzyme or can be nonnative and is encoded by the effector element. In some examples, the method 440 can further include, in response to the extracellular antigen binding domain of the CAR binding to the antigen of the tumor cell, activating the effector cell and, in response, synthesizing and secreting a calibrated amount of the enzyme based on the presence of the tumor cell. As previously described, the calibrated amount of the enzyme can be a function of (e.g., is proportional to) an amount of the tumor cell present within the plurality of cells in the environment.

[0094] In some examples, the method 440 further includes detecting expression of the enzyme. Detectable expression of the enzyme can indicate the presence of the target cell. In some examples, as described above, the enzyme can be bound to a detectable reporter protein by a 2A linker peptide. In response to detecting the presence of the enzyme, an additional anti-cancer treatment can be used, such as an antitumor agent. For example, the anti-cancer treatment and/or antitumor agent (as described above) can include chemotherapy, checkpoint inhibitors, antibodies or antibody-drug conjugates, CAR T-cells, cytokines, enzymes, among others.

[0095] As previously described, the enzyme can reduce the tumor-associated ECM and the effector cell can act indirectly on the tumor cell by co-opting other therapeutic proteins or cells in the body. For example, the effector cell can secrete the enzyme to degrade the tumor-associated ECM and stimulate cytokines and/or other immune cells and therapeutic proteins which can provide additional therapeutic effect. In such examples, the method 440 can further include stimulating cytokines by the genetically engineered effector cell, e.g., as activated. [0096] Although the above examples describe effector proteins as an enzyme, examples are not so limited and can include a variety of different types of proteins. Other example effector proteins encoded by effector cells include a detectable reporter protein, a therapeutic protein, a downstream signaling protein, and a combination thereof. As used herein, a detectable reporter protein includes and/or refers to a protein that is detectable upon expression, such as a protein that provides an optical, electrical or other type of detectable signal. Example detectable reporter peptides include luciferase (Luc or Nluc) or a bioluminescent variant thereof, Green Fluorescent Protein (GFP) or a fluorescent variant thereof, and lacZ or a colorimetric variant thereof. A therapeutic protein includes and/or refers to a protein that provides a therapeutic effect to the patient. Example therapeutic proteins include a cytotoxic protein, an immunostimulatory protein, and an immunosuppressive protein. A downstream signaling protein includes and/or refers to a protein that drives downstream elements of a signaling pathway, such as for regulation of cell growth, proliferation, differentiation, and apoptosis.

[0097] Non-limiting examples of effector proteins include cytotoxic polypeptides of bacterial origin (e.g., parasporin, plantaricin A); insect origin (e.g., Polybia-MPl); antiviral polypeptides from viral origin (e.g., a-helical peptide (AHP)); antiviral polypeptides from viral origin (e.g., anti-viral peptides (AVP)); immunosuppressive peptides of fungal origin (e.g., colutellin A); vasodilators (e.g., relaxin, bradykinin) and endopeptidase (e.g., heparanase, relaxin, collagenase); and cell-penetrating cationic peptides (e.g., LL-37, TAT peptide). With respect to colutellin A, self-reactive effector cells can be engineered from effector cells obtained from hosts or other sources having autoimmune disorders (e.g., type 1 diabetes, polymyositis, and lupus). In particular, the effectors cells can be engineered for localized expression of colutellin A upon stimulation by target self-antigens. Systemic infusion of immunosuppressive agents cannot be used in hosts with these conditions due to the risk of other opportunistic infections. With respect to vasodilators and endopeptidases, such effector cells can be used to improve perfusion (see Chauhan VP & Jain RK (2013) Nat. Mater. 12(11):958- 962) and assist in efficient delivery of anticancer agents or antitumor agents that cannot be systemically administered as they damage structural tissues and are tumorigenic. With respect to cell-penetrating cationic peptides, these target peptides can be used to target intracellular bacteria. For example, site-specific overexpression of such peptides can be a potent therapy for tuberculosis. [0098] Various experiments, as further described below, are directed to developing a cell-based therapeutic that induces the desired enzyme response to reduce the ECM associated with tumor cells.

[0099] Various examples are directed to a pharmaceutical composition comprising a genetically engineered effector cell and a pharmaceutically acceptable carrier or excipient, such as the effector cell 100 of FIG. 1, the effector cell 200 of FIG. 2, and/or the population 331 of effector cells 300 of FIG. 3.

[00100] For example, an effector cell composition, such as a pharmaceutical composition, can comprises a plurality of the genetically engineered effector cells described herein and an acceptable carrier, diluents, or excipient (e.g., a pharmaceutically acceptable carrier, diluent, excipient or a combination thereof). The means of making such a composition have been described in the art (see, for instance, Remington's Pharmaceutical Sciences, 16th Ed., Mack, ed. (1980)). Preferably, the composition is prepared to facilitate the administration of the cells into a living organism. In some examples, the pharmaceutical composition comprises a plurality of genetically engineered effector cells as described herein and, for example, a balanced salt solution, preferably Hanks' balanced salt solution, or normal saline.

[00101] Examples are not limited to effector cells which are used for therapeutics. In some examples, the effector cell can include an effector element that encodes a detectable reporter protein (as described above), which can be used for diagnostics. [00102] In some examples, two different cells types can be generated. The first includes an effector cell as described above in connection with FIGs. 1-4. A target tumor cell can additionally be generated, which can be a cell that is modified to express or present a target antigen and is herein referred to as a “genetically engineered tumor cell” or an “antigen-presenting tumor cell”. In some examples, the genetically engineered tumor cells can be referred to as pseudo-tumor cells, in that the cells are modified to present or express the antigen but may not be cancerous. In such examples, the target antigen on the genetically engineered tumor cells (as well as the antigen binding domain of the effector) can include peptides or scFvs that are specific to a segment (e.g., epitope) on the molecule to be detected. The cells can be modified to form genetically engineered tumor cells using an exogenous polynucleotide sequence that encodes an antigen.

[00103] In some examples, the genetically engineered tumor cells can include antigens, such as those described above. In some examples, the genetically engineered tumor cells can be encoded and/or formed using any of SEQ ID NOs: 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, and 45, as well as combinations thereof. Examples are not so limited and the genetically engineered tumor cells and/or antigens of the genetically engineered tumor cells can include other sequences, such as a sequence with at least 80%, 85%, 90%, 95%, or 99% sequence identity to one or more of the sequences set forth in SEQ ID NOs: 34-45, among other sequences.

