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WO2025226662A1 - Cellules effectrices génétiquement modifiées comprenant des éléments régulateurs immunitaires - Google Patents

Cellules effectrices génétiquement modifiées comprenant des éléments régulateurs immunitaires

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Publication number
WO2025226662A1
WO2025226662A1 PCT/US2025/025740 US2025025740W WO2025226662A1 WO 2025226662 A1 WO2025226662 A1 WO 2025226662A1 US 2025025740 W US2025025740 W US 2025025740W WO 2025226662 A1 WO2025226662 A1 WO 2025226662A1
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Prior art keywords
cell
immune
cells
regulator
genetically engineered
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Inventor
Parijat Bhatnagar
Sherri NEWMYER
Harikrishnan RADHAKRISHNAN
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SRI International Inc
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SRI International Inc
Stanford Research Institute
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Publication of WO2025226662A1 publication Critical patent/WO2025226662A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/35—Cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4254—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A61K40/4255—Mesothelin [MSLN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—IL-15
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • Various standard-of-care therapeutics are designed to treat a disease at the time of diagnosis. Although many pathogens and diseased cells undergo dynamic changes in vivo, current drugs are not designed to co-evolve along with the in vivo disease microenvironment. Such therapeutics can include drugs administered in doses that are normalized to the body weight of the patient. However, disease burden can be different for similar-sized patients, and interpatient variability can affect optimal dosing. If drug dosages are administered in excess, the therapeutic agents can end up in systemic circulation which can cause morbidity in normal tissue. In the case of suboptimal delivery, drug resistance may develop. While the patient can be monitored and the dosage adjusted based on health results, continuous monitoring is costly and impractical.
  • CAR chimeric antigen receptor
  • the present invention is directed to overcoming the above-mentioned challenges and others related to therapeutics for treating diseases, among other purposes, such as involving a genetically engineered cell line which can activate in situ to cause synthesis of an engineered protein (effector) and an immune-regulator complex to improve in vivo persistence and reduce dosages for achieving an intended effect on a subject.
  • a genetically engineered cell line which can activate in situ to cause synthesis of an engineered protein (effector) and an immune-regulator complex to improve in vivo persistence and reduce dosages for achieving an intended effect on a subject.
  • a genetically engineered effector cell comprising a cell carrying 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 target cell; an immune-regulator element that encodes an immune-regulator complex including an immune-regulator and a membrane-bound trafficking molecule, wherein the membrane-bound trafficking molecule is configured to localize the immune-regulator to the membrane of the genetically engineered effector cell; an actuator element that encodes a transcription factor binding site that upregulates synthesis of an effector protein and the immune-regulator complex in response to the extracellular antigen binding domain of the CAR binding to the antigen of the target cell; and an effector element that encodes the
  • the immune-regulator being membrane bound is configured to reduce off-target adverse effects as compared to a non-membrane bound immune- regulator.
  • the immune-regulator element encodes the immune- regulator complex that is a hybrid protein and further includes a linker between the immune-regulator and membrane-bound trafficking molecule.
  • the immune-regulator element encodes the immune- regulator with the signal peptide and prodomain of the immune-regulator replaced with a leader sequence derived from an immunoglobulin or a signal peptide derived from a cytokine receptor.
  • the immune-regulator includes a cytokine, a dominant negative receptor, a chimeric inhibitory receptor, or a chimeric death receptor.
  • the cytokine is selected from the group consisting of interleukin (IL)-2, IL-7, IL-12, IL-13, IL-15, and IL-21.
  • the membrane-bound trafficking molecule includes an interleukin (IL) receptor subunit.
  • IL interleukin
  • immune-regulator complex is selected from the group consisting of IL-2 bound to IL-2 receptor subunit, IL-7 bound to IL-7 receptor subunit, IL- 12 bound to IL- 12 receptor subunit, IL- 13 bound to IL- 13 receptor subunit, IL-15 bound to IL-15 receptor subunit, and IL-21 bound to IL-21 receptor subunit.
  • the genetically engineered effector cell is configured to synthesize and secrete a calibrated amount of the effector protein as a function of an amount of the target cell present in a sample or in situ.
  • the intracellular signaling domain is selected from the group consisting of an intracellular signaling portion of a CD28, an intracellular signaling portion of a 4- IBB, an intracellular signaling portion of a CD3 zeta, and a combination thereof.
  • the transcription factor binding site is selected from the group consisting of a nuclear factor of activated T-cell (NF AT) response element, a serum response element (SRE), a cyclic AMP response element (CRE), and a combination thereof.
  • NF AT nuclear factor of activated T-cell
  • SRE serum response element
  • CRE cyclic AMP response element
  • the exogenous polynucleotide sequence includes, in operative association, the receptor element, the immune-regulator element, the actuator element, and the effector element on a single construct.
  • the transmembrane domain is selected from the group consisting of T-cell receptor alpha (a) or beta (P) chain, a CD3 ⁇ chain, CD28, CD3s, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, and a glucocorticoid-induced tumor necrosis factor receptor (GITR).
  • T-cell receptor alpha (a) or beta (P) chain a CD3 ⁇ chain
  • CD28, CD3s, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154 and a glucocorticoid-induced tumor necrosis factor receptor (GITR).
  • GITR glucocorticoid-induced tumor necrosis factor receptor
  • Various aspects of the present disclosure are directed to a single construct configured to form a genetically engineered effector cell with a cell, the single construct comprising an exogenous polynucleotide sequence including, 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 target cell; an immune-regulator element that encodes an immune-regulator complex including an immune-regulator and a membrane-bound trafficking molecule, wherein the membrane-bound trafficking molecule is configured to localize the immune-regulator to the membrane of the genetically engineered effector cell; an actuator element that encodes a transcription factor binding site that upregulates synthesis of an effector protein and the immune-regulator complex in response to the extracellular antigen binding domain of the CAR binding to the antigen of the target cell
  • the single construct further includes a constitutive promoter downstream from the immune-regulator element and upstream from the receptor element.
  • the immune-regulator element includes a sequence with at least 70% sequence identity to a sequence selected from SEQ ID NOs: 5-9.
  • Various aspects of the present disclosure are directed to a method comprising contacting a plurality of cells with a population of genetically engineered effector cells, each of the genetically engineered effector cells of the population comprising a cell carrying an exogenous 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 domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain recognizes an antigen on a surface of a target cell; an immune-regulator element that encodes an immune-regulator complex including an immune-regulator and a membrane-bound trafficking molecule, wherein the membrane-bound trafficking molecule is configured to localize the immune-regulator to the membrane of the genetically engineered effector cell; an actuator element that encodes a transcription factor binding site that upregulates synthesis of an effector protein and the immune- regulator complex in response to the extracellular antigen binding domain
  • the method includes, in response to contacting the plurality of cells with the population of genetically engineered effector cell and a presence of target cells within the plurality of cells, causing binding of the receptor element to an antigen on a surface of the target cells. And, in response to the extracellular antigen binding domain of the CAR binding to the antigen of the target cells, initiating expression of the immune-regulator element by the actuator element to synthesize the immune-regulator complex and cause the immune-regulator to be membrane bound, and initiating expression of the effector element by the actuator element to synthesize and secrete the effector protein.
  • contacting the plurality of cells with the population of the genetically engineered effector cells includes in vivo administering the population of the genetically engineered effector cells to a subject.
  • the method further includes synthesizing and secreting a calibrated amount of the effector protein as a function of an amount of the target cell present in a sample or in situ, and synthesizing and expressing the immune- regulator bound to the membrane in response to the presence of the target cell.
  • FIG. 1 illustrates an example genetically engineered effector cell, in accordance with the present disclosure.
  • FIG. 2 illustrates an example polynucleotide sequence, in accordance with the present disclosure.
  • FIG. 3 illustrates an example population of genetically engineered effector cells in a target environment, in accordance with the present disclosure.
  • FIG. 4 illustrates an example method of using a genetically engineered effector cell, in accordance with the present disclosure.
  • FIGs. 5 A-5D illustrate example polynucleotide sequences used to form genetically engineered effector cells and controls, in accordance with the present disclosure.
  • FIGs. 6A-6G illustrate example growth and functional results of formed genetically engineered effector cells including an IL15-IL15Ra complex, in accordance with the present disclosure.
  • FIGs. 7A-7D illustrate example results of verifying functionality of the genetically engineered effector cells as a protein delivery platform in vitro, in accordance with the present disclosure.
  • FIGs. 8A-8B illustrate example engraftment results of genetically engineered effector cells having a constitutive IL15-IL15Ra complex when administered in vivo, in accordance with the present disclosure.
  • FIG. 9 illustrates the IL15-IL15Ra complex drives reporter protein expression by anti-MSLN CAR with Nluc CD8 T-cells in solid tumors, in accordance with the present disclosure.
  • FIGs. 10A-10J illustrate example results of assessing membrane bound IL- 15 complex promoting protein delivery function and CAR T-cell engraftment in vivo, in accordance with the present disclosure.
  • FIGs. 11 A-l IF illustrate transduction efficiency and viability of genetically engineered effector cells formed from mouse T-cells, in accordance with the present disclosure.
  • FIGs. 12A-12H illustrate antigen meditated antitumor activity of the genetically engineered effector cells, in accordance with the present disclosure.
  • FIGs. 13A-13F illustrate example results from interperitoneally injected CAR- cmb IL 15 -T-cells as contrasted with intravenous injection and assessment of protein delivery and CAR T-cell engraftment (or persistence), in accordance with the present disclosure.
  • FIG. 14 are bioluminescent images taken following mice treated as described in FIG. 10A, in accordance with the present disclosure.
  • FIGs. 15A-15B illustrate example CAR T-cell engraftment results of constitutive membrane bound IL- 15 from genetically engineered effector cells generated from mouse T-cells, in accordance with the present disclosure.
  • FIG. 16 illustrates transplanted CD8 CAR-cmbIL 15 -T-cells exhibit increased PD-1 exhaustion marker expression compared to CD4 CAR-cmbIL 15 -T-cells, in accordance with the present disclosure.
  • CAR T-cell therapies can contribute to manufacturing cost, product variability, and potential for adverse events.
  • a major hurdle to CAR T-cell therapies is preventing cells from becoming exhausted which can decrease therapeutic output and/or otherwise render T-cells ineffective at their intended purpose.
  • multiple infusions can be used to provide CAR T-cells to produce therapeutic proteins.
  • multiple infusions increases the cost of using CAR T-cell therapies and additionally increases the potential for side effects.
  • the technology and techniques for manufacturing and administering CAR T-cells is time-consuming and resources intensive.
  • Embodiments of the present disclosure are directed to a genetically engineered effector cell which includes an immunoregulatory transgene that increases cell viability and potency of a genetically engineered effector cell as compared to prior CAR T-cells and other types of engineered cells.
  • cell lines are genetically engineered with CARs to form genetically engineered effector cells that specifically detect (e.g., bind) antigens expressed on the surface of a target cell.
  • the cell lines are further engineered to encode the immunoregulatory transgene that improves cell persistence.
  • the genetically engineered effector cell can use the natural cell signaling, along with the CAR and the immunoregulatory transgene, to improve cell viability and potency.
  • the effector cells are formed from or include isolated T-cells.
  • the T-cells can be isolated from other types of cells, either prior to engineering the effector cells or during the engineering such as by using a selective expansion process.
  • the genetically engineered effector cells can have improved functionality from natural T-cells.
  • the genetically engineered effector cell can synthesize calibrated amounts of a target protein, and to induce autocrine and paracrine signaling due to artificial cell signaling.
  • Such effector cells can be used, for example, as an in vivo vector for delivery of target proteins in organisms, such as humans.
  • the immunoregulatory transgene can increase survival and expansion of the genetically engineered effector cell and can reduce dosage requirements.
  • CAR T-cell technology such as the engineered T-cells described in PCT Application No. PCT/US2023/073034 and PCT Application No. PCT/US2022/075123, which are each incorporated herein by reference in their entirety for their teachings, require continuous transfusions for drug delivery due to cell exhaustion.
  • Example genetically engineered effector cells of the present disclosure enable the effector cell to last longer than prior solutions, thereby reducing the number of doses delivered to a subject to achieve an intended effect and reducing the burden of manufacturing the genetically engineered effector cells and/or the cost of administering the same.
  • the immunoregulatory transgene is expressed as a complex that includes an immune-regulator and a membrane-bound trafficking molecule, herein referred to as “an immune-regulator complex”.
  • the immune-regulator can include a cytokine or other molecule (e.g., a dominant negative receptor, a chimeric inhibitory receptor, or a chimeric death receptor) that enables the genetically engineered effector cell to last longer by increasing cell viability, thereby improving in vivo persistence and potency, and limiting potential side effects as reduced dosages may be required for achieving an intended effect.
  • the membrane-bound trafficking molecule can cause the immune-regulator to be membrane bound, rather than being secreted into the extracellular environment, which can reduce off-target transgene adverse effects.
  • off-target transgene adverse effects can be additionally reduced by inducible expression of immunoregulatory transgene which limits the immunoregulatory function to when the cell is activated at a target site (e.g., disease site).
  • the immunoregulatory transgene can be expressed either constitutively (herein generally referred to as “constitutive expression” for ease of reference) or inducibly by binding of the CAR to a target cell (herein generally referred to as “induced expression” or “inducible expression” for ease of reference).
  • Constitutive expression includes and/or refers to transcription of a gene independent of external signals, cellular conditions and/or other stimuli.
  • constitutive expression of the immunoregulatory transgene can cause constant and/or continuous synthesis of the immune-regulator complex.
  • Induced or inducible expression includes and/or refers to transcription of a gene which is dependent on stimuli, such as being induced in response to activation of the genetically engineered effector cell caused by the CAR binding to a target cell.
  • induced expression of the immunoregulatory transgene can improve safety of the genetically engineered effector cells by limiting the immunoregulatory function of the transgene (e.g., synthesis of the immune-regulator complex) to when the cell is activated.
  • the immune-regulator complex can advance effector protein delivery by making the genetically engineered effector cell more effective in terms of protein delivery, reducing cell production costs, and improving safety.
  • the genetically engineered effector cells can increase cell viability and potency, and can be applied to a variety of cell types, including but not limited to T- cells, B-cells, natural killer cells, among others.
  • the improved in vivo persistence can directly translate to reduction in dosage needed to exert the intended effect.
  • the immune-regulator is membrane bound which can prevent off target transgene adverse effects, along with the optional induced expression of the immunoregulatory transgene.
  • a cell can be engineered to express genetic elements including transmembrane receptor(s) that autonomously regulate the intracellular transcriptional machinery, herein sometimes referred to as an effector cell or a genetically engineered effector.
