Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2330/00—Production
  • C12N2330/50—Biochemical production, i.e. in a transformed host cell
  • C12N2330/51—Specially adapted vectors

Definitions

• Various standard-of-care therapeutics are designed to treat a disease at the time of diagnosis. Although many pathogens and diseased cells undergo dynamic changes in vivo, current drugs are not designed to co-evolve along with the in vivo disease microenvironment. Such therapeutics can include drugs administered in doses that are normalized to the body weight of the patient. However, disease burden can be different for similar-sized patients, and mterpatient variability can affect optimal dosing. If drug dosages are administered in excess, the therapeutic agents can end up in system circulation which can cause morbidity in normal tissue. In the case of suboptimal delivery, drug resistance may develop. While the patient can be monitored and the dosage adjusted based on health results, continuous monitoring is costly and impractical. Additionally, monitoring strategies and treatments do not exist for many diseases. Thus, static therapeutics often cannot control dynamic pathogens and diseases that evolve and/or persist. The misalignment between the dynamic disease states and static therapeutics imposes a major social and economic burden.
• the present invention is directed to overcoming the above-mentioned challenges and others related to therapeutics for treating diseases, among other purposes, such as involving a genetically engineered CD4 T-cell line which can activate in situ to cause synthesis of an engineered protein (effector) against the target cell.
• the effector element encodes a signal peptide operably linked to the effector protein, the signal peptide being non-native to the effector protein.
• the genetically engineered effector cell is configured to synthesize and secrete an amount of the effector protein as a function of an amount of the target cell present in a sample or in situ.
• the CAR is configured to cause a rise in calcium in response to the extracellular antigen binding domain binding to the antigen of the target cell and the transcription factor binding site is configured to bind to a transcription factor protein that is triggered by the rise in calcium and is translocated into the nucleus of the genetically engineered effector cell.
• the intracellular signaling domain is selected from the group consisting of: an intracellular signaling portion of a 4- IBB, an intracellular signaling portion of a CD3 zeta, and a combination thereof.
• the intracellular signaling domain does not include an intracellular signaling portion of CD28.
• the transcription factor binding site is selected from the group consisting of: a nuclear factor of activated T-cell (NF AT) response element, a serum response element (SRE), a cyclic AMP response element (CRE), and a combination thereof.
• NF AT nuclear factor of activated T-cell
• SRE serum response element
• CRE cyclic AMP response element
• the effector protein is selected from the group consisting of: a detectable reporter protein, a therapeutic protein, a downstream signaling protein, and a combination thereof.
• the exogenous polynucleotide sequence includes, in operative association, the receptor element, the actuator element, and the effector element on a single construct.
• the transmembrane domain is selected from the group consisting of: T-cell receptor a or 0 chain, a CD3 chain, CD28, CD3s, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, and a GITR.
• Various aspects of the present disclosure are directed to a single construct configured to form a genetically engineered effector cell with an isolated CD4 T-cell for secretion of an effector protein upon recognition of an antigen on a surface of a target cell, the single construct comprising an exogenous polynucleotide sequence including, in operative association: a receptor element that encodes a CAR including an extracellular antigen binding domain operably linked to a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain recognizes an antigen on a surface of a target cell; an actuator element that 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 of the target cell; and an effector element that encodes the effector protein, wherein, in response to the extracellular antigen binding domain of the CAR binding to the antigen of the target cell, the genetically engineered effector cell is configured to activate and, to synthe, or
• the single construct is carried by a viral vector or a non-viral carrier.
• the intracellular signaling domain includes each of: an intracellular signaling portion of a 4- IBB and an intracellular signaling portion of a CD3 zeta.
• the intracellular signaling domain does not include an intracellular signaling portion of CD28.
• the transcription factor binding site is selected from the group consisting of: a NF AT response element, a SRE, a CRE, and a combination thereof; and the transmembrane domain is selected from the group consisting of: T-cell receptor a or P chain, a CD3 chain, CD28, CD3s, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, and a GITR.
• the effector protein is selected from the group consisting of: a detectable reporter protein, a therapeutic protein, a downstream signaling protein, and a combination thereof.
• the exogenous polynucleotide sequence includes a sequence with at least 80% sequence identity to a sequence selected from SEQ ID NOs: 1-20.
• V arious aspects of the present disclosure are directed to a population of genetically engineered effector cells, each of the genetically engineered effector cells of the population comprising an isolated CD4 T-cell carrying an exogenous polynucleotide sequence that includes an actuator element bound to an effector element bound to a receptor element, wherein: a receptor element that encodes a CAR including an extracellular antigen binding domain operably linked to a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain recognizes an antigen on a surface of a target cell; an actuator element that 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 of the target cell; and an effector element that encodes the effector protein, where
• the population of engineered effector cells are configured to activate and, in response, to synthesize and secrete a calibrated amount of the effector protein based on a presence of the target cell, the calibrated amount of the effector protein being a function of an amount of the target cell present in a plurality of cells or in a sample.
• each effector element encodes a signal peptide operably linked to the effector protein.
• the intracellular signaling domain is selected from the group consisting of: an intracellular signaling portion of a 4- IBB, an intracellular signaling portion of a CD3 zeta, and a combination thereof.
• the intracellular signaling domain does not include an intracellular signaling portion of CD28.
• the transcription factor binding site is selected from the group consisting of: a NF AT response element, a SRE, a CRE, and a combination thereof; and the transmembrane domain is selected from the group consisting of: T-cell receptor a or (3 chain, a CD3 chain, CD28, CD3s, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, and a GITR.
• the effector protein is selected from the group consisting of: a detectable reporter protein, a therapeutic protein, a downstream signaling protein, and a combination thereof.
• Various aspects of the present disclosure are directed to a method comprising: activating a plurality of CD4 T-cells using a plurality of particles; exposing the plurality of CD4 T-cells to an exogenous polynucleotide sequence to engineer the plurality of CD4 T-cells, wherein the exogenous polynucleotide sequence includes, in operative association: a receptor element that encodes a CAR including an extracellular antigen binding domain operably linked to a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain recognizes an antigen on a surface of a target cell; an actuator element that 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 of the target cell; and an effector element that encodes the effector protein, wherein, in response to the extracellular antigen binding domain of the CAR binding to the antigen of the target cell; and expanding the exogenous
• the effector element further encodes a signal peptide operably linked to the effector protein.
• the intracellular signaling domain includes: an intracellular signaling portion of a 4- IBB and an intracellular signaling portion of a 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: a NF AT response element, a SRE, a CRE, and a combination thereof;
• the transmembrane domain is selected from the group consisting of: T-cell receptor a or (3 chain, a CD3 chain, CD28, CD3s, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, and a GITR.
• activating the plurality of CD4 T-cells includes exposing the plurality of CD4 T-cells to the plurality of particles loaded with anti-human CD3 and anti-human CD28 antibodies.
• the method further includes exposing the plurality of CD4 T-cells to the plurality' of particles at a cell-to-particle ratio of about 6:1 to about 1:6 for a period of time.
• exposing the plurality of CD4 T-cells to the exogenous polynucleotide sequence includes exposing the plurality of CD4 T-cells to a vector carrying the exogenous polynucleotide sequence, wherein the vector is associated with or includes: a viral vector, a non-viral carrier, and/or lipid nanoparticles.
• exposing the plurality of CD4 T-cells to the exogenous polynucleotide sequence includes exposing the activated plurality of CD4 T-cells to a lentivirus carrying the exogenous polynucleotide sequence.
• exposing the plurality of CD4 T-cells to the lentivirus includes exposing between about 0.05xl0 6 cells/milliliter (mL) to about 3xl0 6 cells/mL of the plurality of CD4 T-cells to the lentivirus in a culture medium which is serum-free and contains polybrene.
• exposing the plurality of CD4 T-cells to the exogenous polynucleotide sequence includes providing a total transformation reaction volume including a cell density of betw een about 0.05xl0 6 cells/mL and about 3x10 6 cells/mL of the plurality of CD4 T-cells, a culture medium, and a vector carry ing the exogenous polynucleotide sequence in defined sub-volumes for a period of time.
• expanding the activated plurality of CD4 T-cells includes diluting a total transformation reaction volume with the expansion culture medium containing a cytokine for a period of time and at a cell density of betw een about 0.25xl0 6 cells/mL and about 1x10 6 cells/mL of the activated plurality of CD4 T- cells.
• the cytokine is selected from the group consisting of: interleukin (IL)-2, IL-7, IL-15, and a combination thereof.
• IL interleukin
• Various aspects of the present disclosure are directed a method comprising: activating a plurality of T-cells using a plurality of particles; exposing the plurality of T-cells to an exogenous polynucleotide sequence in a culture medium to engineer the plurality of T-cells, wherein the exogenous polynucleotide sequence includes, in operative association: a receptor element that encodes a CAR comprising an extracellular antigen binding domain operably linked to a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain recognizes an antigen on a surface of a target cell; an actuator element that 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 of the target cell; and an effector element that encodes the effector protein, wherein, in response to the extracellular antigen binding domain of the CAR binding to the antigen of the target cell; and expanding the activate
• the effector element encodes a signal peptide operably linked to the effector protein.
• the intracellular signaling domain includes: an intracellular signaling portion of a 4- IBB and an intracellular signaling portion of a 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: a NF AT response element, SRE, a CRE, and a combination thereof;
• the transmembrane domain is selected from the group consisting of: T-cell receptor a or P chain, a CD3 chain, CD28, CD3a, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, and a GITR.
• the plurality of T-cells include CD3 T-cells, isolated CD4 T-cells, or isolated CD8 T-cells. In some aspects, the plurality of T-cells include isolated CD4 T-cells.
• activating the plurality of T-cells includes exposing the plurality of T-cells to the plurality of particles loaded with anti-human CD3 and anti-human CD28 antibodies.
• the method includes exposing the plurality of T-cells to the plurality of particles at a cell-to-particle ratio of between about 6: 1 and about 1:6 for a period of time.
• the period of time includes between about 10 hours and about 36 hours.
• the method includes resuspending the plurality of T-cells in a complete growth medium and activating the plurality of T-cells by adding the plurality of particles to the complete growth medium.
• exposing the plurality of T-cells to the exogenous polynucleotide sequence includes exposing between about 0.05xl0 6 cells/mL to about 3xl0 6 cells/mL of the plurality of T-cells to a vector including the exogenous polynucleotide sequence in the culture medium which is serum-free and contains polybrene.
• the culture medium contains between about 4 micrograms (pg)/mL and about 8 pg/mL of polybrene.
• exposing the plurality of T-cells to the exogenous polynucleotide sequence includes providing a total transformation reaction volume including a cell density of between about 0.05xl0 6 cells/mL and about 3x10 6 cells/mL of the plurality of T-cells, the culture medium, and the vector in defined sub-volumes for a period of time and at a multiplicity of infection (MOI) of between about 0.1 and about 10.
• MOI multiplicity of infection
• providing the total transformation reaction volume in the defined sub-volumes includes placing aliquots as drop volumes in a tissue-cultured well plate and placing the cultured well plate in an incubator for the period of time. [0054] In some aspects, providing the total transformation reaction volume in the defined sub-volumes includes placing aliquots of the defined sub-volumes on a substrate having a surface which is hydrophobic or hydrophilic.
• the total transformation reaction volume includes between about 0.5 mL and 2 mL and the sub-volumes include between about 0.05 mL and about 0.25 mL.
• the period of time includes between about 10 hours and about 24 hours.
• exposing the plurality of T-cells to the exogenous polynucleotide sequence includes exposing the plurality of T-cells to a vector carrying the exogenous polynucleotide sequence, wherein the vector is associated with or includes: a viral vector, a non-viral carrier, and/or lipid nanoparticles.
• the viral vector includes a lentivirus carrying the exogenous polynucleotide sequence.
• the lentivirus includes lentivirus particles carrying the exogenous polynucleotide sequence and the method further includes resuspending the lentivirus particles in the culture medium sufficient to achieve a MOI of between about 0.1 and about 10.
• expanding the activated plurality of T-cells includes diluting a total transformation reaction volume with the expansion culture medium for a period of time and at a cell density of between about 0.25xl0 6 cells/mL and about IxlO 6 cells/mL of the plurality of T-cells, wherein the expansion culture medium is a complete grow th medium containing a cytokine.
• the cytokine is selected from the group consisting of: IL-2, IL-7, IL-15, and a combination thereof. In some aspects, the cytokine includes IL-7 and IL-15.
• the period of time includes between about 10 days and about 20 days
• the method further includes periodically changing at least a portion of the expansion culture medium over the period of time and while maintaining the cell density of between about 0.25x1 6 cells/mL and about 1x10 6 cells/mL.
• the method includes adding an additive to at least one of the culture medium and the expansion culture medium, the additive being selected from the group consisting of: an antiviral inhibitor, a latency reversal agent, and a combination thereof.
• an additive being selected from the group consisting of: an antiviral inhibitor, a latency reversal agent, and a combination thereof.
• kits comprising: a plurality of T-cells; an exogenous poly nucleotide sequence, wherein the exogenous polynucleotide sequence includes, in operative association: a receptor element that encodes a CAR comprising an extracellular antigen binding domain operably linked to a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain recognizes an antigen on a surface of a target cell; an actuator element that 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 of the target cell; and an effector element that encodes the effector protein, wherein, in response to the extracellular antigen binding domain of the CAR binding to the antigen of the target cell; a culture medium; and an expansion culture medium.
• a receptor element that encodes a CAR comprising an extracellular antigen binding domain operably linked to a transmembrane domain, and an intracellular signaling domain,
• the effector element encodes a signal peptide operably linked to the effector protein.
