Synthetic pathway activators

Abstract

Provided herein are novel synthetic pathway activators comprising transmembrane domains and tiled intracellular signaling domains comprising a combinatorial signaling domain comprising two or more Signal Transducer and Activator of Transcription (STAT)1, STAT3, STAT4, STAT5 and / or Toll-like receptor (TLR) / IL-1R (TIR) signaling domains(s).

Description

Attorney Ref: ANB-223WOSYNTHETIC PATHWAY ACTIVATORSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 690,719, filed September 4, 2024, and U.S. Provisional Application No. 63 / 816,448, filed June 2, 2025, both of which are hereby incorporated in their entirety by reference.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which is hereby incorporated by reference in its entirety. Said XML copy, created on September 3, 2025, is named ANB- 223WO_SL, and is 572,655 bytes in size.BACKGROUND

[0003] Cancer is a disease characterized by uncontrollable growth of cells. Many approaches to treating cancer have been tried, including drugs and radiation therapies. Recent cancer treatments have sought to use the body’s own immune cells to attack cancer cells. One promising approach uses T cells that are taken from a patient and genetically engineered to produce chimeric antigen receptors, or CARs, receptor proteins that give the T cells a new ability to target a specific protein. The receptors are chimeric because they combine antigenbinding and T-cell activating functions into a single receptor.

[0004] Immunotherapy using CAR-T cells is promising because the modified T cells have the potential to recognize cancer cells in order to more effectively target and destroy them.

[0005] After the T cells are engineered with the CARs, the resulting CAR-T cells are introduced into patients to attack tumor cells. CAR-T cells can be either derived from T cells in a patient's own blood (autologous) or derived from the T cells of another healthy donor (allogeneic). Once CAR-T cells are infused into a patient, they come in contact with their targeted antigen on a cell. The CAR-T cells bind to the antigen and become activated. Upon antigen engagement, CAR T cells can proliferate exponentially, initiate antitumor cytokine production, and target tumor cell killing.

[0006] However, there remain some concerns and limitations to CAR T cell-based immunotherapy. Clinically effective adoptive T cell therapy for the treatment of solid tumors will use robust T cell expansion, persistence, and potency. Thus, additional therapies that increase T cell expansion, persistence, and potency remain desirable.IPTS / 200097280.2 1Attorney Ref: ANB-223WOSUMMARY

[0007] In one aspect, provided herein are one or more nucleic acids encoding a synthetic pathway activator (SPA) peptide comprising a chimeric polypeptide comprising: a. optionally, an extracellular domain; b. a transmembrane domain; c. optionally, a costimulatory domain; and d. an intracellular signaling domain comprising: i. one or more Janus Kinase (JAK) signaling domains; and ii. a combinatorial signaling domain comprising two or more Signal Transducer and Activator of Transcription (STAT)l, STAT3, STAT4, STAT5 and / or Toll-like receptor (TLR) / IL-1R (TIR) signaling domains(s), or any combination thereof.

[0008] In one aspect, provided herein are one or more nucleic acids encoding a synthetic pathway activator (SPA) peptide comprising a chimeric polypeptide comprising: a. optionally, an extracellular domain; b. a transmembrane domain comprising an IL-7Ra transmembrane domain, a G- CSFR, transmembrane domain, or a gpl30 transmembrane domain; c. optionally, a costimulatory domain; and d. an intracellular signaling domain comprising: i. one or more Janus Kinase (JAK) signaling domain(s) comprising a gpl30 JAK signaling domain, an IL-7Ra JAK signaling domain, a G- CSFR JAK signaling domain, an IL-12RP2 JAK signaling domain, an IL-9R JAK signaling domain, or an IL-21R JAK signaling domain, or any combination thereof; and ii. one or more Signal Transducer and Activator of Transcription (STAT)l, STAT3, STAT4, STAT5 and / or Toll-like receptor (TLR) / IL- 1R (TIR) signaling domains(s), or any combination thereof.

[0009] In some embodiments, the intracellular signaling domain comprises two or more STAT1, STAT3, STAT4, STAT5 and / or TIR signaling domain(s), or any combination thereof.

[0010] In some embodiments, the STAT1, STAT3, STAT4, and / or STAT5 signaling domain(s) comprises one or more tyrosine phosphorylation motifs comprising YXXQ or YXPQ.IPTS / 200097280.2 2Attorney Ref: ANB-223WO

[0011] In some embodiments, the STAT1, STAT3, STAT4, STAT5 and / or TIR signaling domain(s) induces phosphorylation of one or more of STAT1, STAT3, STAT4, STAT5 and / or TIR upon multimerization of the SPA.

[0012] In some embodiments, the intracellular signaling domain comprises at least one polypeptide sequence from an interleukin receptor or a cytokine.

[0013] In some embodiments, the STAT1, STAT3, STAT4, STAT5 and / or (TIR) signaling domain(s)comprises at least one STAT1 and / or STAT3 signaling domain from at least one of gpl30, IL-21R, G-CSFR, or IL-9R, or any combination thereof.

[0014] In some embodiments, the STAT1, STAT3, STAT4, STAT5 and / or (TIR) signaling domain(s) comprises at least one STAT4 signaling domain from IL-12RP2.

[0015] In some embodiments, the STAT1, STAT3, STAT4, STAT5 and / or (TIR) signaling domain(s)comprises at least one STAT5 signaling domain from at least one of IL-2RP , IL- 7Ra, or IL-15R, or any combination thereof.

[0016] In some embodiments, the STAT1, STAT3, STAT4, STAT5 and / or (TIR) signaling domain(s) comprises at least one TIR domain from IL-18Ra.

[0017] In some embodiments, the STAT1, STAT3, STAT4, STAT5 and / or Toll-like receptor (TLR) / IL-1R (TIR) signaling domain(s) comprises at least one signal adaptor comprising one or more amino acid sequences at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence of: a gpl30 (YXXQ)4 STAT adaptor (SEQ ID NO: 34 or 286), a gpl30_Y759F STAT adaptor (SEQ ID NO: 35), a truncated gpl30(A707-755) with a Y759F mutation (gpl30trunc7_Y759F (SEQ ID NO: 36)), IL9R_310-521 (SEQ ID NO: 37), IL12Rp2_796-801 ((SEQ ID NO: 38), IL12Rp2_714-862 (SEQ ID NO: 39), IL7Ra_316-459 (SEQ ID NO: 40), IL2Rp_333-551 (SEQ ID NO: 41), IL2RP (339-379,393-433,518-551) (SEQ ID NO: 42), IL2Rp_530-551 (SEQ ID NO: 43), IL18R_373-520 (SEQ ID NO: 44), or IL18R_369-541 (SEQ ID NO: 45).

[0018] In some embodiments, the intracellular signaling domain comprises one or more amino acid sequences at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from the sequences provided as SEQ ID NOs: 9, 11, 12, 14, 15, 17, 28, 20, 21, 23, 24, 26, 27, 28, 30, 31, 33, 55, 56, or 284.

[0019] In some embodiments, the intracellular signaling domain induces phosphorylation of at least one of JAK1, JAK2, and / or JAK3 upon multimerization of the SPA.IPTS / 200097280.2 3Attorney Ref: ANB-223WO

[0020] In some embodiments, the one or more JAK signaling domains comprises at least one JAK signaling domain from at least one of gpl30, IL-7Ra, G-CSFR, IL-12RP2, IL-9R, and IL-21R, or any combination thereof.

[0021] In some embodiments, the JAK signaling domain comprises one or more amino acid sequences at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence as set forth in SEQ ID NOs: 10, 13, 16, 19, 22, 25, 29, 32, 47, 48, 49, 50, 51, 52, 53, or 54.

[0022] In some embodiments, the co- stimulatory domain comprises a CD28 co-stimulatory domain.

[0023] In some embodiments, the CD28 co-stimulatory domain comprises a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 46.

[0024] In some embodiments, the transmembrane domain comprises a gpl30 transmembrane domain, an IL-7Ra transmembrane domain, a G-CFSR transmembrane domain, or a G-CFSR transmembrane domain, or a portion thereof.

[0025] In some embodiments, the G-CFSR transmembrane domain comprises a T640N mutation as compared to the full length G-CFSR protein (SEQ ID NO: 263).

[0026] In some embodiments, the transmembrane domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NOs: 6, 7, 8, 261, or 262.

[0027] In some embodiments, the extracellular domain comprises a CD34 epitope, aQB END 10 epitope, a CD34 extracellular domain, or a truncated CD34 extracellular domain.

[0028] In some embodiments, the extracellular domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NOs: 499, 475-478, 1, 2, 3, 4, or 5.

[0029] In some embodiments, the extracellular domain conveys constitutive activity to the intracellular signaling domain upon multimerization of the SPA.

[0030] In some embodiments, the extracellular domain comprises an unpaired cysteine.

[0031] In some embodiments, the SPA peptide comprisesIPTS / 200097280.2 4Attorney Ref: ANB-223WO i. a truncated CD34 extracellular domain, an IL7Ra transmembrane domain, a gpl3O JAK domain, a gpl3O STAT1 and STAT3 signaling domain, and an IL7Ra STAT5 signaling domain; ii. a truncated CD34 extracellular domain, an IL7Ra transmembrane domain, a gpl3O JAK signaling domain, a gpl3O STAT1 and STAT3 signaling domain, and an IL-2RP STAT5 signaling domain; iii. a truncated CD34 extracellular domain, an IL7Ra transmembrane domain, a gpl3O JAK domain, a gpl3O STAT1 and STAT3 signaling domain, and an IL- 12RP2 STAT4 signaling domain; iv. a CD34 epitope extracellular domain, a gpl3O transmembrane domain, a gpl3O JAK domain, a gpl3O STAT1 and STAT3 signaling domain, and an IL7Ra STAT5 signaling domain; v. a CD34 epitope extracellular domain, a gpl3O transmembrane domain, a gpl3O JAK domain, a gpl3O STAT1 and STAT3 signaling domain, and an IL- 2RP STAT5 signaling domain; vi. a CD34 epitope extracellular domain, a gpl3O transmembrane domain, a gpl3O JAK domain, a gpl3O STAT1 and STAT3 signaling domain, and an IL- 12RP2 STAT4 signaling domain; or vii. a CD34 epitope extracellular domain, a gpl3O transmembrane domain, a G- CSFR JAK domain, a G-CSFR STAT1 and STAT3 signaling domain, and an IL-12RP2 STAT4 signaling domain.

