Genetically engineered CD4 T cells for in situ protein synthesis

Abstract

[Solution] An example of a genetically engineered effector cell comprises an isolated CD4 T cell harboring an exogenous polynucleotide sequence comprising a receptor element, an actuator element, and an effector element. The receptor element encodes a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that recognizes an antigen on the surface of a target cell, operably linked to a transmembrane domain, and an intracellular signaling domain. The actuator element encodes a transcription factor binding site that upregulates synthesis of the effector protein. The effector element encodes the effector protein, and the genetically engineered effector cell is configured to activate and synthesize and secrete the effector protein in response to the extracellular antigen-binding domain of the CAR binding to an antigen on the target cell.

Description

[Technical Field]

[0001] This invention was made with government support under Grant Nos. R21CA236640 and R33CA247739, both awarded by the National Cancer Institute of the National Institutes of Health, and Grant No. DP2EB024245, awarded by the National Institute of Biomedical Imaging and Bioengineering, also of the National Institutes of Health. The government has certain rights in this invention.

[0002] This PCT application claims the benefit of U.S. Provisional Application No. 63 / 403,219, filed September 1, 2022, entitled "Primary CD4 T-Cell Biofactory for Antigen-Inducible In Situ Synthesis of Engineered Proteins," which is incorporated herein by reference in its entirety.

[0003] The contents of the electronic sequence listing (S1647176111_sequence _listing.xml; size: 156336 bytes; creation date: August 25, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0004] Various standard therapeutics are designed to treat disease at the time of diagnosis. Many pathogens and disease cells undergo dynamic changes in vivo, yet current drugs are not designed to coevolve with the in vivo disease microenvironment. Such therapeutics include drugs administered at doses standardized for patient weight. However, patients of similar size may have different disease burdens, and patient-to-patient variability can affect optimal dosing. Drug overdose can result in the drug leaking into the systemic circulation and causing normal tissue morbidity. Suboptimal drug delivery can lead to drug resistance. While patients can be monitored and dosages adjusted based on health outcomes, continuous monitoring is costly and impractical. Furthermore, monitoring strategies and treatments do not exist for many diseases. Therefore, static therapeutics often fail to control dynamic pathogens and diseases that evolve and / or persist. The mismatch between dynamic disease states and appropriate treatments imposes a significant societal and economic burden. Summary of the Invention

[0005] The present disclosure is directed to solving these and other related problems of therapeutic agents for treating diseases, including, inter alia, genetically engineered CD4 T cell lines that can be activated in situ to trigger the synthesis of engineered proteins (effectors) against target cells.

[0006] Various aspects of the present disclosure are directed to engineered effector cells comprising isolated CD4+ T cells having an exogenous polynucleotide sequence comprising, in operative association: a receptor element encoding a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain operably linked to a transmembrane domain and which recognizes an antigen on the surface of a target cell, and an intracellular signaling domain; an actuator element encoding a transcription factor binding site that upregulates synthesis of the effector protein in response to the extracellular antigen-binding domain of the CAR binding to an antigen on the target cell; and an effector element encoding the effector protein; wherein the effector cell is configured to activate and synthesize and secrete the effector protein in response to the extracellular antigen-binding domain of the CAR binding to an antigen on the target cell.

[0007] In some embodiments, the effector element encodes a signal peptide that is operably linked to the effector protein, wherein the signal peptide is non-native to the effector protein.

[0008] In some embodiments, the engineered effector cells are configured to synthesize and secrete amounts of effector protein as a function of the amount of target cells present in a sample or in situ.

[0009] In some embodiments, the CAR is configured to elevate calcium in response to the extracellular antigen binding domain binding to an antigen on a target cell, and the transcription factor binding site is configured to bind to a transcription factor protein that is triggered by the calcium elevation and translocates into the nucleus of the engineered effector cell.

[0010] In some embodiments, the intracellular signaling domain is selected from the group consisting of the intracellular signaling portion of 4-1BB, the intracellular signaling portion of CD3 zeta, and combinations thereof.

[0011] In some embodiments, the intracellular signaling domain does not include the intracellular signaling portion of CD28.

[0012] In some embodiments, the transcription factor binding site is selected from the group consisting of a nuclear factor of activated T cells (NFAT) response element, a serum response element (SRE), a cyclic AMP response element (CRE), and combinations thereof.

[0013] In some embodiments, the effector protein is selected from the group consisting of a detectable reporter protein, a therapeutic protein, a downstream signaling protein, and combinations thereof.

[0014] In some embodiments, the exogenous polynucleotide sequence comprises a receptor element, an actuator element, and an effector element in operative association on a single construct.

[0015] In some embodiments, the transmembrane domain is selected from the group consisting of a T cell receptor alpha or beta chain, CD3 zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, and GITR.

[0016] Various aspects of the present disclosure are directed to a single construct comprising an exogenous polynucleotide sequence comprising, in operative association, a receptor element encoding a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain operably linked to a transmembrane domain that recognizes an antigen on the surface of a target cell, and an intracellular signaling domain; an actuator element encoding a transcription factor binding site that upregulates synthesis of the effector protein in response to the extracellular antigen-binding domain of the CAR binding to an antigen on the target cell; and an effector element encoding the effector protein, configured to form an engineered effector cell comprising isolated CD4 T cells that secrete an effector protein upon recognition of an antigen on the surface of a target cell, wherein the engineered effector cell is configured to activate and synthesize and secrete the effector protein in response to the extracellular antigen-binding domain of the CAR binding to an antigen on the target cell.

[0017] In some embodiments, the effector element encodes a signal peptide that operably links to the effector protein.

[0018] In some embodiments, the single construct is carried by a viral vector or a non-viral carrier.

[0019] In some embodiments, the intracellular signaling domain comprises the intracellular signaling portion of 4-1BB and the intracellular signaling portion of CD3 zeta, respectively.

[0020] In some embodiments, the intracellular signaling domain does not include the intracellular signaling portion of CD28.

[0021] In some embodiments, the transcription factor binding site is selected from the group consisting of a nuclear factor of activated T cells (NFAT) response element, a serum response element (SRE), a cyclic AMP response element (CRE), and combinations thereof, and the transmembrane domain is selected from the group consisting of a T cell receptor alpha or beta chain, CD3 zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, and GITR.

[0022] In some embodiments, the effector protein is selected from the group consisting of a detectable reporter protein, a therapeutic protein, a downstream signaling protein, and combinations thereof.

[0023] In some embodiments, the exogenous polynucleotide sequence comprises a sequence having at least 80% sequence identity to a sequence selected from SEQ ID NOs: 1-20.

[0024] Various aspects of the present disclosure are directed to a population of engineered effector cells, each engineered effector cell of the population comprising an isolated CD4 T cell having an exogenous polynucleotide sequence comprising an actuator element that binds to an effector element that binds to a receptor element, the receptor element encoding a CAR comprising an extracellular antigen binding domain that recognizes an antigen on the surface of a target cell, and an intracellular signaling domain operably linked to a transmembrane domain, the actuator element encoding a transcription factor binding site that upregulates synthesis of the effector protein in response to the extracellular antigen binding domain of the CAR binding to the antigen on the target cell, the effector element encoding the effector protein, and wherein the population of engineered effector cells is configured to activate and synthesize and secrete the effector protein in response to the extracellular antigen binding domain of the CAR binding to the antigen on the target cell.

[0025] In some embodiments, a population of engineered effector cells is configured to activate based on the presence of target cells and, in response, synthesize and secrete a calibrated amount of effector protein, the calibrated amount of effector protein being a function of the amount of target cells present in a plurality of cells or sample.

[0026] In some embodiments, each effector element encodes a signal peptide that operably links it to an effector protein.

[0027] In some embodiments, the intracellular signaling domain is selected from the group consisting of the intracellular signaling portion of 4-1BB, the intracellular signaling portion of CD3 zeta, and combinations thereof.

[0028] In some embodiments, the intracellular signaling domain does not include the intracellular signaling portion of CD28.

[0029] In some embodiments, the transcription factor binding site is selected from the group consisting of a nuclear factor of activated T cells (NFAT) response element, a serum response element (SRE), a cyclic AMP response element (CRE), and combinations thereof, and the transmembrane domain is selected from the group consisting of a T cell receptor alpha or beta chain, CD3 zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, and GITR.

[0030] In some embodiments, the effector protein is selected from the group consisting of a detectable reporter protein, a therapeutic protein, a downstream signaling protein, and combinations thereof.

[0031] Various aspects of the present disclosure are directed to methods comprising: activating a plurality of CD4 T cells with a plurality of particles; exposing the plurality of CD4 T cells to an exogenous polynucleotide sequence to engineer the plurality of CD4 T cells; and expanding the activated plurality of CD4 T cells in an expansion culture medium to form a plurality of engineered effector cells comprising the plurality of CD4 T cells bearing the exogenous polynucleotide sequence, wherein the exogenous polynucleotide sequence comprises, in operative association: a receptor element operably linked to a transmembrane domain and encoding a CAR comprising an extracellular antigen-binding domain that recognizes an antigen on the surface of a target cell and an intracellular signaling domain; an actuator element encoding a transcription factor binding site that upregulates synthesis of the effector protein in response to the extracellular antigen-binding domain of the CAR binding to the antigen on the target cell; and an effector element that encodes the effector protein in response to the extracellular antigen-binding domain of the CAR binding to the antigen on the target cell;

[0032] In some embodiments, the effector element further encodes a signal peptide that operably links to the effector protein.

[0033] In some embodiments, the intracellular signaling domain includes an intracellular signaling portion of 4-1BB and an intracellular signaling portion of CD3 zeta and / or does not include an intracellular signaling portion of CD28, the transcription factor binding site is selected from the group consisting of an NFAT response element, an SRE, a CRE, and combinations thereof, and / or the transmembrane domain is selected from the group consisting of a T cell receptor alpha or beta chain, a CD3 zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, and GITR.

[0034] In some embodiments, activating the plurality of CD4 T cells comprises exposing the plurality of CD4 T cells to a plurality of particles carrying anti-human CD3 antibodies and anti-human CD28 antibodies.

[0035] In some embodiments, the method further comprises exposing the plurality of CD4 T cells to a plurality of particles at a cell body to particle ratio of about 6:1 to about 1:6 for a period of time.

[0036] In some embodiments, exposing the plurality of CD4 T cells to the exogenous polynucleotide sequence comprises exposing the plurality of CD4 T cells to a vector comprising the exogenous polynucleotide sequence, wherein the vector is associated with or comprises a viral vector, a non-viral carrier, and / or a lipid nanoparticle.

[0037] In some embodiments, exposing the plurality of CD4 T cells to the exogenous polynucleotide sequence comprises exposing the activated plurality of CD4 T cells to a lentivirus comprising the exogenous polynucleotide sequence.

[0038] In some embodiments, exposing a plurality of CD4 T cells to lentivirus comprises exposing a plurality of CD4 T cells to about 0.05×10 in serum-free, polybrene-containing culture medium. 6 Cells / milliliter (mL) ~ approx. 3 x 10 6 This involves exposing CD4 T cells at a concentration of 1000 cells / mL to lentivirus.

[0039] In some embodiments, exposing a plurality of CD4 T cells to an exogenous polynucleotide sequence is about 0.05×10 6 cells / mL ~ approx. 3×10 6 The method includes providing a total transduction reaction volume comprising a plurality of CD4 T cells at a cell density of 1000 cells / mL, culture medium, and a vector carrying an exogenous polynucleotide sequence in defined sub-amounts for a period of time.

[0040] In some embodiments, expanding the activated CD4 T cells increases the total transduction response dose to about 0.25×10 6 cells / mL ~ approx. 1×10 6 The method involves diluting activated CD4 T cells at a cell density of 1000 cells / mL with cytokine-containing expansion medium for a period of time.

[0041] In some embodiments, the cytokine is selected from the group consisting of interleukin (IL)-2, IL-7, IL-15, and combinations thereof.

[0042] Various aspects of the present disclosure are directed to methods comprising: activating a plurality of T cells with a plurality of particles; exposing the plurality of T cells to an exogenous polynucleotide sequence to engineer a plurality of CD4+ T cells; and expanding the activated plurality of T cells in an expansion culture medium to form a plurality of engineered effector cells comprising the plurality of T cells bearing the exogenous polynucleotide sequence, wherein the exogenous polynucleotide sequence comprises, in operative association: a receptor element operably linked to a transmembrane domain and encoding a CAR comprising an extracellular antigen binding domain that recognizes an antigen on the surface of a target cell and an intracellular signaling domain; an actuator element encoding a transcription factor binding site that upregulates synthesis of the effector protein in response to the extracellular antigen binding domain of the CAR binding to the antigen on the target cell; and an effector element that encodes the effector protein in response to the extracellular antigen binding domain of the CAR binding to the antigen on the target cell;

[0043] In some embodiments, the effector element further encodes a signal peptide that operably links to the effector protein.

[0044] In some embodiments, the intracellular signaling domain includes an intracellular signaling portion of 4-1BB and an intracellular signaling portion of CD3 zeta and / or does not include an intracellular signaling portion of CD28, the transcription factor binding site is selected from the group consisting of an NFAT response element, an SRE, a CRE, and combinations thereof, and / or the transmembrane domain is selected from the group consisting of a T cell receptor alpha or beta chain, a CD3 zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, and GITR.

[0045] In some embodiments, the plurality of T cells comprises CD3 T cells, isolated CD4 T cells, or isolated CD8 T cells. In some embodiments, the plurality of T cells comprises isolated CD4 T cells.

[0046] In some embodiments, activating the plurality of T cells comprises exposing the plurality of T cells to a plurality of particles carrying anti-human CD3 antibodies and anti-human CD28 antibodies.

[0047] In some embodiments, the method further comprises exposing the plurality of T cells to a plurality of particles at a cell body to particle ratio of about 6:1 to about 1:6 for a period of time.

[0048] In some embodiments, the period of time is from about 10 hours to about 36 hours.

[0049] In some embodiments, the method includes resuspending a plurality of T cells in complete growth medium and activating the plurality of T cells by adding a plurality of particles to the complete growth medium.

[0050] In some embodiments, exposing the plurality of T cells to the exogenous polynucleotide sequence comprises culturing the plurality of T cells in a serum-free and polybrene-containing culture medium at a concentration of about 0.05×10 6 cells / mL ~ approx. 3×10 6cells / mL of T cells to a vector carrying an exogenous polynucleotide sequence.

[0051] In some embodiments, the culture medium contains about 4 micrograms (μg) / mL to about 8 μg / mL of polybrene.

[0052] In some embodiments, exposing a plurality of T cells to an exogenous polynucleotide sequence is about 0.05×10 6 cells / mL ~ approx. 3×10 6 The method includes providing a total transfection reaction volume containing a plurality of T cells at a cell density of 1000 cells / mL, culture medium, and vector in defined sub-volumes at a multiplicity of infection (MOI) of about 0.1 to about 10 for a certain period of time.

[0053] In some embodiments, providing the total transformation reaction volume in a defined subvolume includes placing the aliquot as a droplet volume into a tissue-cultured well plate and placing the cultured well plate in an incubator for a period of time.

[0054] In some embodiments, providing the total transformation reaction volume in a defined subvolume comprises disposing an aliquot of the defined subvolume onto a substrate having a hydrophobic or hydrophilic surface.

[0055] In some embodiments, the total transformation reaction volume is about 0.5 mL to 2 mL, and the subvolumes are about 0.05 mL to 0.25 mL.

[0056] In some embodiments, the period of time is from about 10 to about 24 hours.

[0057] In some embodiments, exposing the plurality of T cells to the exogenous polynucleotide sequence comprises exposing the plurality of T cells to a vector comprising the exogenous polynucleotide sequence, wherein the vector is associated with or comprises a viral vector, a non-viral carrier, and / or a lipid nanoparticle.

[0058] In some embodiments, the viral vector comprises a lentivirus carrying the exogenous polynucleotide sequence.

[0059] In some embodiments, the lentivirus comprises lentiviral particles having an exogenous polynucleotide sequence, and the method comprises resuspending the lentiviral particles in sufficient culture medium to achieve an MOI of about 0.1 to about 10.

[0060] In some embodiments, expanding the activated T cells increases the total transduction response dose to about 0.25×10 6 cells / mL ~ approx. 1×10 6 The method comprises diluting a plurality of activated CD4 T cells at a cell density of 1000 cells / mL for a period of time in expansion medium, where the expansion medium is complete growth medium containing cytokines.

[0061] In some embodiments, the cytokine is selected from the group consisting of IL-2, IL-7, IL-15, and combinations thereof. In some embodiments, the cytokine is IL-7 and IL-15.

[0062] In some embodiments, the period of time is about 10 to 20 days, and the method comprises administering about 0.25×10 6 cells / mL ~ approx. 1×10 6 The method further comprises periodically replacing at least a portion of the expansion culture medium while maintaining a cell density of cells / mL.

[0063] In some embodiments, the method further comprises adding an additive to at least one of the culture medium and the expansion medium, wherein the additive is selected from the group consisting of an antiviral inhibitor, a latency reversing agent, and combinations thereof.

[0064] In some aspects, various aspects of the present disclosure are directed to a population of genetically engineered effector cells comprising T cells harboring an exogenous polynucleotide sequence, formed according to any of the claimed methods.

[0065] Various aspects of the present disclosure are directed to kits comprising a plurality of T cells; an exogenous polynucleotide sequence comprising, in operative association: a receptor element encoding a CAR comprising an extracellular antigen-binding domain operably linked to a transmembrane domain and which recognizes an antigen on the surface of a target cell, and an intracellular signaling domain; an actuator element encoding a transcription factor binding site that upregulates synthesis of an effector protein in response to the extracellular antigen-binding domain of the CAR binding to an antigen on the target cell; and an effector element encoding an effector protein in response to the extracellular antigen-binding domain of the CAR binding to an antigen on the target cell; culture medium; and expansion culture medium.

[0066] In some embodiments, the effector element further encodes a signal peptide that operably links to the effector protein.

[0067] In some embodiments, the intracellular signaling domain includes an intracellular signaling portion of 4-1BB and an intracellular signaling portion of CD3 zeta and / or does not include an intracellular signaling portion of CD28, the transcription factor binding site is selected from the group consisting of an NFAT response element, an SRE, a CRE, and combinations thereof, and / or the transmembrane domain is selected from the group consisting of a T cell receptor alpha or beta chain, a CD3 zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, and GITR.

[0068] In some embodiments, the plurality of T cells comprises CD3 T cells, isolated CD4 T cells, or isolated CD8 T cells.

[0069] In some embodiments, the plurality of T cells comprises isolated CD4 T cells.

[0070] In some embodiments, the kit comprises a plurality of particles carrying anti-human CD3 antibodies and anti-human CD28 antibodies.

[0071] In some embodiments, the kit includes another culture medium configured to resuspend the plurality of T cells with the plurality of particles to activate the plurality of T cells.

[0072] In some embodiments, the separate culture medium and the plurality of particles are configured to resuspend the plurality of T cells at a cell body to particle ratio of about 6:1 to about 1:6 for a period of about 10 hours to about 36 hours.

[0073] In some embodiments, the separate culture medium is a complete growth medium.

[0074] In some embodiments, the culture medium is serum-free and contains polybrene and is configured to manipulate a plurality of T cells.

[0075] In some embodiments, the culture medium contains about 4 μg / mL to about 8 μg / mL of polybrene.

[0076] In some embodiments, the kit includes a vector having an exogenous polynucleotide sequence, the vector being associated with or including a viral vector, a non-viral carrier, and / or a lipid nanoparticle.

[0077] In some embodiments, the viral vector comprises a lentiviral particle carrying the exogenous polynucleotide sequence, and the culture medium is configured to resuspend the lentiviral particles in sufficient culture medium to achieve an MOI of about 0.1 to about 10.

[0078] In some embodiments, the kit contains about 0.05×10 6 cells / mL ~ approx. 3×10 6The tissue-cultured well plate is configured to receive a sub-volume of a total transfection reaction volume containing a plurality of T cells at a cell density of 1000 cells / mL, a culture medium, and an exogenous polynucleotide sequence, and to culture the sub-volume for a certain period of time.

[0079] In some embodiments, the total transformation reaction volume is about 0.5 mL to 2 mL, and the period of time is about 10 to about 24 hours.

[0080] In some embodiments, the expansion culture medium is a complete growth medium containing cytokines.

[0081] In some embodiments, the cytokine is selected from the group consisting of IL-2, IL-7, IL-15, and combinations thereof.

[0082] In some embodiments, the cytokines include IL-7 and IL-15.

[0083] In some embodiments, the expansion culture medium contains approximately 0.25×10 6 cells / mL ~ approx. 2×10 6 Dilute the total transformation reaction volume at the cell density in cells / mL.

[0084] In some embodiments, at least one of the culture medium and the expansion medium comprises an additive selected from the group consisting of an antiviral inhibitor, a latency reversing agent, and combinations thereof.

[0085] Various example embodiments can be more fully understood in consideration of the following detailed description in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0086] [Figure 1] FIG. 1 shows an example of genetically engineered effector cells comprising isolated CD4 T cells according to the present disclosure.

[0087] [Figure 2]2A and 2B show examples of engineered effector cells according to the present disclosure.

[0088] [Figure 3] FIG. 3 shows an example of a population of genetically engineered effector cells in a target environment according to the present disclosure.

[0089] [Figure 4-5] 4 and 5 show examples of methods for generating genetically engineered effector cells from T cells according to the present disclosure.

[0090] [Figure 6] FIG. 6 shows an example of a kit for generating genetically engineered effector cells from T cells according to the present disclosure.

[0091] [Figure 7] 7A-7E show examples of polynucleotide sequences used to form engineered effector cells according to the present disclosure.

[0092] [Figure 8] 8A-8H show the effect of altering different factors on the generation and function of effector cells of T cell lines according to the present disclosure.

[0093] [Figure 9] 9A-9C show the results of validation of the functionality of CD4 T cell lineage effector cells as a protein delivery platform in vivo according to the present disclosure.

[0094] [Figure 10] 10A-10H show FRα-specific targeting of tumor cells by CD4 T cells engineered to secrete IFNβ, according to the present disclosure.

[0095] [Figure 11]FIG. 11 shows a flow cytometry plot showing the ratio of CD4 and CD8 in a pan-CD3 T cell population from a healthy donor according to the present disclosure.

[0096] [Figure 12] FIG. 12 shows a comparison of CD4 T cell and CD8 T cell chemotaxis according to the present disclosure.

[0097] [Figure 13] FIG. 13 shows an example of a process for generating genetically engineered effector cells from primary T cells according to the present disclosure.

[0098] [Figure 14-15] 14A-15D show examples of the effect of various parameters of lentivector transduction of primary T cells according to the present disclosure.

[0099] [Figure 16] 16A-16E show examples of the effect of various parameters of primary T cell expansion according to the present disclosure.

[0100] [Figure 17] 17A-17F show validation of functionality of effector cells generated from primary T cells according to the present disclosure.

[0101] [Figure 18] 18A and 18B show examples of strategies for assessing CD3 T cell activation according to the present disclosure.

[0102] [Figure 19] 19A-19D show examples of the effect of additional factors on transduction of primary T cells by lentivectors according to the present disclosure.

[0103] [Figure 20] 20A and 20B show an example of an exploratory screen of chemical additives to improve lentivector transduction of primary T cells according to the present disclosure.

[0104] [Figure 21] FIG. 21 shows an example of changes in the ratio of CD3 T cell subsets in response to cytokines according to the present disclosure.

[0105] [Figure 22] 22A-22C show examples of antigen-specific cell lysis and NFAT-RE-induced delivery functions according to the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0106] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof, and which show by way of illustration specific embodiments in which the present disclosure may be practiced. It will be understood that alternative embodiments may be utilized and that various changes may be made without departing from the scope of the present disclosure. Accordingly, the following detailed description is not to be taken in a limiting sense, and the scope of the present disclosure is defined by the appended claims. It will be understood that the features of the various embodiments described herein can be combined with each other in whole or in part, unless otherwise stated.

[0107] Despite recent approvals for cell therapy by various government agencies, such as the U.S. Food and Drug Administration (FDA), the impact of T cell-based drugs has been limited. This is because adoptive cell therapy is autologous, which can lead to manufacturing costs, product variability, and adverse events. Furthermore, the dynamic nature of cell-based disease states and interpatient variability pose challenges to optimal dosing and necessitate continuous monitoring of individual patients' disease status. As noted above, drug delivery is often normalized to body weight and surface area. However, disease pathology can vary across patients of similar size. Embodiments of the present disclosure are directed to genetically engineered effector cells, comprising isolated CD4 T cells or other types of primary T cells, used as cellular chassis or vectors that function as biofactories for different target proteins. Engineered effector cells can be used to synthesize calibrated amounts of target proteins and induce autocrine and paracrine signaling through artificial cell signaling. Such effector cells can be used as in vivo vectors to deliver target proteins to organisms, such as humans.