  • the genetic elements of the effector cell can be modular and/or the effector cell can include multiple genetic elements to yield an effector cell having the capacity to serve as a vector for a variety of in vitro, ex vivo, and in vivo applications.
  • Such genetically engineered effector cells can be modular in that parts can be conserved, and parts can be changed for different applications.
  • the modularity can be used to combine different receptor elements with different effector elements, and which allows for reprogramming the genetically engineered effector cells to target diseases with known biomarkers, such as cancer, viral infections, and/or autoimmune disorders.
  • the genetically engineered effector cells can be used for therapeutics and treatment methods that self-regulate the therapeutic response upon stimulation by the disease cells and that are applicable to a variety of cell-based diseases, including cancers, emerging pathogens, and others that evade the immune system or involve its malfunction.
  • Multiple types of such genetically engineered effector cells, such as genetically engineered T-cells provide a robust, reproducible cellular system to therapeutically target complex diseases in vivo.
  • Such genetically engineered effector cells also provide a reliable in vivo imaging technology and a reliable, in vitro sensor technology in a variety of applications.
  • the genetically engineered effector cell is modular and antigen-specific. Antigen-specificity can be used to overcome tumor resistance and directs the cytolytic function toward different antigen- presenting target cells, such as subject cells of a human or other organism. Further, the artificial cell-signaling pathway of such genetically engineered effectors cells can introduce the capability to serve as vector by producing calibrated amounts of protein-based therapeutics and inducting intended autocrine and paracrine signaling, upon the genetically engineered effector cell engaging the target antigen.
  • the genetically engineered effector cell can allow for focused synthesis of the biologies at the target site and/or extend treatment duration for better patient outcome by limiting systemic toxicity or other adverse effects. Embodiments are not limited to therapeutics, and other types of effector proteins can be produced.
  • 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) immune-regulator element, (iii) actuator element, and (iv) 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 cells with the different functionalities and for different implementations.
  • 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.
  • the receptor element can be reprogrammed by exchanging the single chain variable fragment (scFV) portion and/or CAR for an extracellular antigen binding domain specific for a different disease- associated antigen or other targets.
  • scFV single chain variable fragment
  • receptor elements that can be used include, without limitation, CARs having specificity for antigens associated with autoimmune disorders, CARs having specificity for antigens associated with neural disorders (e.g., PTSD, Parkinson’s disease, Alzheimer’s disease), ligand-gated GPCRs (e.g., GPR1 Glucose receptor), light-gated ion channels (e.g., melanopsins, rhodopsins, photopsins), pressure sensing ion channels (e.g., TRPV1, TRPV2), and ligand-gated ion channels.
  • CARs having specificity for antigens associated with autoimmune disorders CARs having specificity for antigens associated with neural disorders (e.g., PTSD, Parkinson’s disease, Alzheimer’s disease)
  • ligand-gated GPCRs e.g., GPR1 Glucose receptor
  • light-gated ion channels e.g., melanopsins, r
  • immune-regulator element includes and/or refers to a polynucleotide sequence encoding an immune-regulator complex.
  • the immune- regulator complex can include an immune-regulator and a membrane-bound trafficking molecule.
  • the immune-regulator can include a cytokine, such as interleukin (IL)-2, IL-7, IL-13, or IL-15
  • the membrane-bound trafficking molecule can include an IL receptor subunit, such as an IL receptor alpha subunit (e.g., IL-15Ra).
  • the immune-regulator can include other types of molecules, such as a dominant negative receptor, a chimeric inhibitory receptor, or a chimeric death receptor, as further described herein.
  • the immune-regulator complex can improve cell viability and performance.
  • the immune- regulator can include IL-15, which is a cytokine that maintains cells in a less differentiated state with self-renewing capacity and enhances in vivo persistence.
  • Persistence or cell persistence includes and/or refers to the (durable) survivability (and over a period of time) of the cell(s) with intended functional activity, such as a length of survival and/or a percentage of cells that survive.
  • cell persistence can refer to the ability of transfected or activated genetically engineered effector cells to survive, maintain functionality, and maintain presence in vivo over time after infusion or activation.
  • Example functional aspects of cell persistence in vivo can include survival, proliferation, functional maintenance and trafficking, and localization.
  • IL- 15 can be co-expressed with the membrane-bound trafficking molecule of IL- 15 receptor alpha subunit (IL-15Ra).
  • the immune-regulator complex can be inducibly expressed in response to activation of the genetically engineered effector cell by target cells and can increase the persistence of the genetically engineered effector cell for delivering effector proteins.
  • the inducible immune-regulator complex can also improve safety of the engineered effector cells as the immune-regulator is only expressed when the effector cells are activated by the target cells.
  • constitutive expression of the immune-regulator complex can result in dysfunctional effector cells that can contributed to disease, such autoimmunity or oncogenicity.
  • inducible expression can be used such that the immune-regulator is only functional when the target antigen is present.
  • the persistence of the genetically engineered effector can be strong enough to exert the intended effect (e.g., therapeutic or other effect) but not to cause autoimmunity or oncogenicity.
  • 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 sensing element to a target antigen).
  • a triggering signal e.g., binding of the sensing element to a target antigen.
  • the underlying molecular mechanism of the actuator element is based on the intracellular calcium [Ca 2+ ]i dynamics, a mechanism used by almost all types of cells to regulate their functions.
  • Exemplary response elements include, without limitation, NF AT (“nuclear factor of activated T-cells") response element (NF AT -RE), serum response element (SRE), and cyclic AMP response element (CRE).
  • 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.
  • 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, among others.
  • the genetically engineered effector cell into which the receptor element, the immune-regulator 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).
  • 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 immune-regulator element, the actuator element, and the effector element.
  • the 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 subject or host which is a second human).
  • 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 subject).
  • FIG. 1 illustrates an example genetically engineered effector cell, in accordance with the present disclosure.
  • the genetically engineered effector cell 100 can be modular in that elements 102, 106, 110, 116 can be adjusted for different target cells and to synthesize different effector proteins.
  • the genetically engineered effector cell 100 can comprise an exogenous polynucleotide sequence 101 that includes, in operative association, a receptor element 102, an immune-regulator element 116, an actuator element 106, and an effector element 110.
  • the exogenous polynucleotide sequence 101 can be on a single construct.
  • the genetically engineered effector cell 100 can be formed from and/or include a cell.
  • a variety of different types of cells can be genetically modified to form the effector cell 100.
  • Example cells include 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 genetically engineered effector cell 100 can include a living cell from an organism, such as a basic membrane-bound unit that contains structural and functional elements.
  • the receptor element 102 encodes a CAR 104.
  • a CAR is sometimes called a “chimeric receptor”, a “T-body”, or a “chimeric immune receptor.”
  • a CAR includes and/or refers to an artificially constructed hybrid protein or polypeptide comprising extracellular antigen binding domain(s) 103 of an antibody (e.g., scFv) operably linked to a transmembrane domain 105 and at least one intracellular signaling domain 107.
  • 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 a surface antigen of a target cell, such as a diseased cell of a subject.
  • the CAR 104 can mobilize internal Ca 2+ stores for intracellular Ca 2+ release in response to antigen binding.
  • the extracellular antigen binding domain 103 of the CAR 104 can recognize an antigen on a surface of a target cell.
  • the CAR 104 is configured to cause a rise in calcium in response to the extracellular antigen binding domain 103 binding to the antigen of the target cell and the transmembrane domain 105 is configured to bind to a transcription factor protein that is triggered by the rise in calcium and is translocated into the nucleus of the genetically engineered effector cell 100.
  • the extracellular antigen binding domain 103 includes and/or refers to a polynucleotide sequence that is complementary to the surface antigen of the target cell.
  • the extracellular antigen binding domain 103 can bind to the surface antigen of a target cell, as described above.
  • 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, e.g., the membrane of the genetically engineered 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.
  • a transmembrane domain of a T-cell receptor alpha (a) or beta (P) chain a CD3i chain, CD28, CD3s, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or a glucocorticoid-induced tumor necrosis factor receptor (GITR) can be used.
  • the intracellular signaling domain 107 includes and/or refers to a polynucleotide sequence encoding any oligopeptide or polypeptide known to function as a domain that transmits 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 intracellular signaling portion of a 4- IBB, and an intracellular signal portion of a CD3-zeta.
  • the intracellular signaling domain 107 includes the intracellular signaling portion of CD28, the intracellular signaling portion of 4-1BB, and the intracellular signal portion of CD3- zeta.
  • the intracellular signaling domain 107 does not include an intracellular signaling portion of CD28.
  • the intracellular signaling domain 107 can include the intracellular signaling portion of 4- IBB, the intracellular signal portion of CD3-zeta, or a combination thereof.
  • the intracellular signaling domain 107 includes the intracellular signaling portion of 4-1BB and the intracellular signal portion of CD3-zeta.
  • embodiments are not so limited and can include other types and combinations of intracellular signaling domains.
  • the intracellular signaling domain 107 can 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) an antigen.
  • 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.
  • the extracellular antigen binding domain 103 capable of binding to an antigen includes any oligopeptide or polypeptide that can bind to the antigen, and includes, for example, 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, for example, an antigen present on a cell surface, and thereby imparts specificity to a genetically engineered effector cell 100 expressing the CAR 104.
  • the receptor element 102 encodes a CAR 104 comprising an extracellular antigen binding domain 103 having specificity for mesothelin (MSLN).
  • MSLN mesothelin
  • Other chimeric antigen receptors appropriate for use as the antigen binding portion of the receptor element 102 include those having specificity for a subset of immune cells, for one or more tumor antigens, and/or for one or more viral antigens.
  • the immune-regulator element 116 encodes an immune-regulator complex 117 including an immune-regulator 118 and a membrane-bound trafficking molecule 120.
  • the immune-regulator 118 includes and/or refers to a protein or other type of molecule involved in immune homeostasis and preventing autoimmune diseases, such as molecules that assist with regulation and/or viability of cells including T-cells.
  • the membrane-bound trafficking molecule 120 includes and/or refers to a molecule that is tethered to the cell membrane and can present the immune-regulator signal to other cells without being released into the extracellular environment.
  • the membranebound trafficking molecule 120 can localize the immune-regulator 118 to the membrane of the genetically engineered effector cell 100, such as chaperoning and tethering the immune-regulator 118 to the cell membrane of the genetically engineered effector cell 100.
  • the immune-regulator 118 being membrane bound can reduce off-target adverse effects of the genetically engineered effector cell 100 as compared to a non-membrane bound immune regulator.
  • the immune-regulator complex 117 is synthesized and the immune-regulator 118 is membrane bound.
  • the immune-regulator element 116 is inducibly expressed, sometimes herein referred to as a “inducible immune-regulator element” or a “inducible immune-regulator complex”. That is, the immune-regulator complex 117 is expressed in response to the CAR 104 binding to the target cell, which activates the genetically engineered effector cell 100 as further described below.
  • An inducible immune-regulator element can be upstream of the receptor element 102 and downstream the actuator element 106, such that expression of the immune-regulator complex 117 is upregulated by the transcription factor binding site 108 of the actuator element 106.
  • Embodiments are not so limited and can include an immune-regulator element 116 that is constitutively expressed, sometimes herein referred to as a “constitutive immune-regulator element” or a “constitutive immune-regulator complex”.
  • a constitutive immune-regulator element can be downstream of the CAR 104, such that the genetically engineered effector cell 100 expresses the immune-regulator element 116 independent of the presence of the target cell.
  • the immune-regulator element 116 encodes the immune-regulator complex 117 that is a hybrid protein including the immune-regulator 118 and the membrane-bound trafficking molecule 120.
  • the immune-regulator complex can include additional components, such as a linker between the immune-regulator 118 and the membrane-bound trafficking molecule 120, a tag (e.g., FLAG tag), and/or immunoglobulin (Ig) kappa (IgK) leader sequence (SP) or other Ig SPs or signal peptides.
  • the immune-regulator element 116 can encode the immune-regulator complex 117 that is the hybrid protein and further includes a linker between the immune-regulator 118 and membrane-bound trafficking molecule 120.
  • the linker can include an amino acid linker which links the immune-regulator 118 to the membrane-bound trafficking molecule 120.
  • the immune-regulator 118 can include a protein with the native signal peptide and prodomain removed and replaced with a leader sequence or signal peptide that is non-native to the protein, such as IgK SP.
  • the leader sequence or signal peptide can be derived from any immunoglobulin or cytokine receptor and used for extracellular localization.
  • Example leader sequences derived from an immunoglobulin include IgK SP, IgA SP, IgD SP, IgE SP, immunoglobulin lambda (IgL) SP, and IgM SP, among others.
  • Example signal peptides derived from cytokine receptors include the signal peptide of IL-6R and signal peptide of IL-7R, among others.
  • the signal peptide and prodomain of the protein can be removed and replaced with IgK SP and IgK SP can direct localization of the immune-regulator 118 to the extracellular cell membrane of the genetically engineered effector cell 100.
  • the immune-regulator element 116 can encode the immune-regulator 118 with the native signal peptide and prodomain removed and replaced with the non-native leader sequence or signal peptide and as fused to a full length membrane-bound trafficking molecule 120 with an amino acid linker there between.
  • the immune-regulator element 116 can encode IL- 15 with the IL- 15 signal peptide and prodomain replaced with IgK SP and fused to IL-15Ra with the amino acid linker between.
  • the immune-regulator 118 includes a cytokine, a dominant negative receptor, a chimeric inhibitory receptor, or a chimeric death receptor.
  • Example cytokines include IL-2, IL-7, IL-12, IL-13, IL-15, and IL-21, among others.
  • a dominant negative receptor can be configured to block or otherwise interfere with cell inhibitory signals.
  • a chimeric inhibitory receptor can be configured to convert inhibitory signals to an activation signal or as otherwise mutated.
  • a chimeric inhibitory receptor can include inhibitory signaling domains derived from immune inhibitory receptors, such as PD-1 and CTLA-4.
  • a chimeric death receptor can be configured to convert death signals to growth signals.
  • the dominant negative receptor, chimeric inhibitory receptor, or chimeric death receptor include a protein or polypeptide comprising an extracellular antigen binding domain that when expressed, performs its function, and which can be similar to or include a type of CAR.
  • An example dominant negative receptor includes dominant negative PD1
  • an example chimeric inhibitory receptor includes CTLA4-CD28 chimera
  • an example chimeric death receptor includes truncated Fas receptor or Fas-4-lBB chimera, such as described in: Liu et al., A novel dominant-negative PD-1 armored anti-CD19 CAR T cell is safe and effective against refractory/relapsed B cell lymphoma, Transl Oncol, 14(7)(2021); Shin et al., Positive conversion of negative signaling of CTLA4 potentiates antitumor efficacy of adoptive T-cell therapy in murine tumor modes, Blood, 119(24): 5678-5687 (2012); and Anderson et al., Engineering adoptive T cell therapy to co-opt Fas ligand- mediated death signaling in ovarian cancer enhances therapeutic efficacy, J ImmunoTher Cancer, 10(3) (2022), each of which are incorporated herein in their entireties for their teachings.