[0032] In some embodiments, the SPA peptide comprises a sequence selected from the sequences set forth in SEQ ID NOs: 57-254, 264-283, or 419-474.

[0033] In some embodiments, the SPA peptide comprises a sequence selected from the sequences set forth in SEQ ID NOs: 421, 185, 114, 104, 79, 80, 81, 87, 103, 111, 116, 117, 143, 144, 145, 152, 153, 155, 156, 157, 159, 160, 182, 183, 184, 220, 234, 264, 268, 269, 270, 282, 419, 420, or 422.

[0034] In some embodiments, the SPA peptide induces at least 1.5-fold, 2-fold, or 3-fold more phosphorylation of at least STAT1, STAT3, STAT4 and / or STAT5 upon multimerization of the SPA in a cell as compared to a cell that lacks the SPA peptide.

[0035] In some embodiments, the SPA peptide induces at least 1.5-fold, 2-fold, or 3-fold more phosphorylation of at least STAT1, STAT3, and / or STAT4 upon multimerization of the SPA in a cell as compared to a cell that lacks the SPA peptide.IPTS / 200097280.2 5Attorney Ref: ANB-223WO

[0036] In some embodiments, the SPA peptide induces at least 1.5-fold, 2-fold, or 3-fold more phosphorylation of at least STAT4 upon multimerization of the SPA in a cell as compared to a cell that lacks the SPA peptide.

[0037] In some embodiments, the SPA peptide induces at least 1.5-fold, 2-fold, or 3-fold more phosphorylation of at least STAT5 upon multimerization of the SPA in a cell as compared to a cell that lacks the SPA peptide.

[0038] In one aspect, provided herein are dimer or multimer comprising the SPA peptide disclosed herein.

[0039] In one aspect, provided herein are one or more SPA peptides encoded by the one or more nucleic acid(s) disclosed herein.

[0040] In one aspect, provided herein are at least one vector comprising the at least one nucleic acid disclosed herein.

[0041] In one aspect, provided herein are systems comprising: i. one or more nucleic acid(s) encoding a first chimeric polypeptide comprising a priming receptor; ii. one or more nucleic acid(s) encoding a second chimeric polypeptide comprising a chimeric antigen receptor (CAR); and iii. the one or more nucleic acids encoding a SPA peptide disclosed herein.

[0042] In one aspect, provided herein are systems comprising: i. a first chimeric polypeptide comprising a priming receptor; ii. a second chimeric polypeptide comprising a chimeric antigen receptor (CAR); and iii. the SPA peptide disclosed herein.

[0043] In some embodiments, binding by the CAR or priming receptor to a target induces expression of the SPA peptide.

[0044] In some embodiments, the SPA peptide is constitutively or inducibly expressed.

[0045] In one aspect, provided herein are cells or population of cells comprising the SPA peptide disclosed herein, the multimer or dimer disclosed herein, the nucleic acid disclosed herein, the vector disclosed herein, or the system of disclosed herein.

[0046] In some embodiments, the cell is an immune cell or a stem cell, optionally wherein the immune cell is a primary human immune cell.

[0047] In one aspect, provided herein are pharmaceutical composition comprising the cell or population of cells disclosed herein, and a pharmaceutically acceptable excipient.IPTS / 200097280.2 6Attorney Ref: ANB-223WO

[0048] In one aspect, provided herein are pharmaceutical compositions comprising the nucleic acids disclosed herein or the vectors disclosed herein, and a pharmaceutically acceptable excipient.

[0049] In one aspect, provided herein are methods of engineering a cell, comprising introducing the nucleic acid disclosed herein into the cell.

[0050] In some embodiments, the nucleic acid is introduced into an insertion site in the genome of the cell.

[0051] In some embodiments, the nucleic acid is introduced to the cell non-virally.

[0052] In one aspect, provided herein are methods of editing a cell, comprising: i. providing a nuclease domain and a guide RNA and the nucleic acid of claim 29, and wherein the 5’ and 3’ ends of the nucleic acid comprise nucleotide sequences that are homologous to genomic sequences flanking an insertion site in the genome of the cell; ii. introducing the nuclease domain and nucleic acid into the cell, wherein the guide RNA specifically hybridizes to a target region of the genome of the cell, and wherein the nuclease domain cleaves the target region to create an insertion site in the genome of the cell; and iii. editing the cell via insertion of the nucleic acid into the insertion site in the genome of the cell.

[0053] In some embodiments, the nuclease domain and nucleic acid are introduced to the cell non-virally.

[0054] In one aspect, provided herein are methods of editing an immune cell, comprising: i. providing a ribonucleoprotein complex (RNP) complex wherein the RNP comprises a nuclease domain and a guide RNA, and the nucleic acid disclosed herein, wherein the 5’ and 3’ ends of the nucleic acid comprise nucleotide sequences that are homologous to genomic sequences flanking an insertion site in the genome of the immune cell; ii. non-virally introducing the RNP-nucleic acid complex into the immune cell, wherein the guide RNA specifically hybridizes to a target region of the genome of the primary immune cell, and wherein the nuclease domain cleaves the target region to create the insertion site in the genome of the immune cell; and iii. editing the immune cell via insertion of the nucleic acid disclosed herein into the insertion site in the genome of the immune cell.IPTS / 200097280.2 7Attorney Ref: ANB-223WO

[0055] In some embodiments, non-virally introducing comprises electroporation.

[0056] In some embodiments, the nuclease domain comprises a CRISPR-associated endonuclease (Cas), optionally a Cas9 nuclease.

[0057] In some embodiments, herein the target region of the genome of the cell is a genomic safe harbor (GSH) locus or a T Cell Receptor Alpha Constant (TRAC) locus.

[0058] In some embodiments, the target region is the GS94 locus (locus chrl 1:128340000- 128350000).

[0059] In some embodiments, the nucleic acid is a double- stranded nucleic acid or a singlestranded nucleic acid.

[0060] In some embodiments, the nucleic acid is a linear nucleic acid or a circular nucleic acid, optionally wherein the circular nucleic acid is a plasmid.

[0061] In some embodiments, the cell is an immune cell or a stem cell, optionally a primary human immune cell.

[0062] In some embodiments, the immune cell is an autologous immune cell.

[0063] In some embodiments, the immune cell is an allogeneic immune cell.

[0064] In some embodiments, the immune cell is a natural killer (NK) cell, a T cell, a CD8+ T cell, a CD4+ T cell, a primary T cell, or a T cell progenitor cell.

[0065] In some embodiments, the immune cell is a primary T cell.

[0066] In some embodiments, the immune cell is a primary human T cell.

[0067] In some embodiments, the cell is virus-free.

[0068] In some embodiments, comprising determining CD11c expression in the cell.

[0069] In some embodiments, comprising obtaining the cell from a patient and introducing the nucleic acid in vitro.

[0070] In one aspect, provided herein are methods of treating a disease in a subject comprising administering the nucleic acid disclosed herein, the vector disclosed herein, the cell or population of cells disclosed herein, or the pharmaceutical composition disclosed herein to the subject.

[0071] In some embodiments, the disease is cancer.

[0072] In some embodiments, the cancer is a solid cancer or a liquid cancer.

[0073] In some embodiments, the cancer is kidney cancer, clear cell renal cell carcinoma (ccRcc), colorectal cancer, or lung cancer.

[0074] In one aspect, provided herein are methods of inhibiting a target cell in a subject comprising administering the nucleic acid disclosed herein, the vector disclosed herein, theIPTS / 200097280.2 8Attorney Ref: ANB-223WO cell or population of cells disclosed herein, or the pharmaceutical composition disclosed herein to the subject, wherein the cell inhibits the target cell.

[0075] In one aspect, provided herein are methods of modulating the activity of a cell or an immune cell comprising: i. obtaining a cell or an immune cell comprising ii. SPA peptide disclosed herein; iii. the multimer or dimer disclosed herein; iv. the system disclosed herein; v. the nucleic acid disclosed herein; and / or vi. the vector disclosed herein; and vii. contacting the cell or immune cell with a target cell, wherein the synthetic pathway activator modulates the activity of the cell or immune cell.

[0076] In one aspect, provided herein are methods of modulating the activity of an cell or immune cell comprising: i. obtaining a cell or immune cell comprising ii. SPA peptide disclosed herein; iii. the multimer or dimer disclosed herein; iv. the system disclosed herein; v. the nucleic acid disclosed herein; and / or vi. the vector disclosed herein; and vii. contacting the cell or immune cell with a target cell expressing a priming receptor antigen and a CAR antigen, wherein binding of the priming receptor to the priming receptor antigen on the target cell induces activation of the priming receptor and expression of the chimeric antigen receptor, wherein binding of the chimeric antigen receptor to CAR antigen on the target cell modulates the activity of the cell or immune cell, and wherein the synthetic pathway activator also modulates the activity of the cell or immune cell.

[0077] In some embodiments, the activity comprises inducing the JAK-STAT signaling pathway.

[0078] In some embodiments, the activity comprises an enhanced immune activity as compared to a cell that does not comprise the SPAIPTS / 200097280.2 9Attorney Ref: ANB-223WO

[0079] In some embodiments, the immune activity comprises increased cytotoxicity, immune cell persistence, and / or tumor infiltration as compared to a cell that does not comprise the SPA.

[0080] In some embodiments, the SPA induces phosphorylation of at least STAT1, STAT3, STAT4, STAT5, and / or TIR upon multimerization of the SPA in the cell.

[0081] In some embodiments, the SPA peptide induces at least 1.5-fold, 2-fold, or 3-fold more phosphorylation of at least STAT1, STAT3, STAT4 and STAT5 upon multimerization of the SPA in a cell as compared to a cell that lacks the SPA peptide.

[0082] In some embodiments, the SPA peptide induces at least 1.5-fold, 2-fold, or 3-fold more phosphorylation of at least STAT1, STAT3, and STAT4 upon multimerization of the SPA in a cell as compared to a cell that lacks the SPA peptide.

[0083] In some embodiments, the SPA peptide induces at least 1.5-fold, 2-fold, or 3-fold more phosphorylation of STAT4 upon multimerization of the SPA in a cell as compared to a cell that lacks the SPA peptide.

[0084] In some embodiments, the SPA peptide induces at least 1.5-fold, 2-fold, or 3-fold more phosphorylation of STAT5 upon multimerization of the SPA in a cell as compared to a cell that lacks the SPA peptide.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0085] These and other features, aspects, and advantages of the present disclosure will become better understood with regard to the following description, and accompanying drawings, where:

[0086] FIG. 1A provides a diagram of an exemplary synthetic pathway activator. FIG. IB provides diagrams of exemplary synthetic pathway activators disclosed herein. A figure legend is provided in FIG. 1C.