[0108] Embodiments of the present disclosure include primary T cell lines engineered with chimeric antigen receptors (CARs) to form effector cells that specifically detect (e.g., bind) antigens expressed on the surface of target cells. In some embodiments, the effector cells are formed from or comprise isolated CD4+ T cells. CD4+ T cells can be isolated from other cell types either prior to engineering the effector cells or during the engineering of the effector cells, such as by using a selective expansion process. By binding to the antigen, the engineered effector cells have improved function compared to natural T cells. Surprisingly, in experimental embodiments, CD4+ T cells, when used to form engineered effector cells, showed a stronger propensity for transduction and expansion, among other effects, secretion and activity, compared to CD8+ or CD3+ T cells.

[0109] T cells may be engineered to express genetic elements containing transmembrane receptors that autonomously regulate the intracellular transcriptional machinery, also referred to herein as effector cells or engineered effectors. Furthermore, the genetic elements of effector cells may be modular, and / or effector cells may contain multiple genetic elements, generating engineered effector cells capable of functioning as vectors for a variety of in vitro, ex vivo, and in vivo applications. Such effector cells may be modular in that they are partially storable and partially modifiable for different applications. For example, this modularity can be exploited to combine different receptor elements with different effector elements, allowing engineered effector cells to be reprogrammed to target diseases with known biomarkers, such as cancer, viral infection, and / or autoimmune disorders. Engineered effector cells can self-regulate therapeutic responses in response to stimulation by disease cells and be used for therapeutics and therapies applicable to a variety of cell-based diseases, including cancer, emerging pathogens, and those that evade or misdirect the immune system. Such multiple types of engineered effector cells, such as engineered T cells, provide robust and reproducible cell systems for therapeutically targeting complex diseases in vivo, and offer reliable in vivo imaging and in vitro sensor technologies for a variety of applications.

[0110] Thus, in various embodiments, engineered effector cells are modular and antigen-specific. Antigen specificity may be used to overcome tumor resistance, inducing cytolytic function against different antigen-presenting target cells, such as human or other organism host cells. Furthermore, the artificial cell signaling pathways of such engineered effector cells may confer the ability to function as vectors by producing calibrated amounts of protein-based therapeutic agents and inducing targeted autocrine and paracrine signaling upon engagement of the target antigen. Engineered effector cells may enable focused synthesis of biologics at the target site and / or limit systemic toxicity, allowing for extended treatment durations for better patient outcomes. Embodiments are not limited to therapeutic agents, and other types of effector proteins may be produced.

[0111] Several further embodiments described herein demonstrate the successful implementation of artificial cell signaling pathways in CD4 T cell lines. In some experimental embodiments, CD4 T cell lines are transformed with vectors to engage antigen-presenting target cells and induce the synthesis of calibrated amounts of engineered proteins in situ, often referred to herein as "effector proteins." As described above, engineered effector cells can provide modular, allogeneic, live vectors.

[0112] As used herein, "engineered effector cells" include and / or refer to T cells engineered or modified to comprise (i) a receptor element, (ii) an actuator element, and (iii) an effector element, each of which may be modular. As used herein, the terms "modularized" 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 engineered effector cells. As used herein, "receptor element" includes and / or refers to a polynucleotide sequence encoding a transmembrane receptor, such as a CAR, capable of specifically interacting with a target cell. Depending on the particular application, the receptor element can be reprogrammed by swapping the single-chain variable fragment (scFV) portion and / or the CAR for an extracellular antigen-binding domain specific for a different disease-associated antigen or other target. Other receptor elements that may be used include, but are not limited to, CARs with specificity for antigens associated with autoimmune disorders, CARs with specificity for antigens associated with neurological disorders (e.g., PTSD, Parkinson's disease, Alzheimer's disease), ligand-gated GPCRs (e.g., GPR1 glucose receptor), light-gated ion channels (e.g., melanopsin, rhodopsin, photopsin), pressure-sensing ion channels (e.g., TRPV1, TRPV2), and ligand-gated ion channels.

[0113] As used herein, "actuator element" includes and / or refers to a polynucleotide sequence that encodes a transcription factor binding site that initiates transcriptional and translational events downstream of a trigger signal (e.g., binding of the sensing element to a target antigen). Generally, the molecular mechanism underlying actuator elements is the regulation of intracellular calcium [Ca], a mechanism used in nearly all types of cells to limit their function. 2+ ] iExamples of response elements include, but are not limited to, NFAT ("nuclear factor of activated T cells") response element (NFAT-RE), serum response element (SRE), and cyclic AMP response element (CRE).

[0114] As used herein, "effector element" includes / refers to a polynucleotide sequence encoding an effector protein, in some cases operably linked to a polypeptide. For example, the polynucleotide sequence encoding the effector protein can be 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, and a sequence encoding a therapeutic molecule, among others.

[0115] The engineered effector cells into which the receptor, actuator, and effector elements have been introduced can be any T cell type, including human T cells or non-human T cells (e.g., mammalian, reptilian, plant). In such cases, the genetically engineered cellular "source" of modular elements provides a cellular chassis or frame that provides, among other things, the transcription and translation machinery for expression and presentation of the receptor, actuator, and effector elements. In some embodiments, T cells may be derived from a source (e.g., a first human), recombined, and administered to an organism different from the source (e.g., a host that is a second human). In other embodiments, T cells may be derived from a source (e.g., a first human), recombined, and administered back to the source (e.g., the source is a host).

[0116] Turning to the drawings, Figure 1 shows an example of an engineered effector cell comprising an isolated CD4 T cell according to the present disclosure. The engineered effector cell 100 can be modular in that components can be tailored to synthesize different effector proteins for different target cells.

[0117] As shown in FIG. 1 , engineered effector cells 100 may be formed from and / or include isolated CD4 T cells. The CD4 T cells may be isolated from other cell types prior to engineering the T cells or during engineering the T cells, such as by using a selective expansion process. In some embodiments, CD3 T cells may be used, including a mixture of both CD4 and CD8 T cells, with generally more CD8 T cells than CD4 T cells. The CD4 T cells may then be isolated from the other cells. Surprisingly, as described above, CD4 T cells have shown a stronger propensity for transduction and expansion, among other effects, compared to CD8 or CD3 T cells when forming engineered effector cells, along with higher effector secretion and activity. As further described herein, CD4 T cells expand at a higher rate / efficiency when isolated and expanded than when expanded in a mixture, such as with CD3 T cells.

[0118] Some embodiments include a selective expansion step involving the use of particles coated with proteins (e.g., engineered CD4 T cells bearing a CAR or engineered with a binding element such as a peptide tag or short peptide sequence) and anti-C28 antibodies, which can specifically induce the cells of interest to proliferate further. Additionally, the cells of interest (e.g., CD4 T cells) can be isolated using antibody-based isolation techniques, which can use flow cytometry or magnetic separation.

[0119] The genetically engineered effector cell 100 comprises a receptor element 102, an actuator element 106, and an effector element 110, optionally including a signal peptide 114, in operative association, which may optionally be on a single construct.

[0120] The receptor element 102 encodes CAR104. CARs are often referred to as "chimeric receptors," "T-bodies," or "chimeric immunoreceptors." As used herein, CAR includes and / or refers to an artificially constructed hybrid protein or polypeptide comprising an extracellular antigen-binding domain 103 of an antibody (e.g., scFV) operably linked to a transmembrane domain 105 and at least one intracellular signaling domain 107. For example, CAR104 comprises an extracellular antigen-binding domain 103 operably linked to a transmembrane domain 105 and an intracellular signaling domain 107. CAR104 may be designed to recognize a surface antigen of a target, such as a target cell in a host. CAR104 transduces internal Ca2+ in response to antigen binding. 2+ Store intracellular Ca 2+ For example, the extracellular antigen-binding domain 103 of CAR104 can recognize an antigen on the surface of a target cell, such as a diseased cell of a host. CAR104 is configured to elevate calcium in response to the extracellular antigen-binding domain 103 binding to the antigen on the target cell, and the transmembrane domain 105 is configured to bind to a transcription factor protein that is triggered by the calcium elevation and translocates into the nucleus of the genetically engineered effector cell.

[0121] As used herein, the extracellular antigen-binding domain 103 comprises and / or refers to a polynucleotide sequence that is complementary to a surface antigen of a target cell. As described above, the extracellular antigen-binding domain 103 binds to a surface antigen of a target cell.

[0122] Transmembrane domain 105 includes and / or refers to a polynucleotide encoding a transmembrane segment of a transmembrane protein, a type of membrane protein that spans the membrane of a cell, such as the membrane of the genetically engineered effector cell 100. The transmembrane domain 105 may be derived from a naturally occurring polypeptide or artificially designed. A transmembrane domain 105 derived from a naturally occurring polypeptide can be obtained from any membrane-bound or transmembrane protein. For example, the transmembrane domain of a T cell receptor α or β chain, CD3 ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or GITR may be used.

[0123] The intracellular signaling domain 107 may include and / or refer to any polynucleotide sequence encoding any oligopeptide or polypeptide known to function as a domain transmitting a signal that activates or inhibits a cellular biological process. Examples of intracellular signaling domains include the intracellular signaling portion of CD28, the intracellular signaling portion of 4-1BB, and the intracellular signaling portion of CD3 zeta. In some embodiments, the intracellular signaling domain 107 does not include the intracellular signaling portion of CD28. Surprisingly, the intracellular signaling portion of CD28 does not function well with CD4 T cells as a chassis for the engineered effector cells 100. Without being bound by theory, this was surprising because CD28 is believed to cause the cells to become dysfunctional. For example, the intracellular signaling domain 107 may include the intracellular signaling portion of 4-1BB, the intracellular signaling portion of CD3 zeta, or a combination thereof. In some embodiments, the intracellular signaling domain 107 includes the intracellular signaling portion of 4-1BB and the intracellular signaling portion of CD3 zeta. However, embodiments are not limited and may include other types and combinations of intracellular signaling domains. For example, the intracellular signaling domain 107 may encode any molecule capable of transmitting a signal to a cell when the extracellular antigen binding domain 103 present in the same molecule binds to (interacts with) an antigen.

[0124] Generally, the extracellular antigen-binding domain 103 of CAR104 has specificity for a particular antigen expressed on the surface of a target cell of interest. As described above, the extracellular antigen-binding domain 103 capable of binding to an antigen includes any oligopeptide or polypeptide capable of binding to an antigen, including, for example, the antigen-binding domain of an antibody and the ligand-binding domain of a receptor. The extracellular antigen-binding domain 103 binds to and interacts with an antigen, such as an antigen present on a cell surface, thereby conferring specificity to the engineered effector cell 100 expressing CAR104. In some embodiments, the receptor element 102 encodes CAR104 with an extracellular antigen-binding domain 103 having specificity for folate receptor alpha (FRα), an antigen found to be overexpressed in various cancers, including ovarian, cervical, lung, breast, kidney, and brain cancers. Other chimeric antigen receptors suitable for use as the antigen-binding portion of the receptor element 102 include those having specificity for a subset of immune cells, one or more tumor antigens, and / or one or more viral antigens.

[0125] The actuator element 106 encodes a transcription factor binding site 108. The transcription factor binding site 108 includes and / or refers to a site to which a protein binds that upregulates synthesis of an effector protein 112 in response to the extracellular antigen binding domain 103 of the CAR 104 binding to an antigen on a target cell. The transcription factor binding site 108 may be triggered to bind to a transcription factor by [Ca2+] released in response to antigen binding, as described above. In some embodiments, the transcription factor binding site 108 is selected from a nuclear factor of activated T-cells (NFAT) response element (NFAT-RE), a serum response element (SRE), a cyclic AMP response element (CRE), and combinations thereof. In some embodiments, multiple transcription factor binding sites may be encoded, such as 1-10, 2-10, 3-10, 5-10, 2-8, 2-6, 3-6, 5, or 6 (e.g., 6 NFAT-REs). Thereby, the actuator element 106 can include a sequence for binding a [Ca2+]-triggered factor and can trigger amplified synthesis of an effector protein 112 in response to an increase in [Ca2+].

[0126] In some embodiments, the actuator element 106 encodes a NFAT transcription factor binding site for a transcription factor protein. The NFAT transcription factor family consists of five members: NFATc1, NFATc2, NFATc3, NFATc4, and NFAT5. For reviews, see Sharma et al., PNAS, 108(28) (2011); Hogan PG et al., Ann Rev Immunol, 28 (2010); Rao A, Hogan PG, Immunol Rev, 231(1) (2009); Rao A, Nat Immunol, 10(1) (2009); M. R. Muller and A. Rao, Nature Reviews Immunology, 10, pp. 645-656 (2010); M. Oh-Hora and A. Rao, Curr. Opin. Immunol., 20, pp. 250-258 (2008); and Crabtree & Olson EN, Cell 109 Suppl (2): S67-79 (April 2002), the teachings of which are each incorporated herein in their entirety. NFATc1 to NFATc4 are regulated by calcium signaling. Calcium signaling is essential for NFAT activation because calmodulin, a known calcium-sensing protein, activates the serine / threonine phosphatase calcineurin. The molecular mechanism underlying this strategy is based on intracellular Ca2+ ([Ca2+]i) dynamics (further illustrated in Figure 2A). [Ca2+]i dynamics are common to almost all cell types, thus making this approach broadly applicable. Elevated [Ca2+]i upon stimulation of cells via CAR leads to dephosphorylation of the nuclear factor of activated effector cells 100 protein, which then translocates to the nucleus and interacts with the NFAT-RE to upregulate the expression of effector protein 112. In parallel, the NFAT-RE also performs its inherent function of inducing IL-2 in activated engineered effector cells 100, which controls clonal expansion proportional to disease burden.Expression of the NFAT-RE-induced reporter protein is also used to quantitatively assess the level of activation of the genetically engineered effector cells 100 .

[0127] Effector element 110 encodes an effector protein 112, in some cases, that is operably linked to a signal peptide 114. As further provided herein, in some embodiments, the signal peptide 114 is upstream of the effector protein 112. The signal peptide 114 may be non-native to the effector protein 112. For example, the effector protein 112 may be engineered to include a signal peptide 114 such that it is not secreted into the extracellular environment without the addition of the signal peptide 114, or that it can be secreted more efficiently than if it had the native signal peptide. However, embodiments are not limited, and in some embodiments, the effector protein 112 includes a native signal peptide. For example, the effector protein 112 may (naturally) include the signal peptide 114.

[0128] As used herein, the terms "secretor," "secretory peptide," and "signal peptide" are used interchangeably and include and / or refer to peptides that assist or direct a synthesized effector protein 112 to the extracellular environment (e.g., assist with translocation of the effector element 110). The signal peptide 114 may be operably linked to or fused to the effector protein 112 for release into the extracellular environment. In this case, the signal peptide 114 can direct the translocation of the effector protein 112 out of the engineered effector cell 100. The signal peptide 114 is particularly beneficial when included in an engineered effector cell 100 that expresses an effector protein 112 that is unable to translocate naturally and / or minimally translocates naturally, and that, in the absence of the signal peptide 114, may remain within the engineered effector cell 100 and / or may translocate at a rate below a threshold. Generally, signal peptides are located at the N-terminus of nascent secretory proteins and characteristically have three domains: (1) an N-terminal basic domain, (2) a central hydrophobic core, and (3) a carboxy-terminal cleavage region. Any suitable signal peptide may be used. For example, the signal peptide 114 may be the signal peptide of interleukin-6 (IL-6) or interleukin-2 (IL-2).

[0129] In various embodiments, in response to the extracellular antigen binding domain 103 of the CAR 104 binding to an antigen on a target cell (e.g., a target host cell), the engineered effector cell 100 is configured to activate and synthesize and secrete effector protein 112. For example, the engineered effector cell 100 can synthesize and secrete effector protein 112 as a function of the amount of target cells present in the environment (extracellular environment), such as secreting an amount of effector protein 112 into the environment that is proportional to the number of target cells present in the environment.

[0130] Effector proteins 112 may include a variety of different types of proteins. For example, effector proteins 112 may include detectable reporter proteins, therapeutic proteins, downstream signaling proteins, and combinations thereof. As used herein, detectable reporter proteins include and / or refer to proteins that are detectable upon expression, such as proteins that provide an optical, electrical, or other type of detectable signal. Therapeutic proteins include and / or refer to proteins that provide a therapeutic effect to a host, e.g., a patient. Downstream signaling proteins include and / or refer to proteins that drive downstream elements of a signaling pathway, such as to limit cell growth, proliferation, differentiation, and apoptosis.

[0131] Non-limiting examples of effector proteins include cytotoxic polypeptides derived from bacteria (e.g., parasporin, plantaricin A); cytotoxic polypeptides derived from insects (e.g., Polybia-MP1); antiviral polypeptides derived from viruses (e.g., α-helical peptides (AHP)); antiviral polypeptides derived from viruses (e.g., antiviral peptides (AVP)); immunosuppressive peptides derived from fungi (e.g., cortelin A); vasodilators (e.g., relaxin, bradykinin) and endopeptidases (e.g., heparanase, relaxin, collagenase); and cell-permeable cationic peptides (e.g., LL-37, TAT peptide). Systemic infusion of immunosuppressants cannot be used in hosts with these pathologies due to the risk of other opportunistic infections. For vasodilators and endopeptidases, such effector cells may be used to improve perfusion (see Chauhan VP & Jain RK, Nat. Mater. 12(11):958-962 (2013), incorporated herein in its entirety for its teachings) and aid in the efficient delivery of anticancer drugs that cannot be administered systemically because they damage structural tissue and are tumorigenic. For cell-penetrating cationic peptides, these targeting peptides may be used to target intracellular bacteria. For example, site-specific overexpression of such peptides represents a powerful therapy for tuberculosis.

[0132] As noted above, in some embodiments, the effector protein 112 is a therapeutic protein. In some embodiments, the therapeutic protein may act directly on the target cell. In other embodiments, the therapeutic protein may act on cells adjacent to the target cell or on non-cellular components. Examples of therapeutic proteins include cytotoxic proteins, immunostimulatory proteins, and immunosuppressive proteins.

[0133] Different portions of the genetic elements 102, 106, and 110 of the engineered effector cell 100 may be modular, while other portions may be maintained (e.g., unchanged across different implementations). For example, in some embodiments, the intracellular signaling domain 107, the actuator element 106, and the signal peptide 114 are constant domains, while the extracellular antigen binding domain 103 and the effector protein 112 are variable domains. By way of example, the extracellular antigen binding domain 103 may be altered for different targets and / or the effector protein 112 may be altered for in situ synthesis of different proteins, while the intracellular signaling domain 107, the actuator element 106, and the signal peptide 114 remain the same across different implementations. Retaining portions conserved can shorten production time. However, the embodiments are not limited, and any portion of the engineered effector cell 100 may be altered.

[0134] In some embodiments, engineered effector cell 100 may comprise multiple (e.g., two or more) of some or all of genetic elements 102, 106, 110. For example, engineered effector cell 100 may comprise multiple receptor elements 102, multiple actuator elements 106, and / or multiple effector elements 110. In some embodiments, multiplicity takes the form of providing a host with multiple engineered effector cells (e.g., multiple cells) engineered as described herein to perform multiple tasks for treating or preventing disease and / or other purposes.

[0135] In some embodiments, the actuator element 106 is bound to an effector element 110. In some embodiments, the exogenous polynucleotide sequence 101 comprises an actuator element 106 bound to an effector element 110 which is bound to a receptor element 102. For example, the exogenous polynucleotide sequence 101 comprises an actuator element 106 that is bound to and upstream of the effector element 110, and an effector element 110 that is bound to and upstream of the receptor element 102, and a signal peptide 114 is upstream of the effector protein 112.

[0136] Various embodiments are directed to a (single) construct configured to form an engineered effector cell 100 comprising an isolated CD4 T cell. The single construct comprises an exogenous polynucleotide sequence operatively associated with: (i) a receptor element 102 encoding a CAR 104 comprising an extracellular antigen-binding domain 103 operably linked to a transmembrane domain 105 and which recognizes an antigen on the surface of a target cell, and an intracellular signaling domain 107; (ii) an actuator element 106 encoding a transcription factor binding site 108 that upregulates synthesis of an effector protein 112 in response to the extracellular antigen-binding domain 103 of the CAR 104 binding to an antigen on the target cell; and (iii) an effector element 110 encoding the effector protein 112, such that the engineered effector cell 100 is configured to activate and synthesize and secrete the effector protein 112 in response to the extracellular antigen-binding domain 103 of the CAR 104 binding to an antigen on the target cell. Furthermore, the exogenous polynucleotide sequence may comprise any of the above variations in different combinations. In some embodiments, a single construct is delivered by a viral vector (e.g., a lentivector, an adenovector) or a non-viral carrier or approach (e.g., a transposon-transposase system, a Cluster Regularly Interspaced Short Palindrome Repeats (CRISPR) / Cas system, a Transcription Activator-Like Nuclease (TALEN) system, a Zinc Finger Nuclease (ZFN) system), which may be via a transfection system (e.g., electroporation, lipid nanoparticles).

[0137] In any of the above and below embodiments, the exogenous polynucleotide sequence comprises a sequence having at least 70% sequence identity to a sequence selected from SEQ ID NOs: 2-9 and / or 19-20, or selected from SEQ ID NOs: 1-20, such as including SEQ ID NO: 7 or 20. For example, the exogenous polynucleotide sequence comprises a sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to any of SEQ ID NOs: 1-20.

[0138] 2A and 2B show examples of engineered effector cells according to the present disclosure.

[0139] 2A shows an example of an engineered effector cell 200 according to the present disclosure and a sequence of events 220 that are triggered in a disease environment. The engineered effector cell 200 can be used or functions as a biological vector to synthesize an effector protein 212 or trigger a sequence of events 220 using an artificial cell signaling pathway. As further described herein, the engineered effector cell 200 synthesizes the engineered effector protein 212 in situ upon interaction with an antigen-presenting target cell.

[0140] As described above, engineered effector cell 200 comprises a receptor element 202 encoding an extracellular antigen-binding domain 203, a transmembrane domain 205, and an intracellular signaling domain 207, an actuator element 206 encoding a transcription factor binding site (e.g., NFAT), an effector element 210 encoding an effector protein 212, and an optional signal peptide. Engineered effector cell 200 comprises a single plasmid (e.g., a single construct 222 comprising each of the above) that comprises three constant domains arranged in cis (e.g., actuator element 206, signal peptide 214, and portions of receptor element 202 such as transmembrane domain 205 and intracellular signaling domain 207) and two variable domains (e.g., extracellular antigen-binding domain 203 and effector protein 212).

[0141] The constant domain may be configured to provide functionality to the genetically engineered effector cell 200. The constant domain comprises a transmembrane molecule (e.g., transmembrane domain 205) that forms part of an intracellular signaling pathway and recruits calcium-dependent transcription machinery (e.g., actuator element 206) that upregulates an effector transgene (e.g., effector protein 212) that is fused to a signal peptide 214 that assists in transport of the effector transgene to the extracellular space 223.

[0142] The variable domains are responsible for the applicability of the engineered effector cell 200 to a variety of different diseases, target cells, therapies, and / or other uses. For example, the variable domains can confer specificity to the engineered effector cell 200 for a particular disease. The variable domains can be used to encode molecules (e.g., variable heavy-light chain (VLCC) domains) with specificity for a biomarker on a target cell (e.g., the extracellular antigen-binding domain 203 of the receptor element 202) that recognize an antigen biomarker on the target cell (e.g., a labeled "target disease cell") independent of the peptide-major histocompatibility complex and effector transgene (e.g., the effector protein 212). H -VL ) or scFV, variable domains of the heavy chain (VHH), peptides, other antigens). The variable domains are modular. For example, the extracellular antigen-binding domain 203 may be swapped or modified to reprogram the engineered effector cell 200 to target disease-specific biomarkers in different cell lines. As another example, the effector protein 212 may be swapped or modified with a different therapeutic transgene, essentially to create a ready-made biological vector that neutralizes the pathology that activated the engineered effector cell 200 and is further enhanced by the effector cell's innate cytolytic activity.

[0143] In some embodiments, receptor element 202 encodes a CAR. Features of CARs include the ability to redirect T cell specificity and reactivity to a selected target in a non-MHC-restricted manner, utilizing the antigen-binding properties of monoclonal antibodies. Non-MHC-restricted antigen recognition confers the ability of CAR-expressing effector cells to recognize antigens independently of antigen processing. Referring to FIG. 2A, expression of a transmembrane CAR enables engineered effector cell 200 to sense and bind to target antigens expressed on the surface of target cells. Binding of the CAR to the target surface antigen on the target cell activates engineered effector cell 200, which triggers an activation cascade leading to the expression of effector protein 212, such as an engineered reporter, imaging, and / or therapeutic protein. For example, expression of effector protein 212 is expressed autonomously as part of the activation cascade of effector cell 200 in response to binding of a transmembrane reporter to an antigen displayed on the target cell.