  • the membrane-bound trafficking molecule 120 includes an IL receptor subunit, such as but not limited to IL receptor alpha subunits.
  • the immune-regulator complex 117 can be selected from IL-2 bound to IL-2 receptor subunit, IL-7 bound to IL-7 receptor subunit, IL- 12 bound to IL- 12 receptor subunit, IL- 13 bound to IL- 13 receptor subunit, IL- 15 bound to IL- 15 receptor subunit, and IL-21 bound to IL-21 receptor subunit, among other examples.
  • the IL receptor subunit can include an IL receptor alpha subunit, such as IL-2 receptor alpha subunit (IL2-Ra), IL-7 receptor alpha subunit (IL7-Ra), IL- 12 receptor alpha subunit (IL12-Ra) (e.g., the alpha subunit domain within the IL- 12 receptor beta 2 subunit (IL-12RP2) , IL- 13 receptor alpha subunit (IL13-Ra), such as IL- 13Ral, IL-15 receptor alpha subunit (IL15-Ra), and IL-21 receptor alpha subunit (IL21- Ra), although embodiments are not so limited and can include other receptor subunits, such as an IL receptor beta (RP) subunits and/or IL receptor gamma (y) subunits.
  • IL-2 receptor alpha subunit IL-2 receptor alpha subunit (IL2-Ra)
  • IL-7 receptor alpha subunit IL-7 receptor alpha subunit
  • IL12-Ra e.g., the alpha
  • IL- 12 can be bound to IL-2Ra (CD25), IL-2 receptor beta subunit (IL-2RP) (CD122), or the common gamma (y) chain to all three subunits.
  • IL receptor subunits include IL-2Ra, IL-2RP, common y subunit, IL-7Ra, IL12-Ra, IL12-RP1, IL12-RP2, IL13-Ral, IL13-4Ra, IL15-Ra, IL15-RP, and IL21-Ra, as well as various combinations thereof.
  • the immune-regulator complex 117 can increase survival and expansion of the genetically engineered effector cell 100, such as a population thereof, and can exhibit reduced off-target adverse effects as compared to a non-membrane bound immune-regulator and/or a constitutively expressed immune- regulator complex.
  • the immune-regulator 118 can prolong cell longevity, thereby increasing the persistence of the genetically engineered effector cell 100 and potentially reducing dosage required to achieved an intended effect.
  • the immune-regulator 118 can be involved in regulation of cell homeostasis and differentiations, and can exhibit activity such as growth and migration of activated T- cells and natural killer cells, and induction of B-cell differentiation and proliferation, among other activity.
  • the immune-regulator 118 being membrane bound can reduce off-target transgene adverse effects as compared to immune-regulators which are secreted to the extracellular environment.
  • an inducible immune-regulator complex 117 can further reduce off-target transgene adverse effects by restricting the immune-regulator response to when the genetically engineered effector cell 100 is activated and/or contacts a target cell.
  • the actuator element 106 encodes a transcription factor binding site 108.
  • the transcription factor binding site 108 includes and/or refers to binding site for a protein that upregulates synthesis of an effector protein 112 in response to the extracellular antigen binding domain 103 of the CAR 104 binding to the antigen of the target cell.
  • the transcription factor binding site 108 upregulates synthesis of the effector protein 112 and the immune-regulator complex 117 in response to the extracellular antigen binding domain 103 of the CAR 104 binding to the antigen of the target cell.
  • the transcription factor binding site 108 can bind to transcription factors as triggered by [Ca 2+ ], which as described above, are caused to release in response to the antigen binding.
  • the transcription factor binding site 108 is selected from a nuclear factor of activated T-cell (NF AT) response element (NF AT -RE), a serum response element (SRE), a cyclic AMP response element (CRE), and a combination thereof.
  • NF AT nuclear factor of activated T-cell
  • SRE serum response element
  • CRE cyclic AMP response element
  • a plurality of transcription factor binding sites can be encoded, such as between 1 and 10, between 2 and 10, between 3 and 10, between 5 and 10, between 2 and 8, between 2 and 6, between 3 and 6, 5, or 6 (e.g., 6 NFAT-REs), among other ranges or numbers.
  • the actuator element 106 can thereby include a sequence for binding the factors triggered by [Ca 2+ ], and can trigger amplified synthesis of the effector protein 112 (and optionally the immune-regulator complex 117) in response to the [Ca 2+ ]i rise.
  • the actuator element 106 encodes an NF AT transcription factor binding site for a transcription factor protein.
  • NF AT transcription factor family consists of five members NFATcl, NFATc2, NFATc3, NFATc4, and NFAT5.
  • NFATcl through NFATc4 are regulated by calcium signaling.
  • Calcium signaling is critical to NF AT activation because calmodulin, a well-known calcium sensor protein, activates the serine/threonine phosphatase calcineurin.
  • the underlying molecular mechanism of this strategy is based on intracellular Ca 2+ ([Ca 2+ ]i) dynamics.
  • the [Ca 2+ ]i dynamics are common to almost all cell types, and the approach is thus broadly applicable.
  • the [Ca 2+ ]i rise from CAR-mediated stimulation of cells leads to dephosphorylation of the nuclear factor of an activated effector cell 100 proteins (through Ca 2+ /calmodulin- dependent serine phosphatase calcineurin), which then translocates to the nucleus and interacts with the NF AT -RE to upregulate expression of the effector protein 112 and, optionally, the immune-regulator complex 117.
  • the NFAT-RE also performs its natural function of inducing IL-2 in the activated genetically engineered effector cell 100 that regulates clonal expansion proportional to the disease burden.
  • the expression of a NFAT-RE induced reporter protein can also be used to quantitatively assess the level of activation of the genetically engineered effector cell 100.
  • the effector element 110 encodes the effector protein 112, and in some embodiments, encodes the effector protein 112 operably linked to a signal peptide 114.
  • the signal peptide 114 is upstream of the effector protein 112.
  • the signal peptide 114 can be non-native to the effector protein 112.
  • the effector protein 112 can be unable to secrete into the extracellular environment without the addition of the signal peptide 114 or can be modified to include a signal peptide 114 that allows for the effector protein 112 to secrete more efficiently than with its native signal peptide.
  • the effector protein 112 includes a native signal peptide.
  • the effector protein 112 can (natively) include the signal peptide 114.
  • 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 effector protein 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 effector protein 112 for release into the extracellular environment. In this manner, the signal peptide 114 can direct movement of the effector protein 112 outside of the genetically engineered effector cell 100.
  • a signal peptide 114 is particularly advantageous when included in the genetically engineered effector cell 100 expressing an effector protein 112 that is unable to and/or minimally able to translocate natively, where the effector protein 112 may remain inside the genetically engineered effector cell 100 in the absence of the signal peptide 114 and/or can translocate at a rate below a threshold.
  • signal peptides are located at the N-terminus of nascent secreted proteins and have three domains: (1) a basic domain at the N-terminus, (2) a central hydrophobic core, and (3) a carboxy-terminal cleavage region. Any appropriate signal peptide can be used.
  • the signal peptide 114 can be the signal peptide of Interleukin-6 (IL-6) or Interleukin-2 (IL-2).
  • the genetically engineered effector cell 100 in response to the extracellular antigen binding domain 103 of the CAR 104 binding to the antigen of the target cell (e.g., a target subject cell), is configured to activate, and to synthesize and secrete the effector protein 112 and, in some embodiments, the immune-regulator complex 117.
  • the genetically engineered effector cell 100 can synthesize and secrete an amount of the effector protein 112 as a function of an amount of the target cell present in the environment (e.g., the extracellular environment), such as secreting an amount of the effector protein 112 in the environment that is proportional to the number of target cells present in the environment.
  • the genetically engineered effector cell 100 can synthesize and express the immune-regulator 118 bound to the membrane of the genetically engineered effector cell 100 in response to the presence of the target cell.
  • the immune-regulator complex 117 is constitutively expressed and the immune-regulator 118 is bound to the membrane of the genetically engineered effector cell 100 independent of (e.g., prior to) activation of the genetically engineered effector cell 100.
  • the immune-regulator 118 is membrane bound.
  • the immune- regulator 118 can improve cell persistence.
  • the effector protein 112 can include a variety of different types of proteins.
  • the effector protein 112 can include a detectable reporter protein, a therapeutic protein, a downstream signaling protein, and a combination thereof.
  • 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.
  • a therapeutic protein includes and/or refers to a protein that provides a therapeutic effect to the subject, e.g., a patient.
  • 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.
  • Non-limiting example 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); antiviral polypeptides from viral origin (e.g., anti-viral peptides); 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).
  • cytotoxic polypeptides of bacterial origin e.g., parasporin, plantaricin A
  • insect origin e.g., Polybia-MPl
  • vasodilators and endopeptidases such effector cells can be used to improve perfusion (see Chauhan VP & Jain RK, Nat. Mater. 12(11) :958-962 (2013), which is incorporated herein by reference in its entirety for its teaching) and assist in efficient delivery of anticancer agents that cannot be systemically administered as they damage structural tissues and are tumorigenic.
  • cell-penetrating cationic peptides these target peptides can be used to target intracellular bacteria. For example, sitespecific overexpression of such peptides can be a potent therapy for tuberculosis.
  • the effector protein 112 is a therapeutic protein.
  • the therapeutic protein can act directly on the target cell, in some embodiments. In other embodiments, the therapeutic protein can act on cells adjacent to the target cell or on non-cellular components.
  • Example therapeutic proteins include a cytotoxic protein, an immunostimulatory protein, and an immunosuppressive protein.
  • Different parts of the genetic elements 102, 106, 110, 116 of the genetically engineered effector cell 100 can be modular and other parts can be conserved (e.g., may not change for different implementations).
  • the intracellular signaling domain 107, the immune-regulator element 116, the actuator element 106, and the signal peptide 114 are constant domains
  • the extracellular antigen binding domain 103 and the effector protein 112 are variable domains.
  • the extracellular antigen binding domain 103 can be changed for different targets and/or the effector protein 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.
  • embodiments are not so limited, and any part of the genetically engineered effector cell 100 can be modified.
  • the genetically engineered effector cell 100 can include multiple (e.g., two or more) of some or all of the genetic elements 102, 106, 110, 116.
  • the genetically engineered effector cell 100 can include multiple receptor elements 102, multiple immune-regulator elements 116, multiple actuator elements 106, and/or multiple effector elements 110.
  • multiplicity takes the form of providing multiple genetically engineered effector cells (e.g., a plurality of cells) modified as described herein to a subject to provide more than one task for treating or preventing a disease and/or for other purposes.
  • the exogenous polynucleotide sequence 101 includes the actuator element 106 bound to the effector element 110 bound to immune- regulator element 116 bound to the receptor element 102.
  • the exogenous polynucleotide sequence 101 can include the actuator element 106 bound to and upstream from the effector element 110, and the effector element 110 and the immune- regulator element 116 bound to and upstream from the receptor element 102.
  • the immune-regulator complex 117 and effector protein 112 are inducibly expressed, with the CAR 104 being constitutively expressed.
  • the exogenous polynucleotide sequence 101 can include the actuator element 106 bound to and upstream from the effector element 110, and effector element 110 bound to and upstream from the receptor element 102, and the immune-regulator element 116 bound to and upstream from the receptor element 102.
  • the immune-regulator complex 117 (along with the CAR 104) is constitutively expressed and effector protein 112 is inducibly expressed. Accordingly, in various embodiments, the placement of the immune-regulator complex 117 in the exogenous polynucleotide sequence 101 can be used to provide inducible verses constitutive expression of the immune-regulator complex 117.
  • the exogenous polynucleotide sequence 101 can include additional components, which are not illustrated by FIG. 1.
  • Example additional components include linkers between respective elements 102, 106, 110, 116 and promoters.
  • Example linkers can include such as 2A peptides and amino acid linkers.
  • a constitutive promoter such as EF-l-apha core promoter
  • a constitutive promoter includes and/or refers to a sequence that upregulates (e.g., drives) expression of a transgene independent of cellular conditions and/or other stimuli.
  • the constitutive promoter can cause continuous expression of the transgene (e.g., the CAR is active and not regulated by other external factors).
  • FIG. 2 illustrates an example polynucleotide sequence, in accordance with the present disclosure.
  • Various embodiments are directed to a (single) construct that is configured to form the genetically engineered effector cell (e.g., 100 of FIG. 1) with a cell (e.g., T-cell).
  • a cell e.g., T-cell
  • the single construct can comprise the exogenous polynucleotide sequence 201 including, in operative association: (i) the receptor element 202 that encodes the CAR including the extracellular antigen binding domain operably linked to the transmembrane domain, and the intracellular signaling domain, wherein the extracellular antigen binding domain recognizes an antigen on a surface of a target cell; (ii) the immune-regulator element 216 that encodes an immune-regulator complex including an immune-regulator and a membrane-bound trafficking molecule, wherein the membrane-bound trafficking molecule is configured to localize the immune- regulator to the membrane of the genetically engineered effector cell; (iii) the actuator element 206 that encodes the transcription factor binding site that upregulates synthesis of an effector protein and, optionally, the immune-regulator complex, in response to the extracellular antigen binding domain of the CAR binding to the antigen of the target cell; and (iv) the effector element 210 that encodes the effector protein, wherein,
  • the exogenous polynucleotide sequence 201 can include any of the above described variations and in different combinations.
  • the single construct is carried by a viral vector (e.g., lentivector, adenovector) or a non-viral carrier or approach (e.g., a Transposon-Transposase system, Cluster Regularly Interspaced Short Palindrome Repeats (CRISPR)/Cas system, Transcription Activator-Like Nuclease (TALEN) system, Zinc Finger Nuclease (ZFN) system) that may be mediated by a transfection system (e.g., electroporation, lipid nanoparticles).
  • a transfection system e.g., electroporation, lipid nanoparticles.
  • the immune-regulator element 216 is upstream from the receptor element 202 and downstream from the actuator element 206.
  • the exogenous polynucleotide sequence 201 can further include a constitutive promoter downstream from the immune-regulator element 216 and upstream from the receptor element 202.
  • the constitutive promoter can drive expression of the receptor element 202 independent of a presence of the target cell.
  • embodiments are not so limited and the constitutive promoter can be upstream from the immune-regulator element 216 and receptor element 202, such that both the immune-regulator element 216 and receptor element 202 are constitutively expressed.