[00104] Some examples are directed to methods of forming the genetically engineered effector cells, such as genetically engineering or modifying an effector cell to include the components and features as described by the effector cell 100 of FIG. 1.

[00105] The genetically engineered effector cells and cell compositions provided herein have properties advantageous for use in a variety of in vitro, ex vivo, and in vivo applications. For example, in vitro uses of the effector cells and cell compositions provided herein include, without limitation, detecting target tumor cells on the basis of antigens expressed on the surface of the target cells. Also, the target (host) tumor cell can be a cell type associated with any other pathology for which the affected (host) cell having aberrant expression of a cell surface antigen relative to an unaffected (host) cell. Methods for using the genetically engineered effector cells or cell compositions for in vitro target cell detection are described above and further below.

[00106] Ex vivo uses of the genetically engineered effector cells and cell compositions provided herein include, without limitation, early cancer detection and companion diagnostic or therapeutic applications for the disease tumor cells identified on the basis of antigens expressed on the surface of the disease tumor cells. For example, the cells can be used for ex vivo applications in companion diagnostics for cancer immunotherapy. By way of example, the effector cell engineered with NFAT RE6X with Nluc-2A-GFP can be engineered to express different types of CARs. The expression of Nluc when CAR engages its target antigen versus the non-specific Nluc expression can inform on the comparative and quantitative robustness of each CAR for its efficiency to cause the intended on-target effect versus unintended off-target effects. Methods for using the genetically engineered effector cells or cell compositions in ex vivo therapeutic applications are described further below.

[00107] In vivo applications of the genetically engineered effector cells and cell compositions provided herein include, without limitation, in vivo methods for localized therapy at a disease site (e.g., targeted therapy for ovarian cancer).

[00108] Various examples are directed to a panel of different types of genetically engineered effector cells, such as a plurality of effector cells engineered with different effector proteins and/or extracellular antigen binding domains (among other differences), and which are used to simultaneously target tumor cells and/or secrete different effector proteins.

[00109] In some examples, a method of detecting a target tumor cell comprises (a) contacting a genetically engineered effector cell to a cell population, and (b) detecting expression of the enzyme, wherein detectable expression of the enzyme indicates the presence of the target tumor cell of interest. In some examples, the effector cell includes aNFAT-RE and a detectable reporter protein, and in the presence of the target tumor cell in the contacted cell population, the genetically engineered effector cell binds to a surface molecular antigen on the target tumor cell and activates the NFAT-RE; and (b) detecting expression of the detectable reporter protein, wherein detectable expression of the reporter protein indicates the presence of the target tumor cell.

[00110] Some examples are directed to methods of treating or preventing cancer using genetically engineered effector cells expressing a CAR as a therapeutic agent. For example, provided herein are methods comprising administering a genetically engineered effector cell expressing the CAR as an active therapeutic agent. The disease against which the effector cell expressing the CAR is administered is not particularly limited as long as the disease shows sensitivity to the effector cell. In some examples, a genetically engineered effector cell expressing the CAR binds to an antigen expressed on the surface of a target tumor cell that targeted to be decreased or eliminated for treatment of the aforementioned diseases, that is, an enzyme for effector, is administered to treat or prevent such diseases. The terms “treat,” and “prevent” as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, methods described herein can provide any amount of any level of treatment or prevention of cancer in a mammal or other host. Furthermore, the treatment or prevention provided by example methods can include treatment or prevention of one or more conditions or symptoms of cancer. Also, for purposes herein, “prevention” can encompass delaying the onset of the disease, or a symptom or condition thereof.

[00111] In some examples, genetically engineered effector cells are administered to a host (e.g., subject) in need thereof as a composition comprising the genetically engineered effector cells and a suitable carrier, diluent, or excipient as described herein. Any appropriate method of providing modified CAR-expressing cells to a host can be used for methods described herein. In some examples, methods for providing effector cells to a host can be adapted from clinical protocols for cellular and adoptive immunotherapy for infusion of donor-derived immune cells into a human host. In some examples, an adapted clinical protocol suitable for methods provided herein comprises obtaining effector cells from a host, genetically engineering (e.g., modifying) effector cells to express a CAR and NFAT-RE regulated enzyme transgene as described above, and infusing the genetically engineered effector cells back into the host. A host, as used herein, includes and/or refers to any organism, such as a human, an animal (e.g., mammal, reptile, bird), insect, plant, among others, and which can be a subject of a study or test and/or a patient. A “subject” is sometimes interchangeably used with “host”. Host cells include cells obtained from the host.

[00112] Administration of the genetically engineered effector cells provided herein can be administered by any appropriate route, including, without limitation, administration intravenously, intratumorally, intramuscularly, subcutaneously, intraperitoneally, intraarterially, or into an afferent lymph vessel, by parenteral administration, for example, by injection or infusion. In some examples, where genetically engineered effector cells or populations of such effector cells are administered, the effector cells can be cells that are allogeneic or autologous to the host, such as a mammal. Preferably, the effector cells are autologous to the host.

[00113] In some examples, the genetically engineered effector cells comprise a CAR that detects an antigen on a tumor cell, and a NF AT response element to induce expression of a reporter polypeptide. Such examples can be used for transfusion medicine to detect the presence of cancer cells and/or to identify and track the progress of cancer within a subject.

[00114] As used herein, a target tumor cell (sometimes herein interchangeably referred to as a “target cell of a host”, “target cell of interest”, “a diseased cell”, or “a target disease cell”) includes and/or refers to a tumor cell of interest associated with a living organism (e.g., a biological component of interest) or a modified living cell in a test environment (e.g., genetically modified tumor cells or other antigen-present cells in solution). An antigen of the target tumor cell includes and/or refers to a structure (e.g., binding site) of the target tumor cell which the antigen binding domain of the receptor element can bind to (e.g., has an affinity for). The effector cell can be from a variety of different type of cells, such as human and non-human cells, and sometimes herein referred to as “the source”. As used herein, the terms “genetically modified” and “genetically engineered” are used interchangeably and include and/or refer to a prokaryotic or eukaryotic cell that includes an exogenous polynucleotide, regardless of the method used for insertion. In some examples, the effector cell is modified to comprise a non-naturally occurring nucleic acid molecule that is created or modified by the hand of man (e.g., using recombinant deoxyribonucleic acid (DNA) technology) or is derived from such a molecule (e.g., by transcription, translation, etc.). An effector cell that contains an exogenous, recombinant, synthetic, and/or otherwise modified polynucleotide is considered to be a genetically engineered effector cell.