[0144] More specifically, genetically engineered effector cells 200 expressing a CAR can bind to a specific antigen via the CAR, and are activated in response to a signal transmitted to the effector cells 200. Activation of CAR-expressing effector cells 200 varies depending on the type of target cell and the intracellular domain of the CAR, and can be confirmed by indicators such as cytokine release, increased cell proliferation rate, and changes in cell surface molecules. For example, cytotoxic cytokines (e.g., tumor necrosis factor, lymphotoxin, etc.) released from activated effector cells 200 destroy antigen-expressing target cells. Furthermore, cytokine release or changes in cell surface molecules stimulate other immune cells, such as B cells, dendritic cells, natural killer cells, and macrophages.

[0145] As shown in FIG. 2A, an example sequence of events 220 triggered by or associated with engineered effector cell 200 includes: (1) active migration of effector cell 200 to diseased cells, (2) engagement of a CAR on the surface of effector cell 200 with an antigen of the target cell, (3) activation of effector cell 200, (4) upregulation of effector protein 212 by signal peptide 214 via NFAT, and (5) cleavage of signal peptide 214 and transport of effector protein 212 to the extracellular space 223.

[0146] 2B shows effector cells formed from CD4 T cells 221-A and CD4 T cells 221-B in an environment containing target cells 225 bearing antigens 227-A and 227-B on their surfaces. As shown, effector protein 212 is produced in response to binding of the antigen-binding domains of CAR204-A and CAR204-B to effector cells 221-A and 221-B. As further shown, CD4 T cell line effector cells 221-A are transduced at least three times, expand twice as fast, and express five times more effector protein 212 than CD8 T cell line effector cells 221-B. Without being bound by theory, this is believed to be due to the expression of the NFAT transcription activation domain of antigen-stimulated killer CD8 T cell line effector cells 221-B. 53 SSPS 56 This is likely due to impaired phosphorylation of the motif, limiting the ability of the NFAT transcription machinery to signal through the NFAT-RE.

[0147] 3 shows an example of a population of genetically engineered effector cells in a disease setting according to the present disclosure. Population 341 may include a plurality of genetically engineered effector cells 300-1, 300-2, 300-3, 300-4, 300-5, 300-6, 300-N (referred to generally herein as "genetically engineered effector cells" for ease of reference). Each genetically engineered effector cell 300 may include at least substantially the same features and elements as genetically engineered effector cell 100 of FIG. 1, and the details will not be repeated here.

[0148] In the example shown in FIG. 3 , the environment is an extracellular space 340 containing (the presence of) target cell(s) 342, and the space 340 may be referred to as a disease environment. A population 341 of engineered effector cells 300 can bind to an antigen on the target cell(s) 342 via the antigen-binding domain of the CAR. In response to binding, the engineered effector cells 300 may become activated and, in response, synthesize and secrete an effector protein. For example, the engineered effector cells 300 may synthesize and secrete a calibrated amount of effector protein based on the presence of the target cell(s) 342. The calibrated amount of effector protein may be a function of the amount of target cell(s) 342 present in the extracellular space 340 or in a plurality of (host) cells, such as in a sample. As described above, the calibrated amount of effector protein may be proportional to the amount of target cells 342. Although the extracellular space 340 shows the engineered effector cells 300 and the target cells 342, the extracellular space 340 and the plurality of (host) cells may further include normal and / or diseased cells, among other non-cellular elements. In some embodiments, each engineered effector cell 300 may synthesize and secrete the same effector protein, while in other embodiments, they may synthesize and secrete different proteins and / or combinations.

[0149] 4 and 5 illustrate exemplary methods for generating genetically engineered effector cells from T cells according to the present disclosure. Methods 450, 560 may be implemented to generate a population 341 of genetically engineered effector cells 100 shown in FIG. 1 or genetically engineered effector cells 300 shown in FIG. 3. As illustrated in method 560 shown in FIG. 5, embodiments are not limited to CD4 T cells. Methods 450, 560 may be used to generate genetically engineered effector cells from primary T cells using selected processing parameters that optimize delivery performance (e.g., effector protein synthesis and secretion). As further described below, such processing parameters may include or relate to the type of T cells used, T cell activation, transformation parameters, and expansion parameters, including, but not limited to, transformation and expansion techniques, amounts, and / or times, among other parameters.

[0150] Referring to FIG. 4 , at 452, method 450 includes activating a plurality of CD4 T cells using a plurality of particles. In some embodiments, activating a plurality of CD4 T cells includes exposing the plurality of CD4 T cells to a plurality of particles carrying anti-human CD3 antibodies and anti-human CD28 antibodies. As used herein, particle includes and / or refers to a localized physical object that has or exhibits particular particle characteristics, such as size, shape, and / or dielectric properties. For example, particle may include beads or nanoparticles, such as Dynabeads®. Activating T cells includes and / or refers to T cells expressing chimeric antigen receptors on their surface, thereby stimulating the T cells to increase in number. Activation can occur in response to antigen exposure. In some embodiments, the T cells can be frozen and thawed prior to activation. In other embodiments, the T cells can be fresh. In some embodiments, the T cells can include a plurality of CD4 T cells isolated from pan-CD3 T cells (e.g., depleted of CD8 T cells). In another embodiment, the T cells may comprise a plurality of CD3 T cells, and the CD4 T cells are isolated by selective expansion, which effectively removes CD8 T cells. In some embodiments, the plurality of CD4 T cells may be resuspended in complete growth medium and activated by adding a plurality of particles to the complete growth medium.

[0151] As noted above, in various embodiments, frozen T cells may be used, while in other embodiments, fresh T cells may be used. Frozen cells are easier to use because good donors can be pre-selected. In terms of starting cell number, fresh cells are preferable to frozen cells because frozen cells may result in cell loss, such as at least a 20% loss. However, in terms of transformation efficiency, frozen and fresh cells are similar. Therefore, if the starting cell number is unlimited, frozen cells may be used to reduce logistical complexity.

[0152] Activation involves exposing a plurality of CD4 T cells to a plurality of particles at a particular cell-body to particle ratio for a period of time. In some embodiments, the cell-body to particle ratio may include about 6:1 to about 1:6. In some embodiments, the cell-body to particle ratio may include about 1:2 to about 1:6, about 1:1 to about 1:6, about 1:1 to about 1:5, about 1:2 to about 1:5, about 1:3 to about 1:4, and about 1:4, or about 1:3, among other ratios. In various embodiments, the cell-body to particle ratio may affect, for example, the progression of T cells to peak activation. The cell-body to particle ratio may define the intensity of stimulation. For example, by increasing the number of particles coated with activation molecules, T cells can be stimulated at a higher intensity. As further described in the experimental embodiments, data showed that T cells were activated more quickly using a high cell-body to particle ratio of 1:3, while a similar activation level was obtained with a cell-body to particle ratio of 1:1 when stimulated for a longer period of time. Therefore, the cell body particle ratio may be adjusted to activate T cells more quickly. As an additional factor, excessive activation of T cells can cause activation-induced cell death. Therefore, selecting the correct cell body particle ratio is useful for optimizing activation while minimizing cell death.

[0153] In some embodiments, the time period for activating CD4 T cells may include, among other times, about 10 hours to about 36 hours, about 15 hours to about 36 hours, about 20 hours to about 36 hours, about 25 hours to about 36 hours, about 30 hours to about 36 hours, about 10 hours to about 30 hours, about 10 hours to about 24 hours, about 10 hours to about 20 hours, about 15 hours to about 30 hours, about 20 hours to about 24 hours, about 36 hours, about 30 hours, about 24 hours, about 20 hours, or about 15 hours.

[0154] At 454, method 450 includes exposing the plurality of CD4 T cells to an exogenous polynucleotide sequence to engineer (e.g., transform and / or introduce the exogenous polynucleotide sequence) the plurality of CD4 T cells. In various embodiments, the plurality of CD4 T cells may be engineered before or after activation of the CD4 T cells. The exogenous polynucleotide sequence may include at least some substantially the same features and elements as the engineered effector cell 100 of FIG. 1 above, and the details will not be repeated here.

[0155] In some embodiments, exposing the plurality of CD4 T cells to an exogenous polynucleotide sequence comprises using a vector carrying the exogenous polynucleotide sequence, which may involve or include viral or non-viral carriers or approaches, such as transposon-transposase systems, CRISPR / Cas systems, TALEN systems, ZFN systems, etc., which may be via a transfection system, as described above.

[0156] In some embodiments, exposing the plurality of CD4 T cells to the exogenous polynucleotide sequence comprises using a virus (e.g., a viral vector), such as a lentivirus or an adenovirus. In some embodiments, the CD4 T cells are exposed to a lentivirus having the exogenous polynucleotide sequence. In some embodiments, the lentivirus may comprise a lentiviral particle having the exogenous polynucleotide sequence. For example, the plurality of activated CD4 T cells may be exposed to the lentivirus in serum-free and polybrene-containing culture medium.

[0157] Embodiments are not limited to the use of lentiviruses. Other types of viruses or vectors may be used, such as adenoviruses. Additionally, exogenous polynucleotide sequences may be introduced into CD4 T cells by techniques other than transduction.

[0158] The culture medium used to expose CD4 T cells to exogenous polynucleotide sequences may be referred to as a "transformation culture medium." In some embodiments, the (transformation) culture medium may be serum-free. Serum contains numerous excess proteins that can nonspecifically bind to surfaces and mask protein epitopes important for viral binding and subsequent entry into target cells, thereby inhibiting the adhesion of vectors (e.g., lentivectors) to cells. Therefore, in some embodiments, it may be beneficial to avoid serum to enhance cell-to-virus interactions and improve viral transduction efficiency. In another embodiment, the (transformation) culture medium may contain serum (e.g., 10% serum) and include a complete growth medium that can be used to transform T cells.

[0159] In some embodiments, the serum-free culture medium may contain about 4 micrograms (μg) / milliliter (mL) to about 8 μg / mL of polybrene, although embodiments are not limited thereto and may contain about 5 μg / mL to about 12 μg / mL, about 5 μg / mL to about 8 μg / mL, about 6 μg / mL to about 8 μg / mL, or about 8 μg / mL of polybrene, among other ranges. Additionally, in some embodiments, the culture medium may be a complete growth medium, as described above.

[0160] In some embodiments, the transformation step is carried out at a specific concentration (e.g., 1×10 6The transduction step may include cells at a concentration of approximately 0.05×10 cells / mL (cell density), and / or the transduction step may include limiting the total transduction reaction volume (e.g., T cells + lentivirus or another vector carrying the exogenous polynucleotide sequence + culture medium) to a defined subvolume for a period of time. The total transduction reaction volume includes and / or refers to the total volume of fluid containing the T cells and the exogenous polynucleotide sequence, including the culture medium (transformation culture medium and other fluids), such as the total volume of fluid used to manipulate the T cells. Limiting the cell concentration (e.g., density of activated T cells) and total transduction reaction volume to a subvolume can affect transduction (or other type of transduction) yield by optimizing exposure to the exogenous polynucleotide sequence, such as by enhancing interaction of the virus or other vector with the CD4 T cells. For example, exposing multiple CD4 T cells to a virus or other type of vector may result in a transduction yield of approximately 0.05×10 cells / mL. 6 cells / mL ~ approx. 3×10 6 The method includes providing a total transduction reaction volume comprising a plurality of CD4 T cells at a cell density of about 1×10 cells / mL, culture medium, and a lentivirus (or other type of vector having the sequence) in defined sub-amounts for a period of time. In some embodiments, the total transduction reaction volume is about 1×10 6 It may comprise a plurality of activated T cells at a cell density of 100 cells / mL, culture medium, and lentivirus at a multiplicity of infection (MOI) of about 0.1 to about 10. In another embodiment, the MOI may be about 1 to about 10, about 5 to about 10, about 1 to about 8, about 5 to about 8, about 8 to about 12, or about 10, among other MOIs.

[0161] In some embodiments, the cell density of the T cells used during the transduction process, whether using lentivirus or other viruses or vectors, is about 0.05 x 10, among other ranges. 6 cells / mL ~ approx. 3×10 6 cells / mL, approximately 0.25×10 6 cells / mL ~ approx. 3×10 6 cells / mL, approximately 0.25×10 6 cells / mL ~ approx. 2×10 6 cells / mL, approximately 0.5×106 cells / mL ~ approx. 2×10 6 cells / mL, approximately 0.5×10 6 cells / mL ~ approx. 1×10 6 cells / mL, approximately 0.05×10 6 cells / mL ~ approx. 2×10 6 cells / mL, approximately 0.05×10 6 cells / mL ~ approx. 1×10 6 cells / mL, or approximately 1 x 10 6 The serum may contain a plurality of CD4 T cells (which may or may not be activated) in a concentration of 1000 cells / mL.

[0162] Cell concentration can affect transduction or other transformation yields. Without being bound by theory, this may be due to the random movement of fluid in a given volume, enhancing particle-particle interactions (e.g., collision theory). It may also be primarily due to increased virus-cell contact during transduction. Increasing cell concentration while keeping the MOI constant for a given volume increases transduction yields by enhancing virus-cell interactions for steric reasons (increased virus and cell numbers).

[0163] In some embodiments, the defined sub-volume may comprise a spherical or other shaped droplet volume that maintains contact with a vector, such as a viral vector. In some embodiments, the total transformation reaction volume may comprise about 0.5 mL to about 2 mL, and the defined sub-volume may comprise about 0.05 mL to about 0.25 mL. In some embodiments, the total transformation reaction volume may comprise about 1 mL, and the defined sub-volume may comprise about 0.1 mL. In some embodiments, the period of time may comprise about 10 hours to about 24 hours, about 10 hours to about 20 hours, about 15 hours to about 24 hours, about 15 hours to about 16 hours, or about 16 hours. Embodiments may include other variations, values, and ranges.

[0164] At 456, method 450 comprises expanding the activated plurality of CD4 T cells in an expansion culture medium to form a plurality of engineered effector cells comprising a plurality of CD4 T cells with the exogenous polynucleotide sequence, the plurality of engineered effector cells configured to activate and synthesize and secrete an effector protein in response to the extracellular antigen binding domain of the CAR binding to an antigen on a target cell. As used herein, cell expansion includes and / or refers to cell proliferation.

[0165] In some embodiments, the expansion culture medium may comprise a complete growth medium. The complete growth medium may provide necessary nutrients in optimal ratios to allow optimal cell growth. In some embodiments, the complete growth medium may contain cytokines. In some embodiments, the cytokines may include interleukin (IL)-2, IL-7, IL-15, or a combination thereof. For example, the expansion culture medium may comprise a complete growth medium containing IL-7 and IL-5.

[0166] In some embodiments, expanding the activated CD4 T cells may include diluting the total transduction reaction volume with a cytokine-containing expansion medium to a specific cell density of the activated and transduced CD4 T cells for a period of time. For example, the cell density may be about 0.25 x 10, which is maintained for about 14 days. 6 cells / mL ~ approx. 1×10 6 The activated CD4 T cells may comprise a plurality of activated CD4 T cells per mL. The embodiment is not limited, and the period of time may be from about 10 days to about 20 days. The cell density may be about 0.5 x 10 cells / mL, among other ranges. 6 cells / mL ~ approx. 1×10 6 cells / mL, approximately 0.25×10 6 cells / mL ~ approx. 0.5×10 6 cells / mL, or approximately 0.5 x 10 6The cell density may be measured in cells / mL. Further, expansion may include periodically replacing at least a portion of the expansion culture medium over a period of time while maintaining the cell density. For example, the expansion culture medium may be replaced every day, every two days, or every three days, among other periods of time.

[0167] The embodiments are not limited to the above and may include various modifications. For example, method 450 may include adding additives to the (transformation) culture medium and / or the expansion culture medium. The additives may include antiviral inhibitors and / or latency reversing agents.

[0168] In another embodiment, the T cells are not limited to CD4 T cells, but may include other types of primary T cells, such as CD3 T cells or isolated CD8 T cells. Figure 5 shows an example of such a method 560.

[0169] At 562, method 560 includes activating a plurality of T cells with a plurality of particles. The plurality of T cells may include CD3 T cells, isolated CD4 T cells, or isolated CD8 T cells. The plurality of T cells may be thawed, e.g., frozen and thawed prior to activation, or may be fresh. In any embodiment, method 560 may include resuspending the plurality of T cells in complete growth medium and activating the plurality of T cells by adding a plurality of particles to the complete growth medium. As described above, the plurality of T cells may be activated by exposing the plurality of T cells to a plurality of particles carrying anti-human CD3 antibodies and anti-human CD28 antibodies. For example, the plurality of T cells may be exposed to the plurality of particles at a cell body to particle ratio of about 6:1 to about 1:6 for a period of time, such as about 10 hours to about 36 hours. Embodiments are not limited and may include the above ranges and variations for culture medium, cell body to particle ratio, and / or period of time, among other variations.

[0170] At 564, method 560 includes exposing the plurality of T cells to an exogenous polynucleotide sequence to engineer (e.g., transform and / or introduce the exogenous polynucleotide sequence into) the plurality of T cells. Engineering may occur before or after activation of the T cells. The exogenous polynucleotide sequence may include at least some substantially the same features and elements as engineered effector cell 100 of FIG. 1 above, and for ease of reference, the details will not be repeated here. As noted above, for CD4 T cells, the transcription factor binding site may be NFAT-RE, and the intracellular signaling domain may not include the intracellular portion of CD28 (e.g., may include the intracellular portion of 4-1BB and the intracellular signaling portion of CD3 zeta). For CD8 T cells, the transcription factor binding site may be an SRE and / or a CRE, and the intracellular signaling domain may comprise the intracellular portion of CD28 (e.g., the intracellular portion of CD28 and the intracellular signaling portion of CD3 zeta, or the intracellular portion of CD28, the intracellular portion of m4-1BB, and the intracellular signaling portion of CD3 zeta).

[0171] As described above, the vector may have the exogenous polynucleotide sequence, and the vector may comprise or be associated with a viral vector, a transposon system, or a lipid nanoparticle. In some embodiments, the transformation step may include exposing a plurality of T cells to a virus having the exogenous polynucleotide sequence, such as a viral vector. For example, in some embodiments, method 560 may include exposing a plurality of T cells to a virus, including a lentivirus, such as a lentiviral particle, having the exogenous polynucleotide sequence. In some embodiments, exposing a plurality of T cells to a virus may include exposing a plurality of T cells to a virus, including about 0.05×10 T cells, which may or may not be activated. 6 cells / mL ~ approx. 3×10 6 The method includes exposing a plurality of T cells, at 1000 cells / mL, to a virus in serum-free culture medium containing polybrene. In some embodiments, the culture medium contains about 4 micrograms (μg) / mL to about 8 μg / mL of polybrene, although embodiments are not limited thereto.

[0172] As noted above, transformation (e.g., transduction or other techniques) may be performed using a particular cell concentration and / or defined sub-amount. For example, exposing a plurality of T cells to an exogenous polynucleotide sequence can be performed in a range of about 0.05 x 10, among other ranges. 6 cells / mL ~ approx. 3×10 6 Methods 560 may include providing a total transduction reaction volume including a plurality of T cells at a cell density of cells / mL, culture medium, and a viral or other type of vector in defined sub-amounts for a period of time at an MOI of about 0.1 to about 10. For example, method 560 may include resuspending lentiviral particles in culture medium sufficient to achieve an MOI of about 0.1 to about 10. Embodiments are not limited and may include any of the above ranges for cell concentration, vector, total transduction volume, sub-amount, polybrene concentration, type of culture medium, and / or MOI, among other variations and combinations described herein.

[0173] In some embodiments, providing the defined sub-volumes includes disposing the aliquots as droplets into tissue-cultured well plates and placing the cultured well plates in an incubator for a period of time. In some embodiments, the sub-volumes may additionally or alternatively be disposed on the surface of a hydrophobic or hydrophilic substrate or substrate, and may comprise different shapes, such as spheres.

[0174] At 566, the method 560 includes expanding the activated plurality of T cells in an expansion culture medium to form a plurality of engineered effector cells comprising T cells having the exogenous polynucleotide sequence, the plurality of engineered effector cells configured to activate and synthesize and secrete an effector protein in response to an extracellular antigen-binding domain of the CAR binding to an antigen on a target cell. In some embodiments, expanding the activated and transduced plurality of T cells comprises expanding about 0.25 x 10 T cells for a period of time (e.g., 14 days). 6 cells / mL ~ approx. 1×10 6and diluting the total transduction reaction volume with a plurality of T cells at a cell density of about 0.25 x 10 cells / mL, where the expansion medium is complete growth medium containing cytokines. For example, the cytokines may include IL-2, IL-7, IL-15, or a combination thereof, such as IL-7 and IL-15. As above, the period of time may include about 10 days to about 20 days, and method 560 may include diluting the total transduction reaction volume with a plurality of T cells at a cell density of about 0.25 x 10 cells / mL. 6 cells / mL ~ approx. 1×10 6 and periodically replacing at least a portion of the expansion medium for a period of time while maintaining a cell density of cells / mL. Embodiments are not limited and may include any of the above ranges for cell concentration, expansion medium, and cytokines.

[0175] In some embodiments, method 560 may include adding additives to at least one of the (transformation) culture medium and the expansion culture medium. As described above, additives may include antiviral inhibitors, latency reversing agents, and combinations thereof.

[0176] Figure 6 shows an example of a kit for generating genetically engineered effector cells from T cells according to the present disclosure. Kit 670 includes a plurality of T cells 672, an exogenous polynucleotide sequence 674, a culture medium 676, and an expansion medium 678. The various elements 672, 673, 674, 676, 678 may include those described in any of Figures 1-5 herein. For ease of reference, not all variations will be repeated.

[0177] In some embodiments, for example, the plurality of T cells 672 may comprise CD3 T cells, isolated CD4 T cells, or isolated CD8 T cells. In some embodiments, the plurality of T cells 672 may comprise isolated CD4 T cells.

[0178] In some embodiments, kit 670 further comprises a plurality of particles carrying anti-human CD3 antibodies and anti-human CD28 antibodies. In some embodiments, kit 670 further comprises a separate culture medium configured to resuspend the plurality of T cells 672 with the plurality of particles to activate the plurality of T cells 672. The separate culture medium may comprise complete growth medium. For example, the separate culture medium and the plurality of particles may be configured to resuspend the plurality of T cells at a cell body to particle ratio of about 6:1 to about 1:6 for a period of about 10 hours to about 36 hours (e.g., 24 hours).

[0179] In some embodiments, kit 670 may further comprise a vector (or other carrier) 673 carrying an exogenous polynucleotide sequence 674. As noted above, vector 673 may comprise a viral vector, a transposon system, or a lipid nanoparticle.

[0180] In some embodiments, vector 673 may comprise a viral vector, such as a lentivirus or adenovirus. For example, as shown in FIG. 1 above, vector 673 may comprise a lentivirus having an exogenous polynucleotide sequence. In some embodiments, culture medium 676 may be serum-free and contain polybrene and configured to engineer a plurality of T cells 672. In some embodiments, culture medium 676 contains about 4 μg / mL to about 8 μg / mL of polybrene. In some embodiments, vector 673 comprises lentiviral particles having exogenous polynucleotide sequence 674, and culture medium 676 is configured to resuspend the lentiviral particles in culture medium 676 sufficient to achieve an MOI of about 0.1 to about 10.

[0181] In some embodiments, the kit 670 contains approximately 0.05×10 6 cells / mL ~ approx. 3×10 6The kit further includes a tissue-cultured well plate configured to receive a total transformation reaction volume containing a plurality of T cells at a cell density of 10 ...

[0182] In some embodiments, expansion culture medium 678 is a complete growth medium containing cytokines. The cytokines may include IL-2, IL-7, IL-15, and combinations thereof. In some embodiments, expansion culture medium 678 is added to the total transfection reaction volume and grows approximately 0.25×10 cells / ml for a period of time (e.g., 14 days). 6 cells / mL ~ approx. 1×10 6 Dilute the total transfection reaction volume at multiples of T cell 673 at a cell density of 1000 cells / mL.

[0183] In some embodiments, at least one of the culture medium 676 and the expansion medium 678 comprises an additive selected from the group consisting of an antiviral inhibitor, a latency reversing agent, and combinations thereof.

[0184] Kit 670 of FIG. 6 may include any of the above values ​​and ranges for cell concentration, time period, culture medium used, cytokines, polybrene concentration, vector, MOI, total transfection amount, subamounts, etc., among other variations and combinations described herein.

[0185] The embodiments are not limited to those shown in the drawings and may include various variations in methods of use, different compositions, different systems and kits, etc. For example, different processing parameters used in variations of methods 450, 560 may include adjustments in construct size, transformation method, polybrene concentration in the medium, time period, among other variations.