  • the exogenous polynucleotide sequence 201 can include additional elements, including but not limited to linkers between elements 202, 206, 210, 216, such as self-cleaving 2 A peptides.
  • the immune-regulator element 216 encodes the immune-regulator complex that is the hybrid protein including the immune-regulator and membrane-bound trafficking molecule with a linker between.
  • the immune- regulator encoded by the immune-regulator element 216 can include a modified form of a naturally occurring immune-regulator, such as the immune-regulator with the native signal peptide and prodomain removed and replaced with a non-native leader sequence or signal peptide, as previously described.
  • the immune-regulator element 216 encodes the immune-regulator with the native signal peptide and prodomain removed and replaced with IgK SP and as fused to the full length membrane-bound trafficking molecule with an amino acid linker between the immune-regulator and the full length membrane-bound trafficking molecule.
  • embodiments are not so limited and can include other non-native Ig SPs or signal peptides.
  • the immune-regulator element 216 can be inducibly or constitutively expressed.
  • the actuator element 206 that encodes the transcription factor binding site can upregulate synthesis of an effector protein in response to the extracellular antigen binding domain of the CAR binding to the antigen of the target cell and the genetically engineered effector cell is configured to activate and to synthesize the effector protein, with concurrent or prior synthesis of the immune- regulator complex.
  • the exogenous polynucleotide sequence 201 can includes a sequence with at least 70% sequence identity to a sequence selected from SEQ ID NOs: 1-9, such as being selected from SEQ ID NOs: 3-9, 1-2, 3-4, or 5-9.
  • the exogenous polynucleotide sequence can include at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity any of SEQ ID NOs: 1-9.
  • the immune-regulator element 216 can include a sequence with at least 70% sequence identity to a sequence selected from SEQ ID NOs: 5-9, such as include SEQ ID NO: 9.
  • the immune- regulator element 216 can include at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity any of SEQ ID NOs: 5-9.
  • the genetically engineered effector cell 100 can be activated when in a diseased environment, in accordance with the present disclosure.
  • the genetically engineered effector cell 100 can be used as or act as a living vector to synthesize the effector protein 112 and the immune-regulator complex 117 using the artificial cell-signaling pathway and/or to trigger a sequence of events.
  • the genetically engineered effector cell 100 synthesizes the effector protein 112, and in some embodiments, the immune-regulator complex 117, in situ upon interacting with the antigen-presenting target cell, as further described herein.
  • the genetically engineered effector cell 100 can comprise a single plasmid (e.g., a single construct including each of) comprising constant domains (e.g., the actuator element 106, the optional signal peptide 114, the immune-regulator element 116, and portions of the receptor element 102, such as the transmembrane domain 105 and the intracellular signaling domain 107), and variable domains (e.g., the extracellular antigen binding domain 103 and effector protein 112) arranged in cis.
  • constant domains e.g., the actuator element 106, the optional signal peptide 114, the immune-regulator element 116, and portions of the receptor element 102, such as the transmembrane domain 105 and the intracellular signaling domain 107
  • variable domains e.g., the extracellular antigen binding domain 103 and effector protein 112
  • the constant domains can be configured to provide functionality to the genetically engineered effector cell 100.
  • the constant domains form part of the intracellular signaling pathway and include a transmembrane molecule (e.g., transmembrane domain 105) that mobilizes the calcium-dependent transcriptional machinery (e.g., actuator element 106) to upregulate the effector transgene (e.g., effector protein 112) optionally fused to a signal peptide 114 that assists in transporting the effector transgene into the extracellular space.
  • the calciumdependent transcriptional machinery e.g., actuator element 106 further upregulates the immune-regulator transgene (e.g., the immune-regulator complex 117) which improves cell survival and expansion (e.g., cell viability).
  • the immune-regulator transgene e.g., the immune-regulator complex 117
  • variable domains can be responsible for the applicability of the genetically engineered effector cell 100 to a variety of different diseases, target cells, therapy, and/or other applications.
  • the variable domains can impart specificity to the genetically engineered effector cell 100 against particular diseases.
  • variable domains can include molecules (e.g., a variable heavy -light (VH-VL) chain or scFv, variable domain of the heavy chain (VHH), a peptide, other antigens) with specificity for a biomarker on the target cell (e.g., the extracellular antigen binding domain 103 of the receptor element 102) to identify the antigen biomarker on the target cell independent of the peptide-major histocompatibility complex, and the effector transgene (e.g., effector protein 112).
  • the variable domains are modular.
  • the extracellular antigen binding domain 103 can be exchanged or revised to reprogram the genetically engineered effector cell 100 to target biomarkers specific to different cellbased diseases.
  • the effector protein 112 can be exchanged or revised with different therapeutic transgenes, such as for neutralizing the pathology that activated the genetically engineered effector cell 100 and essentially creating an off- shelf living vector, which is enhanced by the innate cytolytic activity of effector cells.
  • the receptor element 102 encodes a CAR. Characteristics of CARs include their ability to redirect cell specificity and reactivity toward a selected target in a non-major histocompatibility complex (MHC)-restricted manner, exploiting the antigen-binding properties of monoclonal antibodies. The non-MHC-restricted antigen recognition gives effector cells expressing CARs the ability to recognize antigen independent of antigen processing.
  • MHC non-major histocompatibility complex
  • a transmembrane CAR enables a genetically engineered effector cell 100 to sense and bind to the target antigen expressed on the surface of target cell. Binding of the CAR and target surface antigen on the target cell activates the genetically engineered effector cell 100, which triggers an activation cascade leading to the expression of the effector protein 112, such as an engineered reporter, imaging, and/or therapeutic protein.
  • the effector protein 112 such as an engineered reporter, imaging, and/or therapeutic protein.
  • expression of the effector protein 112, and optionally the immune-regulator complex 117 is autonomously expressed as part of the genetically engineered effector cell 100 activation cascade in response to binding of the transmembrane receptor to the antigen on the target cell.
  • the genetically engineered effector cell 100 expressing a CAR can bind to a specific antigen via the CAR, and in response a signal is transmitted into the genetically engineered effector cell 100, and as a result, the genetically engineered genetically engineered effector cell 100 is activated.
  • the activation of the genetically engineered effector cell 100 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.
  • a cytotoxic cytokine e.g., tumor necrosis factor, a lymphotoxin, etc.
  • 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.
  • An example sequence of events related to the genetically engineered effector cell 100 includes: (1) the genetically engineered effector cell 100 actively migrating to the diseased environment, (2) the CAR on the genetically engineered effector cell 100 surface engaging the antigen of the target cell, (3) the genetically engineered effector cell 100 activates, (4) upregulation of the effector protein 112 with the optional signal peptide 114 and optionally the immune-regulator complex 117 through the NF AT, and (5) signal peptide 114 is cleaved off and effector protein 112 is transported into the extracellular space, along with the immune-regulator 118 being synthesized and bound to the membrane of the genetically engineered effector cell 100.
  • FIG. 3 illustrates an example population of genetically engineered effector cells in a target environment, in accordance with the present disclosure.
  • the population 341 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 genetically engineered effector cells 300” for ease of references).
  • Each of the genetically engineered effector cells 300 can include at least substantially the same features and elements as the genetically engineered effector cell 100 of FIG. 1, the details of which are not repeated.
  • the environment is an extracellular space 340 that includes (a presence of) target cell(s) 342, such that the space 340 can be referred to as a target (e.g., diseased) environment.
  • the population 341 of the genetically engineered effector cells 300 can bind to the antigens of the target cell(s) 342 via the antigen binding domain of the CAR. In response to the binding, the genetically engineered effector cells 300 can activate and, in response, synthesize the effector protein and optionally the immune-regulator complex.
  • the genetically engineered effector cells 300 can synthesize and secrete a calibrated amount of the effector protein based on a presence of the target cell(s) 342 and optionally synthesize and express the immune-regulator bound to the membrane in response to the presence of the target cell.
  • the immune-regulator complex is constitutively expressed and the immune-regulator is bound membrane prior to the presence of the target cells 342.
  • the immune-regulator being membrane bound can increase survival and expansion of the population 341 of genetically engineered effector cells 300 and exhibit reduced off-target adverse effects as compared to a non-membrane bound immune-regulator.
  • the immune-regulator can be expressed on the membrane of each of the genetically engineered effector cells 300 in response to a presence of the target cell(s) 342, which further reduces off-target adverse effects as previously described.
  • the calibrated amount of the effector protein can be a function of an amount of the target cell 342 present in a plurality of (subject) cells, such as in an extracellular space 340 or in a sample. As previously described, the calibrated amount of the effector protein can be proportional to the amount of the target cell 342.
  • the extracellular space 340 illustrates genetically engineered effector cells 300 and the target cells 342
  • the extracellular space 340 and the plurality of (subject) cells can further include other normal and/or diseased cells, among other non-cellular components.
  • each of the genetically engineered effector cells 300 can synthesize the same effector protein, different effector proteins, and/or combinations thereof.
  • FIG. 4 illustrates an example method of using a genetically engineered effector cell, in accordance with the present disclosure.
  • the method 450 can be implemented to form the genetically engineered effector cell 100 illustrated by FIG. 1, using the construct illustrated by FIG. 2, and/or the population 341 of genetically engineered effector cells 300 illustrated by FIG. 3.
  • Embodiments can include other methods, including but not limited to, methods of forming genetically engineered effectors cells.
  • method to form genetically engineered effector can include using select process parameters that optimized delivery performance (e.g., synthesis of the effector protein).
  • process parameters can include or relate to the type of cell used, activation of the cell, transformation parameters, and expansion parameters, such but not limited transformation and expansion techniques, volumes, and/or time periods, among other parameters, such as that described in PCT Publication W02024/050325, published on March 7, 2024, and entitled “Genetically Engineered CD4 T-Cells for In Situ Synthesis of Proteins”, which is incorporated herein by reference in its entirety for its teaching.
  • the method 450 includes contacting a plurality of cells with a population of a genetically engineered effector cell.
  • Each of the genetically engineered effector cells of the population comprise a cell carrying an exogenous polynucleotide sequence that includes: (i) 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 target cell; (ii) an immune-regulator element that encodes an immune-regulator complex including an immune-regulator and a membrane-bound trafficking molecule, wherein the membrane-bound trafficking molecule is configured to localize the immune-regulator to the membrane of the genetically engineered effector cell; (iii) an actuator element that encodes a transcription factor binding site that upregulates synthesis of an effector protein and, optionally the immune-regulator complex, in response to the extracellular antigen binding
  • contacting the plurality of cells with the population of the genetically engineered effector cells comprises in vivo administering the population of the genetically engineered effector cells to a subject, such as via infusion or other forms of administering.
  • embodiments are not limited to in vivo methods and can include contacting cell in vitro or otherwise.
  • a sample can be taken from a subject and then exposed to genetically engineered effector cells, such as for diagnostics, research, and other purposes.
  • the method 450 includes, in response to contacting the plurality of cells with the population of genetically engineered effector cell and a presence of target cells within the plurality of cells, causing binding of the receptor element to an antigen on a surface of the target cells. And, at 456, the method 450 includes, in response to the extracellular antigen binding domain of the CAR binding to the antigen of the target cells, optionally, initiating expression of the immune-regulator element by the actuator element to synthesize the immune-regulator complex and cause the immune-regulator to be membrane bound, and initiating expression of the effector element by the actuator element to synthesize and secrete the effector protein.
  • the method 450 can further include synthesizing and secreting, via the population of the genetically engineered effector cells, a calibrated amount of the effector protein as a function of an amount of the target cell present in a sample or in situ, and optionally synthesizing and expressing the immune-regulator bound to the membrane in response to the presence of the target cell.
  • Some embodiments 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 genetically engineered effector cell 100 of FIG. 1.
  • Various embodiments are directed to a pharmaceutical composition
  • a pharmaceutical composition comprising a genetically engineered effector cell and a pharmaceutically acceptable carrier or excipient, such as the genetically engineered effector cell 100 of FIG. 1 and/or the population 341 of genetically engineered effector cells 300 of FIG. 3.
  • a genetically engineered effector cell composition such as a pharmaceutical composition
  • 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).
  • an acceptable carrier diluents, or excipient
  • the composition is prepared to facilitate the administration of the effector cells into a living organism.
  • 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.
  • the genetically engineered effector cells and compositions provided herein can be used in a variety of in vitro, ex vivo, and in vivo applications, including but not limited to use as an in vivo vector for delivery of proteins for a human.
  • in vitro uses of the genetically engineered effector cells and compositions provided herein include, without limitation, detecting target cells on the basis of antigens expressed on the surface of the target cells.
  • the target cell can be a cancer cell (e.g., tumor cell), a cell infected by a pathogen such as a virus or bacterium, a cell type associated with an autoimmune disorder (e.g., Type 1 diabetes, lupus), a cell type associated with a neurodegenerative disease such as Alzheimer's Disease, ALS, or Huntington's Disease.
  • the target (subject) cell can be a cell type associated with any other pathology for which the affected (subject) cell having aberrant expression of a cell surface antigen relative to an unaffected (subject) cell.
  • Ex vivo uses of the genetically engineered effector cells and compositions provided herein include, without limitation, early disease detection and companion diagnostic or therapeutic applications for the disease target cells identified on the basis of antigens expressed on the surface of the disease target cells.
  • the effector cells can be used for ex vivo applications in companion diagnostics for cancer immunotherapy.
  • Other ex vivo applications of the genetically engineered effector cells and cell compositions include applications for companion diagnostics for cell therapies for treating infectious diseases, autoimmune disorders, neurodegenerative disorders, and other cell-based pathologies associated with aberrant expression of a cell surface antigen relative to an unaffected (subject) cell.
  • In vivo applications of genetically engineered effector cells and compositions provided herein include, without limitation, in vivo imaging of disease sites, in vivo methods for localized therapy at a disease site (e.g., targeted therapy for ovarian cancer) or site of pathogen infection (e.g., targeted therapy for cells infected by a virus).
  • a disease site e.g., targeted therapy for ovarian cancer
  • site of pathogen infection e.g., targeted therapy for cells infected by a virus
  • Various embodiments 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 different cells and/or secrete different effector proteins.
  • a method of detecting a target cell comprises (a) contacting a genetically engineered effector cell to a cell population, and (b) detecting expression of the effector protein, wherein detectable expression of the effector protein indicates the presence of the target cell of interest.
  • the effector cell includes a NF AT response element and a reporter protein, and in the presence of the target cell in the contacted cell population, the genetically engineered effector cell binds to a surface molecular antigen on the target cell and activates the NF AT response element; and (b) detecting expression of the reporter protein, wherein detectable expression of the reporter protein indicates the presence of the target cell.