[0087] FIG. 2 shows a cell survival assay (CSA) showing the target cell survival curve after incubating tumor cells with T cells expressing the SPAs provided in Tables 1-12. Control molecules used included a SPA of SEQ ID NO: 259, Lgpl30-trunc7, C7R, a constitutive SPA with added IL2, a constitutive SPA of SEQ ID NO: 259 with added IL-2, a constitutive SPA of SEQ ID NO: 259 with added IL15Ra-IL15 sushi, an inducible SPA of SEQ ID NO: 259 with added IL-2, and an inducible SPA of SEQ ID NO: 259 with added IL15Ra-IL15 sushi.

[0088] FIG. 3 shows the pSTAT profiling of the entire library of SPAs and the top 30 candidates. More potent SPAs are shown at the top of the right panel.IPTS / 200097280.2 10Attorney Ref: ANB-223WO

[0089] FIG. 4A, 4B, 4C, 4D, and 4E shows the correlation of the pSTATl, pSTAT3, pSTAT4, pSTAT5, and pSTAT6 with cytokine independent growth of the edited T cells.

[0090] FIG. 5 A and 5B shows growth of the edited T cells over time after cytokine withdrawal.

[0091] FIG. 6A and 6B show the extended cytokine free survival of T cells expressing the indicated SPAs. 22 SPAs demonstrate increased potency with limited cytokine-free survival.

[0092] FIG. 7 shows a diagram of the increased STAT signaling axis of the SPAs provided herein as compared to the SPA shown in SEQ ID NO: 259. As shown, the SPAs have an increased STAT signaling space as compared to the SPA of SEQ ID NO: 259, with increased STAT 1 signal and additional STAT4 and STAT5 signaling.

[0093] FIG. 8A shows target cell count after incubation with PCT cells expressing the indicate SPA or control T cells. FIG. 8B shows the fold-benefit change in target cell killing at the endpoint of PCT cells expressing the indicate SPA as normalized to PCT cells expressing the SPA of SEQ ID NO: 259 or control constructs.

[0094] FIG. 9A shows secretion of GM-CSF by PCT cells expressing the indicated SPA, a constitutively expressed CAR, or cells engineered with RNP. FIG. 9B shows secretion of IFNy by PCT cells expressing the indicated SPA, a constitutively expressed CAR, or cells engineered with RNP. FIG. 9C shows secretion of TNFa by PCT cells expressing the indicated SPA, a constitutively expressed CAR, or cells engineered with RNP. FIG. 9D shows secretion of IL- 10 by PCT cells expressing the indicated SPA, a constitutively expressed CAR, or cells engineered with RNP. Paired T test for cytokine concentration relative to SPA of SEQ ID NO: 259. * P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001.

[0095] FIG. 10A provides the PCT cell survival for cells from donor 1 after incubation with cytokine-free media for up to 29 days. FIG. 10B provides the PCT cell survival for cells from donor 2.

[0096] FIG. 11A shows the percent change in edited T cell survival over time in cytokine free media over 58 days. FIG. 11B shows the percent change in edited T cell survival over time in media containing 125 pg / ml IL-7 and IL-15 over 58 days. FIG. 11C shows the percent change in edited T cell survival over time in media containing 12.5 ng / ml IL-7 and IL- 15 over 58 days.

[0097] FIG. 12A shows the fold expansion of edited PCT cells from donor 1 expressing the indicated SPA in a repeat stimulation assay over 12 days. FIG. 12B shows the fold expansion of edited PCT cells from donor 2 expressing the indicated SPA in a repeat stimulation assay over 12 days IPTS / 200097280.2 1 1Attorney Ref: ANB-223WO

[0098] FIG. 13A provides a summary of the increased STAT signaling in T cells expressing the SPAs disclosed herein (“new SPA”) as compared to the baseline SPA (“control SPA”) and standard T cells. FIG. 13B provides a summary of increased potency and persistence of T cells expressing the SPAs disclosed herein (“new SPA”) as compared to the baseline SPA (“control SPA”) and standard T cells.DETAILED DESCRIPTIONDefinitions

[0099] Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0100] As used herein, the term “gene” refers to the basic unit of heredity, consisting of a segment of DNA arranged along a chromosome, which codes for a specific protein or segment of protein. A gene typically includes a promoter, a 5’ untranslated region, one or more coding sequences (exons), optionally introns, and a 3’ untranslated region. The gene may further comprise a terminator, enhancers and / or silencers.

[0101] As used herein, the term “locus” refers to a specific, fixed physical location on a chromosome where a gene or genetic marker is located.

[0102] The term “safe harbor locus” refers to a locus at which genes or genetic elements can be incorporated without disruption to expression or regulation of adjacent genes. These safe harbor loci are also referred to as safe harbor sites (SHS). As used herein, a safe harbor locus refers to an “integration site” or “knock-in site” at which a sequence encoding a transgene, as defined herein, can be inserted. In some embodiments the insertion occurs with replacement of a sequence that is located at the integration site. In some embodiments, the insertion occurs without replacement of a sequence at the integration site. Examples of integration sites contemplated are provided in Table D.

[0103] As used herein, the term “insert” refers to a nucleotide sequence that is integrated (inserted) at a target locus or safe harbor site. The insert can be used to refer to the genes or genetic elements that are incorporated at the target locus or safe harbor site using, for example, homology-directed repair (HDR) CRISPR / Cas9 genome-editing or other methods for inserting nucleotide sequences into a genomic region known to those of ordinary skill in the art.

[0104] The term “inserting” refers to a manipulation of a nucleotide sequence to introduce a non-native sequence. This is done, for example, via the use of restrPCTion enzymes and ligases whereby the DNA sequence of interest, usually encoding the gene of interest, can beIPTS / 200097280.2 12Attorney Ref: ANB-223WO incorporated into another nucleic acid molecule by digesting both molecules with appropriate restrPCTion enzymes in order to create compatible overlaps and then using a ligase to join the molecules together. One skilled in the art is very familiar with such manipulations and examples may be found in Sambrook et al. (Sambrook, Fritsch, & Maniatis, “Molecular Cloning: A Laboratory Manual”, 2nded., Cold Spring Harbor Laboratory, 1989), which is hereby incorporated by reference in its entirety including any drawings, figures and tables.

[0105] The “CRISPR / Cas” system refers to a widespread class of bacterial systems for defense against foreign nucleic acid. CRISPR / Cas systems are found in a wide range of eubacterial and archaeal organisms. CRISPR / Cas systems include type I, II, and III subtypes. Wild-type type II CRISPR / Cas systems utilize an RNA-mediated nuclease, Cas9 in complex with guide and activating RNA to recognize and cleave foreign nucleic acid. Guide RNAs having the activity of both a guide RNA and an activating RNA are also known in the art. In some cases, such dual activity guide RNAs are referred to as a small guide RNA (sgRNA).

[0106] Cas9 homologs are found in a wide variety of eubacteria, including, but not limited to bacteria of the following taxonomic groups: Actinobacteria, Aquificae, Bacteroidetes- Chlorobi, Chlamydiae-Verrucomicrobia, Chlroflexi, Cyanobacteria, Firmicutes, Proteobacteria, Spirochaetes, and Thermotogae. An exemplary Cas9 protein is the Streptococcus pyogenes Cas9 protein. Additional Cas9 proteins and homologs thereof are described in, e.g., Chylinksi, et al., RNA Biol. 2013 May 1; 10(5): 726-737 ; Nat. Rev. Microbiol. 2011 June; 9(6): 467-477; Hou, et al., Proc Natl Acad Sci U S A. 2013 Sep 24; 110(39): 15644-9; Sampson et al., Nature. 2013 May 9;497(7448):254-7; and Jinek, et al., Science. 2012 Aug 17;337(6096):816-21. The Cas9 nuclease domain can be optimized for efficient activity or enhanced stability in the host cell.

[0107] As used herein, the term “Cas9” refers to an RNA-mediated nuclease (e.g., of bacterial or archeal orgin, or derived therefrom). Exemplary RNA-mediated nuclases include the foregoing Cas9 proteins and homologs thereof, and include but are not limited to, CPF1 (See, e.g., Zetsche et al., Cell, Volume 163, Issue 3, p759-771, 22 October 2015). Similarly, as used herein, the term “Cas9 ribonucleoprotein” complex and the like refers to a complex between the Cas9 protein, and a crRNA (e.g., guide RNA or small guide RNA), the Cas9 protein and a trans-activating crRNA (tracrRNA), the Cas9 protein and a small guide RNA, or a combination thereof (e.g., a complex containing the Cas9 protein, a tracrRNA, and a crRNA guide RNA).IPTS / 200097280.2 13Attorney Ref: ANB-223WO

[0108] As used herein, the phrase “immune cell” is inclusive of all cell types that can give rise to immune cells, including hematopoietic cells such hematopoietic stem cells, pluripotent stem cells, and induced pluripotent stem cells (iPSCs). In some embodiments, the immune cell is a B cell, macrophage, a natural killer (NK) cell, an induced pluripotent stem cell (iPSC), a human pluripotent stem cell (HSPC), a T cell or a T cell progenitor cell, or dendritic cell. In some embodiments, the cell is an innate immune cell.

[0109] As used herein, the term “primary” in the context of a primary cell or primary stem cell refers to a cell that has not been transformed or immortalized. Such primary cells can be cultured, sub-cultured, or passaged a limited number of times (e.g., cultured 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times). In some cases, the primary cells are adapted to in vitro culture conditions. In some cases, the primary cells are isolated from an organism, system, organ, or tissue, optionally sorted, and utilized, e.g., directly without culturing or sub-culturing. In some cases, the primary cells are stimulated, activated, or differentiated. For example, primary T cells can be activated by contact with (e.g., culturing in the presence of) CD3, CD28 agonists, IL-2, IFN-y, or a combination thereof.