[0186] Various embodiments are directed to medical compositions comprising genetically engineered effector cells, such as the population 341 of genetically engineered effector cells 100 of FIG. 1 and / or the population 341 of genetically engineered effector cells 300 of FIG. 3, and a pharmaceutically acceptable carrier or excipient.

[0187] For example, a composition of engineered effector cells, such as a medical composition, may comprise a plurality of the engineered effector cells described herein and a pharmaceutically acceptable carrier, diluent, or excipient (e.g., a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof). Means for making such compositions are known in the art (see, e.g., Remington's Pharmaceutical Sciences, 16th Ed., Mack, ed. (1980)). Preferably, the composition is formulated to facilitate administration of the effector cells into a living body. In some embodiments, the medical composition comprises the engineered effector cells described herein and a balanced salt solution, preferably Hank's balanced salt solution or normal saline.

[0188] Some embodiments are directed to methods of forming engineered effector cells, such as engineering or modifying effector cells to include elements and characteristics illustrated by engineered effector cell 100 of FIG. 1.

[0189] The genetically engineered effector cells and cell compositions provided herein have beneficial properties for use in a variety of in vitro, ex vivo, and in vivo applications, including, but not limited to, use as in vivo vectors for protein delivery in humans. For example, in vitro uses of the effector cells and cell compositions provided herein include, but are not limited to, detecting target cells based on antigens expressed on their surface. Target cells can be cancer cells (e.g., tumor cells), cells infected with pathogens such as viruses and bacteria, cell types associated with autoimmune diseases (e.g., type 1 diabetes, lupus), and cell types associated with neurodegenerative diseases such as Alzheimer's disease, ALS, and Huntington's disease. Target (host) cells can also be cell types associated with other pathologies in which diseased (host) cells have aberrant expression of cell surface antigens compared to unaffected (host) cells. Below, we describe methods for using genetically engineered effector cells or cell compositions for in vitro target cell detection.

[0190] Ex vivo uses of the genetically engineered effector cells and cell compositions provided herein include, but are not limited to, early disease detection and companion diagnostic or therapeutic applications for disease target cells identified based on antigens expressed on their surface. For example, effector cells may be used ex vivo for companion diagnostics of cancer immunotherapy. As an example, NFAT_RE6X→Nluc-2A-GFP engineered effector cells may be engineered to express different types of CARs. Comparing the expression of Nluc when the CAR engages the target antigen with the expression of nonspecific Nluc can provide a quantitative indication of the relative robustness of each CAR in terms of its efficiency in causing intended on-target effects and unintended off-target effects. Methods for using genetically engineered effector cells or cell compositions in ex vivo therapeutic applications are further described below. Other ex vivo applications of engineered effector cells and cell compositions include, but are not limited to, companion diagnostic applications to cell therapy for treating infectious diseases, autoimmune diseases, neurodegenerative diseases, and other cell-based pathologies associated with aberrant expression of cell surface antigens relative to unaffected (host) cells.

[0191] In vivo applications of the genetically engineered effector cells and cell compositions provided herein include, but are not limited to, in vivo imaging of disease sites, in vivo methods for localized treatment of disease sites (e.g., targeted treatment of ovarian cancer) or pathogen infection sites (e.g., targeted treatment of cells infected with Dengue virus, Zika virus, West Nile virus, yellow fever, HIV, or hepatitis viruses (e.g., HepB, HepC)).

[0192] Various embodiments are directed to panels of different types of engineered effector cells that are used to simultaneously target different cells and / or secrete different effector proteins, such as (among other things) multiple engineered effector cells with different effector proteins and / or extracellular antigen binding domains.

[0193] In some embodiments, a method for detecting target cells comprises (a) contacting a cellular population with a genetically engineered effector cell and (b) detecting expression of an effector protein, wherein detectable expression of the effector protein indicates the presence of the target cell of interest. In some embodiments, the effector cell comprises an NFAT response element and a reporter protein, and in response to the presence of the target cell in the contacted cell population, the genetically engineered effector cell binds to a surface molecule antigen of the target cell and activates the NFAT response element, and (b) detecting expression of the reporter protein, wherein detectable expression of the reporter protein indicates the presence of the target cell.

[0194] In some embodiments, the detected target cells are cancer cells, and the antigen domain of the CAR binds to a cancer cell-specific surface antigen on the target cells. In another embodiment, the detected target cells are virus-infected host cells, such as Zika virus-infected cells. In some such embodiments, the surface molecule antigen expressed on virus-infected cells is a Zika virus-specific envelope glycoprotein (Egp). For example, the antigen recognition portion of the CAR is modified or exchanged to quantitatively evaluate different viral pathogens, such as dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV). In some embodiments, the method utilizes the translation machinery of the infected host cell to process viral ribonucleic acid (RNA) into a virus-specific antigen that can be detected by the engineered effector cells described herein.

[0195] In some embodiments, the present disclosure provides methods for treating or preventing diseases using engineered effector cells expressing a CAR as a therapeutic agent. For example, the present disclosure provides methods comprising administering engineered effector cells expressing a CAR as an active therapeutic agent. The disease to which engineered effector cells expressing a CAR are administered is not particularly limited, as long as the disease is susceptible to the effector cells. Examples of diseases include cancer (e.g., blood cancer (leukemia), solid cancer), inflammatory diseases / autoimmune diseases (e.g., asthma, eczema), hepatitis, infectious diseases caused by viruses, bacteria, or fungi such as Zika virus, influenza, and HIV, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococcus (VRE), and deep mycoses. In some embodiments, engineered effector cells expressing a CAR bind to antigens expressed on the surface of target cells that are targets for reduction or elimination to treat the above-mentioned diseases, i.e., cancer antigens, viral antigens, bacterial antigens, etc., and are administered to treat or prevent such diseases. As used herein, the terms "treatment" and "prevention," and words derived therefrom, do not necessarily mean 100% or complete treatment or prevention. Rather, there are various degrees of treatment or prevention that one of skill in the art will recognize as having potential benefit or therapeutic effect. In this regard, the methods described herein can provide treatment or prevention of cancer in a mammal at any level and in any amount. Furthermore, the treatment or prevention provided by the exemplary methods can include treatment or prevention of one or more conditions or symptoms of the disease, e.g., cancer, being treated or prevented. Also, as used herein, "prevention" can encompass delaying the onset of the disease, or its symptoms or conditions.

[0196] In some embodiments, the engineered effector cells are administered to a host (e.g., a subject) in need thereof as a composition comprising the engineered effector cells described herein and a suitable carrier, diluent, or excipient. Any suitable method of providing engineered CAR-expressing cells to a host may be used in the methods described herein. In some embodiments, the method of providing effector cells to a host can be adapted from cellular immunotherapy and adoptive immunotherapy clinical protocols for injecting donor-derived immune cells into a human host. In some embodiments, an adapted clinical protocol suitable for the methods provided herein comprises obtaining effector cells from a host, genetically engineering (e.g., modifying) the effector cells to express a CAR and NFAT-RE regulatory protein transgene described herein, and re-infusing the engineered effector cells into the host. As used herein, host includes and / or refers to any organism, such as a human, an animal (e.g., a mammal, reptile, bird), insect, plant, etc., which can be the subject and / or patient of research or testing.

[0197] Administration of the genetically engineered effector cells provided herein may be by any suitable route, including, but not limited to, intravenous administration, intratumoral administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, intraarterial administration, or administration to afferent lymphatic vessels by parenteral administration, e.g., injection or infusion. In some embodiments in which genetically engineered effector cells or populations of such effector cells are administered, the effector cells may be allogeneic or autologous to a host, such as a mammal. Preferably, the effector cells are autologous to the host.

[0198] In some embodiments, hosts provided with the engineered effector cells are monitored or evaluated for increased (e.g., improved, more robust) tumor clearance. Accordingly, various embodiments are directed to methods for use in cancer treatment. In some embodiments, hosts provided with the engineered effector cells are monitored or evaluated for clearance of cells expressing a particular antigen.

[0199] Some embodiments are directed to methods for the treatment or prophylaxis of a cell line against a pathogen of interest. For such methods, genetically engineered effector cells comprise a polynucleotide sequence encoding a therapeutic protein, instead of or in addition to a polynucleotide sequence encoding a detectable reporter protein, with a signal peptide (sec) fused to the 3' end of the polynucleotide sequence to aid extracellular transport. Expression of the therapeutic protein is induced upon triggering a cascade of effector cell activation events and activation of an NFAT response element. The method may include localized production of the therapeutic protein at the site of the target cell (e.g., tumor cell, infected cell) and extracellular secretion of the therapeutic protein in the disease microenvironment.

[0200] Some embodiments are directed to methods for using genetically engineered effector cells as sensor technology in various applications. For example, the spread of emerging flavivirus pathogens, such as Zika virus (ZIKV) and dengue virus (DENV), via blood transfusion has been identified as a serious risk. To protect donated blood, donor screening, including blood testing, is recommended. Clinical symptoms appear in only 20% of ZIKV infections, and there are no reliable, commercially available ZIKV diagnostic test kits available outside of clinical laboratories. Therefore, identifying infection is challenging, especially given the similarity of symptoms to other diseases and antibody cross-reactivity with other arboviruses (e.g., dengue fever, chikungunya fever). Accordingly, provided herein are methods comprising contacting a sample containing or suspected to contain cells infected with a virus of interest with a genetically engineered effector cell comprising a CAR having an antigen-binding domain for detecting and binding to an antigen specific to the virus of interest and an NFAT-RE-regulated reporter transgene for signaling the presence of cells infected with the virus of interest.

[0201] Additional uses of the genetically engineered effector cells described herein include:

[0202] To target anticancer chemotherapy prodrugs to tumor sites, genetically engineered effector cells can be loaded with enzymatically activatable prodrugs, where the drug-activating enzyme is synthesized only at the tumor site, thereby locally converting the prodrug to its active form. In some embodiments, the prodrug is not loaded onto the effector cells but can be injected in multiple doses after injection of the genetically engineered effector cells. Alternatively, the prodrug can be attached to imaging nanoparticles or other means of image-guided active drug delivery. By attaching the prodrug to imaging nanoparticles or engineering the effector cells to express an imaging transgene, the genetically engineered effector cells can guide appropriate staging of the patient in preparation for surgery, visually identify and / or image tumor margins, and assist in cytoreductive surgery.

[0203] Some embodiments are directed to a method for locally delivering a chemotherapeutic agent to a site of disease (e.g., a tumor mass, a site of an autoimmune disease) comprising contacting a host cell population with engineered effector cells, the engineered effector cells comprising (i) an exogenous polynucleotide sequence encoding a CAR comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain, and (ii) an NFAT response element operably linked to a polynucleotide sequence encoding an enzyme, wherein, in the presence of a target host cell in the contacted cell population, the engineered effector cells bind to a surface molecule antigen on the target host cell and activate the NFAT response element, initiating expression of the enzyme, which acts on a prodrug pre-designed to be activated by the enzyme and is released at the disease site by utilizing its membrane permeability due to its hydrophobicity.

[0204] For surgical intervention to treat cancer, engineered effector cells may be used to visualize and / or image tumor margins via expression of a detectable reporter protein, such as a fluorescent protein (e.g., GFP, RFP, YFP, and variants thereof) or a bioluminescent enzyme (e.g., luciferase). For example, such engineered effector cells may be used to mark tumor margins to aid in surgical resection and identify remaining positive tumor margins.

[0205] In some embodiments, engineered effector cells are used for non-invasive detection and imaging of tumors based on the expression of imaging enzymes (e.g., thymidine kinase can capture radioactive or otherwise detectable probes; tyrosinase detected by photoacoustic imaging or magnetic resonance imaging) that are expressed when tumor-specific CAR effector cells engage antigens on tumor cells.

[0206] In some embodiments, genetically engineered effector cells may be used to circumvent safety concerns associated with vaccines against flaviviruses. For example, antigenic diversity among the four different dengue virus serotypes contributes to the lack of antibody-mediated immunity, potentially resulting in multiple, consecutive infections. Antibodies are effective in primary infections but fail to neutralize in secondary infections, exacerbating hemorrhagic fever by activating the complement system against large infected cell masses during the acute phase. Additionally, a prior dengue infection has been shown to exacerbate Zika infection. As described herein, the use of effector cells can circumvent these safety concerns related to flaviviruses because they can be engineered to express antiviral proteins of human, non-human, or synthetic origin upon detecting the viral E glycoprotein (EGP) expressed on the surface of virus-infected cells.

[0207] In some embodiments, the engineered effector cell comprises a CAR that detects a cancer-specific antigen (e.g., HPV E6 or E7 antigen in the case of cervical cancer) on a target cancer cell and an NFAT-RE that drives expression of the reporter protein. Such embodiments may be used for early detection of cancer.

[0208] In some embodiments, the engineered effector cell comprises a CAR that detects pathogen-infected cells (e.g., detects ZIKV or DENV glycoproteins on Zika or Dengue virus-infected cells) and an NFAT response element that induces expression of a reporter polypeptide. Such embodiments may be used in transfusion medicine to detect the presence of emerging pathogens (e.g., Zika, Dengue, West Nile, Yellow Fever).

[0209] By using different CARs in engineered effector cells with an NFAT-RE-regulated reporter, it is possible to detect and measure signal-to-noise ratios that guide the selection of appropriate CARs for cell line therapy that exert the intended side effects without exhibiting unintended side effects.

[0210] Mammalian cells may be engineered as effector cells to contain a glucose-sensing GPCR (GPR1) that mobilizes internal Ca2+ stores and an NFAT response element engineered to express insulin. Such engineered effector cells can be used to autonomously synthesize insulin upon sensing glucose. Such embodiments may be used in beta cell replacement therapy.

[0211] Other non-limiting examples of uses of engineered effector cells include: i) imaging of the location of the disease microenvironment to aid surgical resection or monitor disease progression / regression; ii) cytotoxicity to kill disease cells; iii) proliferation to improve T cell persistence; iv) immunostimulation to recruit other immune cells; v) chemokines to recruit other immune cells; vi) immunosuppression to create a local immunosuppressive microenvironment; and vii) regeneration to enhance tissue healing.

[0212] The various ranges provided herein include the stated range and any value or subrange within the stated range. Furthermore, when "about" is used to describe a value and / or range, this includes, refers to, and / or encompasses a variation (up to ±10%) from the stated value and / or range.

[0213] As used herein, a target cell (e.g., a "host target cell," a "target cell of interest," a "disease 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 a target cell includes and / or refers to a structure (e.g., a binding site) on the target cell to which the antigen-binding domain of a reporter element can bind (e.g., has affinity). Effector cells can be from a variety of cell types, including human and non-human cells, and are often referred to herein as "sources." As used herein, the terms "genetically modified" and "genetically engineered" are used interchangeably and include and / or refer to prokaryotic or eukaryotic cells that contain an exogenous polynucleotide, regardless of the method used for insertion. In some embodiments, an effector cell comprises a non-naturally occurring nucleic acid molecule that has been created or modified by the hand of man (e.g., using recombinant deoxyribonucleic acid (DNA) technology) or derived from such a molecule (e.g., by transcription, translation, etc.). Effector cells that contain exogenous, recombinant, synthetic, and / or other modified polynucleotides are considered to be genetically engineered effector cells.

[0214] As used herein, "nucleic acid" includes and / or refers to "polynucleotide," "oligonucleotide," and "nucleic acid molecule," and generally refers to a polymer of DNA or RNA, which may be single-stranded or double-stranded, synthetic or obtained from a natural source (e.g., isolated and / or purified from a natural source), and may contain natural, non-natural, or modified nucleotides, and may contain natural, non-natural, or modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages, in place of the phosphodiesters found between nucleotides in unmodified oligonucleotides. In some embodiments, the nucleic acid does not contain insertions, deletions, inversions, and / or substitutions. However, as described herein, in some instances, the nucleic acid may suitably contain one or more insertions, deletions, inversions, and / or substitutions. In some embodiments, the nucleic acid may encode additional amino acid sequences that do not affect the function of the CAR and the polynucleotide, and which may or may not be translated upon expression of the nucleic acid by a host cell.

[0215] Nucleic acids can be obtained using any suitable method, including those described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY, pp. 280-281 (1982) and / or U.S. Publication No. US 2002 / 0190663, each of which is incorporated herein in its entirety for its teachings. Nucleic acids obtained from biological samples are typically fragmented to generate fragments suitable for analysis.

[0216] Nucleic acids and / or other moieties may be isolated. As used herein, "isolated" includes and / or refers to separation, in whole or in part, from at least some of the components with which it is normally associated, whether from a naturally occurring source or synthetically produced. Nucleic acids and / or other moieties of the present invention may be purified. As used herein, "purified" includes and / or refers to separation from a majority of other compounds or entities. Compounds or moieties may be partially purified or substantially purified. Purity may be stated on a weight basis and may be determined using various analytical techniques, including, but not limited to, mass spectrometry, HPLC, etc.

[0217] Numerous experimental embodiments were performed to generate engineered effector cells and characterize the functionality of the effector cells. Additional experiments were performed to create an optimized process for forming engineered effector cells for in vivo synthesis of engineered proteins. As further described below, the process can optimize transformation (e.g., transduction) yield, expansion rate, and functionality of the engineered effector cells. Exemplary constructs used to generate engineered effector cells include the nucleotide sequences set forth in SEQ ID NOs: 1-20. SEQ ID NOs: 1-20 are each synthetic DNA.

[0218] Figures 7A-7E show examples of polynucleotide sequences used to form engineered effector cells according to the present disclosure. Figure 7A shows an example of a polynucleotide sequence (SEQ ID NO:1) containing a receptor element encoding a CAR, where the CAR contains an antigen-binding domain (e.g., for folate receptor alpha (FRα)), a transmembrane domain (e.g., CD8), and intracellular signaling domains of CD28, 4-1BB, and CD3 zeta (SEQ ID NO:3). Figure 7B shows an example of a polynucleotide sequence (SEQ ID NO:2) containing the receptor element of Figure 7A, an actuator element (e.g., NFAT-RE6x), and an effector element (e.g., Nluc-P2A-GFP, SEQ ID NO:19). Figure 7C shows an example of a polynucleotide sequence (SEQ ID NO:4) comprising a receptor element encoding a CAR and including the actuator element (e.g., NFAT-RE6x) and effector element (e.g., Nluc-P2A-GFP) of Figure 7B, where the CAR comprises an antigen-binding domain (e.g., for FRα), a transmembrane domain (e.g., CD8), and the intracellular signaling domains of CD28 and CD3 zeta (SEQ ID NO:5). Figure 7D shows an example of a polynucleotide sequence (SEQ ID NO:6) comprising a receptor element encoding a CAR and including the actuator element (e.g., NFAT-RE6x) and effector element (e.g., Nluc-P2A-GFP) of Figure 7B, where the CAR comprises an antigen-binding domain (e.g., for FRα), a transmembrane domain (e.g., CD8), and the intracellular signaling domains of 4-1BB and CD3 zeta (SEQ ID NO:7 or SEQ ID NO:20). Figure 7E shows an example of a polynucleotide sequence (SEQ ID NO:8) encoding a CAR containing a receptor element, an actuator element (e.g., NFAT-RE6x), and an effector element of IFNβ (e.g., SEQ ID NO:9), where the CAR contains an antigen binding domain (e.g., for FRα), a transmembrane domain (e.g., CD8), and the intracellular signaling domains of 4-1BB and CD3 zeta (SEQ ID NO:7).

[0219] In several experiments, primary T cells were engineered (e.g., transformed) into primary T cell line effector cells capable of synthesizing engineered proteins proportional to disease burden over time at disease sites. CD4 T cell lines have long persistence, higher transduction rates, faster expansion, and more productive transcriptional machinery. Experimental results show that CD4 T cells engineered with CARs using the 4-1BB intracellular domain but not CD28 transformed approximately three times better than CD8 T cells. These effector cells formed by CD4 T cells showed approximately two-fold expansion, produced five-fold more engineered protein, and exhibited minimal cytolytic activity. Notably, when engineered to induce IFN-β upon interaction with antigen-presenting target cells, CD4 T cell line effector cells outperformed CD8 T cells (e.g., produced more antitumor IFN-β than CD8 T cells) and effectively suppressed ovarian cells grown in vitro and in vivo.

[0220] This technology allows for precise targeting of therapeutic biologics to disease sites while minimizing bioavailability in healthy tissues. By taking advantage of the long-lasting persistence of CD4 T cells, it is possible to improve patient compliance and enhance disease control by reducing the frequency of drug administration and human treatments.

[0221] More specifically, CD4 T cells have an improved ability to deliver specific proteins to disease sites compared to CD8 T cells. As described above, through the incorporation of a CAR with the 4-1BB intracellular domain, which excludes CD28, CD4 T cells demonstrated a three-fold increase in transduction efficiency, a two-fold increase in expansion, and a five-fold increase in target protein expression. In several experiments, the efficacy of the CD4 T cell-based delivery system was validated using a CAR that recognizes (e.g., binds to) FRα as an antigen target and promotes effector cells to produce a bioluminescent reporter enzyme. The modularity of the CAR allows for reprogramming of the platform's specificity to recognize different antigens and induce the expression of desired clinical therapeutic proteins. In further experiments, the CD4 T cell-based effector cells were further engineered and evaluated to deliver functional therapeutics, such as interferon-β (IFNβ), which was used to exert antitumor effects in vivo on intraperitoneally (ip) implanted ovarian cancer cells.

[0222] Further experiments will be directed at assessing the effector T cell phenotype and its impact on therapeutic outcome, including its impact on both the efficient generation of a clinically appropriate dose and the synthesis of effector proteins from cells within the target location. More specifically, the dynamics of CD4 and CD8 T cells, their transduction, expansion, and antigen-guided delivery functions will be assessed in vitro.

[0223] 8A-8H show the effect of altering different factors on the generation and function of effector cells of T cell lines according to the present disclosure.

[0224] While the CD4:CD8 ratio of freshly isolated primary CD3 T cells from peripheral blood was significantly higher in favor of CD4 T cells (CD4:CD8 = 7:1), the final ratio of the two after 25 days of CD3 T cell transduction and expansion was higher in favor of CD8 T cells (CD4:CD8 = 1:2) (data shown in Figures 8A and 8B). While this is not critical for CART cell formation, where the goal is to maximize the generation of cytolytic CD8 T cells, starting with a combined pool of CD4 and CD8 T cells may not be the best strategy when engineering effector cells for delivery functions. As mentioned above, this is because impaired NFAT pathway in CD8 T cells may limit the antigen-directed function of T cells expressing engineered proteins. Figures 8A-8D show the results of assessing the CD4 and CD8 phenotypic distributions at days 5 and 25 for CD3 T cells expanded for 25 days and engineered for delivery functions.

[0225] When T cell cultures contained both CD8 and CD4 T cells, a shift to the CD8 phenotype during expansion was observed. Figure 8C shows the results of numerically expanded donor-matched CD4 and CD3 T cells (containing both CD4 and CD8) after being engineered for delivery function. Holm-Sidak method, ** Statistical analysis was performed using a multiple comparison t-test with p<0.01. For example, Figure 8C shows a 14-fold expansion of the primary CD4 T cell culture 8 days after transduction (p<0.01, all time points after day 0) compared to a 7-fold expansion when starting with a CD3 T cell population from the same donor (CD4:CD8 = 7:1 on day 0). The CD4:CD8 ratio of the CD3 T cell population on day 0 was approximately 7:1 and showed minimal shift by day 10 based on the data (data shown in Figure 10).

[0226] Figures 8D and 8E show T cell transduction efficiency assessed by measuring FRα-CAR expression by flow cytometry 5 days after transduction. The comparison of transduction efficiency between CD4 and CD8 phenotypes further supports the use of the CD4 phenotype for delivery function. The CD3 population was reconstituted by mixing CD4 and CD8 at a 1:1 ratio. More specifically, Figure 8D shows donor-matched CD4 T cells (100%), CD8 T cells (100%), and CD4 and CD8 T cells (50% each) engineered for delivery function (n = 2 healthy donors, triplicate assays). To account for variability due to human genetic diversity, the experiment was repeated three times on different days with two healthy donors (indicated by different symbols). All transductions were performed using the same procedure, and transduction efficiency was determined 5 days later by assessing the proportion of engineered T cells. Compared with CD8 T cells, CD4 T cells not only expanded more rapidly (approximately twofold, Figure 8C) but also transduced more readily (approximately threefold, Figure 8D). Furthermore, compared with CD8 T cells, CD4 T cells were more likely to chemotactically migrate toward the chemokines CCL5 and CCL17 when expanded in the tumor microenvironment (Figure 12). This result is consistent with reports that antigen-reactive CD4 T cells efficiently infiltrate immunologically cold tumors, rendering the tumor microenvironment receptive to cytotoxic CD8 T cells and leading to tumor rejection.