[00115] “Nucleic acid”, as used herein, includes and/or refers to a “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally means a polymer of DNA or ribonucleic acid (RNA), which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, nonnatural or altered intemucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide. In some examples, the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions. It may be suitable in some instances, for the nucleic acid to comprise one or more insertions, deletions, inversions, and/or substitutions. In some examples, the nucleic acid can encode additional amino acid sequences that do not affect the function of the CAR and polynucleotide and which may or may not be translated upon expression of the nucleic acid by a host cell.

[00116] Nucleic acids can be obtained using any suitable method, including those described by Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., pp. 280-281 (1982) and/or U.S. Patent Application Publication No. US2002/0190663, each of which are herein fully incorporated in their entireties for their general and specific teachings. Nucleic acids obtained from biological samples typically are fragmented to produce suitable fragments for analysis.

[00117] Nucleic acids and other moi eties can be isolated. As used herein, “isolated” includes and/or refers to separate from at least some of the components with which it is usually associated whether it is derived from a naturally occurring source or made synthetically, in whole or in part. Nucleic acids and/or other moieties can be purified. As used herein, “purified” includes and/or refers to separate from the majority of other compounds or entities. A compound or moiety can be partially purified or substantially purified. Purity can be denoted by a weight by weight measure and/or determined using a variety of analytical techniques such as but not limited to mass spectrometry and High-performance liquid chromatography (HPLC).

[00118] Various examples are implemented in accordance with the underlying Provisional Application, Ser. No. 63/234,314, entitled “T-cell Biofactory for Degrading Tumor- Associated Extracellular Matrix,” filed August 18, 2021, to which benefit is claimed and which is fully incorporated herein by reference for its general and specific teachings. For instance, examples herein and/or in the Provisional Application can be combined in varying degrees (including wholly). Reference can also be made to the experimental teachings and underlying references provided in the underlying Provisional Application. Examples discussed in the Provisional Application are not intended, in any way, to be limiting to the overall technical disclosure, or to any part of the claimed disclosure unless specifically noted. In some examples, the genetically modified effector cells, diagnostic cells, and/or reporter cells can include at least some of the components or features as described by Repellin CE, et al., entitled “Modular Antigen-Specific T-cell Biofactories for Calibrated In Vivo Synthesis of Engineered Proteins”, Advanced Biosystems, 2(12): 1800210 (2018), and Repellin CE, et al, entitled “NK-Cell Biofactory as an Off-the-Shelf Cell-based Vector for Targeted In Situ Synthesis of Engineered Proteins”, Advanced BioSystems 5(7): 2000398 (2021), each of which are hereby incorporated in their entirety for their general and specific teaching.

EXPERIMENTAL EMBODIMENTS

[00119] A number of experimental examples were conducted to generate genetically engineered effector cells and to characterize functionality. Example constructs used to generate genetically engineered effector cells include the nucleotide sequences set forth in SEQ ID NOs: 1-45. SEQ ID NOs: 1-45 are each synthetic constructs of DNA.

[00120] In some examples, immune cells were modified to form effector cells that, upon engaging the antigen-presenting tumor cells (e.g., natural or genetically modified), produce non-endogenous enzymes for exerting a target effect locally on the TME. [00121] In some examples, two different types of cells, e.g., T-cells, were modified to form effector cells that synthesize enzymes, which are sometimes herein referred to as “T-cell Biofactories” or “cell biofactory”. In some examples, T-cell Biofactories with specificity towards FRa were engineered by using collagenase-2 (e.g., MMP8) as the effector protein. Proof-of-concept was conducted in Acute Lymphoblastic Leukemia T- cell line (Jurkat cells) before translating in primary T-cells derived from a human donor. [00122] FIGs. 5A-5D illustrate example expression constructs associated with genetically engineered effector cells, in accordance with the present disclosure. More particularly, FIG. 5 A is a schematic representation of an expression construct for FRa- specific collagenase-2 T-cell Biofactory. The T-cell Biofactory includes an actuator element, an effector element that encodes collagenase-2, and a receptor element with a CAR specific to FRa. FIG. 5B is a schematic representation of an expression construct for a control cell that includes an actuator element, a receptor element specific to FRa, and an effector element that encodes detectable reporter proteins, e.g., GFP and Nluc. [00123] As used herein, an expression construct includes and/or refers to a nucleic acid sequence (e.g., DNA sequence) including vector(s) or binary vector(s) carrying gene(s). A vector or binary vector includes and/or refers to a DNA sequence that includes a transgene, sometimes referred to as “inserts”, and a backbone. The vector or binary vector can include an expression construct or cassette that includes the transgene and a regulatory sequence to be expressed by a transformed effector cell.

[00124] FIGs. 5C-5D are schematics of different effector elements for a T-cell Biofactory. FIG. 5C is a schematic representation of an effector element that encodes a wild-type collagenase-2. As shown by FIG. 5C, the wild-type collagenase-2 includes a signal peptide, a pro-peptide, and active collagenase-2 (e.g., active protein). As described above, the intracellular synthesis of collagenase-2 is followed by its transport across the cellular membrane assisted by the native secretory signal peptide (1-20 amino acids (aa)). The immature collagenase-2 includes a pro-peptide (21-100aa) and the active protein (101-467aa), then undergoes activation by removing the pro-peptide portion leaving the activated collagenase-2 with an N-terminal MetlOO or LeulOl overhang from the pro-peptide. In physiological in vivo settings, this happens with the help of proteinases in the extracellular milieu. To prepare a therapeutic molecule, it can be achieved in vitro by treating collagenase-2 with proteinases, mercurial compounds and oxidants. The activated form has low half-life when injected. An activated version of collegenase-2 with MetlOO or LeulOl at the N-terminal can be used in some examples, and sometimes herein referred to as “Collagenase-2 T-cell Biofactory”. [00125] FIG. 5D is a schematic representation of an effector element that encodes a modified collagenase-2. In some examples, a modified collagenase-2 is used, which includes a different amino acid overhang including Phe99 at the N-terminal, and which can be achieved by Stromelysin- 1 activation and results in a superactivated version of collagenase-2 expressed from the T-cell Biofactory, sometimes herein referred to as “Act-Collagenase-2 T-cell Biofactory”. The superactivated collagenase-2 has 3.5-fold more activity compared to other active versions of collagenase-2. In various examples, the 369aa superactivated collagenase-2 (Phe99-467aa) was cloned and the signal peptide from Interferon alpha-2a (IFNa2) was added for transporting the superactivated collagenase-2 across the T-cell membrane and into the extracellular space.