[0110] As used herein, the terms “T lymphocyte” and “T cell” are used interchangeably and refer to cells that have completed maturation in the thymus, and identify certain foreign antigens in the body. The terms also refer to the major leukocyte types that have various roles in the immune system, including activation and deactivation of other immune cells. The T cell can be any T cell such as a cultured T cell, e.g., a primary T cell, or a T cell derived from a cultured T cell line, e.g., a Jurkat, SupTl, etc., or a T cell obtained from a mammal. T cells include, but are not limited to, naive T cells, stimulated T cells, primary T cells (e.g., uncultured), cultured T cells, immortalized T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, combinations thereof, or sub-populations thereof. The T cell can be a CD3 + cell. T cells can be CD4+, CD8+, or CD4+and CD8+. The T cell can be any type of T cell, CD4 + / CD8 + double positive T cells, CD4 + helper T cells (e.g. Thl and Th2 cells), CD8 + T cells (e.g. cytotoxic T cells), peripheral Including but not limited to blood mononuclear cells (PBMC), peripheral blood leukocytes (PBL), tumor infiltrating lymphocytes (TIL), memory T cells, naive T cells, T cells, regulatory T cells, y5 T cells, etc. It can be any T cell at any stage of development. Additional types of helper T cells include Th3 (Treg) cells, Thl7 cells, Th9 cells, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tern cells and TEMRA cells). A T cell can also refer to a genetically modified T cell, such as a TIPTS / 200097280.2 14Attorney Ref: ANB-223WO cell that has been modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR). T cells can also be differentiated from stem cells or progenitor cells.

[0111] ‘ ‘CD4 + T cells” refers to a subset of T cells that express CD4 on their surface and are associated with a cellular immune response. CD4 + T cells are characterized by a poststimulation secretion profile that can include secretion of cytokines such as IFN-y, TNF-a, IL-2, IL-4 and IL- 10. “CD4” is a 55 kD glycoprotein originally defined as a differentiation antigen on T lymphocytes, but was also found on other cells including monocytes / macrophages. The CD4 antigen is a member of the immunoglobulin superfamily and has been implicated as an associative recognition element in MHC (major histocompatibility complex) class II restrPCTed immune responses. On T lymphocytes, the CD4 antigen defines a helper / inducer subset.

[0112] ‘ ‘CD8 + T cells” refers to a subset of T cells that express CD8 on their surface, are MHC class I restrPCTed, and function as cytotoxic T cells. The “CD8” molecule is a differentiation antigen present on thymocytes, as well as on cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin superfamily and is an associative recognition element in major histocompatibility complex class I restrPCTion interactions.

[0113] As used herein, the phrase “hematopoietic stem cell” refers to a type of stem cell that can give rise to a blood cell. Hematopoietic stem cells can give rise to cells of the myeloid or lymphoid lineages, or a combination thereof. Hematopoietic stem cells are predominantly found in the bone marrow, although they can be isolated from peripheral blood, or a fraction thereof. Various cell surface markers can be used to identify, sort, or purify hematopoietic stem cells. In some cases, hematopoietic stem cells are identified as c- kit+and lin'. In some cases, human hematopoietic stem cells are identified as CD34+, CD59+, Thyl / CD90+, CD38lo / ', C-kit / CD117+, lin'. In some cases, human hematopoietic stem cells are identified as CD34', CD59+, Thyl / CD90+, CD38lo / ', C-kit / CD117+, lin'. In some cases, human hematopoietic stem cells are identified as CD133+, CD59+, Thyl / CD90+, CD38lo / ', C- kit / CDl 17+, lin'. In some cases, mouse hematopoietic stem cells are identified as CD34lo / ', SCA-1+, Thyl+ / 1°, CD38+, C-kit+, lin'. In some cases, the hematopoietic stem cells are CD150+CD48 CD244'.

[0114] As used herein, the phrase “hematopoietic cell” refers to a cell derived from a hematopoietic stem cell. The hematopoietic cell may be obtained or provided by isolation from an organism, system, organ, or tissue (e.g., blood, or a fraction thereof). Alternatively, an hematopoietic stem cell can be isolated and the hematopoietic cell obtained or provided by IPTS / 200097280.2 1 <Attorney Ref: ANB-223WO differentiating the stem cell. Hematopoietic cells include cells with limited potential to differentiate into further cell types. Such hematopoietic cells include, but are not limited to, multipotent progenitor cells, lineage-restrPCTed progenitor cells, common myeloid progenitor cells, granulocyte-macrophage progenitor cells, or megakaryocyte-erythroid progenitor cells. Hematopoietic cells include cells of the lymphoid and myeloid lineages, such as lymphocytes, erythrocytes, granulocytes, monocytes, and thrombocytes.

[0115] As used herein, the term “construct” refers to a complex of molecules, including macromolecules or polynucleotides.

[0116] As used herein, the term “integration” refers to the process of stably inserting one or more nucleotides of a construct into the cell genome, i.e., covalently linking to a nucleic acid sequence in the chromosomal DNA of the cell. It may also refer to nucleotide deletions at a site of integration. Where there is a deletion at the insertion site, “integration” may further include substitution of the endogenous sequence or nucleotide deleted with one or more inserted nucleotides.

[0117] As used herein, the term “exogenous” refers to a molecule or activity that has been introduced into a host cell and is not native to that cell. The molecule can be introduced, for example, by introduction of the encoding nucleic acid into host genetic material, such as by integration into a host chromosome, or as non-chromosomal genetic material, such as a plasmid. Thus, the term, when used in connection with expression of an encoding nucleic acid, refers to the introduction of the encoding nucleic acid into a cell in an expressible form. The term “endogenous” refers to a molecule or activity that is present in a host cell under natural, unedited conditions. Similarly, the term, when used in connection with expression of the encoding nucleic acid, refers to expression of the encoding nucleic acid that is contained within the cell and not introduced exogenously.

[0118] The term “heterologous” refers to a nucleic acid or polypeptide sequence or domain which is not native to a flanking sequence, e.g., wherein the heterologous sequence is not found in nature coupled to the nucleic acid or polypeptide sequences occurring at one or both ends.

[0119] The term “homologous” refers to a nucleic acid or polypeptide sequence or domain which is native to a flanking sequence, e.g., wherein the homologous sequence is found in nature coupled to the nucleic acid or polypeptide sequences occurring at one or both ends.

[0120] As used herein, a “polynucleotide donor construct” refers to a nucleotide sequence (e.g. DNA sequence) that is genetically inserted into a polynucleotide and is exogenous to that polynucleotide. The polynucleotide donor construct is transcribed into RNA and IPTS / 200097280.2 1 .Attorney Ref: ANB-223WO optionally translated into a polypeptide. The polynucleotide donor construct can include prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. For example, the polynucleotide donor construct can be a miRNA, shRNA, natural polypeptide (i.e., a naturally occurring polypeptide) or fragment thereof or a variant polypeptide (e.g. a natural polypeptide having less than 100% sequence identity with the natural polypeptide) or fragments thereof.

[0121] As used herein, the term “complementary” or “complementarity” refers to specific base pairing between nucleotides or nucleic acids. Complementary nucleotides are, generally, A and T (or A and U), and G and C. The guide RNAs described herein can comprise sequences, for example, DNA targeting sequence that are perfectly complementary or substantially complementary (e.g., having 1-4 mismatches) to a genomic sequence in a cell.

[0122] As used herein, the term “transgene” refers to a polynucleotide that has been transferred naturally, or by any of a number of genetic engineering techniques from one organism to another. It is optionally translated into a polypeptide. As used, transgene can refer to a polynucleotide that encodes a polypeptide.

[0123] The terms “protein,” “polypeptide,” and “peptide” are used herein interchangeably.

[0124] As used herein, the term “operably linked” or “operatively linked” refers to the binding of a nucleic acid sequence to a single nucleic acid fragment such that one function is affected by the other. For example, if a promoter is capable of affecting the expression of a coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under transcriptional control by the promoter), the promoter is operably linked thereto. Coding sequences can be operably linked to control sequences in both sense and antisense orientation.

[0125] As used herein, the term “developmental cell states” refers to, for example, states when the cell is inactive, actively expressing, differentiating, senescent, etc. developmental cell state may also refer to a cell in a precursor state (e.g., a T cell precursor).

[0126] As used, the term “encoding” refers to a sequence of nucleic acids which codes for a protein or polypeptide of interest. The nucleic acid sequence may be either a molecule of DNA or RNA. In preferred embodiments, the molecule is a DNA molecule. In other preferred embodiments, the molecule is a RNA molecule. When present as a RNA molecule, it will comprise sequences which direct the ribosomes of the host cell to start translation (e.g., a start codon, ATG) and direct the ribosomes to end translation (e.g., a stop codon). BetweenIPTS / 200097280.2 17Attorney Ref: ANB-223WO the start codon and stop codon is an open reading frame (ORF). Such terms are known to one of ordinary skill in the art.

[0127] As used herein, the term “subject” refers to a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits, pigs and sheep. In certain embodiments, the subject is a human. In some embodiments the subject has a disease or condition that can be treated with an engineered cell provided herein or population thereof. In some aspects, the disease or condition is a cancer.

[0128] As used herein, the term “promoter” refers to a nucleotide sequence (e.g. DNA sequence) capable of controlling the expression of a coding sequence or functional RNA. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. A promoter can be derived from natural genes in its entirety, can be composed of different elements from different promoters found in nature, and / or may comprise synthetic DNA segments. A promoter, as contemplated herein, can be endogenous to the cell of interest or exogenous to the cell of interest. It is appreciated by those skilled in the art that different promoters can induce gene expression in different tissue or cell types, or at different developmental stages, or in response to different environmental conditions. As is known in the art, a promoter can be selected according to the strength of the promoter and / or the conditions under which the promoter is active, e.g., constitutive promoter, strong promoter, weak promoter, inducible / repressible promoter, tissue specific Or developmentally regulated promoters, cell cycle-dependent promoters, and the like.

[0129] A promoter can be an inducible promoter (e.g., a heat shock promoter, tetracycline- regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor- regulated promoter, HNFla promoter, etc.). The promoter can be a constitutive promoter (e.g., CMV promoter, UBC promoter). In some embodiments, the promoter can be a spatially restrPCTed and / or temporally restrPCTed promoter (e.g., a tissue specific promoter, a cell type specific promoter, etc.). See for example US Publication 20180127786, the disclosure of which is herein incorporated by reference in its entirety.