[0227] The experiments in Figures 8A-8D used a CAR containing the intracellular domains of CD28 and 4-1BB in addition to CD3 zeta, as shown in the sequence in Figure 7A. In further experiments, three combinations of intracellular domains were evaluated for improving the ability of CD4 T cells to function as delivery platforms. The results are shown in Figures 8E and 8F. Three CAR constructs contained intracellular domains from: (i) CD28 alone (28ζ) (e.g., Figure 7C), (ii) 4-1BB alone (BBζ) (e.g., Figure 7D), and (iii) a tandem of CD28 and 4-1BB (28-BB28ζ) (e.g., Figure 7B). Full schematics of the individual CAR constructs are shown in Figures 7A-7D. The results in Figures 8E and 8F demonstrate that while CD4 T cell transduction efficiency (Figure 8E) is independent of the intracellular domain combination used in the CAR, expression levels of the engineered proteins depend on these domains (as indicated by bioluminescent reporter enzymes, Figure 8F). These results indicate that the BBζ CAR, e.g., the CAR containing the 4-1BB intracellular domain but not the CD28 intracellular domain in Figure 7D, more effectively induces the engineered reporter enzyme in both CD4 T cells and effector cells of the CD8 T cell line. Two other CARs (e.g., 28ζ and 28-BBζ in Figures 7B and 7C) induced reporter activity to a similar extent. However, the reporter enzyme activity induced by the BBζ CAR was approximately five-fold higher in CD4 T cells compared to effector cells of the CD8 T cell line, and was specific in response to antigen-presenting target cells (in this case, FRα). + OVCAR3 cells). Further comparison with 28ζCAR revealed that BBζ conferred features such as increased persistence, reduced tonic signaling, and improved patient tolerance of cytokine release syndrome and cell therapy-associated neurotoxicity.

[0228] More specifically, Figure 8E shows donor-matched CD4 T cells (100%) and CD8 T cells (100%) (n = 2 healthy donors) engineered for delivery using CARs with different intracellular domains (28ζ, BBζ, and 28-BBζ), shown in Figures 7B-7D, respectively. As used herein, the intracellular domain of 28ζ comprises the intracellular domains of CD28 and CD3 zeta (and engineered effector cells were generated by transducing T cells with the sequence shown in Figure 7C), the intracellular domain of BBζ comprises the intracellular domains of 4-1BB and CD3 zeta (and engineered effector cells were generated by transducing T cells with the sequence shown in Figure 7D), and the intracellular domain of 28-BBζ comprises the intracellular domains of CD28, 4-1BB, and CD3 zeta (and engineered effector cells were generated by transducing T cells with the sequence shown in Figure 7B). The results in Figure 8E show that the process yield when transducing CD4 T cells is approximately 3-fold higher compared to CD8 T cells and approximately 1.5-fold higher compared to the exemplified CD3 population (CD:CD8 = 1:1).

[0229] Figure 8F shows donor-matched CD4 T cells (100%) and CD8 T cells (100%) engineered for delivery function using CARs with different intracellular domains (28ζ, BBζ, and 28-BBζ) cocultured (E:T = 10:1) with OVCAR3 target cells (n = 2 healthy donors, triplicate assays). NFAT-RE-inducible reporter (Nluc) activity was quantified to assess the impact of the different intracellular domains on delivery function for both CD4 T cell and CD8 T cell phenotypes. For Figures 8D-8F, statistical analysis and p values ​​were calculated using one-way ANOVA and Tukey's multiple comparison test. * p<0.05, **** Determined by p<0.0001.

[0230] Further experiments were aimed at assessing whether the reduced expression of engineered effector proteins in CD8 T cell lineage effector cells using the NFAT-RE transcription start site was due to impaired NFAT-based transcription machinery. Alternatively, this could be due to reduced antigen stimulation resulting from the cytolytic effect of the FRα-specific CD8 T cell delivery platform on antigen-presenting target cells. To assess this, FRα-presenting target OVCAR3 cells were contacted with microparticles (2.8 μm diameter) functionalized with human FRα antigen and anti-human CD28 antibody. Microparticles similarly functionalized with anti-CD3 and anti-CD28 antibodies served as positive controls, and unstimulated (engineered for delivery function) CD4 T cells and CD8 T cells served as negative controls. The results, shown in Figure 8G, demonstrated approximately 10-fold higher bioluminescent reporter activity (e.g., higher expression), verifying that CD4 T cell lineage effector cells possess a stronger NFAT-based transcription machinery for robust engineered function, which is impaired in CD8 T cells and not due to a reduced number of antigen-presenting target cells. More specifically, Figure 8G shows a comparison of Nluc activity between engineered CD4 T cells (containing BBζCAR) and CD8 T cells for delivery function when stimulated with microparticles. Microparticles were functionalized with anti-CD3 and CD28 antibodies or with FRα antigen and anti-CD28 antibodies. Unstimulated CD4 and CD8 T cells served as negative controls. Nluc activity is expressed as a function of increasing numbers of engineered CD4 and CD8 T cells, with a constant cell-to-particle ratio (E:T) of 10:1. For Figure 8G, statistical analysis and p-values ​​were calculated using two-way ANOVA and Tukey's multiple comparison test. ** Determined by p<0.01.

[0231] Figure 8H shows the cytolytic function of donor-matched (engineered for delivery function) FRα-CAR CD4 and FRα-CAR CD3 T cells against FRα tumor cells (A2780cis-FRα+Luc2+). Unmodified CD3 T cells served as a negative control. Statistical analysis and p values ​​were calculated using the Holm-Sidak method.*** p<0.001 was determined using a multiple comparison t-test. All results are expressed as mean ± SD. The data shown in Figure 8H confirm that CD4 T cells engineered for delivery function exhibit minimal cytolytic activity. Compared to CD8 T cells, effector cells from the CD4 T cell line can be administered at a higher maximum recommended starting dose in first-in-human clinical trials. In the experiment, CD4 T cells and CD8 T cells, both engineered for FRα specificity, were incubated with target cells engineered to present FRα (e.g., FRα+A2780cis cells, as described in C.E. Repellin et al., Engineered Ovarian Cancer Cell Lines for Validation of CAR T-Cell Function, Advanced Biosystems 4, 1900224 (2020), the entire teachings of which are incorporated herein by reference). Target cells were also engineered to express the Luc2® enzyme, an ATP-dependent bioluminescent reporter that serves as a live cell marker. Unengineered CD3 T cells served as a negative control. The results confirmed that the cytolytic activity of CD4 T cell-based effector cells was significantly lower than that of CD3 T cell-based effector cells (CD4:CD8 = 1.5:1) (all E:T p < 0.01), demonstrating that CD4 T cells are a suitable phenotype for use in cell-based delivery systems. Thus, engineered CD4 T cells can be delivered with increased capacity to robustly express desired proteins without the undesirable side effect of killing healthy cells that may express target antigens at basal levels.

[0232] Various experiments were conducted to validate the function of CD4 T cell-based effector cells as a protein delivery system in vivo. For these experiments, immunocompromised mice bearing intraperitoneal tumors were treated with CD4 T cell-based effector cells engineered for delivery functions (e.g., protein delivery).

[0233] 9A-9C show the results of the present disclosure, demonstrating the functionality of effector cells from a CD4 T cell line as a protein delivery platform in vivo. FIG. 9A details the experimental schedule, and FIGS. 9B and 9C show the results. A 12-day-old xenograft tumor (antigen-positive FRA) was + MSLN neg A2780cis, antigen negative FRα neg MSLN + A2780cis) were cultured at days 0, 1, 2, 3, and 4 in 2 × 10 engineered cells for in situ delivery (CAR-BBζ-Nluc). 6 The engineered CD4 T cells were treated with 1000 CD4 T cells. Target-specific delivery function, such as Nluc activity (Figure 9B), was imaged and quantified not only at baseline (day 0) but also on days 1, 2, 3, 4, and 5 (Figure 9C). The engineered CD4 T cells specific for the FRα antigen expressed FRα. + When stimulated with tumor cells, the Nluc reporter activity demonstrated higher delivery capability than when stimulated with non-target cells, e.g., FRα-negative tumors, confirming target-specific in situ delivery. Negative controls included CD4 T cells with the same inducible delivery capability but without a CAR (e.g., no CAR-Nluc) and FRα-specific CD4CAR (CAR-BBζ) T cells without NFAT-RE inducible delivery capability. Increased delivery was observed in Nluc-generated FRα-CAR T cells compared to control groups, as indicated by increased inducible reporter activity (Figures 9B and 9C). These results demonstrate that the reporter-expressing CD4 T cell platform can be used for in vivo sensing. This technology may be used to monitor disease progression, evaluate treatment response, and deliver therapeutic proteins in situ.

[0234] As shown in Figures 9A-9C, effector cells from engineered primary CD4 T cell lines demonstrated antigen-specific delivery function in vivo (n = 6 mice per group). FRα-specific primary T cells engineered for NFAT-RE-induced delivery function were expressed as FRα. + MSLN neg A2780cis or FRα neg MSLN+ NSG mice bearing A2780cis tumors were intraperitoneally injected at 24-hour intervals for 5 days, and NFAT-RE-inducible effector (Nluc) activity was measured for 6 days, including the day of injection, as a baseline to assess delivery function. NFAT-RE-inducible effector (Nluc)-engineered CD4 T cells without CAR or FRα-specific primary CAR T cells (engineered without NFAT-RE-inducible effector (Nluc)) were treated with FRα-specific CAR T cells. + MSLN neg A2780cis tumors served as the control group. Figure 9A is a schematic diagram of the administration, treatment, and imaging schedule. Figure 9B contains representative bioluminescence images, and Figure 9C shows the quantitative results. All results are expressed as mean ± SEM. Statistical analysis and p values ​​were calculated using two-way analysis of variance and Tukey's multiple comparison test. * Determined by p<0.05.

[0235] Further experiments involved replacing the nucleotide sequence of the bioluminescent reporter enzyme with that of the antitumor cytokine interferon-β (IFNβ) (Figures 10A-10D and the polynucleotide sequence in Figure 7). Using microparticles functionalized with FRα antigen and anti-CD28 antibody, we reaffirmed the use of CD4 T cells in the synthesis of engineered therapeutic proteins via the NFAT transcriptional machinery (Figure 10A). Compared to pan-CD3 T cells and killer CD8 T cells engineered for delivery, the helper CD4 T cell-based delivery platform produced twice as much IFNβ, confirming enhanced antigen-specific delivery. IFNβ secretion was minimal from unstimulated CD3 T cells, CD4 T cells, and CD8 T cells (engineered for IFNβ delivery), as shown in Figure 10A. The results in Figures 10B to 10D show the dose-dependent growth inhibitory effect of IFNβ on various cell lines (OVCAR3, A2780cis, and HEK293T / 17) when the cell lines were treated with supernatants from CD4 T cells and CD8 T cells engineered to produce IFNβ. Growth inhibitory effects were observed for OVCAR3 (Figure 10B) and A2780cis (Figure 10C), but not for HEK293T / 17 (Figure 10D).

[0236] More specifically, Figures 10A-10H show FRα-specific targeting of tumor cells by CD4 T cells engineered to secrete IFNβ, according to the present disclosure. Figure 10A shows IFNβ secretion from T cells (CD4, CD8, and CD3) engineered for delivery function upon stimulation with FRα-specific / anti-CD28 Dynabeads (cell-to-particle ratio 1:3). For Figure 10A, statistical analysis and p-values ​​were calculated using two-way ANOVA and Tukey's multiple comparison test. **p<0.01. Figures 10B-10D show the growth inhibitory effects of IFNβ secreted from effector cells of CD4 T cells and CD8 T cell lines, assessed on OVCAR3 (Figure 10B), A2780cis (Figure 10C), and HEK293T / 17 (Figure 10D) cell lines. Cells were cultured for 3 days in the presence of serially diluted supernatants obtained from engineered CD4 T cells and CD8 T cells stimulated with FRα-specific / anti-CD28 Dynabeads. All results are expressed as mean ± SD. For Figures 10B-10D, statistical analysis and p values ​​were calculated using the Holm-Sidak method. ** Determined using a multiple comparison t test with p<0.01.

[0237] As shown in Figures 10E-10H, IFNβ effector cells from a primary CD4 T cell line were effective in inhibiting tumor growth in vivo and increasing mouse survival (n = 6 mice per group). + Luc2 + NSG mice bearing A2780cis tumors were intraperitoneally injected with FRα-specific primary T cells engineered for NFAT-RE-inducible IFNβ delivery at 24-hour intervals for 6 days, and tumor luminescence was measured every 3–4 days to assess tumor growth. Control groups received intraperitoneal injections of FRα-specific primary CAR T cells (engineered without NFAT-RE-inducible IFNβ delivery) or rhIFNβ (0.25 μg in 100 μL). Figure 10E shows a schematic diagram of the administration, treatment, and imaging schedule, Figure 10F includes representative bioluminescence images, and Figure 10G shows quantitative results. All results are expressed as mean ± SEM. Statistical analysis and p values ​​were calculated using two-way analysis of variance and Tukey's multiple comparison test. * p<0.05. Figure 10H shows the survival rate of mice in each group (n=6). Statistical analysis was performed using the log-rank Mantel-Cox test.

[0238] To compare the administration of IFNβ delivered by CD4 T cells with the direct administration of rhIFNβ, calculations were performed at the last point on the x-axis in Figure 10A. The engineered cells produced IFNβ when 125,000 CD4 T cells were stimulated for 72 hours at a cell-body-particle ratio of 1:3. Using rhIFNβ as a standard, the equivalent IFNβ activity in the culture supernatant was calculated and determined to be 70.436 picograms (pg) / mL, as shown on the y-axis. Therefore, the total amount of IFNβ in a total supernatant volume of 300 μL should be 70.436 pg / mL × 300 μL = 21 pg of IFNβ. Each mouse received 5 × 10 6 of IFNβ-producing CD4 T cells were injected, which resulted in 21 pg of IFNβ (50 × 10 6 Because the number of CD4 T cells used to secrete 840 pg of IFNβ (21 pg × 40 = 840 pg = 84 ng) is 40 times the number of CD4 T cells used to secrete 840 pg of IFNβ in vivo (21 pg × 40 = 840 pg = 84 ng), it was estimated that effector cells could secrete approximately 0.84 ng of IFNβ in vivo (21 pg × 40 = 840 pg = 84 ng). The amount of rhIFNβ injected per dose per mouse is 0.25 μg = 250 ng. This is approximately 300 times the amount of IFNβ estimated to be delivered by CD4 T cells (e.g., 250 ng / 0.84 ng = 297.62).

[0239] More specifically, Figures 10E-10H show the therapeutic utility of effector cells from IFNβ-producing CD4 T cell lines. To demonstrate the therapeutic utility in vivo, IFNβ-producing CD4 T cells were cultured in a 100% IFNβ-positive (FRα) ... + Luc2 + The experimental schedule is shown in Figure 10E, and the results are shown in Figures 10F to 10H. + Luc2 + A2780cis cells (2 × 10 6 ) were implanted intraperitoneally into NSG mice. 12-day-old xenograft tumors were grown at 5 × 10 6 FRα-CAR + T cells (with NFAT-RE-inducible IFNβ) were intraperitoneally treated daily for 6 days (days 13-19). The therapeutic effect was assessed by comparing the control group (e.g., 5 × 10 T cells without NFAT-RE-inducible IFNβ). 6 FRα-CAR +T cells; 0.25 μg recombinant human IFNβ (rhIFNβ)) (Figure 10F) and quantitative comparison of tumor luminescence (Figure 10G). + A significant reduction in tumor burden, as indicated by decreased luminescence, was observed in IFNβ-producing FRα-CAR compared to T cells + This was observed in T cells, indicating that CD4 T cell effector cells produced functional therapeutic agents (Figure 10G). The results in Figure 10H show that rhIFNβ and the control FRα-CAR, which did not produce IFNβ, were significantly different from those in the control FRα-CAR. + IFNβ-producing FRα-CAR compared with direct injection of T cells + This shows a statistically significant increase in survival in mice treated with T cells. rhIFNβ treatment had no observed benefit on survival, and all mice suffered weight loss or succumbed to distress.

[0240] FIG. 11 shows flow cytometry plots showing the proportions of CD4 T cells and CD8 T cells in pan-CD3 T cell populations from healthy donors (e.g., three healthy donors on day 10 of in vitro expansion), according to the present disclosure.

[0241] FIG. 12 shows a comparison of chemotaxis of CD4 T cells and CD8 T cells according to the present disclosure. CD4 T cells and CD8 T cells were compared for migration towards the chemotactic gradient of CCL5 and CCL7. Statistical analysis was performed using a two-tailed Student's t-test. **** p<0.0001.

[0242] The following describes the chemokine-induced T cell migration assay used in Figure 12. To evaluate chemokine-induced migration, a Boyden chamber Transwell® migration assay was performed. Donor-matched CD4 and CD8 T cells were thawed and stimulated with anti-CD3 / CD28 Dynabeads (cell-to-particle ratio 1:3). Three days after stimulation, the cells were de-beaded and serum-starved overnight in medium containing 2% heat-inactivated FBS. Transwell permeable inserts with a pore size of 5 μM were used for the assay and pre-soaked in serum-free RPMI for 30 minutes in a cell culture incubator (37°C, 5% CO2, 95% humidity). Serum-starved CD4 and CD8 T cells were counted and 5 x 10 per insert. 5 Cells were resuspended in 100 μL of serum-free RPMI and seeded into the top chamber of the insert. 650 μL of complete growth medium supplemented with 25 nanograms (ng) / mL of CCL55 or CCL7 was added to the bottom chamber as a chemoattractant. T cell migration was carried out for 4 hours in a cell culture incubator (37°C, 5% CO2, 95% humidity). After incubation, the insert (top chamber) was removed, and cells in the bottom chamber were quantified using CellTiter-Glo® reagent. All samples were performed in triplicate wells, and data are expressed as the mean ± SD of n-fold migration corresponding to CD4 T cell and CD8 T cell migration in the absence of chemoattractant.

[0243] I. Synthetic and Experimental Information on CD4 T Cells as a Chassis for Genetically Engineered Effector Cells

[0244] (1) Materials and reagents Table 1 lists the sources of materials, supplies, services, and equipment used in the above experiments. [Table 1]

[0245] (2) Preparation Transfer plasmids carrying different gene payloads were designed using SnapGene software and subcloned into lentivector plasmids. Plasmid preparation services (chemical synthesis of DNA insert sequences, subcloning into each vector backbone, and amplification) were provided by Epoch Life Science, Inc. (Texas, MO). Target cells engineered to express modified firefly luciferase (Luc2) were used as described in (i) C.E. Repellin et al., Modular Antigen-Specific T-cell Biofactories for Calibrated In Vivo Synthesis of Engineered Proteins, Advanced Biosystems 2, 1800210 (2018); and (ii) C.E. Repellin et al., Engineered Ovarian Cancer Cell Lines for Validation of CAR T Cell Function, Advanced Biosystems 4, 1900224 (2020), each of which is incorporated herein by reference in its entirety. + OVCAR3 and FRα + A2780cis were maintained in complete growth medium [RPMI 1640, 10% heat-inactivated FBS, and 1x penicillin-streptomycin solution]. Primary T cells were maintained in complete growth medium [RPMI 1640, 10% heat-inactivated FBS, 1x penicillin-streptomycin solution, and 2x GlutaMAX®]. To minimize cell clumping, Ca +2 and Mg +2 Phosphate-buffered saline (PBS) without ATP was used. Puromycin N-acetyltransferase was used as a selection marker when applicable, and puromycin dihydrochloride (puromycin) was used for selection of stable cell lines. Biotinylated human FRα protein was used to analyze FRα CAR expression on engineered primary T cells.

[0246] (3) Method for generating lentivector particles Lentivector particles were produced as described in H. Radhakrishnan, H.S. Javitz, P. Bhatnagar, Lentivirus Manufacturing Process for Primary T-Cell Biofactory Production, Advanced Biosystems 4, 1900288 (2020), the entire teachings of which are incorporated herein by reference. Briefly, a second-generation lentivector packaging system was used to prepare lentivector particles. HEK293T / 17 producer cells (12 x 10 6 ) were seeded in 21 mL of complete DMEM supplemented with 10% heat-inactivated FBS and 1x penicillin-streptomycin solution in a tissue-culture-treated T150 flask and placed in a cell culture incubator (37 °C, 5% CO2, 95% humidity). After 24 h, the transfer plasmid was co-transfected with second-generation packaging plasmids (psPAX2, pMD2.G) and pAdVAntage plasmid at a weight (wt) ratio of 4:3:1:0.4 (transfer plasmid: 12 μg, pxPAX2: 9 μg, pMD2.G: 3 μg, pAdV: 1.2 μg). Transporter 5® transfection reagent was used (100 μL) according to the manufacturer's protocol. The pseudoviral particle-rich cell culture supernatant was collected and replenished (30 mL) every 24 h for 3 days. The lentivector-rich cell culture supernatant was clarified using a 0.45 μM filter. The supernatant was transferred to a polypropylene conical ultracentrifuge tube and clarified by centrifugation at 20,700 g for 2 hours at 4°C in a Beckman Coulter Optima XPN-90 ultracentrifuge using an SW32-Ti rotor. The resulting pellet was resuspended in 400 μL of serum-free RPMI and aliquoted. The lentivector particles generated in this process were used to generate 1 × 10 6 When transducing 10 cells, the MOI is 10. Aliquots of the lentivector were stored at -80°C until use.

[0247] (4) Methods for generating engineered primary T cell line delivery systems Primary T cells engineered with the NFAT-RE-inducible drug delivery system and used in the in vivo validation experiments (Figures 9A–9C and 10E–10H) were generated using the following procedure. Briefly, human primary T cells (CD3, CD4, or CD8) were purchased from Stanford Blood Center (Palo Alto, CA). T cells were immediately counted and used fresh, or cryopreserved for future use using freezing medium [90% heat-inactivated fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO)] in liquid nitrogen. Biotin-anti-human CD3 and biotin-anti-human CD28 antibodies were coupled to Dynabead Biotin Binder paramagnetic particles (anti-CD3 / CD28 Dynabeads) according to the manufacturer's instructions. Human primary T cells were thawed (day 0), resuspended in complete growth medium, and activated with anti-CD3 / CD28 Dynabeads (cells:particles = 1:3). After 24 hours (day 1), 1 × 10 6 Activated primary T cells were transduced with the appropriate lentivector particles resuspended in 0.1 mL of serum-free RPMI at an MOI of approximately 10 and in the presence of 8 μg / mL polybrene. A 0.1 mL aliquot of the transduction reaction mix was dispensed dropwise into a tissue-culture-treated 6-well plate and placed in a cell culture incubator (37°C, 5% CO2, 95% humidity) for 16 hours. After 16 hours of culture (approximately day 2), transduced primary T cells were cultured at 1 × 10 in complete growth medium supplemented with recombinant human IL-7 (25 IU / mL), recombinant human IL-15 (25 IU / mL), and 8 μg / mL polybrene. 6 After a further 24 hours (day 3) and daily thereafter, cells were counted using acridine orange and propidium iodide (AOPI) staining in a Nexcelom K2 cell counter. They were cultured at 0.5 × 10 cells / mL in complete growth medium supplemented with recombinant human IL-7 (25 IU / mL) and recombinant human IL-15 (25 IU / mL). 6 The cells were maintained at a concentration of 1000 cells / mL, and half of the medium was replaced every 2–3 days. Polybrene was not added after the third day.

[0248] (5) Flow cytometry analysis The generation yield of genetically engineered primary T cells was determined by the expression of FRα-CAR on T cells engineered for drug delivery (%FRα-CAR + Five days after transduction, approximately 1 × 10 6 T cells were harvested and de-beaded by placing the T cell suspension on a DynaMag-2 sample rack for 2 minutes to remove the Dynabead biotin-conjugated particles. The de-beaded T cells were washed with cell staining buffer and stained for 1 hour at 4°C using an antibody cocktail containing biotinylated human FRα protein (FOLR1-His Tag-Avi Tag), PerCR / Cy5.5 anti-human CD3 antibody, and the LIVE / DEAD Fixable Aqua Dead Cell Stain Kit. The cells were washed and secondary stained for 1 hour at 4°C using APC-streptavidin. The samples were washed, resuspended in 200 μL of Cell Staining Buffer, and analyzed on a BD FACS Symphony A3 (BD ​​Biosciences). Data were further processed using FlowJo software. To determine the proportion of CD4 and CD8 phenotypes in CD3 T cell cultures (Figures 8A and 8B), de-beaded T cells were washed and stained for 1 hour at 4°C using an antibody cocktail containing PerCR / Cy5.5 anti-human CD3, Brilliant Violet 421 anti-human CD8, PE anti-human CD4, and the LIVE / DEAD Fixable Aqua Dead Cell Stain Kit®, according to a single-step protocol. All samples were analyzed using a BD FACS Symphony A3 (BD ​​Biosciences), and data were further processed using FlowJo® software.