  • the detected target cell is a cancer cell and the antigenbinding domain of the CAR binds a cancer cell-specific surface antigen on the target cell.
  • the detected target cell is a virus-infected subject cell.
  • the antigen-recognizing portion of the CAR is modified or exchanged to quantitatively assess different viral pathogens such as dengue virus (DENV), West Nile (WNV), and Yellow Fever (YFV).
  • the methods harness the translational machinery of the infected subject cell to process viral ribonucleic acid (RNA) into a virus-specific antigen that is detectable by the genetically engineered effector cell as described herein.
  • Some embodiments are directed to methods of treating or preventing a disease using genetically engineered effector cells as a therapeutic agent.
  • methods can comprise 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.
  • the disease examples include a cancer (e.g., blood cancer (leukemia), solid tumor), an inflammatory disease/autoimmune disease (e.g., asthma, eczema), hepatitis, and an infectious disease, the cause of which is a virus, a bacterium, or a fungus, for example, tuberculosis, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and deep mycosis.
  • a cancer e.g., blood cancer (leukemia), solid tumor
  • an inflammatory disease/autoimmune disease e.g., asthma, eczema
  • hepatitis hepatitis
  • infectious disease the cause of which is a virus, a bacterium, or a fungus, for example, tuberculosis, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and deep mycosis
  • a genetically engineered effector cell expressing the CAR binds to an antigen expressed on the surface of a target cell that targeted to be decreased or eliminated for treatment of the aforementioned diseases, that is, a tumor antigen, a viral antigen, a bacterial antigen or the like, is administered to treat or prevent such diseases.
  • a tumor antigen a viral antigen, a bacterial antigen or the like
  • a genetically engineered effector cell expressing the CAR binds to an antigen expressed on the surface of a target cell that targeted to be decreased or eliminated for treatment of the aforementioned diseases, that is, a tumor antigen, a viral antigen, a bacterial antigen or the like, is administered to treat or prevent such diseases.
  • treatment or prevention can include treatment or prevention of one or more conditions or symptoms of the disease, e.g., cancer, being treated or prevented. Also, for purposes herein, “prevention” can encompass delaying the onset of the disease, or a symptom or condition thereof.
  • genetically engineered effector cells are administered to a subject (e.g., a host) in need thereof as a composition comprising the genetically engineered effector cells and a suitable carrier, diluent, or excipient as described herein.
  • a suitable carrier e.g., a carrier for modifying CAR-expressing cells
  • methods for providing effector cells to a subject can be adapted from clinical protocols for cellular and adoptive immunotherapy for infusion of donor-derived immune cells into a human subject.
  • an adapted clinical protocol suitable for methods provided herein comprises obtaining effector cells from a subject, genetically engineering (e.g., modifying) effector cells to express a CAR and NF AT -RE regulated protein transgene as described herein, and infusing the genetically engineered effector cells back into the subject.
  • a subject 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.
  • 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.
  • the effector cells can be cells that are allogeneic or autologous to the subject (e.g., a host), such as a mammal.
  • the effector cells are autologous to the subject.
  • a subject, to which genetically engineered effector cells are provided is monitored or assessed for increased (e.g., improved, more robust) tumor clearance. Accordingly, various embodiments are directed to methods used for cancer therapies. In some embodiments, a subject to which genetically engineered effector cells are provided is monitored or assessed for clearance of cells expressing an antigen. [00124] Some embodiments are directed to a method for cell-based treatment or prevention against a pathogen of interest.
  • the genetically engineered effector cell comprises a polynucleotide sequence encoding a therapeutic protein place of, or in addition to, the polynucleotide sequence encoding the detectable reporter protein; and is fused with a signal peptide (sec) on the 3’ end of the polynucleotide sequence to assist in extracellular transport.
  • a signal peptide sec
  • expression of a therapeutic protein is induced.
  • the method can include the localized production of a therapeutic protein at the site of the target cell (e.g., a tumor cell, infected cell) and extracellular secretion of the therapeutic protein in the disease microenvironment.
  • Some embodiments are directed to methods for using genetically engineered effector cells as a sensor technology.
  • transfusion-mediated spread of emerging virus pathogens can be a serious risk.
  • Such an example method can comprise contacting a genetically engineered effector cell comprising a CAR having an antigen binding domain for binding to an antigen specific to the virus to a sample comprising or suspected of comprising cells infected with the virus of interest, and NF AT -RE regulated reporter transgene to inform the presence of the cells infected with the virus.
  • Additional applications of the genetically engineered effector cells described herein include the following:
  • genetically engineered effector cells can be loaded with enzymatically activatable prodrugs, where the drug-activating enzyme is synthesized only at the tumor location, thus providing localized transformation of the prodrug into its active form.
  • the prodrug may not be loaded into the effector cells, and can be infused in multiple doses subsequent to the infusion of the genetically engineered effector cells.
  • the prodrug can alternatively be bound to an imaging nanoparticle or other means of image-guided means of active drug delivery. Attaching the prodrug to an imaging nanoparticle or engineering the effector cells to express imaging transgenes enables the effector cells to guide appropriate staging of the patient in preparation of surgery and for visually identifying and/or imaging tumor margins to assist in cytoreductive surgery.
  • Some embodiments are directed to methods of localized delivery of a chemotherapeutic agent to a disease site (e.g., tumor mass, site of autoimmune disease) comprises contacting a genetically engineered effector cell to a subject cell population, wherein the genetically engineered effector cell comprises (i) an exogenous polynucleotide sequence encoding a CAR comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain; and (ii) a NF AT response element operably linked to a polynucleotide sequence encoding an enzyme, wherein, in the presence of the target subject cell in the subject cell population, the genetically engineered effector cell binds to a surface antigen on the target subject cell and activates the NF AT response element to initiate expression of the enzyme, which acts on the prodrug predesigned to be activated by this enzyme and uses it membrane permeability due to its hydrophobicity to be released at the site of the disease.
  • a disease site e.g
  • genetically engineered effector cells can be used for visualizing and/or imaging tumor margins via the expression of detectable reporter protein such as fluorescent proteins (e.g., GFP, RFP, YFP, and variants thereof) or bioluminescent enzymes (e.g., luciferase).
  • detectable reporter protein such as fluorescent proteins (e.g., GFP, RFP, YFP, and variants thereof) or bioluminescent enzymes (e.g., luciferase).
  • detectable reporter protein such as fluorescent proteins (e.g., GFP, RFP, YFP, and variants thereof) or bioluminescent enzymes (e.g., luciferase).
  • detectable reporter protein such as fluorescent proteins (e.g., GFP, RFP, YFP, and variants thereof) or bioluminescent enzymes (e.g., luciferase).
  • luciferase e.g., luciferase
  • genetically engineered effector cells are used for detection and imaging of tumors based on expression of an imaging enzyme (e.g., thymidine kinase is capable of trapping a radioactive probe or otherwise detectable probe; tyrosinase detected by photoacoustic imaging or magnetic resonance imaging) expressed when tumor-specific CAR effector cells engage the antigen on tumor cells.
  • an imaging enzyme e.g., thymidine kinase is capable of trapping a radioactive probe or otherwise detectable probe; tyrosinase detected by photoacoustic imaging or magnetic resonance imaging
  • genetically engineered effector cells comprise a CAR that detects a cancer-specific antigen on a target cancer cell (e.g., a HPV E6 or E7 antigen in case of cervical cancer) and a NF AT -RE to drive the expression of a reporter protein as described above.
  • the genetically engineered effector cells comprise a CAR that detects an antigen on a pathogen-infected cell and a NF AT response element to induce expression of a reporter polypeptide.
  • a CAR that detects an antigen on a pathogen-infected cell
  • a NF AT response element to induce expression of a reporter polypeptide.
  • Such embodiments can be used for transfusion medicine to detect the presence of emerging pathogens.
  • Different CARs can be used in genetically engineered effector cells with NF AT -RE regulated reporters to detect and measure signal-to-noise ratio to guide the selection of appropriate CARs for a cell-based therapy that exert the intended therapeutic effect without exhibiting unintended side-effects.
  • Mammalian cells can be engineered as effector cells to comprise a glucose- sensing GPCR (GPR1) which mobilizes internal Ca 2+ stores and NF AT response element-regulated to express engineered insulin.
  • GPR1 glucose- sensing GPCR
  • Such engineered effector cells can be used for autonomous synthesis of insulin upon sensing glucose.
  • Such embodiments can be used for beta-cell replacement therapy.
  • Other non-limiting example uses of the genetically engineered effector cells include: i) imaging of the location of disease microenvironments to assist in surgical resection or monitor disease progression/regression; ii) cytotoxicity to kill the disease cells; iii) proliferation to enhance T-cell persistence; iv) immune-stimulation to recruit other immune cells; v) chemokine to recruit other immune cells; vi) immunosuppression to create localized immunosuppressive microenvironment; and vii) regeneration to enhance tissue healing.
  • a target cell (sometimes interchangeably referred to as a “target cell of a subject”, “target cell of interest”, “a diseased cell”, or “a target disease cell”) includes and/or refers to a cell of interest associated with a living organism (e.g., a biological component of interest).
  • An antigen of the target cell includes and/or refers to a structure (e.g., binding site) of the target 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”.
  • 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.
  • 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.).
  • “Native” includes and/or refers to a component that is naturally occurring to the species.
  • “Nonnative” includes and/or refers to a non-naturally occurring component, such as a component introduced by human or other organism activity.
  • An effector cell that contains an exogenous, recombinant, synthetic, and/or otherwise modified polynucleotide is considered to be a genetically engineered effector cell.
  • Nucleic acid includes and/or refers to a “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally means a polymer of DNA or 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, non-natural or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide.
  • the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions. However, it may be suitable in some instances, as discussed herein, for the nucleic acid to comprise one or more insertions, deletions, inversions, and/or substitutions.
  • 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 subject cell.
  • 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. Publication No. US2002/0190663, each of which are incorporated herein by reference in their entireties for their teachings. Nucleic acids obtained from biological samples typically are fragmented to produce suitable fragments for analysis.
  • Nucleic acids and/or other moieties 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 of the invention can be purified. As used herein, “purified” includes and/or refers s 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 can be determined using a variety of analytical techniques such as but not limited to mass spectrometry, HPLC, etc.
  • a number of experimental embodiments were conducted to generate genetically engineered effector cells that include immune-regulator elements and to characterize the functionality of the genetically engineered effector cells. Additional experiments were conducted to generate an optimized process for forming genetically engineered effector cells for in vivo synthesis of effector proteins.
  • Example constructs used to generate genetically engineered effector cells include the nucleotide sequences set forth in SEQ ID NOs: 1-11. SEQ ID NOs: 1-11 are each synthetic DNA.
  • the genetically engineered effector cells were generated using T-cells, and are sometimes herein referred to as “T-cells modified with CAR” or “CAR T-cells”.
  • a transgene that encodes a hybrid protein containing IL- 15 and a domain of the IL-15Ra was engineered and expressed as a functional membrane bound IL- 15 molecule.
  • the IL15-IL15Ra complex was co- engineered with a genetically engineered effector cell that drives NanoLuc® (Nluc) reporter protein expression in response to a tumor antigen.
  • the IL15-IL15Ra complex enabled the genetically engineered effector cells to circulate in vivo and expand 13 days post injection with reduced number of injections. For example, the data in FIGs. 8A-8B was generated with two injections on day 9 and day 14 after tumor implantations.
  • incorporating the immune-regulator complex into the genetically engineered effector cell can boost efforts to drive cell-based therapeutic protein delivery, such as by using proteins that directly combat the immunosuppressive tumor microenvironment.
  • using immune-regulator complexes that are inducible expressed in response to the active disease provided a mechanism to limit the immune-regulator adverse effects.
  • IL15-IL15Ra complex to drive cell expansion and therapeutic protein delivery in vivo, two different types of IL15-IL15Ra complex were engineered into the genetically engineered effector cell: (i) the inducible IL15-IL15aa complex that is expressed in response to the CAR binding to a target cell, and (ii) the constitutive IL 15- IL15Ra complex that is expressed independent of the presence of target cells. Both IL15-IL15Ra complexes included the IL-15Ra domain that chaperones IL-15 to the cell surface of the genetically engineered effector cell to promote cell persistence.
  • IL-15Ra can localize and anchor IL- 15 to the cell surface, such that IL- 15 is membrane bound and not secreted into the surrounding extracellular environment. Rendering IL- 15 membrane bound can reduced potential off-target adverse effects.
  • the inducible IL15-IL15Ra complex can further improve the potential for safe administration of the genetically engineered effector cells because the immune-regulator is expressed only when the genetically engineered effector cells are activated by target cells, e.g., the disease antigen.
  • the disclosed IL15-IL15Ra complex is incorporated into the genetically engineered effector cell to improve cell potency and decrease production costs associated with manufactory large numbers of genetically engineered effector cells to overcome cell exhaustion.
  • the reporter protein is replaced with therapeutic proteins and by strategically targeting the immunosuppressive mechanism, effective solid tumor treatments can be identified and advanced.
  • the IL15-IL15Ra complexes offer the potential to safely administer the immunoregulator function to genetically engineered effector cells.
  • Some advantages of the IL15-IL15Ra complex include: i) administered CAR T-cell survival and expansion increased in vivo so that fewer cells and injections (e.g., less labor and resources) are needed; and ii) durable, cell based, therapeutic protein delivery can increase to enable assessment of anti-tumor treatments in vivo.
  • drug toxicity or other adverse effects can be reduced by the membrane bound nature of the immune-regulator complex that limits its localization to the genetically engineered effector cell and the added features of inducible expression, which offer two means to improve safety of such genetically engineered effector cells.
  • mouse primary T-cells were engineered as a platform using CARs to induce the synthesis of desired proteins at the disease site.
  • This approach allows for the use of immunocompetent syngeneic tumor models to evaluate the CAR T- cells function within the context of a fully functioning immune system.
  • Current efforts to evaluate cell-based technologies typically rely on xenograft tumor models in immunodeficient mice, which provide early feasibility data but may not fully capture the immune effects present in the tumor microenvironment.
  • a primary T- cell-based effector cell for site-specific protein expression was translated from human T- cells to mouse T-cells, allowing for the use of an immunocompetent syngeneic tumor model.
  • Co-expression of membrane-bound interleukin 15 (mbIL15) on the T-cells enhanced intra-tumoral accumulation of both CD4 and CD8 CAR T-cells and supported delivery function.
  • Validation of the platform in syngeneic models shows efficacy assessments beyond solid tumors and allow for the evaluation of immune-related toxicities arising from interactions between the therapeutic protein, genetically engineered effector cells, and the subject immune system.