[00126] FIGs. 6A-6F illustrate plots characterizing genetically engineered effector cells producing enzymes as a function of time, target, and cell number, in accordance with the present disclosure.

[00127] FIG. 6A is a fluorescence-activated cell sorting (FACS) plot showing FRa- CAR positivity in collagenase-2 T-cell Biofactory (e.g., a Jurkat T-cell generated using an expression construct as shown by FIG. 5 A and 5C) as compared to the control CAR T-cell (e.g., generated using an expression construct as shown by FIG. 5B) and an unmodified T-cell. FIG. 6B is a plot showing collagenase-2 expression from the T-cell Biofactory or control cells when stimulated by FRa+ and FRaneg target cells as a function of time. FIG. 6C is a plot showing collagenase-2 expression from the T-cell Biofactory or control cells when stimulated by FRa+ and FRaneg target cells as a function of target tumor cell number. FIG. 6D is a plot showing collagenase-2 expression from the T-cell Biofactory (generated using plasmid 1) or control cells (generated using plasmid 5) when stimulated by FRa+ and FRaneg target cells as a function of effector cell number or control cell number.

[00128] FIG. 6E is a plot showing collagenase-2 expression from a T-cell Biofactory in comparison with control cells (e.g., Car T-cells and unmodified T-cells) when stimulated with 5 different FRa+ and FRaneg target cells for 48 hours at an Effector: Target (E:T) ratio of 10:1. Baseline collagenase-2 expression from the respective effector cells and target cells are shown as “No target” and “No Effector”. Effector cell is either T-cell Biofactory or control CAR T-cell. Collagenase-2 expression was quantified using total human collagenase-2 ELISA on cell culture supernatants from co-cultures of 48 hours (unless otherwise specified).

[00129] FIG. 6F is a plot showing collagenase-2 activity in the cell culture supernatants from a T-cell Biofactory when stimulated chemically (e.g., with PMA/Io) in comparison with control CAR T-cells. The collagenase-2 activity was measured as the rate of change of fluorescence observed when the synthetic collagenase substrate Mca-PLGL- DPA-AR-NH2 was cleaved by the expressed collagenase-2.

[00130] As shown by FIGs. 6A-6F, it was demonstrated that the specificity of ECM- degrading T-cell Biofactory was successfully directed toward the FRa antigen (FIG. 6A). Further, the antigen specific stimulation of the T-cell Biofactory to produce collagenase-2 was confirmed (FIG. 6F). While the collagenase-2 production from the T- cell Biofactory was proportional to the number of T-cell Biofactory (FIG. 6D) and the duration of stimulation (FIG. 6B), it was also demonstrated that it was proportional to the number of target cells (FIG. 6C).

[00131] FIGs. 7A-7B illustrate plots characterizing expression of enzymes by genetically engineered effector cell responsive to stimulation by PMA/Io as a function of cell number, in accordance with the present disclosure.

[00132] FIG. 7A is a plot showing collagenase-2 expression from Collagenase-2 T-cell Biofactory (generated using plasmid 1) or control cells (generated using plasmid 5) when chemically stimulated by PMA/Io as a function of cell number (both cell lines used Jurkat cell as the chassis). Collagenase-2 expression was quantified using total human collagenase-2 ELISA on cell culture supernatants from co-cultures of 48 hours. FIG. 7B is a FACS plot showing FRa-antigen positivity in 5 ovarian cancer cell lines (generated using plasmids 7-10) including: (i) OVCAR-3, (ii) A2780cis, (iii) A1847, (iv) A2780cis-Luc2, and (v) A2780cis-FRa-Luc2.

[00133] FIGs. 8A-8F illustrate plots characterizing genetically engineered effector cell function, in accordance with the present disclosure. Specifically, FIGs. 8A-8F illustrate the functional characterization of Act-Collagenase-2 T-cell Biofactory (using Jurkat cell as the chassis and plasmid 2 and/or the expression construct as illustrated by FIG. 5D). [00134] FIG. 8A is a FACS plot of FRa-CAR expression in Act-Collagenase-2 T-cell Biofactory (cell line). FIG. 8B is a plot showing collagenase-2 activity in the cell culture supernatants from Act-Collagenase-2 T-cell Biofactory (e.g., generated using the expression construct illustrated by FIG. 5D and/or plasmid 2) and Collagenase-2 T-cell Biofactory (e.g., generated using the expression construct illustrated by FIG. 5C and/or plasmid 1) when stimulated chemically (with PMA/Io) in comparison with control CAR T-cells (e.g., generated using the expression construct illustrated by FIG. 5B and/or plasmid 5) as a function of cell number. The Collagenase-2 activity was measured as the rate of change of fluorescence observed when the synthetic collagenase substrate Mca- PLGL-DPA-AR-NH2 was cleaved by the expressed collagenase-2. FIG. 8C is a plot showing collagenase-2 expression from Act-Collagenase-2 T-cell Biofactory and Collagenase-2 T-cell Biofactory (produces full-length collagenase-2) in comparison to control CAR T-cells when stimulated with 5 different FRa+ and FRaneg target cells (e.g., as associated with plasmids 7-10) for 48 hours at an E:T ratio of 1:1. Baseline collagenase-2 expression from the respective effector cells are shown as “No target”. The effector cells include (i) T-cell Biofactory that produces superactivated collagenase- 2 (e.g., Act-Collagenase-2 T-cell Biofactory), (ii) T-cell Biofactory that produces the full length collagenase-2 (e.g., Collagenase-2 T-cell Biofactory), and (iii) CAR T-cells. [00135] FIGs. 8D-8E are plots showing active collagenase-2 from Act-Collagenase-2 T- cell Biofactory degrades rat tail collagen- 1 coating upon chemical, PMA/Io, and antigenic, beads loaded with a-CD3 and a-CD28 (CD3/28), stimulation compared to control CAR T-cells. The un-degraded collagen-1 left in the plate was quantified by Coomassie staining and absorbance measurement at 595nm. The Act-Collagenase-2 T- cell Biofactory mediated collagen-1 degradation can be inhibited by pan-collagenase inhibitor (lOpM GM6001). FIG. 8F is a plot showing ex-vivo degradation of collagen-1 in tumor explants derived from KPCY tumor (this cell line produced collagen-1 rich ECM) bearing C57/BL6 mice by conditioned media derived from Act-Collagenase-2 T- cell Biofactory in comparison with the conditioned media derived from control CAR T- cells. Collagen degradation was quantified by assessing the presence of hydroxyproline, a degradation byproduct in supernatants of tumor explants treated with conditioned media. Conditioned media was derived by co-culturing Act-Collagenase-2 T-cell Biofactory and control CAR T-cells with OVCAR3 cells for 5 days at an E:T ratio of 10:1. The OVCAR3 cells have endogenous expression of FRa antigen, and the OVCAR3 cells have been modified to express the reporter proteins Luc2 and E2 Crimson for traceability and tumor clearance analysis.