[0249] (6) In vitro evaluation of the delivery function of engineered T cells (engineered for delivery function) FRα-CAR (with NFAT-RE-inducible delivery) +CD4 T cells and CD8 T cells were co-cultured with targets (OVCAR3 or FRα-antigen / anti-CD28 Dynabeads) at an effector-to-target ratio (E:T) of 10:1 in 200 μL of complete growth medium in wells of a 96-well plate. After 24 hours of co-culture, reporter activity, such as NanoLuc (Nluc) activity, was measured in the engineered primary T cells using the Nano-Glo® assay according to the manufacturer's protocol. Briefly, Nluc substrate was diluted in the cell lysis buffer provided with the Nano-Glo assay and added to the co-cultured cells in the 96-well plate to assess enzyme (Nluc) activity. After a short 3-minute incubation, bioluminescence was read using a microplate reader.

[0250] (7) In vitro evaluation of the cytolytic function of engineered T cells (engineered for delivery function) FRα-CAR (with NFAT-RE-inducible delivery) + CD3 T cells and CD4 T cells were cultured in wells of a 96-well plate in 200 μL of complete growth medium (FRα + Luc2-2A-E2Crimson + The cells were co-cultured with 2500 A2780cis cells. After 24 hours of co-culture, reporter activity, such as Luc2 activity, in the A2780cis cells was measured using the One-Glo® assay according to the manufacturer's protocol. Briefly, Luc2 substrate was diluted in the cell lysis buffer provided with the One-Glo assay and added to the co-cultures in a 96-well plate to assess Luc2 activity. After a short 10-minute incubation, bioluminescence was read using a microplate reader.

[0251] (8) Quantification of IFNβ delivery by effector cells of CD4 T cell lines T cells (CD3, CD4, CD8) engineered for IFNβ delivery were stimulated with microparticles functionalized with FRα antigen and anti-CD28 antibody at a cell-to-particle ratio of 1:3. Three days after stimulation, IFNβ activity in the cell culture supernatant was assayed using QUANTI-Blue™ according to the manufacturer's instructions. TMMeasurements were performed using an assay kit with IFN-α / β reporter HEK 293 cells. Recombinant human IFNβ standard was measured in parallel to measure equivalent IFNβ activity in the culture supernatant. Unmodified T cells and T cells engineered to express FRα-CAR (without NFAT-RE-inducible delivery function) were used as controls. All experiments were performed in triplicate, and data are expressed as mean ± SD.

[0252] (9) In vivo evaluation of the antitumor function of IFNβ-delivering effector cells from CD4 T cell lines The growth inhibitory effects of IFNβ secreted by effector cells from CD4 T cells and CD8 T cell lines were evaluated using ovarian cancer cell lines (OVACR3 and A2780cis) and HEK293T / 17 cells. IFNβ-enriched medium was serially diluted in a 96-well plate, and 5,000 cells / well of each cell line were added to a cell culture incubator (37°C, 5% CO2, 95% humidity) and cultured for 3 days. Growth inhibition was determined by the CellTiter-Glo® assay according to the manufacturer's protocol. All experiments were performed in triplicate, and data are expressed as mean ± SD.

[0253] (10) In vivo validation of the delivery function of engineered T cells (engineered for delivery function using the NFAT-RE delivery system) In vivo validation of effector CD4 T cell lines was performed in mice at SRI International in accordance with the guidelines of the Institutional Animal Care and Use Committee (approval number 22001). Six- to eight-week-old female NOD.Cg-Prkdc mice were obtained from the Jackson Laboratory. scid Il2rg tm1Wjl Twenty-four NSG / SzJ mice were purchased. After quarantine, the NSG mice were anesthetized and 2 × 10 mice were cultured in 100 μL of 1 × PBS. 6 FRα + MSLN Neg Luc2-2A-E2Crimson + A2780cis cells (18 animals) or FRα neg MSLN+ Luc2-2A-E2Crimson + A2780cis cells (6 mice) were implanted intraperitoneally in 100 μL of 1× PBS. For the next 10 days, 150 mg of D-luciferin dissolved in 1× PBS per kilogram (kg) of mouse was injected intraperitoneally every 3–4 days, and tumor growth was monitored. On day 11 after implantation, FRα + MSLN Neg Luc2-2A-E2Crimson + Mice transplanted with A2780cis cells were randomly divided into three groups (6 mice each). + MSLN Neg Luc2-2A-E2Crimson + A2780cis cells and FRα neg MSLN + Luc2-2A-E2Crimson + A2780cis cells) were engineered for delivery function and then cultured at 2 × 10 6 Primary CD4 T cells (e.g., FRα-CAR with an NFAT-RE-inducible Nluc reporter), control CD4 T cells (without FRα-CAR and with an NFAT-RE-inducible Nluc reporter), or CD4 T cells without a CAR (with an NFAT-RE-inducible Nluc reporter) were treated daily for 5 days. Bioluminescent reporter (Nluc) activity was measured daily, including day 0, after treatment, by intraperitoneal injection of Nano-Glo substrate (a 1:20 dilution of substrate in 1x PBS, equivalent to 0.5 mg per kg of mouse). Imaging was performed with an IVIS Lumina X5 imaging system. Data were quantified by analyzing ROIs using Living Image software. Tumor luminescence was plotted as the mean ± SEM of total luminous flux (photons / second) versus days after treatment.

[0254] (11) In vivo challenge of primary CD4 T cell lines with tumors using an IFN-β delivery system In vivo tumor challenge mouse studies were performed at SRI International in accordance with the guidelines of the Institutional Animal Care and Use Committee (approval number 22001). Eighteen 6- to 8-week-old male NOD.Cg-Prkdc mice were obtained from the Jackson Laboratory. scid Il2rg tm1Wjl NSG mice were anesthetized and 2 × 10 mice were cultured in 100 μL of 1 × PBS. 6 FRα + Luc2-2A-E2Crimson + A2780cis cells were intraperitoneally transplanted. After 12 days, the mice were randomly divided into three groups (n = 6 each) and 5 × 10 6 FRα-CAR + CD4 T cells (NFAT-RE-induced delivery function) or FRα-CAR + A T cell-based IFN-β delivery system (with NFAT-RE-directed delivery function) was administered daily for 6 days. For group 3, 0.25 μg of rhIFN-β was administered per mouse daily in 100 μL of 1x PBS for 6 days. Tumor growth was monitored every 3–4 days by intraperitoneal injection of 150 mg of D-luciferin per kg of mouse in 1x PBS. Luminescence imaging was performed using an IVIS Lumina X5 imaging system. Data were quantified by analyzing ROIs using Living Image software. Tumor luminescence was plotted as the mean ± SEM of total luminous flux (photons / second) versus days after tumor implantation.

[0255] (12) Statistical analysis Unless otherwise stated, results were expressed as arithmetic mean ± SD. In each experiment, each sample was measured in technical replicates. A value of p<0.05 was considered to indicate statistical significance as determined using one-way analysis of variance and Tukey's multiple comparison test or as indicated ( * p<0.05, ** p<0.01, *** p<0.001, ****p<0.0001). Analyses were performed using Prism version 8.0 (GraphPad Software).

[0256] As described above, various experimental embodiments are directed to optimizing the process of generating genetically engineered effector cells from primary T cells. In these experimental embodiments, various process parameters were evaluated to increase the generation yield of engineered primary T cells for delivery functions. Compared to typical spinoculation-based methods, limiting the transduction reaction volume to maximize lentivector exposure to T cells resulted in a 2.5-fold increase in transduction yield. The cell density and cytokines used in the expansion process were configured to expand T cell line effector cells by more than 100-fold in 14 days, and the functionality of these cells was validated in vivo using intraperitoneally implanted tumor cells. Primary T cell line effector cells can be scaled and administered to express a wide range of therapeutic proteins (e.g., cytokines, interferons, enzymes, agonists, and antagonists) at disease sites, eliminating the need for systemic administration of large amounts of these proteins, making them applicable to humans. In these experimental embodiments, primary T cells have been converted into a platform for direct synthesis of complex biologics at disease sites at the appropriate time and location. Unlike current transient drug delivery systems, which exhibit systemic biodistribution and potentially adversely affect healthy tissues, this technology can be used to synthesize engineered proteins that exert their therapeutic effects solely through autocrine or paracrine signaling at disease sites without adversely affecting healthy tissues. In vivo experiments confirmed the synthesis of functional proteins by engineered cells.

[0257] As shown above in Figure 2A, various embodiments use a process to efficiently scale the generation of effector cells from primary T cell lines to engineer T cells and use a CAR with specificity for FRα and a reporter enzyme to express a desired biologic. The DNA template for the antigen-sensing scFv domain and reporter enzyme can be swapped to redirect the specificity of this platform to other clinically relevant antigens and express proteins for sensing and / or therapeutic functions.

[0258] 13 shows an example of a process for generating genetically engineered effector cells from primary T cells according to the present disclosure. As shown, isolated CD3 T cells were activated with anti-CD3 / CD28 Dynabeads (cells:particles = 1:3) for 24 hours, the activated T cells were transduced by increasing the contact of the lentivector with the T cells in a volume of 0.1 mL for 16 hours, and the transduced cells were cultured at 0.5 x 10 in complete growth medium supplemented with IL-7 and IL-15. 6 Cells were expanded at 1000 cells / mL and half of the medium was changed every 2-3 days for 14 days. Various experiments were conducted to evaluate different process parameters and their impact on the transduction of primary T cells with lentivectors.

[0259] 14A-14D show examples of the effect of various parameters of lentivector transduction of primary T cells according to the present disclosure. More specifically, the effect of lentivector transduction of primary T cells on the early activation of CD3 T cells (CD69 + CD25 - ) (Figure 14A), peak activation (CD69 + CD25 + ) (Figure 14B), and late activation (CD69 - CD25 + ) (Figure 14C) (see also Figures 18A-18B for gating strategies with representative fluorescence-activated cell sorting (FACS) plots). Figure 14B shows that after particle stimulation at 1:3 cells to particles, 60% of CD3 T cells reached peak activation (CD69) within 24 hours. + CD25 +) as shown in Figure 14D. Thus, this ratio is used in the formation and / or manufacturing process in various experiments. Figure 14D shows a comparison of the transduction efficiency of particle-activated T cells at a 1:3 ratio with chemically activated T cells (phorbol 12-myristate 13-acetate and ionomycin, PMA / Io). While the transduction efficiencies were similar (42% vs. 40%, n = 3), the engineered FRα-CAR + The yield of T cells generated was higher after particle activation compared with PMA / Io (75% vs. 60%, n = 3). Transduction efficiency of non-activated T cells was approximately 15%.

[0260] As shown in Figures 14A-14B, the expression of T cell activation markers (CD25, CD69) in CD3 T cells (n=3 donors) was assessed by flow cytometry 24 or 48 hours after stimulation with chemicals (phorbol 12-myristate 13-acetate (30 nM) and ionomycin (1 μM), PMA / Io) or at different cell-to-particle (Dynabeads carrying anti-human CD3 and anti-human CD28) ratios. The assessment strategy for CD3 T cell activation is shown in Figures 18A and 18B. Different stages of T cell activation were assessed by the expression of T cell activation markers (CD25, CD69) in early activation (CD69). + CD25 - ) (Figure 14A), peak activation (CD69 + CD25 + ) (Figure 14B), and late activation (CD69 - CD25 + ) (Figure 14C).

[0261] As shown in Figure 15, FRα-CAR expression (%FRα-CAR on the left Y-axis) + T cells) and T cell viability (% viability on the right Y-axis) were assessed by flow cytometry after transduction of stimulated and unstimulated primary T cells. As shown in Figures 15B-15D, FRα-CAR expression (%FRα-CAR on the left Y-axis) and T cell viability (% viability on the right Y-axis) were assessed by flow cytometry after transduction of stimulated and unstimulated primary T cells. +T cell viability (% viability on the right Y-axis) and T cell viability (% viability on the right Y-axis) were assessed by flow cytometry after varying factors affecting transduction, including (i) gene payload size (chimeric antigen receptor (CAR) alone, 5.6 kb, vs. delivery system for T cell lines with CAR and NFAT-RE-inducible transgene, 7.2 kb), as outlined in Figures 7A-7D (Figure 15B), (ii) lentivector pseudotype (RD114 vs. VSV-g) (Figure 15C), and (iii) lentivector transduction method (method a: 1.5 and 14.5 hours in a cell culture incubator, 0.5 mL reaction volume, spinoculation at 800 gravity (G); method b: 16 hours in a cell culture incubator, 1.0 mL reaction volume; method c: 16 hours in a cell culture incubator, 0.1 mL reaction volume) (Figure 15D). Transduction efficiency was determined after 5 days. All results are expressed as mean ± SD. Statistical analysis and p values ​​for Figures 15A and 15D were determined by one-way analysis of variance and Tukey's multiple comparison test. Statistical analysis and p values ​​for Figure 14C were determined by two-tailed Student's t-test. * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001. Figure 15B was determined by a two-tailed Student's t-test. Figure 15C was analyzed by a Student's t-test, but the results were not significant.

[0262] Figures 15B-15D show a comparison of other parameters that affect the transduction of primary T cells. The experiments were performed on the percentage of modified primary T cells (%FRα-CAR on the left Y-axis). + Improvement is assessed as the number of viable primary T cells (% viability on the right Y-axis) and the number of live primary T cells in culture 5 days after transduction.

[0263] Figure 15B shows the effect of gene payload size (LTR vs. LTR) of 7.2 kb (for a T cell line delivery system with a CAR and an NFAT-RE-inducible transgene) versus 5.6 kb (CAR only) using the same plasmid vector. Although no difference in cell viability was observed, the transduction yield for transducing primary T cells with the 7.2 kb gene payload was approximately 50% lower than that with the 5.6 kb gene payload (62.85 ± 14% vs. 33.8 ± 15%, n = 4 donors). This is consistent with reports that the efficiency of lentivector transduction decreases with increasing transgene length.

[0264] To address the issue of reduced yields in generating effector cells from primary T cell lines, two pseudotyped lentiviral vectors for transducing T cells were compared: (i) the VSV-g envelope protein derived from vesicular stomatitis virus, and (ii) the RD114 envelope protein derived from infectious feline endogenous retrovirus. These were described in Zhang et al., "Transduction of Bone-Marrow-Derived Mesenchymal Stem Cells by Using Lentivirus Vectors Pseudotyped with Modified RD114 Envelope Glycoproteins," Journal of Virology 78(3):1219-1229 (2004), the teachings of which are incorporated herein in their entirety. VSV-g envelope protein has been shown to be effective in engineering T cells, and RD114 has been reported to improve the efficiency of engineering CD34 hematopoietic cells and CAR T cells. The results are shown in Figure 15C. No significant differences were observed in the transduction efficiency (RD114: 37.1 ± 9.5%; VSV-g: 34 ± 10.6%) or viability of engineered primary T cells. Because of its acceptance, VSV-g pseudotyped lentivectors were used in various experiments.

[0265] Experiments were further directed to assessing the effect of (i) cell concentration (FIG. 19A), (ii) MOI (FIG. 19B), (iii) transduction reaction volume (FIG. 19C), and (iv) polybrene concentration (FIG. 19D) on the transduction of primary T cells. Based on the test range for each parameter studied, a spinoculation step was used to transduce one million primary T cells at an MOI of 10 with 8 μg / mL polybrene in 500 μL.

[0266] To further enhance process yield, two different classes of chemical additives, AVI and LRA, were evaluated. Detailed results are shown in Figures 20A and 20B. When lentiviral vectors are used, intracellular antiviral responses interfere with the transduction efficiency of primary T cells. To address this issue, we evaluated the use of AVI, which suppresses intracellular immunity to infection from lentiviral vectors and potentially increases transduction yield. Inhibition of intracellular antiviral signaling improved T cell transduction.

[0267] Several AVIs were evaluated to inhibit three different antiviral pathways, and the results are shown in Figure 20. These included: (i) the TANK-binding kinase 1 (TBK1) pathway: BX795 and (5Z)-7-oxozeaenol; (ii) the RNA-dependent protein kinase (PKR) pathway: 2-aminopurine (2-AP) and C16; and (iii) other pathways, such as STAT (ruxolitinib) and Rho (Y-27632) signaling. Combined treatment of AVIs targeting the PKR or TBK1 pathway during T cell transduction increased the transduction of primary T cells (Figure 19A).

[0268] Figure 20B shows the results of LRA as an additive in T cell transduction and expansion medium. LRA promotes the development of chromatin structure, which determines DNA accessibility and retroviral gene integration in the host genome. Gammaretroviruses, deltaretroviruses, and lentiviruses, which insert DNA into transcriptionally active chromatin, showed a strong preferential bias for gene insertion sites. A subset of LRAs, including protein kinase C (PKC) agonists and / or their combinations with bromoextraterminal (BET) or histone deacetylase (HDAC) inhibitors, were evaluated for their ability to enhance T cell transduction with large lentiviral constructs. The LRA romidepsin increased the percentage of engineered T cells (55%) relative to vehicle controls (42%) (Figure 20B). However, the percentage of live cells was only 40% compared to 75% in controls, making romidepsin unsuitable for use with lentivectors.

[0269] The results above were used to develop an integrated process flow, which was then evaluated by transducing primary T cells from three human donors (e.g., n=3). For improved transduction, a volume-limiting approach was used to limit the transduction reaction volume before adding IL-2 to each reaction mix. Three different methods were evaluated to limit the transduction volume: (method a) 0.5 mL, spinoculation at 800G in a 24-well plate well for 1.5 hours, followed by incubation of the reaction for an additional 14.5 hours in a cell culture incubator (37°C, 5% CO2, 95% humidity); (method b) using a fixed reaction volume of 1.0 mL in a 6-well plate well for 16 hours in a cell culture incubator; and (method c) limiting the volume to a 0.1 mL droplet in a 6-well plate well for 16 hours in a cell culture incubator. All three methods were evaluated using a 1x10 6Primary T cells were transduced with lentivector particles at an MOI of 10 in serum-free medium supplemented with 8 μg / mL polybrene. After 16 hours, the transduction reaction was diluted with complete growth medium supplemented with 50 U / mL IL-2. Process efficiency was assessed 5 days after transduction (Figure 15D). Results showed 32% transduction with 44% viability using (method a) spinoculation, 53% transduction with 8% viability using (method b) a 1.0 mL reaction volume, and 60% transduction with 60% viability using (method c) a 0.1 mL droplet restriction reaction. By limiting the reaction volume to increase contact between the lentivector and the cells, method c produced approximately 2.5 times more engineered primary T cells than the spinoculation method (method a). Based on these results, method c, which confines transduction to 0.1 mL droplets, was selected as part of an optimized production process.

[0270] Various experiments were directed to assessing factors that influence the expansion of effector cells of engineered primary T cell lines. Figures 16A-16E show examples of the effect of various parameters of primary T cell expansion according to the present disclosure. In particular, T cell density and cytokines were evaluated for their impact on the in vitro expansion of engineered primary T cells. Numerical expansion of engineered primary T cells was assessed at different cell densities (Figure 16A) and with supplementation with different cytokines (IL-2, IL-7, IL-15) (Figure 16B). Figure 16C shows the naive / memory T cell phenotype (naive / memory) assessed by flow cytometry of CD4 T cells and CD8 T cells at days 7 and 14 of in vitro expansion using the same cytokine combinations. N :CD45RA + / CCR7 + ), Central Memory (T CM :CD45RA - / CCR7 + ), Effector Memory (T EM :CD45RA - / CCR7 - ), and terminal differentiation effector memory (T EMRA :CD45RA + / CCR7+ Figure 16D shows that FRα-specific engineered primary T cells expanded with the same cytokine combinations were able to express FRα-specific T cells. + Luc2-2A-E2Crimson + Figure 16D shows the dose-dependent induction of cytolysis in A2780cis target cells. Figure 16E shows the dose-dependent induction of effector function, e.g., delivery function, as represented by NFAT-RE-inducible NanoLuc (Nluc) reporter activity, in FRα-specific engineered primary T cells expanded with the same cytokine combination. FRα-specific engineered primary T cells were stimulated with particles conjugated to target FRα antigen and CD28 costimulatory particles or with OVCAR3 cells endogenously expressing FRα at an effector-to-target ratio of 5:1 (E:T). All results are expressed as mean ± SD. Statistical analysis and p values ​​in Figure 16E were calculated using one-way analysis of variance and Tukey's multiple comparison test. * Determined by p<0.05.

[0271] Figure 16A shows the effect of cell density on the numerical expansion of these engineered primary T cells in the presence of 50 IU / mL of IL-2. Over 14 days of culture, engineered primary T cells expanded to 1 x 10 6 0.5 × 10 cells / mL compared to approximately 16-fold expansion 6 cells / mL and 0.25 x 10 6 The cell density was approximately 45-fold expanded at 100 cells / mL.

[0272] Figure 16B shows the expansion results of engineered primary T cells in the presence of different cytokines (IL-2, IL-7, IL-15, and their combinations). Engineered primary T cells were expanded to 0.5 x 10 cells / mL by supplementing every other day with complete medium and including each cytokine complement (50 IU / mL IL-2, 25 IU / mL IL-7, 25 IU / mL IL-15). 6 Over 14 days of culture, a proliferation trend ranging from approximately 70-fold with IL-15 to approximately 110-fold with the combination of IL-2, IL-7, and IL-15 was observed.

[0273] Figure 16C shows the phenotypic changes of engineered primary T cells when cultured in different cytokine cocktails, as detailed in Figure 16B. CD4 and CD8 subsets were analyzed at days 7 and 14 (see Figure 21 for CD4 / CD8 ratios) using CD45RA and CCR7 markers, as compared to naive (T N ), Central Memory (T CM ), Effector Memory (T EM ), and terminal differentiation effector memory (T EMRA At day 7, over 90% of both CD4 and CD8 T cells were analyzed. N (CD45RA + / CCR7 + Approximately 50%) compartment and T CM (CD45RA - / CCR7 + After 7-14 days of expansion, the CD4 T cells were composed of the T compartment (approximately 42%), as shown in Figure 16B. N The compartment was reduced to less than 25% and T CM The compartment was reduced to approximately 6%. EM (CD45RA - / CCR7 - ) increased from approximately 3% on day 7 to approximately 13% on day 14, and T EMRA (CD45RA + / CCR7 + ) increased from approximately 5% on the 7th day to approximately 60% on the 14th day.

[0274] Similar observations were made for CD8 T cell subsets. At day 7, the CD8 T cell subset comprised approximately 58% of T N , about 37% T CM , about 3% T EM , and about 2% T EMRA At day 14, the composition had a T N , about 4% T CM , about 3% T EM , and approximately 53% of T EMRA T of engineered primary T cellsN With the exception of IL-7-supplemented medium, which showed no significant changes in the compartment, all other cytokines induced T cell expansion and had similar effects on the four phenotypes. This is consistent with results demonstrating the use of IL-7 to generate less differentiated CAR T cells with a stem-like T phenotype. EMRA compartment, whereas T CM The compartment was significantly reduced, and this effect was minimal in IL-7-supplemented cultures. IL-7-only cultures reduced expansion by 11-fold (Figure 16B), but generated more naive and central memory T cells, known for their superior antitumor efficacy. Indeed, IL-7-treated T cells have been used to reduce the number of CAR T cells required for clinical responses due to their high antigen-stimulated proliferation capacity, persistence, and superior efficacy. In addition to proliferation, the cytolytic function of primary T cells engineered into a delivery platform targeted to the FRα antigen-expressing A2780cis tumor cell line (Figure 16D) was confirmed to be proportional to the effector-to-target ratio (E:T), regardless of the cytokine composition used to expand the different T cell cultures. The target-specific cytolytic function of FRα-CAR cells was further supported by the use of two independent cell lines (A2780cis and KPCY) differentially engineered to express the FRα antigen, compared with the respective antigen-negative controls (see Figures 22A and 22B).

[0275] Figure 16E shows engineered effector function, e.g., delivery function, as represented by NFAT-RE-induced NanoLuc (Nluc) reporter protein activity in the same T cell culture. Engineered primary T cells were expanded for 14 days and stimulated with beads (conjugated to FRα antigen and anti-CD28 antibody, e.g., FRα antigen / anti-CD28) or with OVCAR3 cells (which endogenously express FRα antigen). 24 hours after stimulation, a two-fold higher delivery function was observed for engineered primary T cells compared to OVCAR3 cells when FRα antigen / anti-CD28 particles were used for stimulation, representing a six-fold increase in delivery function compared to that observed from unstimulated engineered primary T cells.