[0130] Gene editing, as contemplated herein, may involve a gene (or nucleotide sequence) knock-in or knock-out. As used herein, the term “knock-in” refers to an addition of a DNA sequence, or fragment thereof into a genome. Such DNA sequences to be knocked- in may include an entire gene or genes, may include regulatory sequences associated with a gene or any portion or fragment of the foregoing. For example, a polynucleotide donor construct encoding a protein may be inserted into the genome of a cell carrying a mutant gene. In some embodiments, a knock-in strategy involves substitution of an existing sequence with the IPTS / 200097280.2 1 OAttorney Ref: ANB-223WO provided sequence, e.g., substitution of a mutant allele with a wild-type copy. On the other hand, the term “knock-out” refers to the elimination of a gene or the expression of a gene. For example, a gene can be knocked out by either a deletion or an addition of a nucleotide sequence that leads to a disruption of the reading frame. As another example, a gene may be knocked out by replacing a part of the gene with an irrelevant (.e.g., non-coding) sequence.

[0131] As used herein, the term “non-homologous end joining” or NHEJ refers to a cellular process in which cut or nicked ends of a DNA strand are directly ligated without the need for a homologous template nucleic acid. NHEJ can lead to the addition, the deletion, substitution, or a combination thereof, of one or more nucleotides at the repair site.

[0132] As used herein, the term “homology directed repair” or HDR refers to a cellular process in which cut or nicked ends of a DNA strand are repaired by polymerization from a homologous template nucleic acid. Thus, the original sequence is replaced with the sequence of the template. The homologous template nucleic acid can be provided by homologous sequences elsewhere in the genome (sister chromatids, homologous chromosomes, or repeated regions on the same or different chromosomes). Alternatively, an exogenous template nucleic acid can be introduced to obtain a specific HDR-induced change of the sequence at the target site. In this way, specific mutations can be introduced at the cut site.

[0133] As used herein, a single- stranded DNA template or a double- stranded DNA template refers to a DNA oligonucleotide that can be used by a cell as a template for HDR. Generally, the single- stranded DNA template or a double-stranded DNA template has at least one region of homology to a target site. In some cases, the single-stranded DNA template or doublestranded DNA template has two homologous regions flanking a region that contains a heterologous sequence to be inserted at a target cut site.

[0134] The terms “vector” and “plasmid” are used interchangeably and as used herein refer to polynucleotide vehicles useful to introduce genetic material into a cell. Vectors can be linear or circular. Vectors can integrate into a target genome of a host cell or replicate independently in a host cell. Vectors can comprise, for example, an origin of replication, a multicloning site, and / or a selectable marker. An expression vector typically comprises an expression cassette. Vectors and plasmids include, but are not limited to, integrating vectors, prokaryotic plasmids, eukaryotic plasmids, plant synthetic chromosomes, episomes, cosmids, and artificial chromosomes.

[0135] As used herein, the phrase “introducing” in the context of introducing a nucleic acid or a complex comprising a nucleic acid, for example, an RNP-DNA template complex, refers to the translocation of the nucleic acid sequence or the RNP-DNA template complex from IPTS / 200097280.2 1 QAttorney Ref: ANB-223WO outside a cell to inside the cell. In some cases, introducing refers to translocation of the nucleic acid or the complex from outside the cell to inside the nucleus of the cell. Various methods of such translocation are contemplated, including but not limited to, electroporation, contact with nanowires or nanotubes, receptor mediated internalization, translocation via cell penetrating peptides, liposome mediated translocation, and the like.

[0136] As used herein the term “expression cassette” is a polynucleotide construct, generated recombinantly or chemically synthesized, comprising regulatory sequences operably linked to a selected polynucleotide to facilitate expression of the selected polynucleotide in a host cell. For example, the regulatory sequences can facilitate transcription of the selected polynucleotide in a host cell, or transcription and translation of the selected polynucleotide in a host cell. An expression cassette can, for example, be integrated in the genome of a host cell or be present in an expression vector.

[0137] As used herein, the phrase “subject in need thereof’ refers to a subject that exhibits and / or is diagnosed with one or more symptoms or signs of a disease or disorder as described herein.

[0138] A “chemotherapeutic agent” refers to a chemical compound useful in the treatment of cancer. Chemotherapeutic agents include “anti-hormonal agents” or “endocrine therapeutics” which act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer.

[0139] The term “composition” refers to a mixture that contains, e.g., an engineered cell or protein contemplated herein. In some embodiments, the composition may contain additional components, such as adjuvants, stabilizers, excipients, and the like. The term “composition” or “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective in treating a subject, and which contains no additional components which are unacceptably toxic to the subject in the amounts provided in the pharmaceutical composition.

[0140] The term “in situ” refers to processes that occur in a living cell growing separate from a living organism, e.g., growing in tissue culture.

[0141] The term “in vivo” refers to processes that occur in a living organism.

[0142] As used herein, the term “ex vivo” generally includes experiments or measurements made in or on living tissue, preferably in an artificial environment outside the organism, preferably with minimal differences from natural conditions.IPTS / 200097280.2 20Attorney Ref: ANB-223WO

[0143] The term “mammal” as used herein includes both humans and non-humans and include but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0144] The term “percent identity,” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the “percent identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.

[0145] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0146] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc’ NatT. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

[0147] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov / ).

[0148] The term “sufficient amount” means an amount sufficient to produce a desired effect, e.g., an amount sufficient to modulate protein aggregation in a cell.

[0149] The term “therapeutically effective amount” is an amount that is effective to ameliorate a symptom of a disease.IPTS / 200097280.2 21Attorney Ref: ANB-223WO

[0150] The term “ameliorating” refers to any therapeutically beneficial result in the treatment of a disease state, e.g., a cancer disease state, lessening in the severity or progression, remission, or cure thereof.

[0151] As used herein, the term “effective amount” refers to the amount of a compound (e.g., a compositions described herein, cells described herein) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.

[0152] As used herein, the term “treating” includes any effect, e.g., lessening, reducing, modulating, ameliorating or eliminating, that results in the improvement of the condition, disease, disorder, and the like, or ameliorating a symptom thereof.

[0153] The terms “modulate” and “modulation” refer to reducing or inhibiting or, alternatively, activating or increasing, a recited variable.

[0154] The terms “increase” and “activate” refer to an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater in a recited variable.

[0155] The terms “reduce” and “inhibit” refer to a decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50- fold, 100-fold, or greater in a recited variable.

[0156] With regard to the binding of an antibody to a target molecule, the terms “bind,” “specific binding,” “specifically binds to,” “specific for,” “selectively binds,” and “selective for” a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen mean binding that is measurably different from a non-specific or non-selective interaction (e.g., with a non-target molecule). For example, an antibody that “selectively binds” or “specifically binds” an antigen is an antigen-binding moiety that binds the antigen with high affinity and does not significantly bind other unrelated antigens. Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. In that case, specific binding is indicated if the binding of the antibody to the target molecule is competitively inhibited by the control molecule.

[0157] “Affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or epitope). Unless indicated otherwise, as used herein, “affinity” refers to intrinsic IPTS / 200097280.2 99Attorney Ref: ANB-223WO binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen or epitope). The affinity of a molecule X for its partner Y can be represented by the dissociation equilibrium constant (KD). The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including, but not limited to, surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®).

[0158] The term “hypervariable region” or “HVR”, as used herein, refers to each of the regions of an antibody variable domain which are hypervariable in sequence and / or form structurally defined loops (“hypervariable loops”). Generally, native four-chain antibodies comprise six HVRs; three in the VH (Hl, H2, H3), and three in the VL (LI, L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops and / or from the complementarity determining regions (CDRs), the latter being of highest sequence variability and / or involved in antigen recognition. With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. Hypervariable regions (HVRs) are also referred to as “complementarity determining regions” (CDRs), and these terms are used herein interchangeably in reference to portions of the variable region that form the antigen-binding regions. This particular region has been described by Kabat et al., U.S. Dept, of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and by Chothia et al., J Mol Biol 196:901-917 (1987), where the definitions include overlapping or subsets of amino acid residues when compared against each other.Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.

[0159] The amino acid sequence boundaries of a CDR can be determined by one of skill in the art using any of a number of known numbering schemes, including those described by Kabat et al., supra (“Kabat” numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (“Chothia” numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732- 745 (“Contact” numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (“IMGT” numbering scheme); and Honegge and Pliickthun, J. Mol. Biol., 2001, 309:657-70 (“AHo” numbering scheme); each of which is incorporated by reference in its entirety.IPTS / 200097280.2 23Attorney Ref: ANB-223WO

[0160] Table A provides the positions of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR- H2, and CDR-H3 as identified by the Kabat and Chothia schemes. For CDR-H1, residue numbering is provided using both the Kabat and Chothia numbering schemes.

[0161] CDRs may be assigned, for example, using antibody numbering software, such as Abnum, available at bioinf.org.uk / abs / abnum / , and described in Abhinandan and Martin, Immunology, 2008, 45:3832-3839, incorporated by reference in its entirety.

[0162] The “EU numbering scheme” is generally used when referring to a residue in an antibody heavy chain constant region (e.g., as reported in Kabat et al., supra). Unless stated otherwise, the EU numbering scheme is used to refer to residues in antibody heavy chain constant regions described herein.

[0163] As used herein, the term “single chain” refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In a particular such embodiment, the C- terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain in the single-chain Fab molecule. As described in more detail herein, an scFv has a variable domain of light chain (VL) connected from its C-terminus to the N-terminal end of a variable domain of heavy chain (VH) by a polypeptide chain. Alternately the scFv comprises of polypeptide chain where in the C-terminal end of the VH is connected to the N-terminal end of VL by a polypeptide chain.

[0164] The “Fab fragment” (also referred to as fragment antigen-binding) contains the constant domain (CL) of the light chain and the first constant domain (CHI) of the heavy chain along with the variable domains VL and VH on the light and heavy chains respectively. The variable domains comprise the complementarity determining loops (CDR, also referred to as hypervariable region) that are involved in antigen-binding. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region.

[0165] “F(ab’)2” fragments contain two Fab’ fragments joined, near the hinge region, by disulfide bonds. F(ab’)2 fragments may be generated, for example, by recombinant or synthetic methods or by pepsin digestion of an intact antibody. The F(ab’) fragments can be dissociated, for example, by treatment with B-mercaptoethanol.

[0166] ‘ ‘Fv” fragments comprise a non-covalently-linked dimer of one heavy chain variable domain and one light chain variable domain.

[0167] The “Single-chain Fv” or “scFv” includes the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In one embodiment, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which IPTS / 200097280.2 24Attorney Ref: ANB-223WO enables the scFv to form the desired structure for antigen-binding. For a review of scFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994). HER2 antibody scFv fragments are described in WO93 / 16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458.