  • the delivery platform was transformed from human T-cells encoding human transgenes to mouse T-cells encoding mouse transgenes and tested the delivery platform in a syngeneic model with mouse tumor cells implanted in C57BL/6 immunocompetent mice.
  • MSLN mesothelin
  • MSLN-CAR was used to activate the T-cell and mobilize the NF AT transcription machinery to express a reporter effector protein.
  • Mouse CD4 and CD8 T- cells were both engineered to evaluate potential delivery function among T-cell subsets because similarly engineered human CD4 T-cells gave 5-fold increased effector protein expression compared to CD8 T-cells. See Radhakrishnan, H., et al., Engineered CD4 T cells for in vivo delivery of therapeutic proteins. Proc Natl Acad Sci U S A, 2024.
  • FIGs. 5 A-5D illustrate example polynucleotide sequences used to form genetically engineered effector cells and controls, in accordance with the present disclosure.
  • FIG. 5 A illustrates an example polynucleotide sequence (SEQ ID NO: 10) that includes a receptor element encoding a CAR, with the CAR including an antigen binding domain (e.g., against mesothelin (MSLN)), a transmembrane (e.g., CD8), intracellular signal domains of CD28, 4-1BB and CD3 zeta, an actuator element (e.g., NFAT-RE 6x) and an effector element that includes green fluorescent protein (GFP) and Nluc with a 2A linker between.
  • MSLN mesothelin
  • CD8 transmembrane
  • intracellular signal domains of CD28, 4-1BB and CD3 zeta e.g., CD8
  • an actuator element e.g., NFAT-RE 6x
  • an effector element that includes green fluorescent protein (GFP) and Nluc with a 2A linker between.
  • GFP green fluorescent protein
  • FIG. 5 A is used to generate a negative control genetically engineered effector cell that does not include an immune-regulator element (e.g., negative control for the influence of the immune- regulator complex).
  • FIG. 5B illustrates an example polynucleotide sequence (SEQ ID NO: 1 or SEQ ID NO: 2) that includes the receptor element of FIG.
  • an immune- regulator element e.g., SEQ ID NO: 9 including IL-15 (SEQ ID NO: 6) and IL15-Ra (SEQ ID NO: 8), along with IgK SP (SEQ ID NO: 5) and an amino acid linker (SEQ ID NO: 7)
  • an actuator element e.g., NF AT -RE 6x
  • an effector element e.g., GFP- 2A-Nluc.
  • the polynucleotide sequence of FIG. 5B is used to generate a genetically engineered effector cell that constitutively expresses the immune-regulator complex, herein sometimes generally referred to as a “constitutive mbIL15 expression system” or “cmbIL15”.
  • FIG. 5B illustrates an example polynucleotide sequence (SEQ ID NO: 3 or SEQ ID NO: 4) that includes the receptor element of FIG.
  • the actuator element e.g., NF AT -RE 6x
  • an effector element e.g., GFP-P2A-Nluc
  • immune-regulator element e.g., SEQ ID NO: 9 including IL-15 (SEQ ID NO: 6) and IL15-Ra (SEQ ID NO: 8), along with IgK SP (SEQ ID NO: 5) and an amino acid linker (SEQ ID NO: 7) of FIG. 5B, with the immune-regulator element being upstream of the constitutive promoter for the receptor element.
  • FIG. 5C is used to generate genetically engineered effector cell that inducible expresses the immune-regulator complex, herein sometimes generally referred to as a “inducible mbIL15 expression system” or “imbIL15”.
  • FIG. 5D illustrates an example polynucleotide sequence (SEQ ID NO: 11) that includes the actuator element (e.g., NF AT -RE 6x), effector element (e.g., GFP-2A- Nluc), and immune-regulator element (e.g., SEQ ID NO: 9 including IL- 15 (SEQ ID NO: 6) and IL15-Ra (SEQ ID NO: 8), along with IgK SP (SEQ ID NO: 5) and an amino acid linker (SEQ ID NO: 7)) of FIG.
  • the actuator element e.g., NF AT -RE 6x
  • effector element e.g., GFP-2A- Nluc
  • immune-regulator element e.g., SEQ
  • the polynucleotide sequence of FIG. 5D is used to generate a control genetically engineered effector cell that does not include a receptor element including a CAR.
  • FIGs 6A-6G illustrate example growth and functional results of forming genetically engineered effector cells including IL15-IL15Ra complex, in accordance with the present disclosure.
  • FIG. 6A is a summary of mouse genetically engineered effector production and expansion. For example, experiments were directed to engineering and expanding mouse primary T-cells using the polynucleotide sequences of FIGs. 5A-5D to transition a cell-based delivery platform from a xenogeneic mouse tumor model to an immunocompetent, syngeneic tumor model (FIG. 6A).
  • the lentiviral-packaged genetic circuit included a second-generation CAR containing the SSI single-chain variable fragment (scFv) that targets both human and mouse MSLN orthologs.
  • scFv single-chain variable fragment
  • Engineered T-cells were activated through CAR signaling of NF AT transcription machinery and monitored with the reporter protein Nluc as previously described in Repellin, C.E., et al., Modular Antigen-Specific T-cell Biofactories for Calibrated In Vivo Synthesis of Engineered Proteins. Adv Biosyst, 2018. 2(12), which is incorporated herein by reference in its entirety for its teaching.
  • An armoring option included CAR-inducible Nluc expressed in cis via self-cleaving 2 A peptide with IL- 15 fused with IL- 15 Receptor a subunit (IL- 15Ra) to express mbIL15.
  • IL- 15 signaling was incorporated to improve in vivo persistence, and IL- 15 was fused to the transmembrane receptor, IL15Ra, to enhance IL- 15 signaling while restricting its systemic exposure.
  • cmbIL15 constitutive
  • imbIL15 inducible
  • cmbIL15 improves genetically engineered effector cell proliferative capacity and antitumor function.
  • imbIL15 where mbIL15 expression is driven by NF AT signaling in response to CAR binding to antigen, this restricted expression has the potential to limit oncogenesis stemming from unrestricted IL- 15 hyperproliferative signaling.
  • CD4 CAR T-cell and CD8 CAR T-cell e.g., genetically engineered effector cells
  • splenocyte T-cell subsets were isolated with negative selection kits and simultaneously transduced and activated with CD3/CD28 beads (FIG. 6A). It is believed that activating the splenocytes prior to transduction was not needed since activation poorly induces expression of low-density lipoprotein receptor (LDL-R), the cell receptor that binds VSV-G lentiviral envelope protein, in mouse T-cells.
  • LDL-R low-density lipoprotein receptor
  • transduction reagent synperonic F108 circumvents the requirement for lentivirus to bind LDL-R, and performing the activation and transduction simultaneously reduced cell handling.
  • mouse T-cells were transduced in a limited volume, with IxlO 6 cells concentrated per 100 microliters (pL) volume. See Radhakrishnan, H., et al., Primary T- cell-based delivery platform for in vivo synthesis of engineered proteins, Bioeng Transl Med, 2024. 9(1): p. e!0605, which is incorporated herein by reference in its entirety for its teaching.
  • the resulting mouse CD4 and CD8 T-cell cultures expanded around 20- fold and around 75-fold, respectively, within 9 days (FIGs. 6B-6C). This expansion rate was 2-4-fold increased than those obtained using a 500 pL transduction volume (FIGs. 6B-6C insets). Concentrating the activation/transduction volume also increased the transduction efficiency by 14-24 percent (%) for CD4 CAR T-cells and 5-7% for CD8 CAR T-cells, respectively (FIGs. 11 A-l ID), and improved CD4 and CD8 CAR T-cell viability during expansion (FIGs. 1 IE-1 IF). The reduced volume likely increased virus, cell, and bead contact, resulting in around 40-100xl0 6 transduced cells per spleen after 9 days in culture.
  • CD4 CAR T-cells are more abundant among splenocytes than CD8 CAR T-cells, and CD4 CAR T-cells transduced well but expanded at a reduced rate. Conversely, CD8 CAR T-cell transduction efficiency was decreased but cells expanded more rapidly. The net result is that a similar mouse splenocyte number was routinely used to manufacture the same number of CD4 and CD8 CAR T-cells.
  • cmbIL15 and imbIL15 engineering did not impact T-cell growth rates; but the ability for mbIL15 to benefit ex vivo expansion was likely masked by the culturing conditions that included IL-2, IL-7, and IL- 15 supplementation.
  • Antigen-specific cytolytic potential of the engineered genetically engineered effector cells was determined to assess MSLN-CAR signaling and genetically engineered effector cell function (FIGs. 6D-6E). Both MSLN-CAR CD4 T-cells and MSLN-CAR CD8 T-cells preferentially lysed MSLN + Luc2 + A2780cis ovarian tumor cells compared to Folate Receptor a (FRa + ) Luc2 + A2780cis cells, supporting that MSLN binding triggered the SSI scFv CAR and drove tumor cell cytolysis (FIGs. 6C- 6E and FIGs. 12A-12H).
  • human MSLN neg FRa neg A2780cis ovarian cancer cells was used and engineered to express MSLN or FRa, for testing antigen specificity because they better controlled for the expression of target antigen.
  • Mouse KPC cells endogenously express MSLN, target of SSI CAR.
  • Antigen-specific tumor cell cytolysis was similar among the three engineered CD4 CAR T-cell mbIL15 variants (FIG. 6D), demonstrating the mbIL15 intervention did not impact CD4 CAR T-cell cytolytic potential in this in vitro assay.
  • cmbIL15 intervention reproducibly enhanced CD8 CAR T-cell cytolytic function around 20-fold compared to tumoricidal function mediated independent of antigen-CAR signaling (FIG. 6E) and cmbIL15 signaling independent of CAR expression (FIG. 12H).
  • imbIL15 armoring did not enhance CD8 CAR T- cell cytolysis.
  • STAT5 phosphorylation (Phospho-STAT5), a proliferative signal that is activated downstream of IL-15, was analyzed by phosflow cytometry to assess mbIL15 functionality (FIGs. 6F-6G). Serum and cytokines were withdrawn from the cultures for 16 hours to reduce IL- 15 signaling. Serum and cytokine starvation of CD4 and CD8 CAR T-cells resulted in basal levels of Phospho-STAT5 (solid black lines) that increased with treatment of soluble IL- 15 (dashed black lines).
  • cmbIL15 engineered CAR T-cells demonstrated increased Phospho-STAT5 expression as observed for mbIL15 neg CAR T-cells treated with soluble IL-15 (FIGs. 6F and 6G).
  • inducible mbIL15 expression resulted in basal Phospho-STAT5 expression seen in untreated mbIL15 neg CAR T-cells.
  • This phosflow cytometry analysis demonstrated that mbIL15 mediated STAT5 signaling in both CD4 and CD8 T-cell subsets and that inducible mbIL15 expression prevented tonic IL- 15 signaling that can promote unrestricted proliferation in the absence of antigen.
  • FIGs. 6F-6G show the membrane bound IL15-IL15Ra complex is functional in supporting the phosphorylation of STAT5 (PSTAT5), a T-cell proliferation marker. All cells (both CD4 and CD8 subsets) were serum starved overnight.
  • Adding soluble IL- 15 to genetically engineered effector cells for one hour increased PSTAT5 levels as detected through FACS analysis system (see solid (no-IL-15) and dashed (soluble IL-15 treatment) black lines).
  • T-cells engineered with the CAR and the constitutively expressed IL15-IL15Ra complex (cmbIL15) (blue line) exhibited increased PSTAT5 labeling (e.g., increased STAT5 phosphorylation) that is comparable to the level observed with soluble IL-15 treatment.
  • T-cells engineered with inducible IL15-IL15Ra complex (imbIL15) exhibited basal PSTAT5 levels.
  • FIGs. 6A-6G thereby illustrate lentiviral transduction and expansion of mouse CD4 and CD8 CAR T-cells engineered with functional MSLN scFv (SSI) and membrane bound IL- 15 complex.
  • FIG. 6A is a summary of mouse CAR T-cell production and expansion.
  • FIGs. 5A-5D are schematics of MSLN-CAR (or missing CAR) and mbIL15 constructs (inducible, constitutive, or missing mbIL15) used to engineer T-cells with NF AT activated GFP-2A-Nluc dual reporter expression (Effector).
  • FIG. 6B shows CD4 T-cell and FIG.
  • FIG. 6D shows CD4 MSLN-CAR T-cell and FIG.
  • FIG. 6E shows CD8 MSLN-CAR T-cell cytolysis of MSLN + Luc2 + A2780cis (closed symbols) ovarian cancer cells compared to FRa + Luc2 + A2780cis (open symbols) cells, demonstrating functional MSLN-CAR signaling and effector function.
  • E:T represents the ratio of 2500 CAR T-cells to increasing A2780cis target cell number.
  • FIGs. 6F-6G mbIL15 signaling function was demonstrated by phosflow cytometry analysis of PSTAT5 in CD4 (FIG.
  • CAR T-cells were serum and cytokine starved for 16 hours and assessed for PSTAT5 expression. Soluble hIL15 was added to serum starved CAR T cells for 1 hour as a positive control to stimulate PSTAT5 expression (dashed black line). A trace for each CAR construct representative of a duplicated experiment is shown. Error bars for all experiments indicate ⁇ 1 SD.
  • FIGs. 7A-7D illustrate example results of verifying functionality of the genetically engineered effector cells as a protein delivery platform in vitro, in accordance with the present disclosure.
  • Various experiments were conducted to assessment antigen triggering protein delivery by CD4 and CD8 CAR T-cell subsets.
  • the genetically engineered effector cells were cultured with magnetic beads bound to biotinylated MSLN and anti-CD28 (MSLN-aCD28) (10: 1 bead:cell ratio) and assessed for Nluc activity.
  • CD4 and CD8 CAR T-cells expressed Nluc in response to antigen challenge, with CD8 CAR T-cells (engineered with CAR alone or CAR-cmbIL15) expressing 1.5- 3 -fold increased Nluc among repeated experiments compared to similarly engineered CD4 CAR T-cells (FIGs. 7A-7B).
  • cmbIL15 co-expression did not impact Nluc expression compared to that observed with MSLN-aCD28-activated T-cells engineered with CAR alone.
  • CD4 and CD8 MSLN-CAR T-cells were cocultured with either MSLN + or FRa + A2780cis target cells to assess tumor associated antigen triggering of Nluc expression by the engineered CAR T-cells (FIGs. 7C-7D).
  • the A2780cis ovarian cancer cell lines, engineered to express MSLN or FRa, enabled the assessment of antigen- induced and leaky Nluc expression, respectively.