[0276] Engineered primary T cells expanded with IL-7 showed peak delivery function, but not significantly different from engineered primary T cells expanded with other cytokine combinations. Unlike engineered primary T cells expanded with IL-7 alone, those expanded with a combination of IL-7 and IL-15 showed enhanced proliferation. IL-7 and IL-15 are believed to support long-term persistence and memory responses of T cells, and such cytokines were selected as the cytokine supplement for the expansion of engineered primary T cells for cellular delivery of proteins.

[0277] Taken together, the above experiments encompassed optimizing multiple process parameters and evaluating their effect on the in vitro performance of the T cell line delivery platform. Specifically, thawed T cells were activated with anti-CD3 / CD28 Dynabeads (1:3 cell-to-particle ratio) for 24 hours, and the activated T cells were transduced for 16 hours by increasing the contact of the lentivector with the T cells in a 0.1 mL volume. The transduced cells were cultured at approximately 0.05 x 10 cells in complete growth medium supplemented with IL-7 and IL-15, with half of the medium replaced every 2-3 days for 14 days. 6 Excellent results were obtained when cells were expanded at 1000 cells / mL for 24 hours. These optimized parameters were used in subsequent in vivo studies, which are described in detail below.

[0278] Various experiments are directed to verifying the functionality of the engineered primary T cell line delivery platform. Figures 17A-17F show the validation of functionality of effector cells formed from primary T cells according to the present disclosure. For example, Figures 17A-17F show the results of in vitro validation of target-specific delivery function proportional to disease burden. FRα-specific primary T cells engineered for NFAT-RE-induced delivery function delivered target FRα. + A2780cis (Figure 17A) and FRα +When cocultured with KPCY cells (Figure 17B), they showed a proportional increase in reporter activity compared to their respective non-targeting (FRα) control cells. Figure 17C shows the results of CAR T cells generated using the above procedure and developed for the effector cells of the T cell lineage to reduce tumor burden. Tumor regression was observed in intraperitoneal KPCY tumors in NSG mice when treated with FRα-specific CAR T cells in a dose-dependent manner (n = 5 mice per group). Bioluminescence (Luc2 activity) from intraperitoneal tumors was used to assess in vivo tumor burden. Statistical analysis was performed using two-way ANOVA and Tukey's multiple comparison test. There was a statistically significant interaction between the number of days and administration of FRα-specific CAR T cells on tumor burden [F(18,96) = 4.595, p < 0.0001] (Figures 17D-17F).

[0279] Effector cells from primary T cell lines formed using the same procedure were antigen-specific and functional in vivo (n = 5 mice per group). FRα-specific primary T cells engineered for NFAT-RE-induced delivery were injected intraperitoneally with FRα at 24-h intervals for 5 days. + NSG mice bearing A2780cis tumors were intraperitoneally injected, and NFAT-RE-inducible effector (Nluc) activity was measured for 6 days, including the day of injection, as a baseline for assessing delivery function. A control group was included to assess any background signal that may arise from the Nluc substrate. A control group was injected with FRα-specific primary CAR T cells (operated without NFAT-RE-inducible effector (Nluc)) equipped with Luc2+ tumor cells to maintain comparable tumor burden. More specifically, Figure 17D shows a schematic diagram of the administration, treatment, and imaging schedule, Figure 17E includes representative bioluminescence images, and Figure 17F shows quantitative results. All results are expressed as mean ± SEM. Statistical analysis and p values ​​for Figures 17A, 17B, and 17F were calculated using the Holm-Sidak method. * p<0.05, ** p<0.01, *** Determined using a multiple comparison t-test with p<0.001.

[0280] More specifically, target-specific delivery function proportional to disease burden was assessed in vitro by co-culturing FRα-specific primary T cells engineered for NFAT-RE-induced delivery to target cells A2780cis (Figure 17A) and KPCY cells (Figure 17B). neg A2780cis, FRα neg Compared to non-targeting cells (KPCY), co-culture with antigen-positive target cells demonstrated a proportional and significant increase in delivery function and Nluc reporter activity with increasing numbers of target cells. However, control cells (e.g., primary T cells engineered for NFAT-RE-induced delivery function without a CAR) did not demonstrate delivery function even when co-cultured with the same target and non-targeting cells (Figure 122C). Further experiments verified that the above steps did not impair the intrinsic cytolytic function of CAR T cells.

[0281] To assess tumor growth, KPCY2838c3 pancreatic ductal adenocarcinoma cells derived from KPCY mice were transfected with human FRα antigen and Luc2 (FRα + Luc2 + LPCY cells) and 0.5 × 10 6 The FRα-CAR was intraperitoneally transplanted into NSG mice. + T cells (without the NFAT-RE inducible Nluc reporter) were expanded for 16 days and injected i.p. with 10-day-old FRα + Luc2 + LPCY tumors. The results in Figure 17C show the effect of FRα-CAR on tumor regression. + T cells (1×10 6 pieces, 3×10 6 pieces, and 10 x 10 6 FRα-CAR + The dose-escalation effect of 10×10 T cell vesicles is shown. 6 FRα-CAR + Treatment with T cells (p<0.01) resulted in approximately 60% tumor regression at day 21, with 3×10 6 FRα-CAR +Approximately 35% tumor regression was observed on day 21 in the treated cells (p<0.01).

[0282] In various experiments, FRα-CAR + T cells are generated with delivery capabilities, and upon engaging the target FRα antigen, for example, the FRα-CAR activates the NFAT-RE signaling pathway to induce expression of the desired protein. Figure 17D shows the details of the experimental schedule, and Figures 17E and 17F show the results. More specifically, Figures 17E and 17F show the results of 2 x 10 T cells intraperitoneally implanted into NSG mice. 6 FRα + Luc2 + Results for A2780cis cells are shown. 12-day-old xenograft tumors were cultured at 2 × 10 6 FRα-CAR + T cells (with an NFAT-RE inducible Nluc reporter) were treated intraperitoneally. + A control group was included to assess any background signal due to the use of Nluc substrate on tumor cells. This group contained an NFAT-RE-inducible Nluc reporter (control FRα-CAR) to maintain comparable tumor burden. + FRα-CAR without T cells + T cells were treated by intraperitoneal injection. Effector (Nluc) activity was measured not only at baseline (day 0) but also on days 1, 2, 3, 4, and 5 (FIG. 17E) and quantified (FIG. 17F). FRα-CAR with delivery function (e.g., with an NFAT-RE inducible Nluc reporter) + A significant increase in the activity of engineered effectors was observed in the T cell-treated group, confirming the targeted in situ delivery function of engineered primary T cells.

[0283] 18A and 18B show examples of strategies for assessing CD3 T cell activation according to the present disclosure. FIG. 18A shows the early activation of CD3 T cells (CD69 + CD25 - ), peak activation (CD69 + CD25 +), and late activation (CD69 - CD25 + Figure 18B is a schematic diagram of the gating strategy used to assess CD69 and CD25 expression in stimulated and unstimulated CD3 T cells by flow cytometry.

[0284] 19A-19D show examples of the effect of additional factors on lentivector transduction of primary T cells according to the present disclosure. FRα-CAR expression (&FRα-CAR on the left Y-axis) + T cell density (T cells) and T cell viability (% viability on the right Y-axis) were assessed by flow cytometry after varying factors affecting transduction. Figure 19A shows T cell concentration in the transduction reaction results, Figure 19B shows lentivector MOI results, Figure 19C shows transduction reaction volume results, and Figure 19D shows polybrene concentration results. Transduction efficiency was determined after 5 days. All results are expressed as mean ± SD.

[0285] 20A and 20B show an example of an exploratory screen of chemical additives to improve lentivector transduction of primary T cells according to the present disclosure. FRα-CAR expression (&FRα-CAR on the left Y-axis) + T cell) and T cell viability (% viability on the right Y-axis) were assessed by flow cytometry after combined treatment with AVI (Figure 20A) and LRA (Figure 20B) during lentivector transduction. Transduction efficiency was determined after 5 days. All results are expressed as mean ± SD.

[0286] Figure 21 shows an example of changes in the ratio of CD3 T cell subsets in response to cytokines, according to the present disclosure. The CD4 / CD8 ratio was assessed by flow cytometry of CD3 T cells on days 7 and 14 of in vitro expansion when the growth medium was supplemented with different cytokines (IL-2, IL-7, IL-15, and their combinations).

[0287] 22A-22C show examples of antigen-specific cytolysis and NFAT-RE-induced delivery functions according to the present disclosure. FRα-specific CAR T cells generated using the above steps demonstrated FRα-specific CAR T cells compared to their respective antigen-negative target cells. + Luc2-2A-E2Crimson + KPCY (Figure 22A) target cells and FRα + Luc2-2A-E2Crimson + Nluc induced cytolysis of A2780cis (Figure 22B) target cells in a dose-dependent manner. Figure 22C shows Nluc activity in primary T cells engineered for NFAT-RE-directed delivery function when co-cultured with antigen-positive and antigen-negative target cells for 24 hours.

[0288] II. Methods for evaluating the constitutive parameters for generating genetically engineered effector cells from primary T cells

[0289] (1) Materials and reagents Table 2 lists the sources of materials, supplies, services, and equipment used in the above experiments. [Table 2] JPEG2025530782000004.jpg40139

[0290] (2) Preparation Transfer plasmids carrying different gene payloads were designed using SnapGene software and subcloned into lentivector plasmids. Plasmid preparation services (chemical synthesis of DNA insert sequences, subcloning into each vector backbone, and amplification) were provided by Epoch Life Science, Inc. (Texas, Missouri). Target cells engineered to express modified firefly luciferase (Luc2) [FRα] were used as described in Repellin et al., Modular Antigen-Specific T-cell Biofactories for Calibrated In Vivo Synthesis of Engineered Proteins, Advanced Biosystems 2(12), 1800210 (2018); and Repellin et al., Engineered Ovarian Cancer Cell Lines for Validation of CAR T Cell Function, Advanced Biosystems 4(1), 1900224 (2020), the teachings of which are incorporated herein by reference in their entirety. + A2780cis (gender: female), FRα + OVCAR3 (sex: female) was maintained in RPMI medium [RPMI1640, 10% heat-inactivated FBS, and 1x penicillin-streptomycin solution]. Mouse pancreatic cancer cell line [KPCY2838c3 (sex: female)] was maintained in DMEM medium [DMEM, 10% heat-inactivated FBS, 1x GlutaMAX, and 1x penicillin-streptomycin solution]. To minimize cell clumping, Ca +2 and Mg +2Phosphate-buffered saline (PBS) without ATP was used. Puromycin N-acetyltransferase was used as a selectable marker, where applicable, and puromycin dihydrochloride (puromycin) was used for selection of stable cell lines. Chemical activation of T cells was achieved by treatment with 1 μM phorbol 12-myristate 13-acetate and ionomycin (PMA / Io). Biotinylated human FRα protein was used to analyze FRα CAR expression on engineered primary T cells.

[0291] (3) Method for generating lentivector particles Lentivector particles were produced as described in H. Radhakrishnan, H.S. Javitz, P. Bhatnagar, Lentivirus Manufacturing Process for Primary T-Cell Biofactory Production, Advanced Biosystems 4(6), 1900288 (2020), the entire teachings of which are incorporated herein by reference. Lentivirus production and its use in engineered cells was performed at SRI International in accordance with the guidelines of the approved Biological Use Authorization (BUA 17-05). Briefly, lentivector particles were prepared by packaging the corresponding transfer plasmid using a second-generation lentivector packaging system. HEK293T / 17 (sex: female) producer cells (12 × 10 6) were seeded in 21 mL of complete DMEM supplemented with 10% heat-inactivated FBS and 1x penicillin-striptomycin solution in a tissue-culture-treated T150 flask and placed in a cell culture incubator (37°C, 5% CO2, 95% humidity). After 24 h, the transfer plasmid was co-transfected with second-generation packaging plasmids (psPAX2, pMD2.G) and pAdVAntage plasmid at a weight ratio of 4:3:1:0.4 (transfer plasmid: 12 μg, pxPAX2: 9 μg, pMD2.G: 3 μg, pAdV: 1.2 μg). Transporter 5 transfection reagent was used (100 μL) according to the manufacturer's protocol. The pseudoviral particle-enriched cell culture supernatant was collected and replenished (30 mL) every 24 h for 3 days. The lentivector-enriched cell culture supernatant was clarified using a 0.45 μM filter. The supernatant was transferred to a polypropylene conical ultracentrifuge tube and clarified by centrifugation at 20,700 g for 2 hours at 4°C in a Beckman Coulter Optima XPN-90 ultracentrifuge using an SW32-Ti rotor. The resulting pellet was resuspended in 400 μL of serum-free RPMI and aliquoted. The lentivector particles generated in this process were used to generate 1 × 10 6 When transducing 10 cells, it is expected to achieve an MOI of 10. Aliquots of the lentivector were stored at -80°C until use.

[0292] (4) Methods for manipulating primary T cells Primary T cells engineered with the NFAT-RE-inducible drug delivery system and used in the in vivo validation studies (Figures 16A-16F) were generated using the developmental protocol reported herein. Human primary T cells were purchased from the Stanford Blood Center (Palo Alto, CA). T cells were immediately counted and used fresh, or cryopreserved for future use using freezing medium [90% heat-inactivated fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO)] in liquid nitrogen. Biotin-anti-human CD3 and biotin-anti-human CD28 antibodies were coupled to Dynabead Biotin Binder paramagnetic particles (anti-CD3 / CD28 Dynabeads) according to the manufacturer's instructions. Human primary T cells were thawed (day 0), resuspended in complete growth medium, and activated with anti-CD3 / CD28 Dynabeads (cells:particles = 1:3). After 24 hours (day 1), 1 × 10 6 Activated primary T cells were transduced with the appropriate lentivector particles resuspended in 0.1 mL of serum-free RPMI at an MOI of approximately 10 and in the presence of 8 μg / mL polybrene. A 0.1 mL aliquot of the transduction reaction mix was dispensed dropwise into a tissue-culture-treated 6-well plate and placed in a cell culture incubator (37°C, 5% CO2, 95% humidity) for 16 hours. After 16 hours of culture (approximately day 2), transduced primary T cells were cultured at 1 × 10 in complete growth medium supplemented with recombinant human IL-7 (25 IU / mL), recombinant human IL-15 (25 IU / mL), and 8 μg / mL polybrene. 6 After a further 24 hours (day 3) and daily thereafter, cells were counted using acridine orange and propidium iodide (AOPI) staining in a Nexcelom K2 cell counter. They were cultured at 0.5 × 10 cells / mL in complete growth medium supplemented with recombinant human IL-7 (25 IU / mL) and recombinant human IL-15 (25 IU / mL). 6 The cells were maintained at a concentration of 1000 cells / mL, and half of the medium was replaced every 2–3 days. Polybrene was not added after the third day.

[0293] (5) Approaches to improve the generation of delivery systems for primary T cell lines The above steps are devoted to exploring factors that improve the generation of cell-based delivery systems. For transduction optimization, the following were varied: (i) activation time (Figures 13A-13C), (ii) cell-to-particle ratio (Figures 14A-14C and Figure 15A), (iii) construct size (Figure 15B), pseudotyped lentivector (Figure 15C), and (iv) transduction method (method a, method b, method c). For transduction optimization, the following were varied: (i) concentration of activated primary T cells (number of cells varied in a 0.5 mL volume) (Figure 18A), (ii) MOI (1 x 10 in a 0.5 mL volume) (Figure 18B). 6 (iii) the transduction reaction volume (MOI of 10, 1 × 10 6 (iv) the number of cells (0.5 mL volume, MOI of 10, 1 x 10) (Figure 19C), and (iv) the polybrene concentration (0.5 mL volume, MOI of 10, 1 x 10) 6 The transduction buffer composition was modified by adding AVIs [PKR inhibitors 2-aminopurine (2-AP) and C16, TANK-binding kinase inhibitors BX-795 and (5z)-7-oxozeaenol, ROCK inhibitor Y-27632, and STAT inhibitor ruxolitinib] and LRAs [romidepsin, bryostatin, prostratin, and (+)-JQ1] to further explore the effects of these chemicals. To optimize the expansion of transduced T cells, the starting cell concentration of transduced cells (Figure 16A) and proliferation cytokines (Figures 16B-16E) were modified.

[0294] (6) Flow cytometry analysis The generation yield of genetically engineered primary T cells was determined by the expression of FRα-CAR on T cells engineered for drug delivery (%FRα-CAR + T cells) and cell viability. Five days after transduction, approximately 1 × 10 6T cells were harvested and de-beaded by placing the T cell suspension on a DynaMag-2 sample rack for 2 minutes to remove the Dynabead biotin-conjugated particles. The de-beaded T cells were washed with cell staining buffer and stained for 1 hour at 4°C using an antibody cocktail containing biotinylated human FRα protein (FOLR1-His Tag-Avi Tag), PerCR / Cy5.5 anti-human CD3 antibody, and the LIVE / DEAD Fixable Aqua Dead Cell Stain Kit. The cells were washed and secondary stained for 1 hour at 4°C using APC-streptavidin. The samples were washed, resuspended in 200 μL of Cell Staining Buffer, and analyzed on a BD FACS Symphony A3 (BD ​​Biosciences). Data were further processed using FlowJo software. To determine T cell activation (Figures 14A-14C), debeaded T cells were washed and stained for 1 hour at 4°C using an antibody cocktail containing PerCR / Cy5.5 anti-human CD3, Brilliant Violet 510 anti-human CD25, and Alexa Flour 700 anti-human CD69, according to a single-staining-step protocol. Gating was performed using unstained control samples after fluorescence compensation. To assess the naive / memory phenotype of expanded T cells (Figure 15C), a human naive / memory T cell ID panel kit was used. All samples were analyzed using a BD FACS Symphony A3 (BD ​​Biosciences), and data were further processed using FlowJo software.

[0295] (7) In vitro evaluation of the cytolytic function of engineered T cells (engineered for delivery function) FRα-CAR (with NFAT-RE-inducible delivery) + T cells were cultured in wells of a 96-well plate in 200 μL of complete growth medium with target (FRα + Luc2-2A-E2Crimson +A2780cis cells were co-cultured with Nluc2-containing IgG1 ...

[0296] (8) In vitro evaluation of the delivery function of engineered T cells (engineered for delivery function) FRα-CAR (with NFAT-RE-inducible delivery) + T cells were co-cultured with targets (OVCAR3 or FRα-antigen / anti-CD28 Dynabeads) at an effector-to-target ratio of 10:1 in 200 μL of complete growth medium in wells of a 96-well plate. After 24 h of co-culture, reporter activity, such as Nluc activity, was measured in the engineered primary T cells using the Nano-Glo assay according to the manufacturer's protocol. Briefly, Nluc substrate was diluted in the cell lysis buffer provided with the Nano-Glo assay and added to the co-cultured cells in the 96-well plate to assess Nluc activity. After a short 3-minute incubation, bioluminescence was read using a microplate reader.

[0297] (9) In vivo tumor challenge with CAR T cells (control cells with NFAT-RE-inducible delivery function) In vivo tumor challenge mouse studies were performed at SRI International in accordance with the guidelines of the Institutional Animal Care and Use Committee (approval number 22001). Twenty 6- to 8-week-old male NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ (NSG) mice were purchased from the Jackson Laboratory. The mice were transfected with a lentiviral vector (FRα). + Luc2-2A-E2Crimson +KPCY2838c3 cells, engineered to express human FRα, Luc2, and E2Crimson using A2780cis, were selectively expanded in the presence of puromycin. NSG mice were anesthetized and cultured at 1 × 10 in 100 μL of 1 × PBS. 5 FRα + Luc2-2A-E2Crimson + KPCY2838c3 cells were intraperitoneally transplanted. After 10 days, the mice were randomly divided into four groups (n=5 each), and three groups were divided into 1×10 6 pieces, 3×10 6 Pieces, or 10 x 10 6 Each group was treated with FRα-CAR+ T cells. A fourth, untreated group served as a negative control. Tumor growth was monitored every 3–4 days by intraperitoneal injection of 150 mg of D-luciferin per kg of mouse in 1x PBS. Luminescence imaging was performed on an AMI HTX Spectral instrument with a 60-second exposure. Data were quantified by region-of-interest (ROI) analysis using Aura Image software. Tumor luminescence was plotted as the mean ± SEM of total luminous flux (photons / second) versus days after tumor implantation.

[0298] (10) In vivo validation of the delivery function of engineered T cells (engineered for delivery function using an NFAT-RE inducible delivery system). In vivo validation of the T cell delivery system was performed in mice at SRI International, following the guidelines of the Institutional Animal Care and Use Committee (approval number 22001). Twenty-four 6- to 8-week-old female NOD.Cg-Prkdc mice were obtained from the Jackson Laboratory. scid Il2rg tm1Wjl After mandatory quarantine, NSG mice were anesthetized and 2 × 10 mice were injected with 100 μL of 1 × PBS. 6 FRα + Luc2-2A-E2Crimson +A2780cis cells were implanted intraperitoneally. Tumor growth was monitored every 3-4 days for the next 12 days using 150 mg of D-luciferin dissolved in 1x PBS per kilogram (kg) of mouse injected intraperitoneally. Eleven days after implantation, mice were randomly divided into two groups (n=5 each). The two groups were then treated daily for 5 days with 2x10 engineered cells for delivery. 6 Primary T cells (e.g., FRα-CAR with an NFAT-RE-inducible Nluc reporter) or control cells (e.g., FRα-CAR alone without an NFAT-RE-inducible Nluc reporter) were treated. Luminescence reporter (Nluc) activity was measured by intraperitoneal injection of Nano-Glo substrate (1:20 dilution of substrate in 1x PBS, equivalent to 0.5 mg per kg of mouse) on days 0, 1, 2, 3, 4, and 5 after treatment. Imaging was performed with an IVA Lumina X5 imaging system. Data were quantified by analysis of ROIs using Living Image software. Tumor luminescence was plotted as the mean ± SEM of total luminous flux (photons / second) versus days after treatment.

[0299] (11) Statistical analysis Unless otherwise stated, results were expressed as arithmetic mean ± SD. In each experiment, each sample was measured in technical replicates. A value of p<0.05 was considered to indicate statistical significance as determined using one-way analysis of variance and Tukey's multiple comparison test or as indicated ( * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001). Analyses were performed using Prism version 8.0 (GraphPad Software).

[0300] The various experiments described above used a variety of different plasmids to form effector cells and controls. Table 3 below provides a list of the plasmids and associated sequences. [Table 3] JPEG2025530782000006.jpg229100

[0301] In experimental embodiments, we have created various genetically engineered effector cells that harness CD4 T cell biology and transform them into cell line platforms capable of assessing and responding proportionally to disease burden by expressing precisely engineered proteins at disease sites. Helper CD4 T cells, when engineered for protein-delivering cell line effector cells, have been demonstrated to offer a distinct competitive advantage over killer CD8 T cells or combined pools of CD4 and CD8 T cells (e.g., pan-CD3 T cells). Overall, properly engineered CD4 T cells can bridge two gaps that hinder the clinical response of this technology: scaling up cell production to generate clinical doses and increasing synthesis of therapeutic proteins from cells, thereby reducing the clinical dose required to produce the desired effect.

[0302] In various embodiments, as described in further detail below, a scalable process for forming or manufacturing lentiviral vector particles was used at high titers to support the transduction of primary T cells with large gene inserts and a simple, efficient process for engineering primary T cells. In experimental embodiments, CD4 T cells were compared to CD8 T cells, and when engineered for delivery function, CD4 T cells were favored for multiple reasons. For example, CD4 T cells engineered with a CAR containing the 4-1BBζ intracellular domain but without the CD28 intracellular domain demonstrated approximately 30-fold improved performance (approximately 3-fold transduction, approximately 2-fold expansion, and approximately 5-fold activity) compared to similarly engineered CD8 T cells. Surprisingly, when starting with pan-CD3 T cells, a combined population of CD4 and CD8 T cells, the final population after cell expansion was primarily composed of CD8 T cells.

[0303] Non-cytolytic CD4 T cells have been experimentally confirmed as the appropriate phenotype for this function. The low cytolytic nature of CD4 T cell delivery offers substantial advantages, such as the possibility of increasing the maximum tolerated dose of engineered CD4 T cells without exposing them to healthy tissue. CD4 T cells are believed to have an advantage over CD8 T cells because the NFAT transcriptional machinery is more productive in CD4 T cells. Furthermore, CD4 T cells produce more Th1 cytokines and proliferate faster than CD8 T cells. Compared to non-therapeutic doses of pan-CD3 CAR T cells, administering the same number of T cells, half from each subset (CD4, CD8), more effectively treats tumors. Helper CD4 T cells then persist for a long time and induce stemness in CD8 T cells, expanding the memory T cell pool and improving treatment outcomes. Unlike CD8 T cells, CD4 T cells infiltrate cold tumors and recruit other CD8 T cells. CD4 T cells also aid in vascular normalization, inhibit hypoxia, and reduce metastasis. CD4 T cells have been shown to persist for over 10 years. In fact, when passaged in vivo, CD4 T cells outlive their host mice by four times and expand at least 1040-fold.