• the intracellular signaling domain includes: an intracellular signaling portion of a 4- IBB and an intracellular signaling portion of a 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: a NF AT response element, a SRE, a CRE, and a combination thereof;
• the transmembrane domain is selected from the group consisting of: T-cell receptor a or (3 chain, a CD3 ⁇ chain, CD28, CD3s, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, and a GITR.
• the plurality of T-cells include CD3 T-cells, isolated CD4 T-cells, or isolated CD8 T-cells.
• the plurality of T-cells includes isolated CD4 T-cells.
• the kit includes a plurality of particles loaded with antihuman CD3 and anti-human CD28 antibodies.
• 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.
• the other culture medium and the plurality of particles are configured to resuspend the plurality of T-cells at a cell-to-particle ratio of between about 6: 1 and about 1 :6 for a period of time of about 10 hours to about 36 hours.
• the other culture medium is a complete growth medium.
• the culture medium is serum-free and contains polybrene, and is configured to engineer the plurality of T-cells.
• the culture medium contains between about 4 pg/mL and about 8 pg/mL of polybrene.
• the kit includes a vector carrying the exogenous polynucleotide sequence, wherein the vector is associated with or includes: a viral vector, anon-viral carrier, and/or lipid nanoparticles.
• the viral vector includes lentivirus particles carrying the exogenous polynucleotide sequence and the culture medium is configured to resuspend the lentivirus particles in the culture medium sufficient to achieve a MOI of between about 0.1 and about 10.
• the kit includes a tissue-cultured well plate configured to receive a total transformation reaction volume including a cell density of between about 0.05xl0 6 cells/mL and about 3xl0 6 cells/mL of the plurality of T-cells, the culture medium, and the exogenous polynucleotide sequence in sub-volumes and to culture the sub-volumes for a period of time.
• the total transformation reaction volume includes between about 0.5 mL and about 2 mL and the period of time includes between about 10 hours and about 24 hours.
• the expansion culture medium is a complete growth medium containing a cytokine.
• the cytokine is selected from the group consisting of: IL-2, IL-7, IL-15, and a combination thereof.
• the cytokine includes IL-7 and IL-15.
• the expansion culture medium is configured to dilute a total transformation reaction volume for a period of time and at a cell density of between about 0.25xl0 6 cells/mL and about 2xl0 6 cells/mL of the plurality of T-cells.
• FIG. 1 illustrates an example genetically engineered effector cell comprising an isolated CD4 T-cell, in accordance with the present disclosure.
• FIGs. 2A-2B illustrate example genetically engineered effector cells, in accordance with the present disclosure.
• FIGs 8A-8H illustrate effects of varying different factors on production and function of the T-cell based effector cell, in accordance with the present disclosure.
• FIGs. 9A-9C illustrate the results of verifying the functionality the CD4 T-cellbased effector cells as a protein delivery platform in vivo, in accordance with various embodiments.
• FIGs. 10A-10H illustrate FRa-specific targeting of tumor cells by a CD4 T-cell engineered to secrete IFN(3, in accordance with the present disclosure.
• FIG. 11 illustrates flow cytometry plots showing the proportion of CD4 and CD8 T-cells in the pan CD3 T-cell population from healthy donors, in accordance with the present disclosure.
• FIG. 12 illustrates a comparison of CD4 and CD8 T-cell chemotaxis, in accordance wdth the present disclosure.
• FIG. 13 illustrates an example process for forming genetically engineered effector cells from primary T-cells, in accordance with the present disclosure.
• FIGs. 14A-15D illustrate example effects of various parameters on the lentivector transduction of primary T-cells, in accordance with the present disclosure.
• FIGs. 16A-16E illustrate example effects of various parameters on the expansion of primary T-cells, in accordance with the present disclosure.
• FIGs. 17A-17F illustrate functional validation of the effector cell formed from a primary T-cell, in accordance with the present disclosure.
• FIGs. 18A-18B illustrate an example strategy for evaluating CD3 T-cell activation, in accordance with the present disclosure.
• FIGs. 19A-19D illustrate example effects of additional factors on transduction of primary T-cells with lenti vectors, in accordance with the present disclosure.
• FIGs. 20A-20B illustrate an example exploratory screen of chemical additives for improving transduction of primary T-cells with lentivectors, in accordance with the present disclosure.
• FIG. 21 illustrates an example change in the proportion of CD3 T-cell subsets in response to cytokines, in accordance with the present disclosure.
• FIGs. 22A-22C illustrate example antigen-specific cytolysis and NFAT-RE inducible delivery function, in accordance with the present disclosure.
• Embodiments in accordance w ith the present disclosure are directed to genetically engineered effector cells comprising isolated CD4 T-cells, or other types of primary T-cells, which are used as a cellular chassis or vector to act as a biofactory for different target proteins.
• the engineered effector cell can be used to synthesize calibrated amounts of the target protein, and to induce autocrine and paracrine signaling due to artificial cell signaling.
• Such effector cells can be used, for example, as an in vivo vector for delivery target proteins in organisms, such as humans.
• Embodiments of the present disclosure include primary T-cell lines that are genetically engineered with chimeric antigen receptors (CARs) to form effector cells that specifically detect (e.g., bind) antigens expressed on the surface of a target cell.
• the effector cells are formed from or include isolated CD4 T- cells.
• the CD4 T-cells can be isolated from other types of cells, either prior to engineering the effector cells or during the engineering such as by using a selective expansion process. By binding to the antigen, the genetically engineered effector cells can have improved functionality from natural T-cells.
• CD4 T-cells had higher propensity for transduction and expansion, among higher effector secretion and activity, as compared to CD8 or CD3 T-cells when forming genetically engineered effector cells.
• a T-cell can be engineered to express genetic elements including transmembrane receptor(s) that autonomously regulate the intracellular transcriptional machinery, herein sometimes referred to as an effector cell or a genetically engineered effector.
• the genetic elements of the effector cell can be modular and/or the effector cell can include multiple genetic elements to yield an engineered effector cell having the capacity to serve as a vector for a variety of in vitro, ex vivo, and in vivo applications.
• Such effector cells can be modular in that parts can be conserved, and parts can be changed for different applications.
• the modularity can be used to combine different receptor elements with different effector elements, and which allows for reprogramming the genetically engineered effector cells to target diseases with known biomarkers, such as cancer, viral infections, and/or autoimmune disorders.
• the genetically engineered effector cells can be used for therapeutics and treatment methods that self-regulate the therapeutic response upon stimulation by the disease cells and that are applicable to a variety of cell-based diseases, including cancers, emerging pathogens, and others that evade the immune system or involve its malfunction.
• Multiple types of such genetically engineered effector cells, such as genetically engineered T-cells provide a robust, reproducible cellular system to therapeutically target complex diseases in vivo.
• Such genetically engineered effector cells also provide a reliable in vivo imaging technology and a reliable, in vitro sensor technology in a variety of applications.
• the genetically engineered effector cell is modular and antigen-specific. Antigen-specificity can be used to overcome tumor resistance and directs the cytolytic function toward different antigen- presenting target cells, such as host cells of a human or other organism. Further, the artificial cell-signaling pathway of such genetically engineered effectors cells can introduce the capability to serve as vector by producing calibrated amounts of protein-based therapeutics and inducting intended autocrine and paracrine signaling, upon the genetically engineered effector cell engaging the target antigen.
• the genetically engineered effector cell can allow for focused synthesis of the biologies at the target site and/or extend treatment duration for better patient outcome by limiting systemic toxicity. Embodiments are not limited to therapeutics, and other types of effector proteins can be produced.
• CD4 T-cell line was transformed into a vector for engaging antigen-presenting target cells and to trigger the synthesis of calibrated amounts of engineered proteins in situ, herein sometimes referred to as “effector proteins”.
• effector proteins The genetically engineered effector cell can provide an allogenic living vector that is modular, as described above.
• a “genetically engineered effector cell” includes and/or refers to a T-cell that is genetically engineered or modified to comprise a (i) receptor element, (ii) actuator element, and (iii) effector element, each of which can be modular.
• the terms “modular” and “modularity” include and/or refer to the versatility associated with recombinant sequence domains and the resulting recombinant polypeptides when assembled in various combinations for introduction into an engineered effector cell.
• receptor element includes and/or refers to a polynucleotide sequence encoding a transmembrane receptor, such as a CAR, capable of a specific interaction with a target cell.
• the receptor element can be reprogrammed by exchanging the single chain variable fragment (scFV) portion and/or of CAR for an extracellular antigen binding domain specific for a different disease-associated antigen or other targets.
• scFV single chain variable fragment
• receptor elements that can be used include, without limitation, CARs having specificity for antigens associated with autoimmune disorders, CARs having specificity for antigens associated with neural disorders (e.g., PTSD, Parkinson’s disease, Alzheimer’s disease), ligand-gated GPCRs (e.g., GPR1 Glucose receptor), light-gated ion channels (e.g., melanopsins, rhodopsins, photopsins), pressure sensing ion channels (e g., TRPV1, TRPV2), and ligand-gated ion channels.
• CARs having specificity for antigens associated with autoimmune disorders CARs having specificity for antigens associated with neural disorders (e.g., PTSD, Parkinson’s disease, Alzheimer’s disease)
• ligand-gated GPCRs e.g., GPR1 Glucose receptor
• light-gated ion channels e.g., melanopsins, r
• actuator element includes and/or refers to a polynucleotide sequence encoding a transcription factor binding site that initiates transcription and translation events downstream of a triggering signal (e.g., binding of the sensing element to a target antigen).
• a triggering signal e.g., binding of the sensing element to a target antigen.
• the underlying molecular mechanism of the actuator element is based on the intracellular calcium [Ca 2+ ]i dynamics, a mechanism used by almost all types of cells to regulate their functions.
• exemplary response elements include, without limitation, NF AT (“nuclear factor of activated T-cells") response element (NFAT-RE), serum response element (SRE), and cyclic AMP response element (CRE).
• effector element includes and/or refers to a polynucleotide sequence encoding an effector protein, and in some instances, an effector protein operably linked to a signal peptide.
• the polynucleotide sequence encoding the effector protein can be, for example, a sequence derived from a human gene, a sequence derived from a gene of a non-human species, a recombinant sequence, a sequence encoding a detectable reporter molecule, a sequence encoding a detectable imaging molecule, a sequence encoding a therapeutic molecule, among others.
• the genetically engineered effector cell into which the receptor element, the actuator element, and the effector element are introduced can be any T-cell ty pe including human T-cells or non-human T-cells (e.g., mammal, reptiles, plants, among others).
• the genetically modified cellular "source" of the modular elements provides a cellular chassis or frame providing, among other things, transcriptional and translational machinery' for expression and presentation of the receptor element, the actuator element, and the effector element.
• the T-cells can be from a source (e.g., a first human), modified, and administered to an organism that is different than the source (e.g., the host which is a second human).
• the T-cells can be from the source (e.g., a first human), modified, and administered back to the source (e.g., the source is the host).
• FIG. 1 illustrates an example genetically engineered effector cell comprising an isolated CD4 T-cell, in accordance with the present disclosure.
• the genetically engineered effector cell 100 can be modular in that elements can be adjusted for different target cells and to synthesize different effector proteins.
• the genetically engineered effector cell 100 can be formed from and/or include an isolated CD4 T-cell.
• the CD4 T-cells can be isolated from other types of cells prior genetically modifying the T-cells or during, such as byusing a selective expansion process.
• CD3 T-cells can be used, which contain a mixture of both CD4 T-cells and CD8 T-cells generally with a higher volume of CDS T-cells as compared to CD4 T-cell.
• the CD4 T-cells can then be isolated from the other cells.
• CD4 T-cells have higher propensity for transduction and expansion, among higher effector secretion and activity, as compared to CD8 T-cells or CD3 T-cells when forming genetically engineered effector cells.
• CD4 T-cells grow at greater rates and/or efficiencies when isolated and then grown than when grown in a mixture, such as with CD3 T-cells.
• Some embodiments include a selective expansion process involving use of particles (e.g., beads) coated with a protein (e.g., against which the engineered CD4 T- cell has a CAR or is engineered with a binding component, such as a peptide tag or short peptide sequence) and anti-CD28 antibody.
• particles e.g., beads
• a protein e.g., against which the engineered CD4 T- cell has a CAR or is engineered with a binding component, such as a peptide tag or short peptide sequence
• anti-CD28 antibody e.g., anti-CD28 antibody
• the genetically engineered effector cell 100 comprises an exogenous polynucleotide sequence that includes, in operative association, a receptor element 102, an actuator element 106, and an effector element 110, which can optionally include the signal peptide 114 and which optionally be on a single construct.
• the receptor element 102 encodes a CAR 104.
• a CAR is sometimes called a “chimeric receptor”, a “T-body”, or a “chimeric immune receptor.”
• a CAR includes and/or refers to an artificially constructed hybrid protein or polypeptide comprising extracellular antigen binding domain(s) 103 of an antibody (e.g., scFv) operably linked to a transmembrane domain 105 and at least one intracellular signaling domain 107.
• the CAR 104 includes an extracellular antigen binding domain 103 operably linked to the transmembrane domain 105, and the intracellular signaling domain 107.
• the CAR 104 can be designed to identify a surface antigen of a target, such as a target cell of a host.
• the CAR 104 can mobilize internal Ca +2 stores for intracellular Ca +2 release in response to antigen binding.
• the extracellular antigen binding domain 103 of the CAR 104 can recognize an antigen on a surface of a target cell, such as diseased cells of a host.
• the CAR 104 is configured to cause a rise in calcium in response to the extracellular antigen binding domain 103 binding to the antigen of the target cell and the transmembrane domain 105 is configured to bind to a transcription factor protein that is triggered by the rise in calcium and is translocated into the nucleus of the genetically engineered effector cell 100.
• the extracellular antigen binding domain 103 includes and/or refers to a polynucleotide sequence that is complementary to the surface antigen of the target cell.