[0168] The term “single domain antibody” or “sdAb” refers to a molecule in which one variable domain of an antibody specifically binds to an antigen without the presence of the other variable domain. Single domain antibodies, and fragments thereof, are described in Arabi Ghahroudi et al., FEBS Leters, 1998, 414:521-526 and Muyldermans et al., Trends in Biochem. Sci., 2001, 26:230-245, each of which is incorporated by reference in its entirety. Single domain antibodies are also known as sdAbs or nanobodies. Sdabs are fairly stable and easy to express as fusion partner with the Fc chain of an antibody (Harmsen MM, De Haard HJ (20“7). "Properties, production, and applications of camelid single-domain antibody frag”ents". Appl. Microbiol Biotechnol. 77(1): 13-22).

[0169] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dPCTates otherwise.Synthetic pathway activators

[0170] In various aspects, cells disclosed herein comprise one or more nucleic acids encoding one or more “synthetic pathway activator” (SPA) peptides. In various aspects, systems disclosed herein employ one or more “synthetic pathway activator” (SPA) peptides. Immune cells (e.g., CAR-expressing cells) can be limited by the necessity for in vivo expansion following infusion. To achieve robust expansion, T cells use three signals: antigenstimulation, co- stimulation, and cytokine-induced stimulation. Activation of CARs is sufficient to induce the first two signals, but cannot recapitulate cytokine signaling. Furthermore, the tumor microenvironment is often immunosuppressive and devoid of pro- inflammatory cytokines. SPA peptides can thus be used to stimulate robust in vivo expansion and enhance desirable properties (i.e., increased survival, persistence, and potency) of immune cells such as T cells, e.g., immune cells expressing priming receptors and / or CARs as described herein. The synthetic pathway activators described herein provide enhanced antitumor killing and anti-tumor function without lymphodepletion, such as cytotoxicity, persistence and tumor-infiltration to immune cells. Without wishing to be bound by theory, this increased function is due to the use of combinatorial STAT signaling by incorporating additional JAK / STAT signaling motifs (e.g., STAT1, STAT4, STAT5, Toll-like receptorIPTS / 200097280.2 25Attorney Ref: ANB-223WO(TLR) and / or IL-1R (TIR)) in addition to or instead of a STAT3 signaling motif. For example, the SPA’s provided herein have increased STAT1, STAT3, STAT4, and / or STAT5 signaling as compared to chimeric cytokine receptors (e.g., synthetic pathway activators) comprising single STAT signaling domains.

[0171] In one aspect, provided herein are one or more nucleic acids encoding a synthetic pathway activator (SPA) peptide comprising a chimeric polypeptide comprising: optionally, an extracellular domain; a transmembrane domain; optionally, a costimulatory domain; and an intracellular signaling domain comprising: one or more Janus Kinase (JAK) signaling domains; and a combinatorial signaling domain comprising two or more Signal Transducer and Activator of Transcription (STAT)l, STAT3, STAT4, STAT5 and / or Toll-like receptor (TLR) / IL-1R (TIR) signaling domains(s), or any combination thereof.

[0172] In one aspect, provided herein are one or more nucleic acids encoding a synthetic pathway activator (SPA) peptide comprising a chimeric polypeptide comprising: optionally, an extracellular domain; a transmembrane domain comprising an IL-7Ra transmembrane domain, a G-CSFR, transmembrane domain, or a gpl30 transmembrane domain; optionally, a costimulatory domain; and an intracellular signaling domain comprising: one or more Janus Kinase (JAK) signaling domain(s) comprising a gpl30 JAK signaling domain, an IL-7Ra JAK signaling domain, a G-CSFR JAK signaling domain, an IL-12RP2 JAK signaling domain, an IL-9R JAK signaling domain, or an IL-21R JAK signaling domain, or any combination thereof; and one or more Signal Transducer and Activator of Transcription (STAT)l, STAT3, STAT4, STAT5 and / or Toll-like receptor (TLR) / IL-1R (TIR) signaling domains(s), or any combination thereof.

[0173] In one aspect, provided herein are synthetic pathway activator (SPA) peptides comprising a chimeric polypeptide comprising: a lipid anchor or a transmembrane domain; an intracellular signaling domain; and a multimerization region. In another aspect, provided herein are synthetic pathway activator (SPA) peptides comprising a chimeric polypeptide comprising: an extracellular domain, a lipid anchor or a transmembrane domain; an intracellular signaling domain; and a multimerization region. In some embodiments, a SPA peptide further comprises a CD8-alpha hinge domain. In some embodiments, the SPA peptide is constitutively expressed in a cell. In some embodiments, expression of the SPA peptide in a cell is induced, e.g., by T cell activation. For example, an inducible SPA can be expressed after engagement of a CAR T cell with the CAR cognate ligand on a target cell. In some embodiments, expression of the SPA peptide in a cell is induced by the priming receptor and / or CAR signaling.IPTS / 200097280.2 n .Attorney Ref: ANB-223WOSPA Structure

[0174] In various embodiments, a SPA peptide mimics activation of interleukin signaling. Interleukin receptors are cytokine receptors that signal through Signal Transducer and Activator of Transcription (STAT) transcription factors (e.g., STAT1, STAT3, STAT4, and / or STAT5). Interleukin receptors typically function by dimerization in response to ligand binding. Once dimerized, receptors can bind janus-associated kinases (JAKs) to induce JAK cross-phosphorylation and downstream “JAK / STAT” signaling. Accordingly, induced receptor agonism or ligand-independent dimerization of receptors can be utilized in the Synthetic Pathway Activators to induce constitutive receptor activity and thus, constitutive cytokine signaling. Additional exemplary SPAs are described in W02024192100, hereby incorporated by reference in its entirety.

[0175] In some embodiments, the SPAs disclosed herein comprise an optional extracellular domain (ECD), a transmembrane domains (TMD), an optional intracellular costimulatory domain, and an intracellular domain (ICD) comprising one or more JAK signaling motif(s) or domain(s) and / or a combinatorial signaling motif(s) or domain(s) comprising two or more Signal Transducer and Activator of Transcription (STAT)l, STAT3, STAT4, STAT5 and / or Toll-like receptor (TLR) / IL-1R (TIR) signaling motif(s) or domain(s), or any combination thereof. Exemplary intracellular domains can comprise a full length intracellular domain from a receptor that signals via STAT transcription factors, or STAT and / or TIR signaling adaptors that comprise portions of the signaling motif(s) or domain(s) (e.g., one or more STAT1, STAT3, STAT4, STAT5 and / or TIR signaling motif(s) or domain(s)). Such signaling adaptors allow for shorter length, better defined biology, and diversified signaling intensity. Such STAT signaling adaptors comprise one or more of the tyrosine phosphorylation motifs comprising the sequence YXXC, YENC, YXXQ, YVLQ, or YXPQ.

[0176] In various embodiments, SPA peptides comprise interleukin receptors or functional fragments thereof. In some embodiments, SPAs comprise or are derived from interleukin receptor intracellular signaling domains or functional fragments thereof. Exemplary interleukin or cytokine receptors that can be used in the SPA disclosed herein are, but are not limited to, interleukin-6 signal transducer (IL6ST) protein, an Interleukin-7 receptor alpha (IL-7Ra) protein, a Granulocyte Colony Stimulating Factor Receptor (G- CSFR) protein, an Interleukin- 12 receptor (IL-12R) protein, an Interleukin-9 receptor (IL-9R) protein, an Interleukin-21 receptor (IL-21R) protein, an Interleukin- 18 receptor (IL-18R)IPTS / 200097280.2 27Attorney Ref: ANB-223WO protein, and / or an Interleukin-2 receptor beta (IL-2RP) protein. In some embodiments, SPA peptides comprise or are derived from the an interleukin-6 signal transducer (IL6ST) protein, an Interleukin-7 receptor alpha (IL-7Ra) protein, a Granulocyte Colony Stimulating Factor Receptor (G-CSFR) protein, an Interleukin- 12 receptor (IL-12R) protein, an Interleukin-9 receptor (IL-9R) protein, an Interleukin-21 receptor (IL-21R) protein, an Interleukin- 18 receptor (IL-18R) protein, an Interleukin-2 receptor beta (IL-2RP) protein, or a combination or functional fragment thereof. Interleukin-6 signal transducer (IL6ST) is also known as glycoprotein 130 (gpl30).

[0177] Granulocyte Colony Stimulating Factor Receptor (G-CSFR), is also known as Colony Stimulating Factor 3 Receptor (HGNC: 2439, NCBI Gene: 1441, UniProtKB / Swiss- Prot: Q99062) is a cytokine receptor encoded by the CSF3R gene, and binds to colony stimulating factor 3, a cytokine that controls the production, differentiation, and function of granulocytes.

[0178] Interleukin-2 receptor beta subunit (IL-2RP, IL2RP, IL2-Rb) is also known as CD122, Interleukin- 15 Receptor Subunit Beta, and IL-15RP (HGNC: 6009, NCBI Gene: 3560, UniProtKB / Swiss-Prot: P14784) is an interleukin encoded by the IL2RB gene. The IL- 2R and IL-15R protein complexes share the same beta subunit. The full IL-2 Receptor is comprised of the IL-2RP / IL-15RP subunit, the common y subunit chain IL2RG, and the IL- 2a subunit. The IL-2 receptor is involved in T cell-mediated immune responses. IL-2R signals via JAK1, JAK3, STAT3 and STAT5.

[0179] Interleukin-7 receptor alpha (IL-7Ra, IL7Ra, IL7Ra), is also known as CD25 (HGNC: 6024, NCBI Gene: 3575, UniProtKB / Swiss-Prot: Q01113) and is encoded by the IL7R gene. The IL-7 Receptor is comprised of the IL-7Ra subunit and the IL2R common y subunit chain (IL2RG) and signals via JAK1, JAK3, STAT1, STAT3 and STAT5. The IL- 7Ra subunit can also pair with the TSLPR in lieu of the y subunit and signal via JAK1, JAK2, and STAT5.

[0180] Interleukin-9 receptor (IL-9R), is also known as CD 129 (HGNC: 6030, NCBI Gene: 3581, UniProtKB / Swiss-Prot: P16871) and is encoded by the IL9R gene. The IL-9 Receptor is comprised of the IL-9R subunit and the IL-2R common y subunit chain (IL2RG) and signals via JAK1, JAK3, STAT1, STAT3 and STAT5.