[00136] Various experiments were conducted to functionally characterize and compare the Act-Collagenase-2 T-cell Biofactory (using primary human T-cells). FIGs. 9A-9D, FIGs. 10A-10E, and FIGs. 11A-11C illustrates examples experimental results of generating effector cells using human primary T-cells.

[00137] FIGs. 9A-9D illustrate plots characterizing different types of genetically engineered effector cells as compared to control cells, in accordance with the present disclosure. More specifically, FIGs. 9A-9D are FACS plot showing FRa-CAR expression in primary human Act-Collagenase-2 T-cell Biofactory (FIG. 9B and generated using plasmid 2), Collagenase-2 T-cell Biofactory (FIG. 9D and generated using plasmid 1), and control T-cells (FIG. 9 A is unmodified T-cells and FIG. 9C is control CAR T-Cell as generated using plasmid 5).

[00138] FIGs. 10A-10E illustrate plots characterizing therapeutic activity of example genetically engineered effector cells, in accordance with the present disclosure. FIG. 10A is a plot showing collagenase-2 expression from Act-Collagenase-2 T-cell Biofactory (generated using plasmid 2) in comparison with control CAR T-cells (generated using plasmid 5) and unmodified T-cells when stimulated with 5 different FRa+ and FRaneg target cells (generated using plasmids 7-10) for 48 hours at an E:T ratio of 6:1. In this figure, the Effector = primary T-cell Biofactory or primary CAR T- cells. Baseline collagenase-2 expression from the respective effector cells are shown as “No target”. FIG. 10B is a plot showing collagenase-2 expression from Act- Collagenase-2 T-cell Biofactory and Collagenase-2 T-cell Biofactory (generated using plasmid 1) in comparison with control CAR T-cells as a function of effector cell numbers when stimulated with FRa+ target cells (OVCAR3) for 48 hours. FIG. 10C is a plot showing collagenase-2 activity in the cell culture supernatants from Act- Collagenase-2 T-cell Biofactory and Collagenase-2 T-cell Biofactory when stimulated with FRa+ target cells (OVCAR3) (E:T ratio of 1 : 1) for 48 hours in comparison with control CAR T-cells. The collagenase-2 activity was measured as the rate of change of fluorescence observed when the synthetic collagenase substrate Mca-PLGL-DPA-AR- NH2 was cleaved by the expressed collagenase-2.

[00139] FIGs. 10D-10E (as well as FIGs. 11 A-l 1C) demonstrate that encoding the T- cells with Biofactory function does not compromise other T-cell functions. FIG. 10D is a plot showing Interleukin-2 (IL-2) secretion and FIG. 10E is a plot showing Interferon y (I FNy ) secretion from Act-Collagenase-2 T-cell Biofactory in comparison with control CAR T-cells and unmodified T-cells when stimulated by FRa+ (Al 847) and FRaneg (A2780cis) target cells for 24 hours at an E:T ratio of 1:1.

[00140] FIGs. 11A-11C illustrate plots characterizing cytolytic activity of example genetically engineered effector cells, in accordance with the present disclosure. As noted above, FIGs. 11 A-l 1C demonstrate that encoding the T-cells with Biofactory function does not compromise other T-cell functions. FIGs. 11A-11C are plots showing cytolytic activity of Act-Collagenase-2 T-cell Biofactory in comparison with control CAR T-cells and unmodified T-cells on Luc2 expressing FRa+ (FIG. 11 A is Al 847 and FIG. 11C is A2780cis-FRa) and FRaneg (FIG. 11B is A2780cis) target cells when co-cultured for 24 hours. Luc2 activity was normalized to 100% using target cell only and to 0% when target cells were treated with 1% Tween20. The curves were fit using a four-parameter logistic model, Luc2 = Luc2min + {Luc2max - Luc2min}/{1 + 10^A [b* (logl0[h(E:T)50] - X)]}.

[00141] Various experiments were conducted to assess collagen producing mouse tumor cell line engineered to express human FRa tumor antigen to stimulate primary human T- cells engineered to express collagenase-2.

[00142] FIGs. 12A-12B illustrate plots characterizing example genetically engineered tumor cells which are modified to express human FRa tumor antigens, in accordance with the present disclosure. The KPC mouse tumor cell line with yellow fluorescent protein (YFP) expression was engineered to express E2 Crimson, Luc2, and FRa, and used as a target tumor cell. FIG. 12A is a FACS plot showing E2 Crimson expression in two engineered KPC mouse tumor cells lines (generated using plasmids 10-11) with YFP expression. FIG. 12B is a plot showing Luc2 activity from the engineered tumor cell lines as a function of cell number. Luc2 activity was measured using OneGlo® reagent from Promega following manufacturer instructions.

[00143] FIGs. 13A-13B illustrate plots characterizing response of genetically engineered effector cells to the genetically engineered tumor cells, in accordance with the present disclosure. FIG. 13 A is a plot showing collagenase-2 expression form Act- Collagenase-2 T-cell Biofactory (Effector Chassis: Jurkat cell line), Collagenase-2 T- cell Biofactory (Effector Chassis: Jurkat cell line) and control CAR T-cell (Effector Chassis: Jurkat cell line) line when stimulated by two KPC tumor cell lines engineered to express FRa for 48 hours at an E:T ratio of 1 : 1. FIG. 13B is a plot showing collagenase-2 expression from Act-Collagenase-2 T-cell Biofactory (Effector Chassis: primary T-cell) in comparison with control CAR T-cells (Effector Chassis: primary T cell) when stimulated with two KPC tumor cell lines engineered to express FRa for 48 hours at an E:T ratio of 6:1. Baseline collagenase-2 expression from the respective effector cells are shown as “No target”.