• the extracellular antigen binding domain 103 can bind to the surface antigen of a target cell, as described above.
• the transmembrane domain 105 includes and/or refers to a polynucleotide sequence encoding a transmembrane segment of a transmembrane protein, e.g., a type of membrane protein that spans the membrane of a cell, such as the membrane of the genetically engineered effector cell 100.
• the transmembrane domain 105 can be derived from a natural polypeptide, or can be artificially designed.
• a transmembrane domain 105 denved from a natural polypeptide can be obtained from any membrane-binding or transmembrane protein.
• a transmembrane domain of a T-cell receptor a or P chain a CD3 chain, CD28, CD3e, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or a GITR can be used.
• the intracellular signaling domain 107 includes and/or refers to a polynucleotide sequence encoding any oligopeptide or polypeptide known to function as a domain that transmits a signal to cause activation or inhibition of a biological process in a cell.
• Example intracellular signaling domains include an intracellular signaling portion of a CD28, an intercellular signaling portion of a 4-1BB, and an intracellular signal portion of a CD3-zeta.
• the intracellular signaling domain 107 does not include an intracellular signaling portion of CD28.
• the intracellular signaling portion of CD28 does not work well with the CD4 T-cell as the chassis for the genetically engineered effector cell 100.
• the intracellular signaling domain 107 can include the intercellular signaling portion of 4-1BB, the intracellular signal portion of CD3-zeta, or a combination thereof.
• the intracellular signaling domain 107 includes the intercellular signaling portion of 4- IBB and the intracellular signal portion of CD3-zeta.
• embodiments are not so limited and can include other types and combinations of intracellular signaling domains.
• the intracellular signaling domain 107 can encode any molecule that can transmit a signal into a cell when the extracellular antigen binding domain 103 present within the same molecule binds to (interacts with) an antigen.
• the extracellular antigen binding domain 103 of a CAR 104 has specificity for a particular antigen expressed on the surface of a target cell of interest.
• the extracellular antigen binding domain 103 capable of binding to an antigen includes any oligopeptide or polypeptide that can bind to the antigen, and includes, for example, an antigen-binding domain of an antibody and a ligand-binding domain of a receptor.
• the extracellular antigen binding domain 103 binds to and interacts with the antigen, for example, an antigen present on a cell surface, and thereby imparts specificity to an genetically engineered effector cell 100 expressing the CAR 104.
• the receptor element 102 encodes a CAR 104 comprising an extracellular antigen binding domain 103 having specificity for Folate-Receptor alpha (FRa), which is an antigen found to be overexpressed on vanous cancers including ovarian, cervical, lung, breast, kidney, and brain.
• FRa Folate-Receptor alpha
• Other chimeric antigen receptors appropriate for use as the antigen binding portion of the receptor element 102 include those having specificity for a subset of immune cells, for one or more tumor antigens, and/or for one or more viral antigens.
• the actuator element 106 encodes a transcription factor binding site 108.
• the transcription factor binding site 108 includes and/or refers to binding site for a protein that upregulates synthesis of an effector protein 112 in response to the extracellular antigen binding domain 103 of the CAR 104 binding to the antigen of the target cell.
• the transcription factor binding site 108 can bind to transcription factors as triggered by [Ca 2+ ], which as described above, are caused to release in response to the antigen binding.
• the transcription factor binding site 108 is selected from a nuclear factor of activated T-cell (NF AT) response element (NFAT- RE), a serum response element (SRE), a cyclic AMP response element (CRE), and a combination thereof.
• NF AT nuclear factor of activated T-cell
• SRE serum response element
• CRE cyclic AMP response element
• a plurality of transcription factor binding sites can be encoded, such as between 1 and 10, between 2 and 10, between 3 and 10, between 5 and 10, between 2 and 8, between 2 and 6, between 3 and 6, 5, or 6 (e.g., 6 NFAT-REs), among other ranges or numbers.
• the actuator element 106 can thereby include a sequence for binding the factors triggered by [Ca 2+ ], and can trigger amplified synthesis of the effector protein 112 in response to the [Ca 2+ ]irise.
• the actuator element 106 encodes an NF AT transcription factor binding site for a transcription factor protein.
• NF AT transcription factor family consists of five members NFATcl, NFATc2, NFATc3, NFATc4, and NFAT5.
• NFATcl through NFATc4 are regulated by calcium signaling.
• Calcium signaling is critical to NF AT activation because calmodulin, a well-known calcium sensor protein, activates the serine/threonine phosphatase calcineurin.
• the underlying molecular mechanism of this strategy' is based on intracellular Ca 2+ ([Ca 2+ ]i) dynamics (as further shown by FIG. 2A).
• the [Ca 2+ ]i dynamics are common to almost all cell types, and the approach is thus broadly applicable.
• Ca 21 1 rise from CAR-mediated stimulation of cells leads to dephosphorylation of the nuclear factor of an activated effector cell 100 proteins (through Ca +2 /calmodulin-dependent serine phosphatase calcineurin), which then translocates to the nucleus and interacts with the NFAT-RE to upregulate expression of the effector protein 112.
• the NFAT-RE also performs its natural function of inducing IL-2 in the activated genetically engineered effector cell 100 that regulates clonal expansion proportional to the disease burden.
• the expression of a NFAT-RE induced reporter protein can also be used to quantitatively assess the level of activation of a genetically engineered effector cell 100.
• the effector element 110 encodes the effector protein 112, and in some instances, encodes the effector protein 112 operably linked to a signal peptide 114.
• the signal peptide 114 is upstream of the effector protein 112.
• the signal peptide 114 can be non-native to the effector protein 112.
• the effector protein 112 can be unable to secrete into the extracellular environment without the addition of the signal peptide 114 or can be modified to include a signal peptide 114 that allows for the effector protein 112 to secrete more efficiently than with its native signal peptide.
• the effector protein 112 includes a native signal peptide.
• the effector protein 112 can (natively) include the signal peptide 114.
• the terms “secretor”, “secretory peptide”, and “signal peptide” are used interchangeable and include and/or refer to a peptide that assists or directs the synthesized effector protein 112 into the extracellular environment (e.g., assists with translocating the effector element 110).
• the signal peptide 114 can be operably linked or fused to the effector protein 112 for release into the extracellular environment. In this manner, the signal peptide 114 can direct movement of the effector protein 112 outside of the genetically engineered effector cell 100.
• a signal peptide 114 is particularly advantageous when included in the genetically engineered effector cell 100 expressing an effector protein 112 that is unable to and/or minimally-able to translocate natively, where the effector protein 112 may remain inside the genetically engineered effector cell 100 in the absence of the signal peptide 114 and/or can translocate at a rate below a threshold.
• signal peptides are located at the N-terminus of nascent secreted proteins and characteristically have three domains: (1) a basic domain at the N-terminus, (2) a central hydrophobic core, and (3) a carboxy-terminal cleavage region. Any appropriate signal peptide can be used.
• the signal peptide 114 can be the signal peptide of Interleukin-6 (IL-6) or Interleukin-2 (IL-2).
• the effector protein 112 is a therapeutic protein.
• the therapeutic protein can act directly on the target cell, in some embodiments. In other embodiments, the therapeutic protein can act on cells adjacent to the target cell or on non-cellular components.
• Example therapeutic proteins include a cytotoxic protein, an immunostimulatory protein, and an immunosuppressive protein.
• the actuator element 106 is bound to the effector element 110.
• the exogenous polynucleotide sequence 101 includes the actuator element 106 bound to the effector element 110 bound to the receptor element 102.
• the exogenous polynucleotide sequence 101 can include the actuator element 106 bound to and upstream from the effector element 110, and the effector element 110 bound to and upstream from the receptor element 102, wherein the signal peptide 114 is upstream from the effector protein 112.
• FIG. 2A illustrates an example of a genetically engineered effector cell 200 and a sequence of events 220 triggered when in a diseased environment, in accordance with the present disclosure.
• the genetically engineered effector cell 200 can be used as or act as a living vector to synthesize the effector protein 212 using the artificial cell-signaling pathway and/or to trigger a sequence of events 220.
• the genetically engineered effector cell 200 synthesizes the engineered effector protein 212 in situ upon interacting with the antigen-presenting target cell, as further described herein.
• FIG. 2B illustrates effector cells formed from a CD4 T-cell 221 -A and from a CD8 T-cell 221-B in an environment containing a target cell 225 with antigens 227-A, 227-B on the surface of the target cell 225.
• the effector proteins 212 are generated.
• the CD4 T-cell-based effector cell 221 -A can transduce at least 3 times more, expand two times faster, and express five times more effector protein 212 than the CD8 T-cell-based effector cell 221-B.
• exposing the plurality of CD4 T-cells to the exogenous polynucleotide sequence can include use of a vector carrying the exogenous polynucleotide sequence.
• the vector can be associated with or include a viral or a non-viral carrier or approach, such as a Transposon-Transposase system, CRISPR/Cas system, TALEN system, ZFN system that may be mediated by a transfection system (e.g., electroporation, lipid nanoparticles), as previously described.
• the serum-free culture medium can contain between about 4 micrograms (pg)/milliliter (mL) and about 8 pg/mL of polybrene; however, embodiments are not so limited and can include between about 5 pg/mL to about 12 pg/mL, about 5 pg/mL and 10 pg/mL, about 5 pg/mL and about 8 pg/mL, about 6 pg/mL and about 8 pg/mL, or about 8 pg/mL of polybrene, among other ranges.
• the culture medium can be a complete grow th medium.
• the transformation process can include a particular concentration of cells (e.g., cell density of IxlO 6 cells/mL) and/or the transformation process can include confining the total transformation reaction volume (e.g., T-cells + lentivirus or other vector carrying the exogenous polynucleotide sequence + culture medium) in defined sub-volumes for a period of time.
• the total transformation reaction volume can include and/or refer to a total volume of fluid containing the T- cells, the exogenous polynucleotide sequence, and including culture medium (e.g., transformation culture medium and others fluid), such as the total volume of fluid(s) used to engineer the T-cells.
• the cell concentration e.g., density' of activated T- cells
• confinement of the total transformation reaction volume to sub-volumes can impact the transduction (or other type of transfomiation) yield by optimizing exposure to the exogenous polynucleotide sequence, such as by increasing the interaction of the virus or other vector with the CD4 T-cells.
• exposing the plurality of CD4 T-cells to the virus (e.g., lentivirus) or other type of vector can include providing a total transformation reaction volume including a cell density of between about 0.05xl0 6 cells/mL and about 3x10 6 cells/mL of the plurality of CD4 T- cells, the culture medium, and the lentivirus (or other vector carrying the sequence) in defined sub-volumes for a period of time.
• the total transformation reaction volume can include the cell density of about 1x10 6 cells/mL of the activated plurality of T-cells, the culture medium, and the lentivirus at a multiplicity of infection (MOI) of between about 0.1 and about 10.
• the MOI can be between about 1 and about 10, about 5 and about 10, about 1 and about 8, about 5 and about 8, about 8 and about 12, or about 10, among other MOIs.
• the cell density of T-cells used during the transformation process can include between about 0.05xl0 6 cells/mL and about 3xl0 6 cells/mL, about 0.25xl0 6 cells/mL and about 3xl0 6 cells/mL, about 0.25xl0 6 cells/mL and about 2xl0 6 cells/mL, about 0.5xl0 6 cells/mL and about 2xl0 6 cells/mL, about 0.5xl0 6 cells/mL and about IxlO 6 cells/mL, about 0.05xl0 6 cells/mL and about 2x10 s cells/mL, about 0.05xl0 6 cells/mL and about 1x10 s cells/mL, or about 1x10 6 cells/mL of the plurality of CD4 T- cells (which may be activated or not), among other ranges.
• the concentration of cells can impact the transduction or other transformation yield. Without being bound by theory, this may be due to the random movement of particles in a given volume of fluid increasing interactions between particles (e.g., the collision theory). It may also be central to increasing the virus-cell contact during transduction. Increasing cell concentration while keeping MOI constant in a given volume can increase virus-cell interaction due to steric reasons (with increase in vims and cell number) and can increase transduction yield.
• the defined sub-volumes can include drop volumes, such as spheres or other shapes which are kept in contact with the vector, such as a viral vector.
• the total transformation reaction volume can include between about 0.5 mL and about 2 mL and the defined sub-volume can include between about 0.05 mb and about 0.25 mb In some embodiments, the total transformation reaction volume can include about 1 mb and the defined sub-volume can include about 0.1 mL.
• the period of time can be between about 10 hours and about 24 hours, about 10 hours and about 20 hours, about 15 hours and about 24 hours, about 15 hours and about 16 hours, or about 16 hours. Embodiments can include other variations, values, and ranges.
• the method 450 includes 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 carrying the exogenous polynucleotide sequence, the plurality of genetically engineered effector cells being configured to activate and, to synthesize and secrete the effector protein responsive to the extracellular antigen binding domain of the CAR binding to the antigen of the target cell.
• cell expansion can refer to and/or include cell proliferation.
• the expansion culture medium can include a complete growth medium.
• Complete growth medium can provide the necessary nutrients at optimal proportion that enables optimal cell growth.
• the complete growth medium can contain a cytokine.
• the cytokine can include interleukin (IL)-2, IL-7, IL- 15, or a combination thereof.
• the expansion culture medium can include a complete growth medium containing IL-7 and IL-15.
• expanding the activated plurality of CD4 T-cells can includes diluting a total transformation reaction volume with the expansion culture medium containing the cytokine for a period of time and at a particular cell density of the plurality of activated and transduced plurality' of CD4 T-cells.
• the cell density can include between about 0.25xl0 6 cells/mL and about IxlO 6 cells/mL of the plurality of activated plurality of CD4 T-cells and which is maintained over a period of time of about 14 days.