  • Nluc expression was only induced when the target cell expressed MSLN and not when it expressed FRa, demonstrating that antigen binding mediates CAR signaling and induces transgene expression.
  • FIGs. 7A-7D show CD4 and CD8 CAR T-cells express reporter protein in response to antigen challenge in vitro. Nluc reporter protein expressed by mouse CD4 and CD8 CAR T-cells was measured 24 hours after incubation with (FIG. 7A, CD4; FIG. 7B, CD8) MSLN-aCD28 bound beads or (FIG. 7C, CD4; FIG. 7D, CD8) with A2780cis target cells.
  • FIG. 7A shows results of CD4 and FIG. 7B shows results of CD8 CAR T-cells (3xl0 4 cells) that were stimulated with MSLN-aCD28 bound beads (10: 1 bead:cell ratio) and serially diluted 1 :2.
  • FIG. 7A shows results of CD4
  • FIG. 7B shows results of CD8 CAR T-cells (3xl0 4 cells) that were stimulated with MSLN-aCD28 bound beads (10: 1 bead:cell ratio) and serially diluted 1 :2.
  • FIG. 7C shows results of CD4 and FIG. 7D shows results of CD8 CAR T-cells (3xl0 4 cells) that were cocultured with increasing MSLN + Luc2 + A2780cis (closed symbols) or FRa + Luc2 + A2780cis (open symbols).
  • MSLN + Luc2 + A2780cis activates the genetically engineered effector cells.
  • FIGs. 8A-8B illustrate example engraftment results of genetically engineered effector cells having a constitutive IL15-IL15Ra complex when administered in vivo, in accordance with the present disclosure.
  • 9 days post subcutaneous injection of 2xl0 5 KPCY pancreatic tumor cells in C57BL/6 mice 5xl0 6 genetically engineered effector cells were injected intraperitoneally verses intravenously to compare administration routes and monitor cell persistence.
  • FIG. 8A shows the results of CD4- based genetically engineered effector cells and FIG.
  • CD8-based genetically engineered effector cells having a constitutive IL15-IL15Ra complex and showing the cells expanded and accumulated in blood, the spleen, and the tumor of C57BL/6 mice 13 days post injection.
  • the CD45.1 marker distinguishes the injected cells from that of the subject (e.g., host). Similar cells accumulated amount the two T- cell subsets of CD4 and CD8, with both intraperitoneal and intravenous injection. The greater accumulation of adoptively transferred CD45.1+CD8 cells suggest that CD8 T- cells may expand better than CD4 T-cells.
  • FIG. 9 illustrates the IL15-IL15Ra complex drives reporter protein expression by anti-MSLN CAR with Nluc CD8 T-cells in solid tumors, in accordance with the present disclosure.
  • FIGs. 10A-10J illustrate example results of assessing membrane bound IL-15 complex promoting protein delivery function and CAR T-cell engraftment in vivo, in accordance with the present disclosure.
  • Various experiments were conducted to assess the impact of mbIL15 on the ability of the genetically engineered effector cells to delivery an effector protein in vivo.
  • mouse CAR T-cells were evaluated in immunocompetent C57BL/6 mice (FIG. 10A).
  • Luc2 + KPCY pancreatic tumor cells were implanted subcutaneously to generate solid tumors prior to mouse CAR T-cell treatment.
  • CAR T-cells were injected intraperitoneally because it was identified that that intravenous and intraperitoneal administration yielded similar adoptive cell engraftment (FIGs. 13A-13C) as previously described.
  • CD4 and CD8 CAR-cmbIL 15 -T-cell administered with intraperitoneal injection also demonstrated increased protein delivery function (FIGs. 13D-13F).
  • FIG. 10A In assessing CARs, CAR-imbIL15, and CAR-cmbIL15 constructs, two genetically engineered effector cells (5xl0 6 ) injections spaced four days apart were administered following tumor implantation (FIG. 10A). Bioluminescent imaging was performed before and after genetically engineered effector cell injection to monitor Nluc delivery in vivo (FIGs. 10B-10D). Nluc activity steadily increased over a 13 day period following the first injection of CD4 and CD8 CAR-cmbIL 15 -T-cells (as compared to background signal measured in tumor-implanted mice receiving no T-cell treatment).
  • CAR T-cell numbers increased with cmbIL15 co-engineering, which is consistent with improved genetically engineered effector cell persistence with mbIL15 armoring.
  • imbIL15 armoring did not improve genetically engineered effector cell abundance, suggesting a threshold of mbIL15 expression and signaling is required to improve cell persistence.
  • Tumor-associated CD8 CAR-cmbIL 15 -T-cell numbers were 2-fold increased among panCD45 + CD3 + cells compared to CD4 CAR-cmbIL 15 -T-cells, likely driven by the increased propensity for CD8 T-cells to expand in response to antigen.
  • CD4 CAR-cmbIL 15 -T-cell numbers were increased 2- fold compared to CD8 CAR-cmbIL 15 -T-cells.
  • the abundance of CD4 CAR-cmbIL 15- T-cells in the spleen was consistent with the ability of cmbIL15 armoring to increase genetically engineered effector cell proliferation and enhance systemic cell circulation.
  • the CD8 CAR-cmbIL 15 -T-cells were less abundant in the spleen, indicating the tumor- associated CD8 T-cells expanded better in the tumor.
  • decreased systemic circulation of the CD8 CAR T-cells might indicate T-cell dysregulation.
  • CD8 T-cells intratumorally and may explain the enriched abundance of CD8 CAR-cmbIL 15 -T-cells observed. It is not clear if CD4 T-cells are prone to exhaustion like CD8 T-cells, but there was increased expression of the PD-1 exhaustion marker in tumor-associated CD8 CAR-cmbIL 15 -T- cells compared to CD4 CAR-cmbIL15-T-cells (FIG. 16). Regardless of the extent to which CD4 and CD8 CAR T-cells expand in vivo, mbIL15 signaling enhanced accumulation of both CAR T-cell subsets.
  • CD4 and CD8 CAR T-cells were successfully transplanted without co-administration of the other CAR T-cell subset.
  • Some experiments were directed to assessing whether mbIL15 supports intratumoral protein delivery function. Nluc expression in the dissociated tumor was measured to evaluate intratumoral effector protein delivery (FIG. 101).
  • cmbIL15 co-engineering significantly increased tumor associated Nluc expression (2 fold for CD4 CAR-cmbIL 15 -T-cell compared to control CD4 CAR (without mbIL15); 9 fold for CD8 CAR-cmbIL 15 -T-cell compared to control CD8 CAR (without mbIL15)).
  • CAR-cmbIL 15 -T-cells Tumor associated Nluc activity from CAR-cmbIL 15 -T-cells was compared with tumor cell number, which varied with heterogenous tumor growth (FIG. 10J).
  • This positive correlation demonstrates that findings of tumor burden driving proportional expression of desired effector protein from the genetically engineered effector cells is also relevant in vivo.
  • FIGs. 10A-10J show cmbIL15 armored mouse genetically engineered effector cells expand and express reporter protein in vivo.
  • CP Cyclophosphamide
  • FIG. 10B includes bioluminescent images taken following injection of Nano-Gio® Fluorofurimazine In Vivo Substrate to tumor bearing mice to measure total flux corresponding to effector Nluc expression. Four representative images are shown. BLI performed prior to CAR T- cell injection provided individual mouse background measurements that are reported as “0 Days Post CAR T cell injection.”
  • FIGs. 10C-10D show the time course of Nluc activity following injection of CD4 (FIG. 10C) and CD8 (FIG. 10D) CAR T-cells.
  • FIGs. 10C show the time course of Nluc activity following injection of CD4 (FIG. 10C) and CD8 (FIG. 10D) CAR T-cells.
  • FIG. 10E-10J show the results of tumor and spleen tissues that were processed 14 days post CAR T-cell injection to assess CD45.1 + CAR T-cell persistence with flow cytometry and Nluc expression with the Nano-Gio® Luciferase Assay.
  • FIG. 10E, CD4; FIG. 10F, CD8 CD45.1 + T-cell abundance in tumor among panCD45 + CD3 + cells.
  • FIG. 10G, CD4; FIG. 10H, CD8) CD45.1 + T-cell abundance in splenocytes among panCD45 + CD3 + cells.
  • FIG. 101 shows the results of tumor associated Nluc expression as measured and normalized to the number of viable, dissociated tumor cells using duplicate plating of the digested tissue.
  • FIG. 10E, CD4; FIG. 10F, CD8 CD45.1 + T-cell abundance in tumor among panCD45 + CD3 + cells.
  • FIG. 10G, CD4; FIG. 10H, CD8 CD45.1 + T-cell abundance in splenocyte
  • a one-way ANOVA was conducted to test overall significance (p ⁇ 0.001) followed by post hoc two-sample unpaired T test with Welch’s correction to compare treatments to the negative control (No Treatment) (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001).
  • FIGs. 10C-10D a one-way ANOVA was conducted to test overall significance (p ⁇ 0.001) followed by post hoc two-sample unpaired T test with Welch’s correction to compare treatments to the negative control (No Treatment) (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001).
  • a genetically engineered effector cell was transformed from human T-cells to mouse T-cells, improved their in vivo persistence, and validated successful engraftment and protein delivery function in tumor bearing immunocompetent mice.
  • these genetically engineered effector cells can be used to evaluate efficacy in treating a range of diseases.
  • a lentiviral-based transduction process was used that aligns with clinical manufacturing of human CAR T- cell treatments.
  • a process to expand sufficient mouse CD4 and CD8 CAR T-cells to preclinically evaluate their delivery function was used.
  • mouse CD4 and CD8 CAR T-cells separately supported persistent intratumoral protein delivery.
  • the development and implementation of drug delivering mouse CD4 and CD8 CAR T-cells in an immunocompetent system provides the ability to evaluate the localization, tumor infiltration, persistence, efficacy, and potential toxicity of therapeutic payloads in the context of a functional immune system.
  • human CAR T-cell formulations have focused on advancing CD8 CAR T-cell cytolytic potential and using CD4 CAR T- cells in a “helper” role.
  • CD4 CAR T-cells can directly drive successful tumor elimination and can outperform CD8 CAR T-cells due to a propensity for the latter to become exhausted with high tumor burden.
  • Successful clinical treatment also highlights CD4 CAR T-cell efficacy in treating cancer, in which CAR T-cell administration, blended with CD4 and CD8 T-cell subsets, resulted in long term engraftment of CD4 CAR T-cells with a cytolytic phenotype persisting beyond 10 years in leukemia patients in remission.
  • mouse CD4 CAR T-cells also exhibit improved persistence in syngeneic mouse leukemia models.
  • adoptive cell persistence and overall protein expression are better efficacy benchmarks than cytolytic function; and as determined in the case of human T- cells, CD4 CAR T-cells express around 5-fold more protein compared to similarly engineered CD8 CAR T-cells when stimulated by target tumor cells.
  • mouse CD8 CAR T-cells demonstrated increased antigen-triggered effector protein expression in vitro, compared to mouse CD4 CAR T-cells, suggesting that NF AT signaling may be different in mouse and human T-cells.
  • Mouse CD4 CAR T-cells showed comparable protein delivery function in vivo compared to CD8 CAR T-cells, supporting that the persistent nature of the CD4 T-cell promotes effector protein delivery in mice despite modified NF AT signaling.
  • Using a similar syngeneic, immunocompetent mouse model implanted with a less aggressive tumor allows for longitudinal studies and contrast CD4 and CD8 CAR T-cell engraftment and effector protein delivery as well as evaluate potential toxicity, including cytokine release syndrome attributed to CD4 T-cells.
  • mbIL15 into the cell-based delivery platform was instrumental in improving its persistence and protein delivery. Multiple IL-15 armoring efforts have increased clinical CAR T-cell and CAR-NK treatment efficacy by improving adoptive cell engraftment and anti -tumor efficacy. The strategy behind mbIL15 armoring is to improve CD8 T-cell and NK cell survival signaling through fL2Py receptors which are expressed on these cells. In various experiments, mbIL15 engineering improved CD8 CAR T-cell expansion in vivo, but also observed CD4 CAR T-cells, which express decreased IL2P receptor levels, persist in response to engineered mbIL15 signaling.
  • mbIL15 to support CD4 CAR T-cell proliferation is likely due to the ability of IL15Ra to augment IL2P receptor expression and to cis present IL-15.
  • the net effect is that CD4 CAR T-cells armored with IL- 15 signaling can be employed to deliver therapeutic proteins in response to disease antigen in vivo.
  • the combination of increased engraftment and improved effector protein output enabled sustained protein delivery with only two CAR T-cell injections, alleviating manufacturing resources and time.
  • One caveat of armoring therapeutic cells with constitutive proliferative IL-15 signaling is the potential for the engineered cells to become oncogenic.
  • Inducible mbIL15 expression within the tumor microenvironment offers a means to control mbIL15 signaling and prevent hyperproliferation.
  • Using inducible mbIL15 signaling suggests a threshold of IL-15 signaling is needed for CAR T-cell expansion and that minimal signaling is needed to see engrafted cells exhibit protein delivery function.
  • mbIL15 supports inducible protein delivery by the CD8 and CD4 T-cell chassis, and its regulated expression can mitigate any potential concerns due to oncogenicity of the genetically engineered effector cell.
  • T-cells were modified to deliver proteins in response to tumor antigen in immunocompetent mice implanted with pancreatic solid tumor model, and which can include therapeutic proteins that neutralize the immunosuppressive tumor microenvironment. Many of these proteins are likely to be immunomodulatory, the intent being to turn a “cold” tumor into a “hot” tumor. Delivering these therapies can provide in situ delivery of therapeutic proteins to the disease site and enable interrogating their antitumor efficacy in vivo where crosstalk between a functioning immune system, CAR T-cells, and the tumor is intact.
  • HEK293T/17 adherent cells were used for lentivirus production and maintained in DMEM media (DMEM, 10% heat-inactivated FBS, 100 units (U)/milliliter (mL) penicillin, and 100 microgram (pg)/mL streptomycin).
  • Target cell lines included the human derived A2780cis ovarian cancer cell line and the mouse derived KPCY2838c3 (KPCY) pancreatic ductal adenocarcinoma cell line.
  • FRa + Luc2 + E2Crimson + A2780cis and MSLN + Luc2 + E2Crimson + A2780cis cell lines were engineered as described in Repellin, C.E., et al., Modular Antigen-Specific T-cell Biofactories for Calibrated In Vivo Synthesis of Engineered Proteins. Adv Biosyst, 2018, 2(12), and maintained in RPMI media (RPMI, 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 pg/mL streptomycin).