[00144] FIGs. 14A-14D illustrate plots characterizing cytolytic activity of example genetically engineered effector cells in response to the genetically engineered tumor cells, in accordance with the present disclosure. More specifically, FIGs. 14A-14D are plots showing cytolytic activity of Act-Collagenase-2 T-cell Biofactory (Effector Chassis: primary T-cell) in comparison with control CAR T-cells (Effector Chassis: primary T-cell) and unmodified T-cells on Luc2 expressing FRaneg (FIG. 14A is KPC2838c3, and FIG. 14C is KPC6694c2) and FRa+ (FIG. 14B is KPC2838c3-FRa, and FIG. 14D is KPC6694c2-FRa) target cells when co-cultured for 24 hours. Luc2 activity was normalized to 100% using target cell only and to 0% when target cells were treated with 1% Tween20. The curves were fit using a four-parameter logistic model, Luc2 = Luc2min + {Luc2max - Luc2min}/{1 + 10^A [b* (log!0[h(E:T)50] - X)]}. [00145] Various experiments were conducted to assess FRa-specific T-cell Biofactory used to express different ECM-degrading enzymes (chassis: Jurkat cell line).

[00146] FIGs. 15A-15C illustrate plots characterizing different enzymes expressed by example genetically engineered effector cells, in accordance with the present disclosure. The FRa-specific primary T-cell Biofactory was used to synthesize HPSE (FIG. 15 A, generated using plasmid 4), Collagenase-2 (FIG. 15B, FRa-specific Act Collagenase-2 T-cell Biofactory generated using plasmid 2) and b-Sialidase (FIG. 15C, generated using plasmid 3); after their interaction with FRa-positive target tumor cells (OVCAR3 (FRa+) or A2780cis (FRaneg) cell lines, generated using plasmids 7 and 9). Enzyme activity was measured with different kits involving degradation of enzyme-specific substrate. Different degree of activity was observed from each T-cell Biofactory.

[00147] Various plasmids were generated and used to form the T-cell Biofactories and control cells. The table below summarizes the plasmids.

Table of Plasmids and related genetic elements

For the above plasmids 1-4, each of the plasmids includes a receptor element, an actuator element, and an effector element. Each of the receptor elements encodes an antigen binding domain, a transmembrane domain, and an intracellular binding domain. Example antigen binding domains includes the VH and VL binding domains, such as from an anti-FRa antibody and/or those encoded by SEQ ID NO: 5 (scFv including VH binding domain (SEQ ID NO: 6 ), VL binding domain (SEQ ID NO: 8), and a linker (SEQ ID NO: 7). The transmembrane domain and/or an intracellular binding domain include myc (SEQ ID NO: 9), CD8 cytoplasmic (SEQ ID NO: 10), CD28 cytoplasmic (SEQ ID NO: 11), CD28 transmembrane (SEQ ID NO: 21), 4-1BB cytoplasmic (SEQ ID NO: 12), and/or CD3-zeta cytoplasmic (SEQ ID NO: 13). The actuator elements encode transcription factors, such as NFATs as encoded by SEQ ID NO: 3. The effector elements encode an enzyme, and/or an enzyme with a signal peptide that is non-native to the enzyme, such as collagenase-2 (SEQ ID NO: 4), superactivated collagenase-2 (SEQ ID NO: 20), b-Sialidase (SEQ ID NO 26), or HPSE (SEQ ID NO 30). Plasmids 5- 6 were used as controls and plasmids 7-11 were used to generate target tumor cells. The following describes each of plasmids 1-11. Cytoplasmic refers to a cytoplasmic domain, which includes or is interchangeable with an intracellular signaling domain.

[00148] Plasmid 1 (SEQ ID NO: 1) was used to generate a FRa-specific Collagenase-2 T-cell Biofactory that produces active collagenase-2. Plasmid 1 includes a receptor element that encodes aNFAT-Re (x6) (SEQ ID NO: 3), an effector element that encodes an enzyme (SEQ ID NO: 4), and a receptor element that encodes a CAR with an antigen binding domain including a scFv of an anti-FRa antibody (including a VH domain (SEQ ID NO: 6), a linker (SEQ ID NO: 7) and a VL domain (SEQ ID NO: 8)), myc (SEQ ID NO: 9), CD8 cytoplasmic (SEQ ID NO: 10), CD28 cytoplasmic (SEQ ID NO: 11), 4-1BB cytoplasmic (SEQ ID NO: 12), and CD3-zeta cytoplasmic (SEQ ID NO: 13), as well as encoding F2A (SEQ ID NO: 14) and Puromycin N-acetyltransferase (PAC) produced by Streptomyces alboniger (SEQ ID NO: 15), among other genetic elements. Myc serves as a tag that allows preferential sorting/selection of the cells expressing it, as well as specific characterization of the cells expressing it using flow cytometry. PAC is an antibiotic resistance gene that allows selection of transformed cells using the application of Puromycin (Antibiotic) in vitro.