• the period of time can be between about 10 days to about 20 days.
• the cell density can be between about 0.5xl0 6 cells/mL and about 1x10 6 cells/mL, about 0.25xl0 6 cells/mL and about 0.5xl0 6 cells/mL, or about 0.5xl0 6 cells/mL, among other ranges.
• the expansion can include periodically changing at least a portion of the expansion culture medium over the period of time and while maintaining the cell density. For example, the expansion culture medium can be changed every day, every other day, or every third day, among other times and over the period of time.
• the method 450 can include adding an additive to the (transformation) culture medium and/or the expansion culture medium.
• the additive can include an antiviral inhibitor and/or a latency reversal agent.
• T-cells are not limited to CD4 T-cells and can include other types of primary T-cells such as CD3 T-cells or isolated CD8 T-cells.
• FIG. 5 shows such an example method 560.
• the method 560 includes activating a plurality of T-cells using a plurality of particles.
• the plurality of T-cells can include CD3 T-cells, isolated CD4 T-cells, or isolated CD8 T-cells.
• the plurality of T-cells can be thawed, e.g., are frozen and thawed prior to activation, or can be fresh.
• the method 560 can include resuspending the plurality of T-cells in a complete growth medium and activating the plurality of T-cells by adding the plurality of particles to the complete growth medium.
• the plurality of T-cells can be activated by exposing the plurality of T-cells to the plurality of particles loaded with anti-human CD3 and anti-human CD28 antibodies.
• the plurality of T-cells can be exposed to the plurality of particles at a cell-to-particle ratio of between about 6: 1 and about 1:6 for a period of time, such as between about 10 hours and about 36 hours.
• a cell-to-particle ratio of between about 6: 1 and about 1:6 for a period of time, such as between about 10 hours and about 36 hours.
• Embodiments are not so limited and may include any of the above-described ranges and variations for the culture medium, cell-to-particle ratio, and/or periods of time, among other variations.
• the method 560 includes exposing the plurality of T-cells to an exogenous polynucleotide sequence in a culture medium to engineer (e.g., transform and/or introduce the exogenous polynucleotide sequence into) the plurality of T- cells.
• the engineering may occur before or after the activation of the plurality of T- cells.
• the exogenous polynucleotide sequence can include at least some of substantially the same features and components as previously described by the genetically engineered effector cell 100 of FIG. 1, the details of which are not repeated for ease of reference.
• the transcription binding site can be a NFAT-RE and the intracellular signaling domain may not include an intracellular portion of CD28 (e.g., may include an intracellular portion of 4-1BB and an intracellular signaling portion of a CD3 zeta).
• the transcription binding site can be SRE and/or CRE, and the intracellular signaling domain can include intracellular portion of CD28 (e.g., intracellular portion of CD28 and an intracellular signaling portion of a CD3 zeta, or intracellular portion of CD28, an intracellular portion of 4- IBB, and an intracellular signaling portion of a CD3 zeta).
• a vector can carry the exogenous polynucleotide sequence.
• the vector may include or be associated with a viral vector, a transposon system, or lipid nanoparticles.
• the transformation process can include exposing the plurality of T-cells to a vims carrying the exogenous polynucleotide sequence, such as a viral vector.
• the method 560 can include exposing the plurality of T-cells to the virus that includes a lentivirus, such as lentivirus particles carrying the polynucleotide sequence, in some embodiments.
• exposing the plurality of T-cells to the virus includes exposing between about ,05xl0 6 cells/mL and about 3xl0 6 cells/mL of the plurality of T-cells, which may be activated or not, to the vims in the culture medium which is serum- free and contains polybrene.
• the culture medium contains between about 4 pg/mL and about 8 pg/mL of polybrene, however embodiments are not so limited.
• the transformation can occur at particular cell concentrations and/or using defined sub-volumes.
• exposing the plurality of T-cells to the exogenous polynucleotide sequence can include providing a total transformation reaction volume including a cell density of between about 0.05xl0 6 cells/mL and about 3 xlO 6 cells/mL, among the other ranges listing about, of the plurality of T-cells, the culture medium, and the vims or other type of vector in defined sub-volumes for a period of time and at a MOI of betw een about 0.1 and about 10.
• method 560 can include resuspending the lentivims particles in the culture medium sufficient to achieve the MOI of between about 0.1 and about 10.
• Embodiments are not so limited and may include any of the above-described ranges for the cell concentration, vectors, total transformation volume, sub-volumes, polybrene concentration, types of culture medium, and/or MOI, among other variations described herein and combinations thereof.
• providing the defined sub-volumes includes placing aliquots as drop volumes in a tissue-cultured well plate and placing the cultured well plate in an incubator for the period of time.
• the sub-volumes can be placed on surface(s) of a substrate or substrates which are hydrophobic or hydrophilic, and the sub-volumes can include different shapes, such as spheres.
• the method 560 includes expanding the activated plurality of T-cells in an expansion culture medium to form a plurality of genetically engineered effector cells comprising T-cells carrying the exogenous polynucleotide sequence, the plurality of genetically engineered effector cells being configured to activate and, to synthesize and secrete the effector protein responsive to the extracellular antigen binding domain of the CAR binding to the antigen of the target cell.
• expanding the activated and transduced plurality of T-cells comprises diluting a total transformation reaction volume with the expansion culture medium for a period of time (e.g., 14 days) and at a cell density of about 0.25xl0 6 cells/mL and about 1x10 6 cells/mL of the plurality of T-cells, wherein the expansion culture medium is a complete growth medium containing a cytokine.
• the cytokine can include IL-2, IL-7, IL-15, or a combination thereof, such as IL-7 and IL-15.
• the period of time can include between about 10 days and about 20 days and the method 560 further includes periodically changing at least a portion of the expansion culture medium over the period of time and while maintaining the cell density of about 0.25xl0 6 cells/mL and about IxlO 6 cells/mL.
• Embodiments are not so limited and may include any of the above-described ranges for the cell concentration, expansion culture medium, and cytokines.
• the method 560 further include adding an additive to at least one of the (transformation) culture medium and the expansion culture medium.
• the additive can include an antiviral inhibitor, a latency reversal agent, and a combination thereof, as previously described.
• the kit 670 can further include a vector (or other carrier) 673 carry ing the exogenous polynucleotide sequence 674.
• the vector 673 can include a viral vector, a transposon system, or lipid nanoparticles, as previously described.
• the genetically engineered effector cells provided herein can be administered by any appropriate route, including, without limitation, administration intravenously , intratumorally, intramuscularly, subcutaneously, intraperitoneally, intraarterially, or into an afferent lymph vessel, by parenteral administration, for example, by injection or infusion.
• the effector cells can be cells that are allogeneic or autologous to the host, such as a mammal.
• the effector cells are autologous to the host.
• a host to which genetically engineered effector cells are provided is monitored or assessed for increased (e.g., improved, more robust) tumor clearance. Accordingly, various embodiments are directed to methods used for cancer therapies. In some embodiments, a host to which genetically engineered effector cells are provided is monitored or assessed for clearance of cells expressing a particular antigen.
• the genetically engineered effector cell comprises a polynucleotide sequence encoding a therapeutic protein place of, or in addition to, the polynucleotide sequence encoding the detectable reporter protein; and is fused with a signal peptide (sec) on the 3’ end of the polynucleotide sequence to assist in extracellular transport.
• a signal peptide sec
• expression of a therapeutic protein is induced.
• the method can include the localized production of a therapeutic protein at the site of the target cell (e.g., a tumor cell, infected cell) and extracellular secretion of the therapeutic protein in the disease microenvironment.
• Some embodiments are directed to methods for using genetically engineered effector cells as a sensor technology in a variety of applications.
• transfusion-mediated spread of emerging flavivirus pathogens e.g., Zika virus (ZIKV), dengue virus (DENV)
• ZIKV Zika virus
• DENV dengue virus
• Clinical symptoms manifest in only 20% of ZIKV infections, and there are no reliable commercially available ZIKV diagnostic test kits for use outside the clinical laboratory. Identifying the infection is therefore challenging, especially given the similarity of symptoms with those of other diseases and the cross-reactivity of antibodies with other arboviruses (e.g., dengue, chikungunya).
• a method comprising contacting a genetically engineered effector cell comprising a CAR having an antigen binding domain for detection and binding to an antigen specific to the virus of interest to a sample comprising or suspected of comprising cells infected with the virus of interest, and NFAT-RE regulated reporter transgene to inform the presence of the cells infected with the virus of interest.
• genetically engineered effector cells can be loaded with enzymatically activatable prodrugs, where the drug-activating enzyme is synthesized only at the tumor location, thus providing localized transformation of the prodrug into its active form.
• the prodrug may not be loaded into the effector cells, and can be infused in multiple doses subsequent to the infusion of the genetically engineered effector cells.
• the prodrug can alternatively be bound to an imaging nanoparticle or other means of image-guided means of active drug delivery.
• Attaching the prodrug to an imaging nanoparticle or engineering the effector cells to express imaging transgenes enables the engineered effector cells to guide appropriate staging of the patient in preparation of surgery and for visually identifying and/or imaging tumor margins to assist in cytoreductive surgery.
• Some embodiments are directed to methods of localized delivery of a chemotherapeutic agent to a site of the disease (e.g., tumor mass, site of autoimmune disease) comprises contacting a genetically engineered effector cell to a host cell population, wherein the genetically engineered effector cell comprises (i) an exogenous polynucleotide sequence encoding a CAR comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain; and (ii) a NF AT response element operably linked to a polynucleotide sequence encoding an enzyme, wherein, in the presence of the target host cell in the contacted cell population, the genetically engineered effector cell binds to a surface molecular antigen on the target host cell and activates the NF AT response element to initiate expression of the enzyme, which acts on the prodrug predesigned to be activated by this enzyme and uses it membrane permeability due to its hydrophobicity to be released at the site of the disease.
• genetically engineered effector cells are used for non- invasive detection and imaging of tumors based on expression of an imaging enzyme (e.g., thymidine kinase is capable of trapping a radioactive probe or otherwise detectable probe; tyrosinase detected by photoacoustic imaging or magnetic resonance imaging) expressed when tumor-specific CAR effector cells engage the antigen on tumor cells.
• an imaging enzyme e.g., thymidine kinase is capable of trapping a radioactive probe or otherwise detectable probe; tyrosinase detected by photoacoustic imaging or magnetic resonance imaging
• genetically engineered effector cells can be used to circumvent safety concerns associated with vaccines against flaviviruses.
• antigenic diversity among the four different dengue virus serotypes is responsible for the lack of antibody-mediated immunity and allows for multiple sequential infections.
• effector cells can circumvent these safety concerns with flaviviruses because the effector cells, as described herein, can be engineered to express an antiviral protein, from human or non-human or synthetic origin, upon detecting the viral E glycoprotein (Egp) expressed on the surface of cells infected by the virus.
• Egp viral E glycoprotein
• genetically engineered effector cells comprise a CAR that detects a cancer-specific antigen on a target cancer cell (e.g., a HPV E6 or E7 antigen in case of cervical cancer) and aNFAT-RE to drive the expression of a reporter protein as described above. Such embodiments can be used for early detection of cancer.
• the genetically engineered effector cells comprise a CAR that detects an antigen on a pathogen-infected cell (e.g., detecting a ZIKV or DENV E glycoprotein on Zika- or dengue virus-infected cell) and a NF AT response element to induce expression of a reporter polypeptide.
• Such embodiments can be used for transfusion medicine to detect the presence of emerging pathogens (e.g., Zika, dengue, West Nile, Yellow Fever).
• Different CARs can be used in genetically engineered effector cells with NFAT-RE regulated reporters to detect and measure signal-to-noise ratio to guide the selection of appropriate CARs for a cell-based therapy that exert the intended therapeutic effect without exhibiting unintended side-effects.
• Mammalian cells can be engineered as effector cells to comprise a glucose- sensing GPCR (GPR1) which mobilizes internal Ca 2+ stores and NFAT response element-regulated to express engineered insulin.
• GPR1 glucose- sensing GPCR
• Such engineered effector cells can be used for autonomous synthesis of insulin upon sensing glucose.
• Such embodiments can be used for beta-cell replacement therapy.
• Other non-limiting example uses of the genetically engineered effector cells include: i) imaging of the location of disease microenvironments to assist in surgical resection or monitor disease progression/regression; ii) cytotoxicity to kill the disease cells; iii) proliferation to enhance T-cell persistence; iv) immune-stimulation to recruit other immune cells; v) chemokine to recruit other immune cells; vi) immunosuppression to create localized immunosuppressive microenvironment; and vn) regeneration to enhance tissue healing.
• a target cell (sometimes herein interchangeably referred to as a “target cell of a host”, “target cell of interest”, “a diseased cell”, or “a target disease cell”) includes and/or refers to a cell of interest associated with a living organism (e.g., a biological component of interest).
• An antigen of the target cell includes and/or refers to a structure (e.g., binding site) of the target cell which the antigen binding domain of the receptor element can bind to (e.g., has an affinity for).
• the effector cell can be from a variety of different type of cells, such as human and non-human cells, and sometimes herein referred to as “the source”.
• the terms “genetically modified” and “genetically engineered” are used interchangeably and include and/or refer to a prokar otic or eukaryotic cell that includes an exogenous polynucleotide, regardless of the method used for insertion.
• the effector cell is modified to comprise a non-naturally occurring nucleic acid molecule that is created or modified by the hand of man (e.g., using recombinant deoxyribonucleic acid (DNA) technology) or is derived from such a molecule (e.g., by transcription, translation, etc.).
• An effector cell that contains an exogenous, recombinant, synthetic, and/or otherwise modified polynucleotide is considered to be a genetically engineered effector cell.