[0304] Experimental embodiments of cell-mediated drug delivery systems can have a positive impact on various medical fields requiring precise spatiotemporal drug administration, such as solid tumors and viral infections. Based on experiments on cell-based delivery of IFNβ to target solid tumors and viral infections, experiments are aimed at evaluating antitumor efficacy by focusing on localized delivery of IFNβ via CD4 T cell-based effector cells. The resulting data demonstrated that in situ delivery of IFNβ using CD4 T cell-based effector cells substantially exceeded the direct administration of 0.25 μg (70,000 IU) of rhIFNβ daily, a tolerated dose used in mice. Furthermore, intraperitoneal administration of 0.25 μg (70,000 IU) of rhIFNβ daily for 6 days (total dose of 420,000 IU) did not demonstrate significant therapeutic benefit. This is consistent with previous reports showing that daily intraperitoneal administration of rhIFNβ (10,000 IU) for 23 days (total dose of 230,000 IU) failed to produce a therapeutic effect. Indeed, calculations (related to the calculations in Figure 10A above) indicate that the IFNβ dose delivered by CD4 T cells was approximately 300-fold lower than that of directly administered rhIFNβ, yet was more effective. The increased therapeutic effect at reduced dose equivalents suggests the potential of the cell-based platform to minimize undesirable side effects on healthy tissues, thereby increasing the maximum recommended starting dose during subsequent human trials. Such effector cells can continue to be developed to overcome two major problems observed with systemic IFNβ treatment—severe toxicity to healthy tissues and immunosuppression in the local tumor microenvironment—and can be used for synergistic effects with other antitumor agents, such as chemotherapy, radiation therapy, and antibody-based immunotherapy, as well as for bridging the innate and adaptive immune responses.

[0305] This CD4 T cell-based technology functions as a zero-order delivery system, offering a promising solution to the challenges posed by first-order drug delivery systems that use synthetic carriers. Unlike first-order systems, the concentration of therapeutic biologics is concentrated at the disease site and proportional to the disease burden, enhancing efficacy while simultaneously reducing concentrations in healthy tissues and improving safety. Furthermore, CD4 T cells can persist in the body for more than 10 years, significantly reducing the frequency of reinfusions. This not only improves patient compliance but also reduces the burden on healthcare systems and streamlines treatment procedures in the long term.

[0306] In the experiments described below, we used primary T cells to develop a cell-based platform that can be used for site-specific delivery of protein-based drugs. This platform delivery system utilizes T cell activation mechanisms for in situ synthesis, thereby ensuring that cell-mediated synthesis of the desired protein is proportional to the disease burden. Site-specific and proportional synthesis of the desired biologic offers the potential to overcome morbidity issues that can arise from excessive systemic infusion of such drugs and prevent the development of resistance to these drugs. The use of these additives, such as AVI and LRA, can also reduce cellular attrition by decondensing chromatin structure.

[0307] While primary drug delivery systems (e.g., liposomes, nanocarriers, dendrimers, hydrogels, and microparticles) control drug release, their applications remain limited by their short in vivo half-lives, which require multiple injections, and potential toxicity due to their systemic presence. The T cell-based effector cells described above represent a substantial departure from this status quo. This is because T cells chemomigrate through multiple solid tissues to reach disease sites and engage target cells via antigen-specific CARs. At the single-cell level, this signals activation pathways in binary (on / off) events, regardless of the antigen density on the surface of diseased cells, and executes parallel programs that result in the clonal expansion of activated T cells. The integrated effect is a clonal CAR T cell population proportional to the number of target cells. T cell-based drug delivery systems can be engineered to exploit this T cell biology. T cells migrate to disease sites using their cytolytic capabilities, recognize target cells with molecular specificity, and then synthesize protein-based biologics in proportion to the disease burden. Thus, this is an in vivo vector of a living cell line engineered into a stable zero-order drug delivery system. Unlike first-order drug delivery systems, it is capable of sustained in situ production of complex biological agents to perform a wide range of effector functions.

[0308] Furthermore, primary drug delivery approaches are primarily based on synthetic materials and are rapidly cleared by the mononuclear phagocyte system. On the other hand, cell-based systems utilizing T cells have been shown to persist in vivo for over 10 years. In fact, recent findings in mice have concluded that primary T cells, when passaged in vivo in new mice, can outlast the lifespan of the host species by four times and expand at least 1040-fold. This eliminates the need for re-administration in the event of relapse. Therefore, while there is potential for targeting basal expression of target antigens, as seen in the case of CAR T cells, the T cell-based delivery platform represents a pioneering and universal technology, facilitating the delivery of complex biologics over long periods of time without the need for multiple infusions. As a result, this platform technology opens new horizons for the treatment of various diseases.

[0309] While specific embodiments have been illustrated and described herein, various alternative and / or equivalent embodiments may be substituted for the specific embodiments illustrated and described without departing from the scope of the present disclosure. The present application is intended to cover any modifications or variations of the embodiments described herein.

Claims

1. a receptor element encoding a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain operably linked to a transmembrane domain and recognizing an antigen on the surface of a target cell, and an intracellular signaling domain; an actuator element encoding a transcription factor binding site that upregulates synthesis of an effector protein in response to the extracellular antigen-binding domain of the CAR binding to the antigen on the target cell; and an effector element encoding an effector protein; an isolated CD4 T cell having an exogenous polynucleotide sequence comprising, in operative association, and configured to activate and synthesize and secrete the effector protein in response to the extracellular antigen-binding domain of the CAR binding to the antigen on the target cell. Genetically engineered effector cells.

2. 2. The genetically engineered effector cell of claim 1, wherein the effector element further encodes a signal peptide operably linked to the effector protein and non-native to the effector protein.

3. 10. The genetically engineered effector cell of claim 1, configured to synthesize and secrete said effector protein as a function of the amount of said target cells present in a sample or in situ.

4. the CAR is configured to elevate calcium in response to the extracellular antigen-binding domain binding to the antigen on the target cell; the transcription factor binding site is configured to bind to a transcription factor protein that is triggered by the calcium elevation and translocates into the nucleus of the engineered effector cell. The genetically engineered effector cell of claim 1.

5. 2. The genetically engineered effector cell of claim 1, wherein the intracellular signaling domain is selected from the group consisting of the intracellular signaling portion of 4-1BB, the intracellular signaling portion of CD3 zeta, and combinations thereof.

6. The genetically engineered effector cell of claim 1 , wherein the intracellular signaling domain does not include the intracellular signaling portion of CD28.

7. 2. The genetically engineered effector cell of claim 1, wherein the transcription factor binding site is selected from the group consisting of a nuclear factor of activated T cells (NFAT) response element, a serum response element (SRE), a cyclic AMP response element (CRE), and combinations thereof.

8. 2. The genetically engineered effector cell of claim 1, wherein the effector protein is selected from the group consisting of a detectable reporter protein, a therapeutic protein, a downstream signaling protein, and combinations thereof.

9. 2. The genetically engineered effector cell of claim 1, wherein the exogenous polynucleotide sequence comprises the receptor element, the actuator element, and the effector element in operative association on a single construct.

10. 2. The genetically engineered effector cell of claim 1, wherein the transmembrane domain is selected from the group consisting of a T cell receptor alpha or beta chain, CD3 zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, and GITR.

11. A single construct adapted to form genetically engineered effector cells comprising isolated CD4+ T cells that secrete an effector protein upon recognition of an antigen on the surface of a target cell, a receptor element encoding a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain operably linked to a transmembrane domain and recognizing the antigen on the surface of the target cell, and an intracellular signaling domain; an actuator element encoding a transcription factor binding site that upregulates synthesis of the effector protein in response to the extracellular antigen-binding domain of the CAR binding to the antigen on the target cell; an exogenous polynucleotide sequence comprising, in operative association with an effector element encoding said effector protein; and configured to activate and synthesize and secrete the effector protein in response to the extracellular antigen-binding domain of the CAR binding to the antigen on the target cell. A single construct.

12. The single construct of claim 11 , wherein the effector element encodes a signal peptide that is operably linked to the effector protein.

13. 12. The single construct of claim 11, carried by a viral vector or a non-viral carrier.

14. The single construct of claim 11, wherein the intracellular signaling domain comprises the intracellular signaling portion of 4-1BB and the intracellular signaling portion of CD3 zeta, respectively.

15. The single construct of claim 11, wherein the intracellular signaling domain does not include the intracellular signaling portion of CD28.

16. the transcription factor binding site is selected from the group consisting of a nuclear factor of activated T cells (NFAT) response element, a serum response element (SRE), a cyclic AMP response element (CRE), and combinations thereof; the transmembrane domain is selected from the group consisting of a T cell receptor α or β chain, a CD3ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, and GITR; 12. The single construct of claim 11.

17. 12. The single construct of claim 11, wherein the effector protein is selected from the group consisting of a detectable reporter protein, a therapeutic protein, a downstream signaling protein, and combinations thereof.

18. 12. The single construct of claim 11, wherein the exogenous polynucleotide sequence comprises a sequence having at least 80% sequence identity with a sequence selected from SEQ ID NOs: 1-20.

19. A population of genetically engineered effector cells, each said genetically engineered effector cell of said population comprises an isolated CD4+ T cell having an exogenous polynucleotide sequence comprising an actuator element that binds to an effector element that binds to a receptor element; the receptor element encodes a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that recognizes an antigen on the surface of a target cell, operably linked to a transmembrane domain, and an intracellular signaling domain; the actuator element encodes a transcription factor binding site that upregulates synthesis of an effector protein in response to the extracellular antigen-binding domain of the CAR binding to the antigen on the target cell; the effector element encodes the effector protein; and configured to activate and synthesize and secrete the effector protein in response to the extracellular antigen-binding domain of the CAR binding to the antigen on the target cell. A population of genetically engineered effector cells.

20. configured to activate upon the presence of said target cells and, in response thereto, synthesize and secrete a calibrated amount of said effector protein; the calibrated amount of the effector protein is a function of the amount of the target cell present in a plurality of cells or a sample; 20. The population of genetically engineered effector cells of claim 19.

21. 20. The population of genetically engineered effector cells of claim 19, wherein each said effector element further encodes a single signal peptide operably linked to said effector protein.

22. 20. The population of genetically engineered effector cells of claim 19, wherein said intracellular signaling domain is selected from the group consisting of the intracellular signaling portion of 4-1BB, the intracellular signaling portion of CD3 zeta, and combinations thereof.

23. 20. The population of genetically engineered effector cells of claim 19, wherein said intracellular signaling domain does not include the intracellular signaling portion of CD28.

24. the transcription factor binding site is selected from the group consisting of a nuclear factor of activated T cells (NFAT) response element, a serum response element (SRE), a cyclic AMP response element (CRE), and combinations thereof; the transmembrane domain is selected from the group consisting of a T cell receptor α or β chain, a CD3ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, and GITR; 20. The population of genetically engineered effector cells of claim 19.

25. 20. The population of genetically engineered effector cells of claim 19, wherein said effector protein is selected from the group consisting of a detectable reporter protein, a therapeutic protein, a downstream signaling protein, and combinations thereof.

26. activating a plurality of CD4 T cells with a plurality of particles; exposing a plurality of CD4 T cells to an exogenous polynucleotide sequence to manipulate said plurality of CD4 T cells; expanding the activated plurality of CD4+ T cells in an expansion culture medium to form a plurality of genetically engineered effector cells comprising the plurality of CD4+ T cells harboring the exogenous polynucleotide sequence; Equipped with The exogenous polynucleotide sequence a receptor element encoding a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain operably linked to a transmembrane domain and recognizing an antigen on the surface of a target cell, and an intracellular signaling domain; an actuator element encoding a transcription factor binding site that upregulates synthesis of an effector protein in response to the extracellular antigen-binding domain of the CAR binding to the antigen on the target cell; and an effector element encoding the effector protein in response to the extracellular antigen-binding domain of the CAR binding to the antigen on the target cell; operatively related to the plurality of engineered effector cells are configured to activate and synthesize and secrete the effector protein in response to the extracellular antigen-binding domain of the CAR binding to the antigen on the target cell. method.

27. 27. The method of claim 26, wherein the effector element further encodes a single signal peptide operably linked to the effector protein.

28. the intracellular signaling domain comprises an intracellular signaling portion of 4-1BB and an intracellular signaling portion of CD3 zeta and / or does not comprise an intracellular signaling portion of CD28; the transcription factor binding site is selected from the group consisting of a nuclear factor of activated T cells (NFAT) response element, a serum response element (SRE), a cyclic AMP response element (CRE), and combinations thereof; and / or the transmembrane domain is selected from the group consisting of a T cell receptor α or β chain, a CD3ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, and GITR; 27. The method of claim 26.

29. 27. The method of claim 26, wherein activating the plurality of CD4 T cells comprises exposing the plurality of CD4 T cells to a plurality of particles carrying anti-human CD3 antibodies and anti-human CD28 antibodies.

30. 30. The method of claim 29, further comprising exposing said plurality of CD4 T cells to said plurality of particles at a cell body to particle ratio of about 6:1 to about 1:6 for a period of time.

31. exposing the plurality of CD4 T cells to an exogenous polynucleotide sequence comprises exposing the plurality of CD4 T cells to a vector comprising the exogenous polynucleotide sequence; The vector is associated with or comprises a viral vector, a non-viral carrier, and / or a lipid nanoparticle.

27. The method of claim 26.

32. 27. The method of claim 26, wherein exposing the plurality of CD4 T cells to an exogenous polynucleotide sequence comprises exposing the activated plurality of CD4 T cells to a lentivirus comprising the exogenous polynucleotide sequence.

33. Exposing the plurality of CD4 T cells to lentivirus comprises administering to the subject a culture medium that is serum-free and contains polybrene, the culture medium comprising about 0.05 x 10 ６ Cells / milliliter (mL) to approximately 3 x 10 ６ 33. The method of claim 32, comprising exposing said plurality of CD4 T cells to a lentivirus.

34. Exposing the plurality of CD4 T cells to an exogenous polynucleotide sequence comprises exposing the plurality of CD4 T cells to an exogenous polynucleotide sequence in an amount of about 0.05 x 10 ６ cells / mL ~ approx. 3 x 10 ６ 27. The method of claim 26, comprising providing a total transduction reaction volume comprising the plurality of CD4 T cells at a cell density of 1000 cells / mL, culture medium, and a vector comprising the exogenous polynucleotide sequence in defined subamounts for a period of time.

35. Expanding the activated CD4 T cells increases the total transduction response dose to about 0.25 x 10 ６ cells / mL ~ approx. 1 x 10 ６ 27. The method of claim 26, comprising diluting the activated plurality of CD4 T cells at a cell density of 1000 cells / mL with expansion medium containing cytokines for a period of time.

36. 36. The method of claim 35, wherein the cytokine is selected from the group consisting of interleukin (IL)-2, IL-7, IL-15, and combinations thereof.

37. activating a plurality of T cells with a plurality of particles; exposing the plurality of T cells to an exogenous polynucleotide sequence to engineer the plurality of T cells; expanding the activated plurality of T cells in an expansion culture medium to form a plurality of genetically engineered effector cells comprising the plurality of T cells harboring the exogenous polynucleotide sequence; Equipped with The exogenous polynucleotide sequence a receptor element encoding a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain operably linked to a transmembrane domain and recognizing an antigen on the surface of a target cell, and an intracellular signaling domain; an actuator element encoding a transcription factor binding site that upregulates synthesis of an effector protein in response to the extracellular antigen-binding domain of the CAR binding to the antigen on the target cell; and an effector element encoding the effector protein in response to the extracellular antigen-binding domain of the CAR binding to the antigen on the target cell; operatively related to the plurality of engineered effector cells are configured to activate and synthesize and secrete the effector protein in response to the extracellular antigen-binding domain of the CAR binding to the antigen on the target cell. method.

38. 38. The method of claim 37, wherein the effector element further encodes a signal peptide that operably links to an effector protein.

39. the intracellular signaling domain comprises an intracellular signaling portion of 4-1BB and an intracellular signaling portion of CD3 zeta and / or does not comprise an intracellular signaling portion of CD28; the transcription factor binding site is selected from the group consisting of a nuclear factor of activated T cells (NFAT) response element, a serum response element (SRE), a cyclic AMP response element (CRE), and combinations thereof; and / or the transmembrane domain is selected from the group consisting of a T cell receptor α or β chain, a CD3ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, and GITR; 38. The method of claim 37.

40. 38. The method of claim 37, wherein the plurality of T cells comprises CD3 T cells, isolated CD4 T cells, or isolated CD8 T cells.

41. 38. The method of claim 37, wherein the plurality of T cells comprises isolated CD4 T cells.

42. 38. The method of claim 37, wherein activating the plurality of T cells comprises exposing the plurality of T cells to a plurality of particles carrying anti-human CD3 antibodies and anti-human CD28 antibodies.

43. 43. The method of claim 42, further comprising exposing said plurality of T cells to said plurality of particles at a cell body to particle ratio of about 6:1 to about 1:6 for a period of time.

44. 44. The method of claim 43, wherein the period of time is from about 10 hours to about 36 hours.

45. resuspending said plurality of T cells in complete growth medium; activating the plurality of T cells by adding the plurality of particles to the complete growth medium; 45. The method of claim 44, comprising:

46. Exposing the plurality of T cells to an exogenous polynucleotide sequence comprises exposing the plurality of T cells to about 0.05 x 10 T cells in a serum-free and polybrene-containing culture medium. ６ Cells / milliliter (mL) to approximately 3 x 10 ６ 38. The method of claim 37, comprising exposing 100 cells / mL of T cells to a vector comprising the exogenous polynucleotide sequence.

47. 47. The method of claim 46, wherein the culture medium contains about 4 micrograms (μg) / mL to about 8 μg / mL of polybrene.

48. Exposing the plurality of T cells to an exogenous polynucleotide sequence comprises exposing the plurality of T cells to about 0.05 x 10 ６ cells / mL ~ approx. 3 x 10 ６ 47. The method of claim 46, comprising providing a total transduction reaction volume comprising the plurality of T cells at a cell density of 1000 cells / mL, the culture medium, and vector in defined subamounts at a multiplicity of infection (MOI) of about 0.1 to about 10 for a period of time.

49. 49. The method of claim 48, wherein providing the total transformation reaction volume in the defined subvolume comprises placing the aliquot as a droplet volume into a tissue-cultured well plate and placing the cultured well plate in an incubator for a period of time.

50. 49. The method of claim 48, wherein providing the total transformation reaction volume in the defined sub-amount comprises disposing an aliquot of the defined sub-amount on a substrate having a hydrophobic or hydrophilic surface.

51. the total transformation reaction volume is about 0.5 mL to 2 mL; the sub-volume is about 0.05 mL to 0.25 mL; 49. The method of claim 48.

52. 49. The method of claim 48, wherein the period of time is from about 10 to about 24 hours.

53. exposing the plurality of T cells to an exogenous polynucleotide sequence comprises exposing the plurality of T cells to a vector comprising the exogenous polynucleotide sequence; 38. The method of claim 37, wherein the vector is associated with or comprises a viral vector, a non-viral carrier, and / or a lipid nanoparticle.

54. 54. The method of claim 53, wherein the viral vector comprises a lentivirus comprising the exogenous polynucleotide sequence.

55. the lentivirus comprises a lentiviral particle comprising the exogenous polynucleotide sequence; resuspending the lentiviral particles in sufficient culture medium to achieve a multiplicity of infection (MOI) of about 0.1 to about 10.

55. The method of claim 54.

56. Expanding the activated T cells increases the total transduction response dose to about 0.25 x 10 ６ cells / mL ~ approx. 1 x 10 ６ diluting the activated plurality of CD4 T cells at a cell density of 1000 cells / mL with expansion medium for a period of time; The expansion culture medium is a complete growth medium containing cytokines.

38. The method of claim 37.

57. 57. The method of claim 56, wherein the cytokine is selected from the group consisting of interleukin (IL)-2, IL-7, IL-15, and combinations thereof.

58. 57. The method of claim 56, wherein the cytokines are IL-7 and IL-15.

59. The period is about 10 to 20 days, Over the period of time, about 0.25 x 10 ６ cells / mL ~ approx. 1 x 10 ６ 57. The method of claim 56, further comprising periodically replacing at least a portion of said expansion culture medium while maintaining said cell density in cells / mL.

60. further comprising adding a supplement to at least one of the culture medium and the expansion medium; the additive is selected from the group consisting of an antiviral inhibitor, a latency reversal agent, and combinations thereof; 38. The method of claim 37.

61. 61. A population of genetically engineered effector cells comprising T cells harboring the exogenous polynucleotide sequence formed according to the method of any one of claims 37 to 60.

62. a plurality of T cells; an exogenous polynucleotide sequence, an exogenous polynucleotide sequence comprising, in operative association: a receptor element encoding a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain operably linked to a transmembrane domain and recognizing an antigen on the surface of a target cell, and an intracellular signaling domain; an actuator element encoding a transcription factor binding site that upregulates synthesis of an effector protein in response to the extracellular antigen-binding domain of the CAR binding to the antigen on the target cell; and an effector element encoding the effector protein in response to the extracellular antigen-binding domain of the CAR binding to the antigen on the target cell; a culture medium; Expansion culture medium; A kit comprising:

63. 63. The kit of claim 62, wherein the effector element further encodes a signal peptide operably linked to the effector protein.

64. the intracellular signaling domain comprises an intracellular signaling portion of 4-1BB and an intracellular signaling portion of CD3 zeta and / or does not comprise an intracellular signaling portion of CD28; the transcription factor binding site is selected from the group consisting of a nuclear factor of activated T cells (NFAT) response element, a serum response element (SRE), a cyclic AMP response element (CRE), and combinations thereof; and / or the transmembrane domain is selected from the group consisting of a T cell receptor α or β chain, a CD3ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, and GITR; 63. The kit of claim 62.

65. 63. The kit of claim 62, wherein the plurality of T cells comprises CD3 T cells, isolated CD4 T cells, or isolated CD8 T cells.

66. 63. The kit of claim 62, wherein the plurality of T cells comprises isolated CD4 T cells.

67. 63. The kit of claim 62, comprising a plurality of particles carrying the anti-human CD3 antibody and the anti-human CD28 antibody.

68. 68. The kit of claim 67, comprising another culture medium configured to resuspend said plurality of T cells with said plurality of particles to activate said plurality of T cells.

69. 69. The kit of claim 68, wherein the separate culture medium and the plurality of particles are configured to resuspend the plurality of T cells at a cell body to particle ratio of about 6:1 to about 1:6 for a period of about 10 hours to about 36 hours.

70. 69. The kit of claim 68, wherein the separate culture medium is a complete growth medium.

71. 63. The kit of claim 62, wherein the culture medium is serum-free and contains polybrene and is configured to manipulate the plurality of T cells.

72. 72. The kit of claim 71, wherein the culture medium contains about 4 micrograms (μg) / milliliter (mL) to about 8 μg / mL of polybrene.

73. a vector carrying the exogenous polynucleotide sequence, The vector is associated with or comprises a viral vector, a non-viral carrier, and / or a lipid nanoparticle.

63. The kit of claim 62.

74. the viral vector comprises a lentiviral particle carrying the exogenous polynucleotide sequence; the culture medium is configured to resuspend the lentiviral particles in the culture medium sufficient to achieve a multiplicity of infection (MOI) of about 0.1 to about 10; 74. The kit of claim 73.

75. Approximately 0.05×10 ６ cells / mL ~ approx. 3 x 10 ６ 75. The kit of claim 74, comprising a tissue-cultured well plate configured to receive subvolumes of a total transfection reaction volume comprising said plurality of T cells at a cell density of 100000000 cells / mL, said culture medium, and said exogenous polynucleotide sequence, and to culture said subvolumes for a period of time.

76. the total transformation reaction volume is about 0.5 mL to 2 mL; The period of time is from about 10 to about 24 hours.

76. The kit of claim 75.

77. 63. The kit of claim 62, wherein the expansion culture medium is a complete growth medium containing cytokines.

78. 78. The kit of claim 77, wherein the cytokine is selected from the group consisting of interleukin (IL)-2, IL-7, IL-15, and combinations thereof.

79. 78. The kit of claim 77, wherein the cytokines are IL-7 and IL-15.

80. The expansion culture medium is maintained at about 0.25 x 10 ６ cells / mL ~ approx. 1 x 10 ６ 80. The kit of claim 79, configured to dilute the total transformation reaction volume at a cell density of cells / mL.

81. 63. The kit of claim 62, wherein at least one of the culture medium and the expansion medium comprises an additive selected from the group consisting of an antiviral inhibitor, a latency reversing agent, and combinations thereof.

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