[00149] Plasmid 2 (SEQ ID NO: 16) was used to generate a FRa-specific Act Collagenase-2 T-cell Biofactory that produces superactivated collagenase-2. Plasmid 2 includes a receptor element that encodes a NFAT-Re (x6) (SEQ ID NO: 3), an effector element that encodes a IFNalpha2 signal peptide (SEQ ID NO: 19) and a modified enzyme /collagenase-2 (SEQ ID NO: 20), a receptor element that encodes a CAR with an antigen binding domain including a scFv of an anti-FRa antibody (including a VH domain (SEQ ID NO: 6), a linker (SEQ ID NO: 7) and a VL domain (SEQ ID NO: 8)), myc (SEQ ID NO: 9), CD8 cytoplasmic (SEQ ID NO: 10), CD28 transmembrane (SEQ ID NO: 21), 4-1BB cytoplasmic (SEQ ID NO: 12), and CD3-zeta cytoplasmic (SEQ ID NO: 13), as well as encoding F2A (SEQ ID NO: 14) and PAC (SEQ ID NO: 22), among other genetic elements. Examples are not limited to plasmids including all of the listed genetic elements. For example, plasmid 2 may not include the CD28 transmembrane. [00150] Plasmid 3 (SEQ ID NO: 23) was used to generate a FRa-specific Sialidase T- cell Biofactory that produces active b-Sialidase. Plasmid 3 includes a receptor element that encodes aNFAT-Re (x6) (SEQ ID NO: 3), an effector element that encodes a IFNalpha2 signal peptide (SEQ ID NO: 19) and b-Sialidase (SEQ ID NO: 26), a receptor element that encodes a CAR with an antigen binding domain including a scFv of an anti-FRa antibody (including a VH domain (SEQ ID NO: 6), a linker (SEQ ID NO: 7) and a VL domain (SEQ ID NO: 8)), myc (SEQ ID NO: 9), CD8 cytoplasmic (SEQ ID NO: 10), CD28 cytoplasmic (SEQ ID NO: 11), 4-1BB cytoplasmic (SEQ ID NO: 12), and CD3-zeta cytoplasmic (SEQ ID NO: 13), as well as encoding F2A (SEQ ID NO: 14) and PAC (SEQ ID NO: 27), among other genetic elements.

[00151] Plasmid 4 (SEQ ID NO: 28) was used to generate a FRa-specific HPSE T-cell Biofactory that produces the active HPSE. Plasmid 4 includes a receptor element that encodes aNFAT-Re (x6) (SEQ ID NO: 3), an effector element that encodes HPSE (SEQ ID NO: 30), a receptor element that encodes a CAR with an antigen binding domain including a scFv of an anti-FRa antibody (including a VH domain (SEQ ID NO: 6), a linker (SEQ ID NO: 7) and a VL domain (SEQ ID NO: 8)), myc (SEQ ID NO: 9), CD8 cytoplasmic (SEQ ID NO: 10), CD28 (SEQ ID NO: 11), 4-1BB cytoplasmic (SEQ ID NO: 12), and CD3-zeta cytoplasmic (SEQ ID NO: 13), as well as encoding F2A (SEQ ID NO: 14) and PAC (SEQ ID NO: 31), among other genetic elements.

[00152] Plasmid 5 and plasmid 6 were used as controls. Plasmid 5 (SEQ ID NO: 32) was used to generate a Control FRa 28-BB CAR T-cell. Plasmid 5 includes a receptor element that encodes a CAR with an antigen binding domain including a scFv of an anti-FRa antibody (including a VH domain (SEQ ID NO: 6), a linker (SEQ ID NO: 7) and a VL domain (SEQ ID NO: 8)), CD8 (SEQ ID NO: 10), myc (SEQ ID NO: 9), CD28 cytoplasmic (SEQ ID NO: 11), 4-1BB cytoplasmic (SEQ ID NO: 12), and CD3- zeta cytoplasmic (SEQ ID NO: 13), as well as encoding F2A (SEQ ID NO: 14) and PAC (SEQ ID NO: 22) ), among others. Plasmid 6 (SEQ ID NO: 33) can be used to generate a Control FRa BB CAR T-cell. Plasmid 6 includes a receptor element that encodes a CAR with an antigen binding domain including a scFv of an anti-FRa antibody (including a VH domain (SEQ ID NO: 6), a linker (SEQ ID NO: 7) and a VL domain (SEQ ID NO: 8)), myc (SEQ ID NO: 9), CD8 cytoplasmic (SEQ ID NO: 10), CD28 transmembrane (SEQ ID NO: 21), 4-1BB cytoplasmic (SEQ ID NO: 12), and CD3-zeta cytoplasmic (SEQ ID NO: 13), as well as encoding F2A (SEQ ID NO: 14) and PAC (SEQ ID NO: 22), among others. Plasmid 5 encodes for a CAR with an intracellular signaling motif including both CD28 and 4-1BB domains, whereas plasmid 6 encodes for a CAR with an intracellular signaling motif includes 4- IBB domains. Plasmid 5 is a matched control plasmid to plasmid 1, plasmid 3, and plasmid 4. Plasmid 6 is a matched control plasmid to plasmid 2.

[00153] Plasmids 7-11 were used to generate target tumor cells which included detectable reporter proteins, such as Luc2-E2 Crimson expressing tumor cells. Plasmid 7 (SEQ ID NO: 34) was used to generate Luc2-E2 Crimson expressing target tumor (OVCAR3) cells. Various plasmids, including plasmid 7, encode Luc2 (SEQ ID NO: 35), E2 Crimson (SEQ ID NO: 37), and P2A (SEQ ID NO: 36) between the Luc2 and E2 Crimson, as well as encoding PAC, among other genetic elements. Plasmid 8 (SEQ ID NO: 38) was used to generate FRa-specific Luc2-E2 Crimson expressing target tumor (A2780cis) cells. Plasmid 8 encodes a FRa antigen (SEQ ID NO: 44), Luc2 (SEQ ID NO: 35), E2 Crimson (SEQ ID NO: 40), and a promoter of EFl (SEQ ID NO: 39), as well as encoding myc (SEQ ID NO: 45) and PAC, among other genetic elements. Plasmid 9 (SEQ ID NO: 41) was used to generate further Luc2-E2 Crimson expressing target tumor (A2780cis) cells. Plasmid 9 encodes Luc2 (SEQ ID NO: 35), E2 Crimson (SEQ ID NO: 40), and a promoter of EFl (SEQ ID NO: 39), as well as encoding myc (SEQ ID NO: 45) and PAC, among other genetic elements. Plasmid 10 (SEQ ID NO: 42 was used to generate further FRa-specific Luc2-E2 Crimson expressing target tumor (Al 847 and KPC) cells and encodes an FRa antigen (SEQ ID NO: 44), Luc2 (SEQ ID NO: 35), E2 Crimson (SEQ ID NO: 40), and a promoter of EFl (SEQ ID NO: 39), as well as encoding myc (SEQ ID NO: 45) and PAC, among other genetic elements.

Plasmid 11 (SEQ ID NO: 43) was used to generate further Luc2-E2 Crimson expressing target tumor (KPC) cells. Plasmid 11 encodes Luc2 (SEQ ID NO: 35), E2 Crimson (SEQ ID NO: 40), and a promoter of EFl (SEQ ID NO: 39), as well as encoding myc (SEQ ID NO: 45) and PAC, among other genetic elements.