• Nucleic acid includes and/or refers to a “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered intemucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide.
• the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions. However, it may be suitable in some instances, as discussed herein, for the nucleic acid to comprise one or more insertions, deletions, inversions, and/or substitutions.
• the nucleic acid can encode additional amino acid sequences that do not affect the function of the CAR and polynucleotide and which may or may not be translated upon expression of the nucleic acid by a host cell.
• Nucleic acids can be obtained using any suitable method, including those described by Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., pp. 280-281 (1982) and/or U.S. Publication No. US2002/0190663, each of which are herein incorporated in their entireties for their teachings. Nucleic acids obtained from biological samples typically are fragmented to produce suitable fragments for analysis.
• Nucleic acids and/or other moieties can be isolated. As used herein, “isolated” includes and/or refers to separate from at least some of the components with which it is usually associated whether it is derived from a naturally occurring source or made synthetically, in whole or in part. Nucleic acids and/or other moieties of the invention can be purified. As used herein, “purified” includes and/or refers s separate from the majority of other compounds or entities. A compound or moiety can be partially purified or substantially purified. Purity can be denoted by a weight by weight measure and can be determined using a variety of analytical techniques such as but not limited to mass spectrometry, HPLC, etc. EXPERIMENTAL EMBODIMENTS
• FIGs. 7A-7E illustrate example polynucleotide sequences used to form genetically engineered effector cells, in accordance with the present disclosure.
• FIG. 7A-7E illustrate example polynucleotide sequences used to form genetically engineered effector cells, in accordance with the present disclosure.
• FIG. 7A illustrates an example polynucleotide sequence (SEQ ID NO: 1) that includes a receptor element encoding a CAR, with the CAR including an antigen binding domain (e.g., against Folate Receptor alpha (FRa)), a transmembrane (e.g., CD8), and an intracellular signal domain of CD28, 4-1BB and CD3 zeta (SEQ ID NO: 3).
• FIG. 7B illustrates an example polynucleotide sequence (SEQ ID NO: 2) that includes the receptor element of FIG. 7A, an actuator element (e.g., NFAT-RE 6x), and an effector element (e.g., Nluc- P2A-GFP, SEQ ID NO: 19).
• FIG. 1 illustrates an example polynucleotide sequence (SEQ ID NO: 1) that includes a receptor element encoding a CAR, with the CAR including an antigen binding domain (e.g., against Folate Receptor alpha (
• FIG. 7C illustrates an example polynucleotide sequence (SEQ ID NO: 4) that includes the actuator element (e.g., NFAT-RE 6x) and an effector element (e.g., Nluc-P2A-GFP) of FIG. 7B, with a receptor element encoding a CAR, the CAR including an antigen binding domain (e.g., against FRa), a transmembrane (e.g., CD8), and an intracellular signal domain of CD28 and CD3 zeta (SEQ ID NO: 5).
• FIG. 7D illustrates an example polynucleotide sequence (SEQ ID NO.
• FIG. 6 that includes the actuator element (e.g., NFAT-RE 6x) and effector element (e.g., Nluc-P2A-GFP) of FIG. 7B, with a receptor element encoding a CAR, with the CAR including an antigen binding domain (e.g., against FRa) a transmembrane (e.g., CD8), and an intracellular signal domain of 4-1BB and CD3 zeta (SEQ ID NO: 7 or SEQ ID NO: 20).
• the actuator element e.g., NFAT-RE 6x
• effector element e.g., Nluc-P2A-GFP
• FIG. 7E illustrates an example polynucleotide sequence (SEQ ID NO: 8) that includes a receptor element encoding a CAR, with the CAR including an antigen binding domain (e.g., against FRa), a transmembrane (e.g., CD8), and an intracellular signal domain of 4-1BB and CD3 zeta (SEQ ID NO: 7), an actuator element (e.g., NFAT-RE 6x), and an effector element of IFNP (SEQ ID NO: 9).
• an antigen binding domain e.g., against FRa
• a transmembrane e.g., CD8
• an intracellular signal domain of 4-1BB and CD3 zeta SEQ ID NO: 7
• an actuator element e.g., NFAT-RE 6x
• an effector element of IFNP SEQ ID NO: 9
• primary CD4 T-cells were engineered, e.g., transformed, into a zero-order cell-based effector cell capable of synthesizing engineered proteins at the disease site, and proportionate to the disease burden over extended periods of time.
• the CD4 T-cell lines have long persistence and propensities for higher transduction, faster expansion, and more productive transcriptional machinery.
• Experimental results showed that CD4 T-cells modified with a CAR using the 4-1BB intracellular domain and without the CD28 intracellular domain transduce approximately three times better as compared to CD8 T-cells.
• These effector cells, formed with the CD4 T-cells exhibited approximately two-fold expansion rates, produced five times more engineered protein, and displayed minimum cytolytic activity.
• the CD4 T-cellbased effector cell surpassed CD8 T-cells (e.g., produced more anti-tumor IFN-J3 than produced by CD8 T-cells) effectively suppressing ovarian cells grown in vitro and in VIVO.
• This technology allows for precise targeting of therapeutic biologies to disease sites while minimizing bioavailability in healthy tissues. Leveraging CD4 T-cells' extended persistence offers the potential to improve patient compliance and enhance disease management by reducing the frequency of drug administration and for human treatment.
• the CD4 T-cells compared to its CD8 counterpart, contain an enhanced capacity for transporting specific proteins to the disease site.
• the CD4 T-cells exhibited a three-fold improvement in transduction efficiency, doubling the rate of expansion, and a five-fold increase in the expression of target proteins.
• the efficacy of the CD4 T-cell-based delivery system was verified using a CAR that recognized (e.g., bind to) FRa as an antigenic target, prompting the effector cell to produce a bioluminescent reporter enzyme.
• the modular nature of the CAR allows for the redirection of the specificity of the platform to identify another antigen and induce the expression of a desired clinically relevant therapeutic protein.
• the CD4 T-cellbased effector cell was modified and further validated for delivering functional therapeutics, e.g., used to deliver interferon-
• functional therapeutics e.g., used to deliver interferon-
• FIGs. 8A-8H illustrate the effects of varying different factors on production and function of the T-cell based effector cell, in accordance with the present disclosure.
• CD4:CD8 7:1
• FIGs. 8A-8B show the results of CD3 T-cells engineered for delivery function when expanded for 25 days and assessed for distribution of CD4 and CD8 phenotypes on day 5 and day 25.
• FIGs. 8D-8E show the transduction efficiency of T-cells assessed by measuring FRa-CAR expression with flow cytometry 5 days after transduction.
• the results on the comparison of transduction efficiencies of CD4 and CD8 phenotypes further support use the CD4 phenotype for the delivery function.
• FIGs 8A-8D used a CAR that included the intracellular domains of CD28 and 4- IBB in addition to the CD3 zeta domain, as shown by the sequence of FIG. 7A.
• three combinations of the intracellular CAR domains were assessed for improving performance of CD4 T-cells to serve as a delivery platform.
• the results are shown in FIGs. 8E and 8F.
• the three CAR constructs included intracellular domains from (i) CD28 only (e.g., FIG. 7C) (28Q, (ii) 4-1BB only (BBQ (e.g., FIG. 7D), and (iii) CD28 and 4-1BB in tandem (28-BBQ (e.g., FIG. 7B).
• FIGS. 7A-7D Complete schematics of the individual CAR constructs are shown in FIGS. 7A-7D.
• FIGs. 8E-8F shows the results, which indicate that, while transduction efficiency (FIG. 8E) of the CD4 T-cell is independent of the combination of the intracellular domains used in the CAR, the expression level of the engineered protein (represented by a bioluminescent reporter enzyme, FIG. 8F), depends on these domains.
• the results showed that the BB CAR, e.g., the CAR that included the 4-1BB intracellular domain but not the CD28 intracellular domain of FIG. 7D, was more effective in inducing the engineered reporter enzyme in both CD4 and CD8 T-cell-based effector cells.
• the other two CARs (e.g., 28 ⁇ and 28-BB ⁇ of FIGs. 7B-7C) induced the reporter activity to a similar extent.
• the reporter enzyme activity induced by the BB ⁇ CAR was around five-fold more in the CD4 T-cell based effector cell compared to that in the CD8 T-cell based effector and was specific in response to the antigen-presenting target cell (FRa + OVCAR3 cell, in this case).
• the BB ⁇ imparts other traits in the CAR T-cells, such as increased persistence, reduced tonic signaling, and beter toleration by patients in terms of cytokine release syndrome and cell therapy- associated neurotoxicity.
• intracellular domains of 28 includes intracellular domains of CD28 and CD3-zeta (and genetically engineered effector cells were generated by transducing T-cells using the sequence as shown by FIG. 7C)
• intracellular domains of BBC includes 4-1BB and CD3-zeta (and genetically engineered effector cells were generated by transducing T-cells using the sequence as shown by FIG.
• intracellular domains of 28-BB ⁇ includes CD28, 4-1BB, and CD3-zeta (and genetically engineered effector cells were generated by transducing T-cells using the sequence as show n by FIG. 7B).
• the activity of the NFAT-RE-induced reporter (Nluc) was quantified to evaluate the impact of different intracellular domains on the delivery function of both CD4 and CD8 T-cell phenotype.
• the statistical analysis and p values were determined by one-way ANOVA and Tukey’s multiple comparison test, */? ⁇ 0.05, **** ? ⁇ 0.0001.
• FIG. 8G show around ten-fold higher activity (e.g., higher expression) of the bioluminescent reporter, validating that the CD4 T-cell-based effector cell has the stronger NFAT-based transcriptional machinery for a robust engineered function, which is impaired in the CD8 T-cells and is not due to the reduction in the number of antigen-presenting target cells. More particularly, FIG.
• FIG. 8G shows a comparison of Nluc activity in CD4 and CD8 T-cells engineered for the delivery function (with BB ⁇ CAR) when stimulated by microparticles.
• Microparticles were functionalized with anti-CD3 and anti-CD28 antibodies or with FRa antigen and anti- CD28 antibodies.
• Unstimulated CD4 and CD8 T-cells were used as negative controls.
• the Nluc activity is represented as a function of increasing number of engineered CD4 and CD8 T-cells where cell-to-particle ratio (represented as E:T) is constant (10: 1).
• E:T cell-to-particle ratio
• FIG. 8H shows the cytolytic function of donor-matched FRa-CAR CD4 and FRa-CAR CD3 T-cells (engineered for delivery function) against FRa+ tumor cells (A2780cis-FRa+Luc2+). Unmodified CD3 T-cells were used as negative control. Statistical analysis p values were determined by multiple comparison t-test using the Holm-Sidak method, ***p ⁇ 0.001. All results are represented as mean ⁇ SD. The data shown in FIG. 8H verifies that the CD4 T-cells engineered for delivery function exhibit minimum cytolytic activity.
• CD4 T-cell-based effector cells can be administered at an increased dose for a higher maximum recommended starting dose in the first-in-human clinical trials.
• the CD4 and CD8 T- cells both engineered for FRa specificity, were incubated with target cells engineered to present FRa (e.g., FRa + 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), which is incorporated herein by reference in its entirety for its teaching).
• the target cells were also engineered to express the Luc2® enzyme, an ATP-dependent bioluminescent reporter, that served as a live-cell marker.
• Nonengineered primary CD3 T-cells were used as a negative control.
• the results confirmed that the significantly low cytolytic activity in the CD4 T-cell-based effector cells compared to the CD3 T-cell-based effector cells (CD4:CD8 1.5: 1) (p ⁇ 0.01, at all E:T), and attests to selection of the CD4 T-cell as the suitable phenotype for use in a cell-based delivery system.
• the engineered CD4 T-cells can therefore be delivered at an increased tolerated dose to robustly express the desired protein without exhibiting undesired side effect of killing healthy cells that may be expressing basal levels of the target antigen.
• FIGs. 9A-9C illustrate the results of verifying the functionality the CD4 T-cellbased effector cells as a protein delivery platform in vivo, in accordance with various embodiments. The experiment schedule is detailed in FIG. 9A and the results are shown in FIGs. 9B-9C.
• the 12-day old xenograft tumors (antigen positive FRa + MSLN neg A2780cis, antigen negative FRa neg MSLN + A2780cis) were treated with 2xl0 6 CD4 T-cell engineered for in situ delivery (CAR-BB ⁇ -Nluc) on days 0, 1, 2, 3, and 4.
• the target specific delivery function e.g., Nluc activity (FIG. 9B) was imaged and quantified (FIG. 9C) at baseline (day 0) as well as on days 1, 2, 3, 4, and 5.
• the engineered CD4 T-cells with specificity for FRa antigen, exhibited the delivery function by Nluc reporter activity when stimulated by FRa + tumors, compared to when stimulated by non-target cells, e.g., FRa Iieg tumors , confirming the target-specific in situ delivery function.
• Negative controls included CD4 T-cells with the same inducible delivery function but without a CAR (e.g., no CAR-Nluc) and FRa-specific CD4 CAR (CAR-BBQ T-cells without the NFAT-RE inducible delivery function.
• FRa- specific primary T-cells engineered for the NFAT-RE inducible delivery function were i.p. injected in i.p. FRa + MSLN neg A2780cis or FRa neg MSLN + A2780cis tumor-bearing NSG mice at 24-hour interval 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 the delivery function.
• FIG. 9A is a schematic of dosing, treatment, and imaging schedules
• FIG. 9B includes representative bioluminescent images
• FIG. 9C show quantification results. All results are represented as mean ⁇ SEM. Statistical analysis and p values were determined by 2-way ANOVA and Tukey’s multiple comparison test, *p ⁇ 0.05.
• FIG. 10A The secretion of IFNP from the respective unstimulated CD3, CD4, and CD8 T-cells (engineered for IFNP delivery) was minimal and is shown in FIG. 10A.