[0181] Interleukin- 12 receptor beta 2 (IL-12RP2, IL12RP-2, IL12RP2), is also known as Interleukin- 12 Receptor Subunit Beta-2 (HGNC: 5972, NCBI Gene: 3595, UniProtKB / Swiss- Prot: Q99665) and is encoded by the IL12RB2 gene. Co expression of the IL12RP-2 and IL-IPTS / 200097280.2 28Attorney Ref: ANB-223WO12R|3- 1 (HGNC: 5971, NCBI Gene: 3594, UniProtKB / Swiss-Prot: P42701) proteins leads to the formation of high- affinity IL- 12 binding sites. IL-12RP2 signals via JAK2 and STAT4.

[0182] Interleukin- 18 receptor alpha (IL-18Ra) is also known as IL-1RRP, CD218a, and IL-18R1 (HGNC: 5988, NCBI Gene: 8809, UniProtKB / Swiss-Prot: Q13478) is encoded by the IL18R1 gene. Interleukin- 18 receptor beta (IL-18RP) is also known as Interleukin 18 receptor accessory protein, IL18RAP, CD218b (HGNC: 5989, NCBI Gene: 8807, UniProtKB / Swiss-Prot: Q95256) is encoded by the IL18RAP gene. The IL18 Receptor is comprised of the IL-18Ra subunit and the IL-18RP subunit and signals via TIR domains to activate MyD88 signal transduction.

[0183] Interleukin-21 receptor (IL-21R) is also know as CD360 (HGNC: 6006, NCBI Gene: 50615, UniProtKB / Swiss-Prot: Q9HBE5) is encoded by the IL21R gene. The IL-21 Receptor is comprised of the IL-21R protein and the IL-2R common y subunit chain (IL2RG) and signals via STAT1 and STAT3. IL-21R transduces the growth promoting signal of IL21, and is important for the proliferation and differentiation of T cells, B cells, and natural killer (NK) cells.

[0184] Interleukin-6 signal transducer (IL6ST) is also known as gpl30, Interleukin 6 Cytokine Family Signal Transducer, CD130, IL-6R subunit beta, IL-6R beta (HGNC: 6021, NCBI Gene: 3572, UniProtKB / Swiss-Prot: P40189) is encoded by the IL6ST gene. IL6ST dimerizes with IL-6Ra to form the IL-6 receptor that signals via JAK1, STAT1, and STAT3.

[0185] Additional description of JAK / STAT signaling pathways and the structure of JAK and STAT proteins is provided in Kiu, H et al., Biology and significance of the JAK / STAT signalling pathways. Growth Factors 2012 April ; 30(2): 88-106. doi: 10.3109 / 08977194.2012.660936; Wingelhofer, B et al., Implications of STAT3 and STAT5 signaling on gene regulation and chromatin remodeling in hematopoietic cancer. Leukemia 32, 1713-1726 (2018); and Hu, X., et al. The JAK / STAT signaling pathway: from bench to clinic. Sig Transduct Target Ther 6, 402 (2021), each of which are hereby incorporated by reference in their entirety.

[0186] A SPA can comprise multimerized SPA peptides, e.g., two SPA peptides that are dimerized, e.g., homodimerized. Multimerization encompasses dimerization, trimerization, tetramerization, or higher order combinations of SPA peptides that interact with each other.Intracellular signaling domain

[0187] In some embodiments, the STAT1, STAT3, STAT4, STAT5 and / or (TIR) signaling motif(s) or domain(s) comprises at least one STAT1 and / or STAT3 signalingIPTS / 200097280.2 29Attorney Ref: ANB-223WO motif(s) or domain(s) from at least one of gpl3O, IL-21R, G-CSFR, or IL-9R, or any combination thereof. In some embodiments, the STAT1, STAT3, STAT4, STAT5 and / or (TIR) signaling motif(s) or domain(s) comprises at least one STAT4 signaling motif(s) or domain(s) from IL-12RP1 or IL-12RP2. In some embodiments, the STAT1, STAT3, STAT4, STAT5 and / or (TIR) signaling motif(s) or domain(s) comprises at least one STAT5 signaling motif(s) or domain(s) from at least one of IL-2RP, IL-7Ra, or IL-15R, or any combination thereof. In some embodiments, the IL-2RP is IL-2RP-1 or IL-2RP-2. In some embodiments, the STAT1, STAT3, STAT4, STAT5 and / or (TIR) signaling motif(s) or domain(s) comprises at least one TIR domain from IL-18R. The TIR signaling motif(s) or domain(s) can be from IL- 18Ra and / or IL- 18RP .

[0188] In some embodiments, the intracellular signaling domain(s) and / or signaling adaptor(s) induces phosphorylation of STAT1, STAT3, STAT4, and / or STAT5 or TIR. In some embodiments, a functional SPA induces phosphorylation of STAT1, STAT3, STAT4, and / or STAT5.

[0189] In some embodiments, the intracellular signaling domain comprises a type I cytokine receptor superfamily boxl (e.g., IWPNVDP (SEQ ID NO: 288), CSREIPDPA (SEQ ID NO: 289), VWPSLPDHK (SEQ ID NO: 290)) or box2 (e.g., VSVVEIEANDKKP (SEQ ID NO: 291)) peptide motif from Janus kinase (JAK)-interacting cytokine receptors. In some embodiments, the JAK signaling motif(s) or domain(s) of the intracellular signaling domain(s) comprise one or more JAK boxl and / or box2 motif(s). In some embodiments, the JAK signaling motif(s) or domain(s) of the intracellular signaling domain(s) comprise one or more JAK1, JAK2, and / or JAK3 signaling motifs. A description of boxl and box2 motifs in JAK cytokine receptors is provided in Usacheva, A, et al., Journal of Biological Chemistry, Volume 277, Issue 50, 48220 - 48226, hereby incorporated by reference.

[0190] In some embodiments, the intracellular signaling domain comprises one or more STAT3 tyrosine phosphorylation motifs (e.g., minimal signaling motif) comprising the sequence YXXC, YENC, YXXQ, YVLQ, or YXPQ. In some embodiments, the intracellular signaling domain comprises one or more tyrosine phosphorylation motifs comprising YXXQ or YXPQ. In some embodiments, the intracellular signaling domain comprises one, two, three, four, five, or more tyrosine phosphorylation motifs comprising YXXQ.

[0191] In some embodiments, the intracellular signaling domain comprises one or more STAT4 tyrosine phosphorylation motifs comprising the sequence THDGYL (SEQ ID NO: 292), YLPSNID (SEQ ID NO: 293), or THDGYLPSNID (SEQ ID NO: 38). In some embodiments, the intracellular signaling domain comprises one, two, three, four, five, or IPTS / 200097280.2 If)Attorney Ref: ANB-223WO more tyrosine phosphorylation motifs comprising THDGYL (SEQ ID NO: 292), YLPSNID (SEQ ID NO: 293), or THDGYLPSNID (SEQ ID NO: 38).

[0192] In some embodiments, the intracellular signaling domain comprises one or more STAT5 tyrosine phosphorylation motifs comprising the sequence YLSL (SEQ ID NO: 500 or PLNTDAYLSLQELQGQDPTHLV (SEQ ID NO: 501).

[0193] In some embodiments, the intracellular signaling domain comprises one or more minimal JAK / STAT signaling motif(s) (e.g., tyrosine phosphorylation motifs) from a protein comprising a STAT1, STAT3, STAT4, STAT5, and / or TIR signaling motif(s) or domain(s). In some embodiments, the one or more minimal JAK / STAT signaling motif(s) comprises a one or more tyrosine phosphorylation motifs from a STAT1, STAT3, STAT4, STAT5, and / or TIR signaling motif(s) or domain(s).

[0194] In some embodiments, the intracellular signaling domain comprises one or more minimal JAK / STAT signaling motif(s) from a STAT1 and STAT3 signaling motif(s) or domain(s). In some embodiments, the intracellular signaling domain comprises one or more minimal JAK / STAT signaling motif(s) from a STAT1 and STAT5 signaling motif(s) or domain(s). In some embodiments, the intracellular signaling domain comprises one or more minimal JAK / STAT signaling motif(s) from a STAT3 and STAT5 signaling motif(s) or domain(s). In some embodiments, the intracellular signaling domain comprises one or more minimal JAK / STAT signaling motif(s) from a STAT3 and STAT4 signaling motif(s) or domain(s). In some embodiments, the intracellular signaling domain comprises one or more minimal JAK / STAT signaling motif(s) from a STAT1 and STAT4 signaling motif(s) or domain(s). In some embodiments, the intracellular signaling domain comprises one or more minimal JAK / STAT signaling motif(s) from a STAT4 and STAT5, signaling motif(s) or domain(s). In some embodiments, the intracellular signaling domain comprises one or more minimal JAK / STAT signaling motif(s) from a STAT1, STAT3, and STAT4 signaling motif(s) or domain(s). In some embodiments, the intracellular signaling domain comprises one or more minimal JAK / STAT signaling motif(s) from a STAT1, STAT3, and STAT5 signaling motif(s) or domain(s). In some embodiments, the intracellular signaling domain comprises one or more minimal JAK / STAT signaling motif(s) from a STAT3, STAT4, and STAT5 signaling motif(s) or domain(s). In some embodiments, the intracellular signaling domain comprises one or more minimal TIR signaling motifs from IL-18Ra or IL-18Rb.

[0195] Exemplary proteins comprising STAT1 and / or STAT3 signaling motif(s) or domain(s) include, but are not limited to, gpl30, IL-21R, G-CSFR, and IL-9R. Exemplary proteins comprising STAT4 signaling motif(s) or domain(s) include, but are not limited to, IPTS / 200097280.2 1Attorney Ref: ANB-223WOIL-12RP. Exemplary proteins comprising STAT5 signaling motif(s) or domain(s) include, but are not limited to, IL-2RP, IL-7Ra, and IL-15R.

[0196] In some embodiments, the intracellular signaling domain comprises one or more minimal Toll-like receptor (TLR) / IL-1R (TIR) signaling motif or domain. Exemplary proteins with TIR signaling motif(s) or domain(s) include, but are not limited to, IL-18R (e.g., IL-18Ra and / or IL-18RP). The TIR domain of IL-18Ra is provided in SEQ ID NO: 285. In some embodiments, the intracellular signaling domain comprises one or more minimal TIR domain comprising a sequence as set forth in SEQ ID NO: 285. In some embodiments, the intracellular signaling domain comprises one or more Toll-like receptor (TLR) / IL-1R (TIR) tyrosine phosphorylation motifs comprising a sequence as provided in SEQ ID NO: 44.