[00154] Although examples have been illustrated and described herein, a variety of alternate or equivalent implementations can be substituted for the examples shown and described without departing from the scope of the present disclosure. This application is intended to cover adaptations or variations of the specific examples discussed herein.

Claims

1. A genetically engineered effector cell comprising an exogenous polynucleotide sequence that includes, in operative association: a receptor element that encodes a chimeric antigen receptor (CAR) including an extracellular antigen binding domain operably linked to a transmembrane domain and an intracellular signaling domain, wherein the extracellular antigen binding domain recognizes an antigen on a surface of a tumor cell; an actuator element that encodes a transcription factor binding site that upregulates synthesis of an enzyme in response to the extracellular antigen binding domain of the CAR binding to the antigen of the tumor cell, the enzyme configured to reduce a tumor-associated extracellular matrix (ECM) associated with the tumor cell; and an effector element that encodes the enzyme wherein, in response to the extracellular antigen binding domain of the CAR binding to the antigen of tumor cell, the genetically engineered effector cell is configured to activate and, to synthesize and secrete the enzyme to a tumor microenvironment (TME) associated with the tumor cell.

2. The genetically engineered effector cell of claim 1, wherein the genetically engineered effector cell is configured to synthesize and secrete an amount of the enzyme as a function of an amount of the tumor cell present in a sample or in situ.

3. The genetically engineered effector cell of claim 2, wherein the amount of the enzyme is proportional to the amount of tumor cell present in situ.

4. The genetically engineered effector cell of claim 1, wherein the effector element further encodes a signal peptide upstream of the enzyme, the signal peptide being nonnative to the enzyme.

5. The genetically engineered effector cell of claim 1, wherein the enzyme is a modified form of a wild-type enzyme.

43

6. The genetically engineered effector cell of claim 5, wherein the enzyme encoded by the effector element includes an active protein and a sub-portion of a pro-peptide of the wild-type enzyme.

7. The genetically engineered effector cell of claim 5, wherein the enzyme encoded by the effector element includes a removed native signal peptide, and the effector element further includes a signal peptide that is non-native to the enzyme.

8. The genetically engineered effector cell of claim 1, wherein the activation of the genetically engineered effector cell regulates stimulation of cytokines and causes the secretion of the enzyme to the TME.

9. The genetically engineered effector cell of claim 1 , wherein the intracellular signaling domain includes at least one of: an intracellular signaling portion of a CD28, an intracellular signaling portion of a 4- IBB, and an intracellular signaling portion of a CD3 zeta.

10. The genetically engineered effector cell of claim 1, wherein the transcription factor binding site is selected from the group consisting of: a nuclear factor of activated cell (NF AT) response element, a serum response element (SRE), and a cyclic AMP response element (CRE).

11. A population of genetically engineered effector cells, each of the genetically engineered effector cells of the population comprising an exogenous polynucleotide sequence that includes an actuator element, an effector element, and a receptor element, wherein: the receptor element encodes a chimeric antigen receptor (CAR) including an extracellular antigen binding domain operably linked to a transmembrane domain and an intracellular signaling domain, wherein the extracellular antigen binding domain recognizes an antigen on a surface of a tumor cell; the actuator element encodes a transcription factor binding site that upregulates synthesis of an enzyme in response to the extracellular antigen binding domain of the

44 CAR binding to the antigen of the tumor cell, the enzyme configured to reduce a tumor- associated extracellular matrix (ECM) associated with the tumor cell; and the effector element encodes the enzyme, wherein, in response to the extracellular antigen binding domain of the CAR binding to the antigen of the tumor cell, the population of genetically engineered effector cells being configured to activate and, in response, to synthesize and secrete a calibrated amount of the enzyme based on a presence of the tumor cell and, in response, the enzyme reduces the tumor-associated ECM.

12. The population of genetically engineered effector cells of claim 11, wherein the enzyme is a functional fragment of a wild-type enzyme.

13. The population of genetically engineered effector cells of claim 12, wherein the enzyme encoded by the effector element includes an active protein and a sub-portion of a pro-peptide of the wild-type enzyme, the sub-portion including a different amino acid overhang than is present by natural post-translational modifications.

14. The population of genetically engineered effector cells of claim 11, wherein the enzyme is configured to degrade and break down the tumor-associated ECM, as formed in a tumor microenvironment (TME) associated with the tumor cell, to treat or prevent a cancer infection.

15. The population of genetically engineered effector cells of claim 11, wherein the calibrated amount of the enzyme is a function of an amount of the tumor cell present in a plurality of cells or in a sample.

16. A method comprising: contacting a plurality of cells with a volume of a genetically engineered effector cell, wherein the genetically engineered effector cell comprises a polynucleotide sequence that includes: a receptor element that encodes a chimeric antigen receptor (CAR) including an extracellular antigen binding domain operably linked to a transmembrane

45 domain and an intracellular signaling domain, wherein the extracellular antigen binding domain recognizes an antigen on a surface of a tumor cell from the plurality of cells; an actuator element that encodes a transcription factor binding site; and an effector element that encodes an enzyme configured to reduce a tumor-associated extracellular matrix (ECM) associated with the tumor cell; in response to contacting the plurality of cells with the volume of the genetically engineered effector cell and a presence of the tumor cell within the plurality of cells, causing binding of the extracellular antigen binding domain to the antigen of the tumor cell; and in response to the extracellular antigen binding domain of the CAR binding to the antigen of the tumor cell, initiating expression and synthesis of the enzyme by the actuator element; secreting the enzyme by a signal peptide; and reducing the tumor-associated ECM by the enzyme.

17. The method of claim 16, further including detecting expression of the enzyme, wherein detectable expression of the enzyme indicates the presence of the tumor cell.

18. The method of claim 16, further including, in response to the extracellular antigen binding domain of the CAR binding to the antigen of the tumor cell, activating the genetically engineered effector cell and, in response, synthesizing and secreting a calibrated amount of the enzyme based on the presence of the tumor cell.

19. The method of claim 18, wherein the calibrated amount of the enzyme is proportional to an amount of the tumor cell present within the plurality of cells.

20. The method of claim 16, further including stimulating cytokines by the genetically engineered effector cell.