• FIGs. 10B-10D show a dose-dependent growth-inhibitory effect of IFNP on various cell lines (OVCAR3, A2780cis, and HEK293T/17), when the cell lines were treated with the supernatant of CD4 and CD8 T-cells engineered to produce IFNp. While the growth-inhibitory effects were observed on OVCAR3 (FIG. 10B) and A2780cis (FIG. 10C), this effect was not observed on HEK293/T17 cells (FIG. 10D).
• FIGs. 10A-10H illustrate FRa-specific targeting of tumor cells by a CD4 T-cell engineered to secrete IFNP, in accordance with the present disclosure.
• FIG. 10A shows IFNP secretion from T-cells (CD4, CD8 and CD3) engineered for delivery function upon stimulation by FRa-antigen/anti-CD28 DynabeadsTM (cell-to-particle ratio of 1:3).
• FIG. 10A statistical analysis and p values were determined by 2-way ANOVA and Tukey’s multiple comparison test, **p ⁇ 0.01.
• FIGs. 10B-10D show the growth inhibitory effect of the secreted IFNP from CD4 and CD8 T-cell-based effector cells as was assessed on OVACR3 (FIG.
• FIGs. 10B-10D statistical analysis and p values were determined by multiple comparison t-test using Holm-Sidak method, **/? ⁇ 0.01.
• FRa-specific T-cells engineered for NFAT-RE induced IFNP delivery were i.p. injected (5 x 10 6 cells/dose) in i.p. FRa + Luc2 + A2780cis tumor-bearing NSG mice at 24- hr interval for 6 days and tumor luminescence was measured every 3-4 days to assess tumor growth.
• the i.p. administration of FRa-specific primary CAR T-cells (engineered without the NFAT-RE induced IFNP delivery function) or rhIFNP (0.25pg in lOOpL) served as control groups.
• FIG. 10E is a schematic of dosing, treatment, and imaging schedules
• FIG. 10F includes representative bioluminescent images
• FIGs. 10E-10H demonstrate the therapeutic utility of the IFNP producing CD4 T-cell-based effector cell.
• the IFNP producing CD4 T-cells were used to challenge antigen positive (FRa + Luc2 + A2780cis) tumors.
• the experiment schedule is detailed in FIG. 10E and the results are shown in FIGs. 10F-10H.
• FRa + Luc2 + A2780cis cells (2xl0 6 ) were i.p. implanted in NSG mice.
• the 12-day-old xenograft tumors were i.p.
• FRa-CAR + T-cells treated with 5x10 6 FRa-CAR + T-cells (with NFAT-RE inducible IFNP) daily for 6 days (day 13 - day!9).
• the therapeutic efficacy was assessed by imaging (FIG. 10F) and quantitatively comparing the tumor luminescence (FIG. 10G) with control groups (e.g., 5xl0 6 FRa- CAR + T-cells without NFAT-RE inducible IFN
• FIG. 10H show statistically significantly improved survival in mice treated with IFNP producing FRa-CAR + T-cells, when compared to the direct injection of rhIFNP and control FRa- CAR + T-cells that did not produce IFNp.
• the rhIFNP treatment did not show any survival advantage and all mice were sacrificed as a result of weight loss or distress.
• FIG. 11 illustrates flow cytometry plots showing the proportion of CD4 and CD8 T-cells in the pan CD3 T-cell population from healthy donors (e.g., 3 healthy donors at day 10 of in vitro expansion), in accordance with the present disclosure.
• FIG. 12 illustrates a comparison of CD4 and CD8 T-cell chemotaxis, in accordance with the present disclosure. CD4 and CD8 T-cells were compared for their migration toward chemotactic gradients of CCL5 and CCL17. Statistical analysis was performed by two-tailed Student’s t-test, ****/? ⁇ 0.0001.
• chemokine induced T-cell migration assay used in FIG. 12.
• a Boyden chamber Transwell® migration assay was performed to assess chemokine induced migration.
• Donor-matched CD4 and CD8 T-cells were thawed and stimulated with anti-CD3/CD28 DynabeadsTM (at 1 :3 celkparticle ratio).
• the cells were de-beaded and serum starved overnight by keeping the cells in 2% heat inactivated FBS containing media.
• Transwell® permeable inserts with a pore size of 5 pM were used for the assay and were pre-soaked in serum-free RPMI for 30 minutes in a cell culture incubator (37 degrees C, 5% CO2, 95% humidity).
• the serum starved CD4 and CD8 T-cells were counted and 5xl0 5 cells per insert were resuspended in 100 pL serum-free RPMI and seeded on to the top chamber of the insert.
• 650 pL of complete growth medium supplemented with 25 nanograms (ng)/mL CCL5 or 25ng/mL CCL17 was added to the bottom chamber to serve as chemoattractant.
• T- cell migration was performed for 4 hours in a cell culture incubator (37 degrees C, 5% CO2, 95% humidity). After incubation, the insert (top-chamber) was removed and the cells in the bottom chamber were quantified using CellTiter-Glo® reagent.
• Table 1 Resources Table [00244] (2) Preparations. Transfer plasmids with different genetic payloads were designed in SnapGene software and sub-cloned into the lentivector plasmid. Epoch Life Science, Inc. (Missouri City, TX) provided plasmid preparation services (chemical synthesis of DNA insert sequences, sub-cloning into respective vector backbones, and the amplification). Target cells: FRa + OVCAR3 and FRa + A2780cis engineered to express modified firefly luciferase (Luc2), as described in (i) C. E. Repellin et al., Modular Antigen-Specific T-cell Biofactories for Calibrated //?
• Phosphate buffered saline (PBS) without Ca +2 and Mg +2 was used to minimize cell clumping.
• PBS Phosphate buffered saline
• puromycin N-acetyltransferase was used as a selection marker and puromycin dihydrochloride (Puromycin) was used for selecting stable cell lines.
• Biotinylated human FRa protein was used to analyze FRa CAR expression on engineered primary' T-cells.
• HEK293T/17 producer cells (12xl0 6 ) were seeded into tissue culture treated T150 flasks in 21 mL complete DMEM supplemented with 10% heat-inactivated FBS and IX penicillin streptomycin solution and placed in a cell culture incubator (37 degrees C, 5% CO2, 95% humidity).
• transfer plasmid was co-transfected with 2 nd generation packaging plasmids (psPAX2, pMD2.G), and pAdV Antage plasmid at 4:3: 1 :0.4 weight (wt)-ratio, respectively (transfer plasmid: 12 pg, pxPAX2: 9 pg, pMD2.G: 3 pg, pAdV: 1.2 pg).
• Transporter 5TM transfection reagent was used following the manufacturer’s protocol (100 pL). Cell culture supernatant enriched with pseudo-viral particle was collected and replenished every 24 hours for 3 days (30 mL).
• the lentivector-enriched cell culture supernatant was clarified using a 0.45 m filter.
• the supernatant was clarified by transferring it to a polypropylene Konical ultracentrifugation tube and centrifuging at 20,700 Gravitation force (G) in an SW32-Ti rotor using a Beckman Coulter Optima XPN-90 ultracentrifuge at 4 degrees Celsius (C) for 2 hours.
• the resulting pellet was resuspended in 400 pL serum-free RPMI and aliquoted.
• An MOI of 10 when the lenti vector particles produced in this process, was used to transduce 1x10 6 cells.
• the lentivector aliquots were stored at -80 degrees C until use.
• FIGs. 9A-9C and FIGs. 10E-10H The primary T-cells engineered with NFAT-RE inducible drug delivery' system and used in the in vivo validation experiments (FIGs. 9A-9C and FIGs. 10E-10H) were formed using the process described below. Briefly, human primary T-cells (CD3, CD4 or CD8) were purchased from the Stanford Blood Center (Palo Alto, CA). The T-cells were counted and used fresh or were cryostocked using freezing media [90% heat-inactivated fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO)] in liquid nitrogen for future use.
• FBS heat-inactivated fetal bovine serum
• DMSO dimethyl sulfoxide
• Biotin anti-human CD3 and Biotin anti-human CD28 antibody were loaded on Dynabead® Biotin Binder paramagnetic particles following the manufacturer’s instructions (anti-CD3/CD28 Dynabeads).
• Frozen human primary T-cells were thawed (Day 0), resuspended in complete growth medium, and activated by anti-CD3/CD28 Dynabeads (celkparticle of 1:3). After 24 hours (Day 1), IxlO 6 activated primary T-cells were transduced with the appropriate lentivector particles resuspended in 0. 1 mL volume of serum-free RPMI at an MOI of about 10 and in the presence of 8 pg/mL polybrene.
• transduction reaction mix The 0.1 mL aliquots of transduction reaction mix were placed as drops in a tissue-culture treated 6-well plate and placed in a cell culture incubator (37 degrees C, 5% CO2, 95% humidity) for 16 hours. After 16 hours of incubation (around Day 2), the transduced primary T-cells were cultured at IxlO 6 cells/mL in complete grow th medium supplemented with recombinant human IL-7 (25 lU/mL), recombinant human IL- 15 (25 lU/mL), and 8 pg/mL polybrene.
• the cells were counted after another 24 hours (Day 3) and every other day thereafter using acridine orange and propidium iodide (AOPI) staining in aNexcelom K2 cellometer. They were maintained at a concentration of 0.5xl0 6 cells/mL in complete growth medium supplemented with recombinant human IL-7 (25 lU/mL) and recombinant human IL- 15 (25 lU/mL) and the media was replaced every 2-3 days with half media changes. No polybrene was added on Day 3 and beyond. [00247] (5) Flow cytometry analysis.
• AOPI propidium iodide
• the production yield of engineered primary T- cells was determined by assessing the expression of FRot-CAR on the T-cells engineered for drug delivery (% FRa-CAR + T-cells). Five days after transduction, about IxlO 6 T- cells were collected and de-beaded by keeping the T-cell suspension on a DynaMagTM-2 sample rack for 2 minutes to remove the Dynabead biotin binder particles.
• the debeaded T-cells were washed in cell-staining buffer and stained for 1 hour at 4 degrees C using an antibody cocktail containing biotinylated human FRa protein (FOLR1 -His Tag -Avi Tag), PerCR/Cy5 5 anti-human CD3 antibody and the LIVE/DEADTM Fixable Aqua Dead Cell Stain Kit.
• the cells were washed, and a secondary staining was performed for 1 hour at 4 degrees C using APC-streptavidin.
• the samples were washed, resuspended in 200 pL Cell Staining Buffer, and analyzed with a BD FACS Symphony A3 (BD Biosciences). The data was further processed using FlowJo® software.
• FRa-CAR + CD4 and CD8 T-cells were co-cultured with the targets (OVCAR3 or FRa- antigen/anti-CD28 DynabeadsTM) at an effector-to-target ratio (E:T) of 10: 1, in 200 pL of complete growth medium in a single well of a 96-well plate.
• E:T effector-to-target ratio
• the Nluc substrate was diluted in the cell lysis buffer provided with the Nano- Glo® assay and added to the co-culture in 96-well plates to assess the enzyme (Nluc) activity. Following a brief incubation period of 3 minutes, the bioluminescence was read on a microplate reader.
• FRa-CAR + CD3 and CD4 T-cells were co-cultured with target (FRa + Luc2-2A-E2Crimson + A2780cis) cells (2500 cells) in 200 pL of complete growth medium in a single well of a 96-well plate. After a 24-hour co-culture, the manufacturer’s protocol was followed to measure the reporter activity, e.g., Luc2 activity in the A2780cis cells using One-Gio® assay.
• the Luc2 substrate was diluted in the cell lysis buffer provided with the One-Gio® assay and added to co-culture in the 96-well plate for assessing Luc2 activity. Following a brief incubation period of 10 minutes, the bioluminescence was read on a microplate reader.
• T- cells CD3, CD4, CD8 engineered for IFNP delivery was stimulated using microparticles functionalized with FRa antigen and anti-CD28 antibodies at 1 :3 cell-to- particle ratio.
• IFNP activity in the cell culture supernatant was determined using IFN-a/p Reporter HEK 293 Cells using QUANTI-BlueTM assay kit following manufacturer’s instructions.
• Recombinant human IFNP standard was run in parallel to determine the equivalent IFNP activity in the culture supernatant.
• Unmodified T-cells and T-cells engineered to express FRa-CAR were used as control. All experiments were run in triplicate and the data is represented as mean ⁇ SD.
• mice After quarantine, the NSG mice were anesthetized and 2xl0 6 FRa + MSLN Neg Luc2-2A- E2Cnmson + A2780cis cells (in 18 mice) or FRa neg MSLN + Luc2-2A-E2Crimson + A2780cis cells (in 6 mice) in 100 pL lx PBS were i.p. implanted. The tumor growth was monitored every 3-4 days for the next 10 days using i.p. injected 150 mg D- Luciferin per kilogram (kg) of mouse dissolved in lx PBS.
• FRa + MSLN Ncs Luc2-2A-E2Crimson + A2780cis cells were then treated with 2xl0 6 primary CD4 T-cells engineered for delivery function (e.g., FRa-CAR with NFAT-RE inducible Nluc reporter) or the control CD4 T-cells (without FRa-CAR but with NFAT-RE inducible Nluc reporter) or the no CAR CD4 T-cells (with NFAT-RE inducible Nluc reporter) every day for 5 days.