  • Luc2 + E2Crimson + KPCY and MSLN + Luc2 + E2Crimson + KPCY were described previously and maintained in DMEM media supplemented with 2 millimolar (mM) Glutamax.
  • Mouse primary T-cells were cultured in RPMI media supplemented with 4mM Glutamax and 55 micromolar (pM) b- mercaptoethanol (T-cell media). Cytokines were added as described below.
  • the CAR molecule included the MSLN-binding SSI scFv (genbank AF035617.1) and the following mouse domains: CD8a leader sequence and hinge (Uniprot P01731), CD28 transmembrane region (Uniprot P31041), 4-1BB signaling domain (Uniprot P20334) and CD3 ⁇ domain (Uniprot P24161).
  • mbIL15 was synthesized to encode a fusion protein with mouse IL 15 (Uniprot P48346) fused to full length IL15Ra (Uniprot Q60819) with a 26 amino acid (aa) linker.
  • the IL 15 signal peptide and prodomain were also replaced with the IgK leader sequence (SP) to direct trafficking to the extracellular cell membrane. All synthesized DNA was mouse codon optimized. Inducible Nluc (Genbank JQ437370.1) expression was driven by a minimal NF AT response element. mbIL15 expression was either linked to constitutive CAR expression (cmbIL15) or inducible Nluc expression (imbIL15) by fusing the mbIL15 transgene to the CAR or Nluc transgene with a 2A “self-cleaving” peptide, respectively (FIG. 6A). The resulting transfer plasmids were cotransfected with lentiviral packaging plasmids in HEK293T/17 cells to generate concentrated lentiviral stocks as described.
  • SP IgK leader sequence
  • CD4 and CD8 T- cells were isolated through centrifugation and resuspended at IxlO 6 cells/100 pL volume containing concentrated lentivirus (MOI 10), 1 pg synperonic F108 and IxlO 6 Dynabeads® Mouse T-Activator CD3/CD28.
  • the transduction + activation reaction was plated in 100 pL volume per well in a 96 well plate and incubated overnight at 37 degrees Celsius (C).
  • the cells were transferred to a well of a 48-well plate and diluted 1 :5 with T-cell media supplemented with 20 ng/mL hIL-2.
  • cells were magnetically separated from the beads and isolated with centrifugation at 400 x g for 5 minutes.
  • Cells were resuspended in T-cell media. On day 4, the cells were counted and resuspended at 0.5xl0 6 cells/mL in T-cell media supplemented with 10 ng/mL hIL2, 10 ng/mL hIL7, and 10 ng/mL hIL15. The cells were subsequently passaged every 2-3 days maintaining a cell concentration of 0.5-3xl0 6 /mL in T-cell media supplemented with 5 ng/mL hIL2, 10 ng/mL hIL7 and 10 ng/mL hIL15. Cell viability was determined with ViaStainTM AO/PI Cell Staining. Transduction efficiency was evaluated 7 days posttransduction using flow cytometry analysis as described below.
  • Antibodies employed to stain surface molecules for flow cytometry analysis included the following (antibody clone): CD45.1 (A20), panCD45 (30-F11), CD3 (145-2C11), CD4 (RM4-5), CD8 (53-6.7), PD-1 (29F.1A12), and anti-IL15 with anti -Rabbit IgG.
  • the SSI CAR on the surface of transduced cells was labeled with biotinylated MSLN (btMSLN), followed by labeling with fluorochrome-labeled streptavidin.
  • CAR T-cell number/total T-cell number cells were stained 7 days post transduction to eliminate influence of transient transfection on CAR surface expression. Samples were analyzed with the BD FACS Symphony A3 and data were processed with FlowJo 10.9.0 software.
  • MSLN-CAR T-cells (with NFAT- RE inducible delivery function) were cocultured with target cells engineered from the ovarian tumor cell line A2780cis (MSLN + Luc2 + A2780cis and FRa + Luc2 + A2780cis).
  • target cells engineered from the ovarian tumor cell line A2780cis (MSLN + Luc2 + A2780cis and FRa + Luc2 + A2780cis).
  • 30,000 CAR T-cells were cocultured with serially diluted target cells in T-cell media.
  • Luc2 activity associated with A2780cis cell viability was measured with the One-GioTM Luciferase Assay following the manufacturer protocol and signal read on a multilabel plate reader.
  • samples were fixed with BD Cytofix Fixation Buffer and permeabilized with BD Phosflow Perm Buffer III according to manufacturer instructions.
  • Fixed and permeabilized cells were labeled with pStat5 (Tyr694), followed by anti-Rabbit IgG (Poly4064) and fixed with BD Cytofix Fixation buffer.
  • CAR T- cells were cultured with antigen loaded beads or with the ovarian cancer cell line, A2780cis to activate NF AT signaling and Nluc reporter protein delivery.
  • the beads used for activation were prepared using IxlO 7 Dynabeads® Biotin Binder (Invitrogen) beads prewashed with 0.5% HSA/2 mM EDTA/PBS and loaded with 1 pg anti-msCD28 (clone 37.51) and 0.4 pg btMSLN in 100 pL. Prior to T-cell treatment, the beads were washed with T-cell media.
  • 1.5xl0 5 FRa + Luc2 + A2780cis or MSLN + Luc2 + A2780cis cells were serially diluted 1 :2 and incubated with 3xl0 4 CAR T-cells in 200 pL T-cell media in a well of a 96 well plate. After 24 hour coculture, Nluc activity was measured with the Nano-Gio® Luciferase Assay.
  • mice were randomized, to distribute tumor expressing Luc2 bioluminescent intensity among the treatment groups and conditioned with intraperitoneal injection of 120 milligrams (mg)/kilogram (kg) cyclophosphamide.
  • CD45.1 + CD8 + CAR T-cells were washed with PBS and 5xl0 6 cells were administered by intraperitoneal injection.
  • CD45.1 + CD4 + CAR T-cells were washed with PBS and 5xl0 6 cells were administered by intraperitoneal injection.
  • Untransduced cells were added to the CAR T-cell injections to provide the same total cell number to all mice among the CD4 and CD8 CAR T-cell treatment groups.
  • One treatment group was implanted with tumor cells but not injected with CAR T-cells and served as a control group to assess tumor growth and luminescence background. Mice were euthanized if distressed or if tumor ulceration through skin grew greater than 2 millimeter (mm) in diameter. For luminescence measurements, the anesthetized mice were imaged with an IVIS® Lumina X5 imaging station. The tumor luminescence was quantified by analysis of the region-of-interest (ROI) that included the body of the mouse excluding the tail and using Living Image® software to determine the total flux (photons/s).
  • ROI region-of-interest
  • Tumors were minced with a razor blade and digested with 0.1 mg/mL Liberase TL Research Grade, 2.5 mg/mL collagenase D and 0.2 mg/mL DNasel in DMEM/10% FBS for 30 minutes with mixing on an orbital shaker. Following digestion, tumor samples were crushed through a 70 pm cell strainer. Cells resulting from the harvested tissues were resuspended in Cell Stain Buffer, treated with True Stain FcXTM Plus and labeled for flow cytometry. Viable cell numbers of dissociated tumor were determined with ViaStainTM AO/PI Cell Staining. Nluc associated Nluc activity was measured with the Nano-Gio® Luciferase Assay.
  • Nluc and tumor cell number variables were correlated with the Spearman correlation when values were not normally distributed and correlated with the Pearson correlation when values were normally distributed (*p ⁇ 0.05). Normal distribution of Nluc activity and tumor cell number values was assessed with the D’Agostino & Pearson, Anderson-Darling, and Shapiro-Wilk tests.
  • FIGs. 11 A-l IF illustrate transduction efficiency and viability of genetically engineered effector cells formed from mouse T-cells, in accordance with the present disclosure.
  • FIG. 11 A shows CD4 CAR T-cells and
  • FIG. 1 IB shows CD8 CAR T-cell transduction efficiency as determined 7 days post transduction with the activation process when influence of transient transduction is negligible.
  • CD4 and CD8 CAR T- cells were activated and transduced with CAR-cmbIL15, CAR-imbIL15 and CAR (without mbIL15) carrying lentivirus in 100 pL volume.
  • FIG. 11C shows CD4 CAR T-cell and FIG. 1 ID shows CD8 CAR T-cell transduction efficiency as determined by assessing the percentage of cells binding biotinylated MSLN (btMSLN) by flow cytometry analysis.
  • FIG. 1 IE shows CD4 and FIG. 1 IF shows CD8 viability as determined with AOPI staining.
  • CAR-cmbIL15, CAR- imbIL15, and CAR neg -cmbIL15 constructs CD4 T-cells transduced more readily than CD8 T-cells.
  • the CAR-imbIL15 constructs reproducibly demonstrated reduced transduction efficiency in both CD4 and CD8 T-cell subsets.
  • FIGs. 12A-12H illustrate antigen meditated antitumor activity of the genetically engineered effector cells, in accordance with the present disclosure.
  • FIGs. 12A-12B show results of CAR-cmbIL 15 -T-cells
  • FIGs. 12C-12D show results of CAR-imbIL15- T-cells
  • FIGs. 12E-12F show results of CAR-T-cells
  • FIGs. 12G-12H show results of CAR neg -cmbIL 15 -T-cells that were cocultured with MSLN + Luc2 + A2780cis and FRa + Luc2 + A2780cis ovarian cancer cells and Luc2 luminescence was measured to determine tumor cell viability.
  • E:T represents the ratio of 2500 CAR T-cells to increasing A2780cis target cells.
  • FIGs. 13A-13F illustrate example results from interperitoneally injected CAR- cmbIL 15 -T-cells as contrasted with intravenous injection and assessment of protein delivery and CAR T-cell engraftment (or persistence), in accordance with the present disclosure.
  • CD4 or CD8 CAR-cmbIL 15 -T-cells were injected by intraperitoneal or intravenous injection to compare administration routes.
  • FIG. 13B shows CD4 and FIG. 13C shows CD8 CD45.1 + CAR-cmbIL 15 -T-cell accumulation among total CD4 + CD8 + cells in spleen and tumor processed ex vivo for flow cytometry analysis 13 days post CAR T cell injection.
  • FIG. 13D includes bioluminescent images taken following injection of Nano-Gio® Fluorofurimazine In Vivo Substrate to tumor bearing mice to measure total flux corresponding to Nluc effector expression. Imaging performed prior to CAR T-cell injection provided individual mouse background measurements.
  • FIGs. 13E-13F show time course of Nluc activity following injection of CD4 (FIG.
  • FIG. 14 are bioluminescent images taken following mice treated as described in FIG. 10A, in accordance with the present disclosure. Images were taken of the tumor bearing mice described in the schematic FIG. 10A. Luc2 + KPC tumor cells were imaged following injection of D-luciferin (12 days post initial CAR T-cell treatment). Nluc activity produced by activated CAR-cmbIL15-T cells was imaged following injection of Nano-Gio® Fluorofurimazine In Vivo Substrate (13 days post initial CAR T-cell treatment). The red arrow points at the solid tumor that is observed in the “Tumor” panel and as bioluminescence in the “Tumor Luc2” panel. The white arrow indicates “CAR-cmbIL15-T cell Nluc” derived luminescence at the tumor margin that may not overlap with Luc2 + KPC cells. Four representative images are shown for each.
  • FIGs. 15A-15B illustrate examples CAR T-cell engraftment results of cmb IL- 15 from genetically engineered effector cells generated from mouse T-cells, in accordance with the present disclosure.
  • Blood was collected and processed 14 days post CAR T-cell injection to assess CD45.1 + CAR T-cell persistence with flow cytometry (see FIG. 10 A).
  • FIG. 15 A, CD4; FIG. 15B, CD8) CD45.1 + T-cell abundance in blood among panCD45 + CD3 + cells.
  • Error bars indicate ⁇ 1 SEM of the mean average among animal subjects.
  • P values were calculated using one-way ANOVA with Tukey post hoc correction with *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • FIG. 16 illustrates transplanted CD8 CAR-cmbIL 15 -T-cells exhibit increased PD-1 exhaustion marker expression compared to CD4 CAR-cmbIL 15 -T-cells, in accordance with the present disclosure.
  • Ex vivo analysis of tumors harvested 13 days post first CAR T-cell intraperitoneal injection in tumor implanted mice assessed PD-1 expression by flow cytometry.
  • PD-1 expression in tumor dissociated CD45.1 + CAR-cmbIL15-T cells is compared for CD4 and CD8 administered T-Cell subsets. Error bars indicate ⁇ 1 SEM of the mean average among four animal subjects.

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Abstract

L'invention concerne une cellule effectrice génétiquement modifiée donnée à titre d'exemple qui comprend une cellule portant une séquence polynucléotidique exogène qui comprend un élément récepteur qui code pour un récepteur antigénique chimérique (CAR) comprenant un domaine de liaison à l'antigène extracellulaire fonctionnellement lié à un domaine transmembranaire, ainsi qu'un domaine de signalisation intracellulaire, un élément régulateur immunitaire qui code un complexe immunorégulateur comprenant un régulateur immunitaire et une molécule de trafic liée à la membrane, un élément actionneur qui code un site de liaison au facteur de transcription qui régule à la hausse la synthèse d'une protéine effectrice et du complexe immunorégulateur en réponse au domaine de liaison à l'antigène extracellulaire du CAR se liant à un antigène d'une cellule cible et un élément effecteur qui code la protéine effectrice, en réponse au domaine de liaison à l'antigène extracellulaire du CAR se liant à l'antigène de la cellule cible, la cellule effectrice génétiquement modifiée étant conçue pour activer et synthétiser la protéine effectrice et le complexe immunorégulateur.
PCT/US2025/025740 2024-04-22 2025-04-22 Cellules effectrices génétiquement modifiées comprenant des éléments régulateurs immunitaires Pending WO2025226662A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018161038A1 (fr) * 2017-03-03 2018-09-07 Obsidian Therapeutics, Inc. Compositions il12 et méthodes d'immunothérapie
US20220143088A1 (en) * 2019-03-15 2022-05-12 Daegu Gyeongbuk Institute Of Science And Technology Cytokine-based immune cells and immunotherapeutic use thereof
US20240115610A1 (en) * 2015-09-10 2024-04-11 Sri International Genetically engineered diagnostic cells and antigen tests

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20240115610A1 (en) * 2015-09-10 2024-04-11 Sri International Genetically engineered diagnostic cells and antigen tests
WO2018161038A1 (fr) * 2017-03-03 2018-09-07 Obsidian Therapeutics, Inc. Compositions il12 et méthodes d'immunothérapie
US20220143088A1 (en) * 2019-03-15 2022-05-12 Daegu Gyeongbuk Institute Of Science And Technology Cytokine-based immune cells and immunotherapeutic use thereof

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