• 2xl0 6 primary CD4 T-cells engineered for delivery function e.g., FRa-CAR with NFAT-RE inducible Nluc reporter
• control CD4 T-cells without FRa-CAR but with NFAT-RE inducible Nluc reporter
• no CAR CD4 T-cells with NFAT-RE inducible Nluc reporter
• the bioluminescent reporter (Nluc) activity was determined by i.p. injection of the Nano-Gio® substrate (1 :20 dilution of the substrate in lx PBS, equivalent to 0.5 mg per kg of mouse) on all days including on day 0, after treatment. Imaging was performed in an I VIS Lumina X5 imaging system. The data was quantified by analysis of the ROI using Living Image software. The tumor luminescence is plotted as the mean ⁇ SEM of total flux (photons/s) against days after treatment.
• the tumor growth was monitored every 3-4 days using i.p. injected 150 mg D-Luciferin per kg of mouse dissolved in lx PBS.
• the luminescence imaging was performed in an IVIS Lumina X5 imaging system. The data was quantified by analysis of the ROI using Living Image software. The tumor luminescence is plotted as the mean ⁇ SEM of total flux (photons/s) against days after tumor implantation.
• the transduction yield was enhanced about 2.5-fold by restricting the transduction reaction volume for maximizing the lentivector-to-T-cell contact.
• Cell density and cytokines used in the expansion process were adjusted to achieve > 100-fold expansion of the T-cell-based effector cell in 14 days, and the function of these cells was validated in vivo using intraperitoneally implanted tumor cells.
• the primary T-cell-based effector cell has human applications because it can be scaled and administrated to express a broad range of therapeutic proteins (e.g., cytokines, interferons, enzymes, agonists, and antagonists) at the disease site, obviating the need for systemic delivery of large doses of these proteins.
• primary T-cell have been transformed into a platform for synthesizing complex biologies directly at the disease site with precise timing and location.
• this technology can be used to synthesize engineered proteins so as to exert therapeutic effects by autocrine or paracrine signaling only at the disease site without affecting healthy tissues.
• In vivo experiments confirmed the synthesis of functional proteins by the engineered cells.
• FIG. 13 illustrates an example process for forming genetically engineered effector cells from primary T-cells, in accordance with the present disclosure.
• isolated CD3 T-cells were activated with anti-CD3/CD28 DynabeadsTM (celkparticle of 1:3) for 24 hours, transduced the activated T-cells by increasing lentivector-to-T-cell contact in 0.1 mL volume for 16 hours, and expanded the transduced cells at 0.5xl0 6 cells/mL in complete growth medium supplemented with IL-7 and IL- 15, with half-media changes every 2-3 days for 14 days.
• Various experiments were directed to assessing different process parameters and the effect on transducing primary T-cells with lentivectors.
• FIGs. 14A-15D illustrate example effects of various parameters on the lentivector transduction of primary T-cells, in accordance with the present disclosure. More specifically, FIGs. 14A-14C show the effect of the cell-to-particle ratio on early (CD69 + CD25-) (FIG. 14A), peak (CD69 + CD25 + ) (FIG. 14B), and late (CD69’CD25 + ) (FIG. 14C) activation of CD3 T-cells (see also FIGs. 18A-18B for the gating strategyusing a representative fluorescence-activated cell sorting (FACS) plot). FIG.
• FACS fluorescence-activated cell sorting
• FIG 14B shows that 60% of CD3 T-cells progress to peak activation (CD69 + CD25 + ) within 24 hours after stimulation by particles at a cell-to-particle ratio of 1:3. As such, this ratio was used in the formation and/or manufacturing process in various experiments.
• T-cell activation marker (CD25, CD69) expression in CD3 T-cells (n 3 donors) was assessed by flow cytometry- at 24- or 48- hours after stimulation by chemicals (Phorbol 12-myristate 13-acetate (30 nM) and lonomy cin (IpM), PMA/Io) or by different cell-to-particle (DynabeadsTM loaded with anti -human CD3 and anti -human CD28) ratios.
• FIGs. 18A-18B Strategy for evaluating CD3 T-cell activation is presented in FIGs. 18A-18B. Different stages of T-cell activation are shown in early activation (CD69 + CD25‘) (FIG. 14A), peak activation (CD69 + CD25 + ) (FIG. 14B), and late activation (CD69 CD25 + ) FIG. 14C).
• FRa-CAR expression (% FRa-CAR + T-cells on left Y- axis) and T-cell viability (% Viability on right Y-axis) was assessed by flow cytometry after transducing stimulated and unstimulated primary T-cells. As shown by FIGs.
• FRa-CAR expression (% FRa-CAR + T-cells on left Y-axis) and T-cell viability (% Viability on right Y-axis) was assessed by flow cy tometry after varying factors affecting transduction including: (i) size of the genetic payload (chimeric antigen receptor (CAR) only, 5.6 kb vs T-cell-based delivery system comprising of CAR and NFAT-RE inducible transgene, 7.2 kb), see FIGs. 7A-7D for schematics (FIG. 15B), (ii) lentivector pseudotype (RD114 vs VSV-g) (FIG.
• CAR chimeric antigen receptor
• FIG. 15B was determined by Student’s t-test, two-tailed.
• FIG.15C although analyzed by Student's t- test, no significance was obtained.
• FIGs. 15B-15D show a comparison of other parameters that affect the transduction efficiency of primary T-cells. Experiments were directed to assessing improvement as the percentage of modified primary T-cells (% FRa-CAR + T-cells, left y-axis) and the number of live primary T-cells (% viability, right y-axis) in the culture 5 days after transduction.
• VSV-g envelope protein from vesicular stomatitis virus and RD114 envelope protein from infectious feline endogenous retrovirus, as described by 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), which is incorporated herein in its entirety for its teaching.
• VSV-g envelope protein is accepted for engineering T-cells, and RD114 has been reported to improve efficiency in engineering CD34 hematopoietic cells and CAR T-cells.
• the results are presented in FIG. 15C. No significant difference in transduction efficiencies (RD114: 37.1 ⁇ 9.5%; VSV-g: 34 ⁇ 10.6%) or viability of the engineered primary' T-cells was observed. Given the acceptance of VSV-g pseudotyped lentivectors, it was used in various experiments.
• FIGs. 20A-20B The intracellular antiviral response impedes the transduction efficiency of primary T- cells when lentivirus-based vectors are used.
• AVIs was assessed to suppress the intracellular immunity against infection from the lentiviral vectors and potentially increase the transduction yield. Inhibition of intracellular antiviral signaling has improved T-cell transduction.
• RNA-dependent protein kinase (PKR) pathway 2-aminopurine (2-AP) and C16
• PKA RNA-dependent protein kinase
• other pathways such as STAT (ruxolitinib) and Rho (Y-27632) signaling.
• Concomitant treatment with AVIs for PKR or TBK1 pathways during T-cell transduction increased transduction of primary' T-cells (FIG. 19A).
• FIG. 20B shows the results with LRAs as additives in the T-cell transduction and expansion media.
• the LRAs facilitate unfolding of the chromatin structure that determines DNA accessibility of the host genome and retroviral gene integration.
• a preferential bias for the site of gene integration was strongly displayed by gammaretroviruses, delta-retroviruses, and lentiviruses with DNA insertion into transcriptionally active chromatin.
• a subset of LRAs such as protein kinase C (PKC) agonists and/or its combination with inhibitors of bromo extra terminal (BET) or histone deacetylases (HD AC), were assessed for their ability to improve T-cell transduction with large lentiviral constructs.
• PLC protein kinase C
• BET bromo extra terminal
• HD AC histone deacetylases
• the LRA romidepsin increased the percentage of the engineered T-cells (55%) versus vehicle control (42%) (FIG. 20B), but the percentage of live cells was only 40% compared to 75% in the control, rendering romidepsin unfit for use in combination with lentivectors.
• Method a spinoculating in 0.5 mL at 800G in a well of a 24-well plate for 1.5 hours followed by incubating the reaction in a cell culture incubator (37 degrees C, 5% CO2, 95% humidity) for another 14.5 hours;
• Method b using a defined reaction volume of 1.0 mL in a well of a 6-well plate for 16 hours in cell culture incubator;
• Method c restricting the volume within a 0. 1 mL drop in a well of a 6-well plate for 16 hours in cell culture incubator.
• IxlO 6 primary T- cells were transduced with the lenti vector particles at an MOI of 10 in serum-free medium supplemented with 8 pg/mL polybrene and diluted the transduction reaction with complete growth medium supplemented with 50 U/mL IL-2 after 16 hours in all three methods.
• the process efficiency was assessed five days after the transduction (FIG. 15D).
• the results showed 32% transduction at 44% viability with spinoculation (Method a); 53% transduction at 8% viability when using 1.0 mL reaction volume (Method b); and 60% transduction at 60% viability when restricting the reaction volume wi thin the 0. 1 mL drop (Method c).
• Method c Limiting the reaction volume to increase lentivector-to-cell contact allowed Method c to produce about 2.5-fold more engineered primary T-cells compared to the spinoculation method (Method a). Based on these results, Method c was selected, confining the transduction reaction within 0. 1 mL drop, as part of the optimized production process.
• FIGs. 16A-16E illustrate example effects of various parameters on the expansion of primary T-cells, in accordance with the present disclosure.
• T-cell density and cystokins were assessed for the impact on in vitro expansion of engineered primary T-cells.
• Numerical expansion of the engineered primary T-cells was assessed at different cell densities (FIG. 16A) and when supplemented with different cytokines (IL-2, IL-7, IL- 15, and combinations thereof) (FIG. 16B).
• FIG. 16A cell densities
• IL-2, IL-7, IL- 15, and combinations thereof FIG.
• 16C shows naive/memory T-cell phenotype (naive (TN: CD45RA + /CCR7 + ), central memory (TCM: CD45RA /CCR7 1 ).
• effector memory TEM: CD45RA /CCR7
• terminally differentiated effector memory TEMRA: CD45RA'/CCR7
• FIG. 16D shows results of FRa-specific engineered primary T-cells expanded in the same cytokine combinations induced cytolysis in FRa + Luc2-2A-E2Crimson + A2780cis target cells in a dose-dependent manner.
• FIG. 16E shows results of FRa-specific engineered primary T- cells expanded in the same cytokine combinations induced effector function, e.g., the delivery function, as represented by the NFAT-RE inducible NanoLuc® (Nluc) reporter activity in a dose-dependent manner.
• FIG. 16A shows the effect of cell density on numerically expanding these engineered primary' T-cells in the presence of 50 lU/mL IL-2.
• the engineered T-cells expanded around 45-fold at cell densities of 0.5xl0 6 cells/mL and 0.25xl0 6 cells/mL, compared to the around 16-fold expansion at IxlO 6 cells/mL.
• FIG. 16C shows phenotypic changes in the engineered primary T-cells when cultured in different cytokine cocktails, as detailed in FIG. 16B.
• the CD4 and CD8 subsets were analyzed at days 7 and 14 (see FIG. 21 for CD4/CD8 ratios) for naive (TN), central memory' (TCM), effector memory (TEM), and terminally differentiated effector memory (TEMRA) phenotypes using the markers CD45RA and CCR7.
• >90% of both CD4 and CD8 T-cells were composed of TN (CD45RA + /CCR7 + about 50%) and TCM (CD45RA /CCR7 + about 42%) compartments.
• TN naive
• TCM effector memory
• TEMRA terminally differentiated effector memory
• the target specific cytolytic function of the FRa-CAR cells was further supported by two independent tumor cell lines (A2780cis and KPCY) engineered for FRa-antigen expression compared to the respective antigen negative control (see FIG. 22A-22B).
• IL-7-expanded engineered primary T-cells showed peak delivery' function, it was not significantly different from the engineered primary T-cells expanded with other cytokine combinations. Unlike the engineered primary T-cells expanded in IL-7 only, those expanded with the combination of IL-7 and IL- 15 exhibited enhanced proliferation. It is believed that IL-7 and IL-15 support long-term persistence and memory responses of the T-cells, and such cytokines were selected as cytokine supplements for expanding the primary T-cells engineered for cell-based delivery of proteins.
• FIGs. 17A-17F illustrate functional validation of the effector cells formed from a primary T-cell, in accordance with the present disclosure.
• FIGs. 17A-17F show results of in vitro validation of targetspecific delivery function proportionate to the disease burden.
• FRa-specific primary T- cells engineered for the NFAT-RE inducible delivery function showed proportionate increase in reporter activity when co-cultured with target, FRa + A2780cis (FIG. 17A) and FRa + KPCY cells (FIG. 17B), compared to their respective non-target (FRa neg ) control cells.
• 17C shows results of CAR T-cells formed using the process described above and developed for T-cell-based effector cells to reduced tumor burden.
• Bioluminescence (Luc2 activity) from the i.p. tumors was used to assess the tumor burden in vivo.
• FRa-specific primary T-cells engineered for the NFAT-RE inducible delivery function were i.p. injected in i.p. FRa + A2780cis tumor-bearing NSG mice at 24-hour interval 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 the delivery function.
• FIG. 17D shows schematic of dosing, treatment, and imaging schedules
• FIG. 17E shows representative bioluminescent images
• FIG. 17F shows quantification. All results are represented as mean ⁇ SEM.
• Statistical analysis and p values for FIG. 17A, FIG. 17B, and FIG. 17F were determined by multiple t-test using Holm-Sidak method; ⁇ 0.05, **/? ⁇ 0.01, and ***/? ⁇ 0.001.
• the target-specific, delivery function proportionate to the disease burden was assessed in vitro by co-culturing the FRa-specific primary T-cells engineered for the NFAT-RE inducible delivery function against target cells, A2780cis (FIG. 17A) and KPCY cells (FIG. 17B).
• target cells A2780cis
• KPCY cells FIG. 17B
• co-culture with antigen-positive target cells showed a proportionate and significant increase in delivery function, Nluc reporter activity, with increase in target cell number.
• control cells e.g., primary T- cells engineered for the NFAT-RE inducible delivery function but without CAR
• the aforementioned process does not compromise the inherent cytolytic function of CAR T-cells.
• KPCY2838c3 pancreatic ductal adenocarcinoma cells derived from KPCY mice were engineered to express human FRa antigen and Luc2 (FRa + Luc2 + KPCY cells) for assessing tumor growth, and 0.5xl0 6 were i.p. implanted in NSG mice.
• FRa-CAR + T-cells (without the NFAT-RE inducible Nluc reporter) were expanded for 16 days and injected i.p. to challenge 10-day old FRa + Luc2 + KPCY tumors. The results in FIG.
• 17C show a dose-escalation effect of the FRa-CAR + T-cells (IxlO 6 , 3xl0 6 , and 10xl0 6 FRa- CAR + T-cells) on tumor regression.
• FRa-CAR + T-cells IxlO 6 , 3xl0 6 , and 10xl0 6 FRa- CAR + T-cells
• FRa-CAR + T-cells were formed with the delivery function, e.g., upon engaging the target FRa antigen, the FRa-CAR activates the NF AT- RE signaling pathway to induce the expression of desired protein.
• the experiment schedule is detailed in FIG. 17D and the results are shown in FIGs. 17E-17F. More particularly, FIGs. 17E-17F show results of 2xl0 6 FRa + Luc2 + A2780cis cells that were i.p. implanted in NSG mice. The 12-day-old xenograft tumors were i.p.
• FRa-CAR + T-cells treated with 2xl0 6 FRa-CAR + T-cells (with NFAT-RE inducible Nluc reporter) on days 0, 1, 2, 3, and 4.
• a control group was included to assess any background signal from using the Nluc substrate on Luc2 + tumor cells. This group was treated with i.p. injections of FRa- CAR + T-cells without NFAT-RE inducible Nluc reporter (control FRa-CAR + T-cells) to maintain an equivalent tumor burden.
• the effector (Nluc) activity was measured (FIG. 17E) and quantified (FIG. 17F) at baseline (day 0) as well as on days 1, 2, 3, 4, and 5.
• engineered effector activity e.g., delivery function
• the FRa-CAR + T-cells with the delivery function e.g., with NFAT-RE inducible Nluc reporter
• FIGs. 18A-18B illustrate an example strategy for evaluating CD3 T-cell activation, in accordance with the present disclosure.
• FIG. 17A is a schematic of the gating strategy used for assessing early (CD69 + CD25 ), peak (CD69 + CD25-), and late (CD69‘CD25 + ) activated CD3 T-cells by flow cytometry.
• FIG. 18B are representative plots showing CD69 and CD25 expression in stimulated verses non-stimulated CD3 T- cells.
• FIGs. 19A-19D illustrate example effects of additional factors on transduction of primary T-cells with lenti vectors, in accordance with the present disclosure.
• FRa- CAR expression % FRa-CAR + T-cells on left Y-axis
• T-cell viability % Viability on right Y-axis
• FIG. 19A shows T-cell concentration in a transduction reaction results
• FIG. 19B shows lentivector MOI results
• FIG. 19C shows transduction reaction volume results
• FIG. 19D shows polybrene concentration results. Transduction efficiency was determined after 5 days. All results are represented as mean ⁇ SD.
• FIGs. 20A-20B illustrate an example exploratory screen of chemical additives for improving transduction of primary T-cells with lentivectors, in accordance with the present disclosure.
• FRa-CAR expression % FRa-CAR + T-cells on left Y-axis
• T- cell viability % Viability on right Y-axis
• Transduction efficiency was determined after 5 days. All results are represented as mean ⁇ SD.
• FIG. 21 illustrates an example change in the proportion of CD3 T-cell subsets in response to cytokines, in accordance with the present disclosure.
• the CD4/CD8 ratio was assessed by flow cytometry in CD3 T-cells at day 7 and 14 of in vitro expansion when growth media was supplemented with different cytokines (IL-2, IL-7, IL- 15, and combinations thereol).
• FIGs. 22A-22C illustrate example antigen-specific cytolysis and NFAT-RE inducible delivery function, in accordance with the present disclosure.
• FRa-specific CAR T-cells formed using the above-described process induced cytolysis in FRa + Luc2- 2A-E2Crimson + KPCY (FIG. 22A) and FRa + Luc2-2A-E2Crimson + A2780cis (FIG. 22B) target cells in a dose-dependent manner compared to their respective antigen negative target cells.
• FIG. 22C shows Nluc activity from primary T-cells engineered for NFAT-RE inducible delivery function when co-cultured with antigen-positive and antigen-negative target cells for 24 hours.
• Target cells [FRa + A2780cis (Sex: female), FRa + OVCAR3 (Sex: female)] engineered to express modified firefly luciferase (Luc2), 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 (2016), and Repellm et al..
• puromycin N-acetyltransferase was used as a selection marker and puromycin dihydrochloride (Puromycin) was used for selecting stable cell lines.
• Puromycin puromycin dihydrochloride
• a chemical activation of T-cells was achieved by treatment with 1 pM phorbol 12-myristate 13-acetate and 30 nM ionomycin (PMA/Io).
• Biotinylated human FRa protein was used to analyze FRa CAR expression on engineered primary' T-cells.
• lentivector particles were produced as described in Radhakrishnan et al., Lentivirus Manufacturing Process for Primary T-Cell Biofactory Production, Advanced Biosystems 4(6): 1900288 (2020), which is incorporated herein by reference in its entirety for its teaching. Lentivirus manufacturing and its use in engineering cells were performed at SRI International following the guidelines of the approved Biological Use Authorization (BUA 17-05). Briefly, lentivector particles were prepared by packaging the corresponding transfer plasmid using 2 nd -generation lentivector system.
• HEK293T/17 (Sex: female) producer cells (12xl0 6 ) were seeded into tissue culture treated T150 flasks in 21 mL complete DMEM supplemented with 10% heat-inactivated FBS and IX penicillin streptomycin solution and placed in a cell culture incubator (37 degrees C, 5% CO2, 95% humidity). After 24 hours, transfer plasmid was co-transfected with 2 nd generation packaging plasmids (psPAX2, pMD2.G), and pAdV Antage plasmid at 4:3: 1:0.4 wt-ratio.
• psPAX2, pMD2.G 2 nd generation packaging plasmids
• pAdV Antage plasmid at 4:3: 1:0.4 wt-ratio.
• transduction reaction mix The 0.1 mL aliquots of transduction reaction mix were placed as drops in a tissue-culture treated 6-well plate and placed in a cell culture incubator (37 degrees C, 5% COz, 95% humidity) for 16 hours. After 16 hours of incubation (around Day 2), the transduced primary T-cells were cultured at IxlO 6 cells/mL in complete grow th medium supplemented with recombinant human IL-7 (25 lU/mL), recombinant human IL- 15 (25 lU/mL), and 8 ug/mL polybrene.
• the cells were counted after another 24 hours (Day 3) and every other day thereafter using acridine orange and propidium iodide (AOPI) staining in aNexcelom K2 cellometer. They were maintained at a concentration of 0.5xl0 6 cells/mL in complete growth medium supplemented with recombinant human IL-7 (25 lU/mL) and recombinant human IL- 15 (25 lU/mL) and the media was replaced every 2-3 days with half media changes. No polybrene was added on Day 3 and beyond. [00292] (5) Approach for improving the production of primary T-cell-based delivery system. The above process was deviated from when exploring the factors to improve the production of the cell-based delivery system.
• AOPI propidium iodide
• the bioluminescent reporter (Nluc) activity was determined by i.p.
• Nano-Gio® substrate (1 :20 dilution of the substrate in lx PBS, equivalent to 0.5 mg per kg of mouse) on days 0, 1, 2, 3, 4, and 5 after treatment. Imaging was performed in a IVIS Lumina X5 imaging system. The data was quantified by analysis of the ROI using Living Image software. The tumor luminescence is plotted as the mean ⁇ SEM of total flux (photons/s) against days after treatment.
• CD4 T-cells were formed in experimental embodiments that capitalized on the biology of CD4 T-cells and transformed the CD4 T-cells into a cell-based platform that can assess the disease burden and mount a proportional response by expressing engineered proteins precisely at the disease site.
• the helper CD4 T-cell was demonstrated to offer a clear competitive advantage over the killer CD8 T-cell or a combined pool of CD4 and CD8 T-cells (e.g., pan CD3 T-cells) when engineered for cell-based effector cells for delivering proteins.
• the correctly engineered CD4 T-cells offer to bridge two gaps that may prevent the clinical adoption of this technology, e.g., scaling up the cell production to generate a clinical dose and increased synthesis of the therapeutic protein from the cell so as to reduce the size of the clinical dose required to produce the desired effect.
• the non-cytolytic CD4 T-cells were experimentally confirmed as being an appropriate phenotype for performing this function.
• the low cytolysis of CD4 T-cell delivery system offers a substantial advantage, e.g., the maximum tolerated dose of the CD4 T-cell engineered for drug delivery can be high without exposure to the healthy tissues.
• the CD4 T-cells are believed to be advantageous over the CD8 T-cells due to the NFAT-based transcriptional machinery that is more productive in CD4 T-cells. Further, CD4 T-cells produce more Thl cytokines and proliferate faster than CD8 T- cells.
• CD4 T-cells Compared to pan CD3 CAR T-cells with a non-curable dose, the same number of T-cells with half from each subset (CD4, CD8) more effectively treats tumors.
• Helper CD4 T-cells induce sternness in the CD8 T-cells that then persists longer and increases the memory T-cell pool for improved treatment outcome.
• the CD4 T-cells extravasate into cold tumors and recruit other CD8 T-cells.
• the CD4 T-cells also assist vascular normalization, attenuate hypoxia, and reduce metastasis. CD4 T-cells have been found to persist for more than a decade.
• CD4 T-cells when passaged in vivo, CD4 T-cells can outlive the host mouse specie by four times and expand at least 10 40 -fold.
• the cell-mediated drug delivery system of experimental embodiments can positively impact various medical domains that require precise spatiotemporal drug administration, such as for leveraging it for solid tumors and viral infections. Building upon experiments with cell-based delivery of IFNP for targeting solid tumors and viral infections, experiments were directed to assessing the antitumor efficacy by focusing the localized delivery of IFNP through the CD4 T-cell based effector cells.
• IFNP dosage delivered by CD4 T-cells was around 300-fold less than the directly administered rhIFNP, yet more effective.
• the increased therapeutic effect at reduced equivalent dose shows the potential of the cell-based platform to minimize undesired side-effects to the healthy tissues thereby increasing the Maximum Recommended Starting Dose during subsequent human trials.
• effector cells can continue the development for overcoming the two major issues that have been observed with systemic IFNP treatments — severe toxicities to healthy issues and immunosuppression in the local tumor microenvironment; and use it to synergize with other antitumor agents such as chemotherapies, radiotherapies, antibody-based immunotherapies, as well as bridge the innate and adaptive immune responses.
• the CD4 T-cell-based technology acts as a zero-order delivery system, providing a promising solution to the challenges posed by the first-order drug delivery systems with synthetic carriers. Unlike first-order systems, it focusses the concentration of therapeutic biologies at the disease site proportionate to the disease mass, increasing efficacy while reducing concentration in healthy tissues, improving safety. Furthermore, the long-term persistence of CD4 T-cells in the body, which can last for more than a decade, holds great promise in significantly reducing the frequency of re-infusions required. This will not only improve patient compliance but also, over the long term, alleviate the burden on the healthcare system and streamline treatment procedures.
• T-cells were used to develop a cell-based platform that can be used for site-specific delivery of protein-based drugs.
• the platform delivery system utilizes the T-cell’s activation machinery for in situ synthesis, so that the cell-mediated synthesis of desired proteins is proportionate to the disease burden.
• the site-specific and proportionate synthesis of desired biologies offers the potential to overcome morbidity issues that can arise from excess systemic infusion of such drugs, and prevents the development of resistance to these drugs when used in lower amounts
• Additional experiments were directed to assessment of AVIs and LRAs to improve the lentivector transduction yield and increase the starting cell number so as to shorten dose-manufacturing time, thereby improving the affordability of T-cell therapies.
• the use of these additives, such as AVIs and LRAs can also reduce cell exhaustion by decondensing the chromatin structure.
• first-order drug-delivery systems e.g., liposomes, nano-carries, dendrimers, hydrogels, microparticles
• first-order drug-delivery systems offer a controlled release of drugs
• their application is still limited by their short half-life in vivo requirement for multiple infusions, and potential toxicity due to their systemic presence.
• the above described T- cell-based effector cells represents a substantive departure from this status quo. This is because T-cells chemotactically extravasate through multiple solid tissues to the disease sites and engage with the target cells through the antigen-specific CAR.
• the integrated effect is a clonal CAR T-cell population proportionate to the number of target cells.
• the T-cell-based drug delivery system is engineered to leverage this biology of the T-cell.
• T-cells migrate to disease sites with cellular resolution and, upon recognizing the target cells with molecular specificity, can synthesize protein-based biologies proportionate to the disease burden. It is therefore a living cell-based in vivo vector engineered into a stable zero-order drug delivery system. Unlike the first-order drug delivery systems, it can enable sustained in situ production of complex biologic drugs for executing a broad range of effector functions.
• the first-order drug-delivery' approaches are primarily based on synthetic matenals and are thus rapidly cleared by the mononuclear phagocyte system.
• the cell-based system utilizing T-cells have been found to persist in vivo for more than a decade.
• recent findings in mice concluded that the primary T-cell, when passaged in vivo in new mice, can last four times longer than the lifespan of the host species and expand at least 10 40 -fold. This obviates the need for re-dosing even in case of relapse.
• the T-cell-based delivery platform presents a pioneering and universal technology. It facilitates the delivery of intricate biologies over extended periods without the need for multiple infusions. As a result, this platform technology opens new horizons for treating a variety of diseases.

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