[0197] In some embodiments, the intracellular signaling domain comprises a polypeptide sequence from an interleukin receptor. In some embodiments, the intracellular signaling domain comprises a fragment of an intracellular domain (ICD) or the full length ICD of an Interleukin-6 signal transducer (IL6ST) protein, an Interleukin-7 receptor alpha (IL-7Ra) protein, a Granulocyte Colony Stimulating Factor Receptor (G-CSFR) protein, an Interleukin- 12 receptor (IL-12R) protein, an Interleukin-9 receptor (IL-9R) protein, an Interleukin-21 receptor (IL-21R) protein, an Interleukin- 18 receptor (IL-18R) protein, an Interleukin-2 receptor beta (IL-2RP) protein, or a combination or functional fragment thereof. In some embodiments, the SPA comprises one or more STAT domains from the intracellular domain of at least one of gpl30, IL-9R, IL-12RP1, IL-12RP2, IL-7Ra, and / or IL-2RP, or a combination or functional fragment thereof. In some embodiments, the SPA comprises one or more STAT domains from the intracellular domain of IL-2Rp. In some embodiments, the SPA comprises one or more STAT domains from the intracellular domain of IL-12RP1 and / or IL-12RP2. In some embodiments, the SPA comprises one or more STAT domains from the intracellular domain of IL-7Ra.

[0198] In some embodiments, the SPA comprises one or more TIR domains from the intracellular domain of at least IL-18Ra and / or IL-18RP, or a functional fragment thereof. TIR domain sequences are described in Toshchakov VY, et al. A survey of TIR domain sequence and structure divergence. Immunogenetics. 2020 Apr;72(3):181-203, hereby incorporated by reference in its entirety.

[0199] In some embodiments, the intracellular signaling domain comprises a fragment of an interleukin or cytokine receptor provided herein. Such fragments may contain the minimal STAT and / or JAK signaling motif or domain necessary to induce signal transduction. These IPTS / 200097280.2Attorney Ref: ANB-223WO fragments may be termed “signal adaptors.” In some embodiments, the intracellular signaling domain comprises a signal adaptor comprising amino acids 310-521 of IL-9R (IL9R_310-521 (SEQ ID NO: 37)). In some embodiments, the intracellular signaling domain comprises a signal adaptor comprising amino acids 796-801 of IL-12RP2 (IL12Rp2_796-801 ((SEQ ID NO: 38)). In some embodiments, the intracellular signaling domain comprises a signal adaptor comprising amino acids 714-862 of IL-12RP2 (IL 12Rp2_714-862 (SEQ ID NO: 39)). In some embodiments, the intracellular signaling domain comprises a signal adaptor comprising amino acids 316-459 of IL-7Ra (IL7Ra_316-459 (SEQ ID NO: 40)). In some embodiments, the intracellular signaling domain comprises a signal adaptor comprising amino acids 333-551 of IL-2RP (IL2Rp_333-551 (SEQ ID NO: 41)).

[0200] In some embodiments, the intracellular signaling domain comprises a gpl30 intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a polypeptide sequence comprising amino acids 642 to 918 of gpl30.

[0201] In some embodiments, the gpl30 intracellular signaling domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence as set forth in SEQ ID NOs: 9, 10, 11, 28, 29, or 30. In some embodiments, the gpl30 intracellular signaling domain comprises an amino acid sequence as set forth in SEQ ID NOs: 9, 10, 11, 28, 29, or 30.

[0202] In some embodiments, the intracellular signaling domain comprises a truncated gpl30 intracellular signaling domain. In some embodiments, the SPA peptides comprise truncated gpl30 intracellular domains. In some embodiments, the truncated gpl30 intracellular signaling domain comprises the truncated gpl30 intracellular domain of a sequence selected from the group set forth in SEQ ID NOs: 28, 29, 30, 36, 55, or 56. In some embodiments, the truncated gpl30 intracellular signaling domain comprises a sequence with at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the gpl30 intracellular signaling domain as provide in the sequence set forth in SEQ ID NO: 28, 29, 30, 36, 55, or 56.

[0203] In some embodiments, the truncated gpl30 intracellular domain comprises a GP130A771-811 fragment (SEQ ID NO: 55). In some embodiments, the truncated gpl30 intracellular domain comprises a GP130A707-755 fragment (SEQ ID NO 30). In some embodiments, the truncated gpl30 intracellular domain comprises a GP130A818-901 fragment. In some embodiments, the truncated gpl30 intracellular domain comprises one or more truncation selected from the group consisting of GP130A707-755, GP130A771-811, IPTS / 200097280.2Attorney Ref: ANB-223WO and GP130A818-901. In some embodiments, the truncated gpl3O intracellular signaling domain comprises a deletion of amino acids 771 to 811 of gpl3O (SEQ ID NO: 260). In some embodiments, the truncated gpl30 intracellular signaling domain comprises a deletion of amino acids 707 to 755 of gpl30 (SEQ ID NO: 260). In some embodiments, the truncated gpl30 intracellular signaling domain comprises a deletion of amino acids 818 to 901 of gpl30 (SEQ ID NO: 260). In some embodiments, the truncated gpl30 intracellular signaling domain comprises one or more (e.g., one, two, or three) deletions of amino acids selected from the group consisting of amino acids 707 to 755, 771 to 811, and 818-901 of gpl30 (SEQ ID NO: 260). In other embodiments, the gpl30 intracellular domain comprises a Y759F mutation (resulting in a SOCS-proof mutant). In some embodiments, the gpl30 intracellular signaling domain further comprises a Y759F mutation of gpl30 (SEQ ID NO: 260). In other embodiments, the gpl30 intracellular domain comprises a Y759F mutation and a A771-811 truncation (gpl30Y759FA771-811) of gpl30 (SEQ ID NO: 260). In other embodiments, the gpl30 intracellular domain comprises a Y759F mutation and a A707-755 truncation (gpl30Y759FA707-755) of gpl30 (SEQ ID NO: 260). In other embodiments, the gpl30 intracellular domain comprises a Y759F mutation and a A818-901 truncation (gpl30Y759FA818-901) of gpl30 (SEQ ID NO: 260). In some embodiments, the gpl30 intracellular domain comprising a Y759F mutation comprise the sequence as set forth in SEQ ID NOs: 35, 36, or 56. In some embodiments, the full gpl30 ICD sequence is provided in SEQ ID NO: 11. In some embodiments, the STAT signaling motif portion of the gpl30 ICD sequence is provided in SEQ ID NO: 9. In some embodiments, the JAK signaling motif or domain portion of the gpl30 ICD sequence is provided in SEQ ID NO: 10.

[0204] In some embodiments, the IL-7Ra intracellular signaling domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence as set forth in SEQ ID NOs: 12, 13, or 14. In some embodiments, the IL-7Ra intracellular signaling domain comprises an amino acid sequence as set forth in SEQ ID NOs: 12, 13, or 14. In some embodiments, the full IL-7Ra ICD sequence is provided in SEQ ID NO: 14. In some embodiments, the STAT signaling motif portion of the IL-7Ra ICD sequence is provided in SEQ ID NO: 12. In some embodiments, the JAK signaling motif or domain portion of the IL-7Ra ICD sequence is provided in SEQ ID NO: 13.

[0205] In some embodiments, the G-CSFR intracellular signaling domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% IPTS / 200097280.2 34Attorney Ref: ANB-223WO identical to a sequence as set forth in SEQ ID NOs: 15, 16, or 17. In some embodiments, the G-CSFR intracellular signaling domain comprises an amino acid sequence as set forth in SEQ ID NOs: 15, 16, or 17. In some embodiments, the full G-CSFR ICD sequence is provided in SEQ ID NO: 17. In some embodiments, the STAT signaling motif portion of the G-CSFR ICD sequence is provided in SEQ ID NO: 15. In some embodiments, the JAK signaling motif or domain portion of the G-CSFR ICD sequence is provided in SEQ ID NO: 16.

[0206] In some embodiments, the IL-12RP2 intracellular signaling domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence as set forth in SEQ ID NOs: 18, 19, or 20. In some embodiments, the IL-12R P2 intracellular signaling domain comprises an amino acid sequence as set forth in SEQ ID NOs: 18, 19, 20, or 284. In some embodiments, the full IL-12RP2 ICD sequence is provided in SEQ ID NO: 20. In some embodiments, a fragment of the IL-12RP2 ICD sequence is provided in SEQ ID NO: 284. In some embodiments, the STAT signaling motif portion of the IL-12RP2 ICD sequence is provided in SEQ ID NO: 18. In some embodiments, the JAK signaling motif portion of the IL-12RP2 ICD sequence is provided in SEQ ID NO: 19.

[0207] In some embodiments, the IL-9R intracellular signaling domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence as set forth in SEQ ID NOs: 21, 22, or 23. In some embodiments, the IL-9R intracellular signaling domain comprises an amino acid sequence as set forth in SEQ ID NOs: 21, 22, or 23. In some embodiments, the full IL-9R ICD sequence is provided in SEQ ID NO: 23. In some embodiments, the STAT signaling motif portion of the IL-9R ICD sequence is provided in SEQ ID NO: 21. In some embodiments, the JAK signaling motif portion of the IL-9R ICD sequence is provided in SEQ ID NO: 22.

[0208] In some embodiments, the IL-21R intracellular signaling domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence as set forth in SEQ ID NOs: 24, 25, or 26. In some embodiments, the IL-21R intracellular signaling domain comprises an amino acid sequence as set forth in SEQ ID NOs: 24, 25, or 26. In some embodiments, the full IL-21R ICD sequence is provided in SEQ ID NO: 26. In some embodiments, the STAT signaling motif portion of the IL-21R ICD IPTS / 200097280.2Attorney Ref: ANB-223WO sequence is provided in SEQ ID NO: 24. In some embodiments, the JAK signaling motif portion of the IL-21R ICD sequence is provided in SEQ ID NO: 25.

[0209] In some embodiments, the IL-18Ra intracellular signaling domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence as set forth in SEQ ID NO: 27. In some embodiments, the IL-18Ra intracellular signaling domain comprises an amino acid sequence as set forth in SEQ ID NO: 27. In some embodiments, the full IL-18Ra ICD sequence is provided in SEQ ID NO: 27.

[0210] In some embodiments, the IL-2RP2 intracellular signaling domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence as set forth in SEQ ID NOs: 31, 32, or 33. In some embodiments, the IL-2RP2 intracellular signaling domain comprises an amino acid sequence as set forth in SEQ ID NOs: 31, 32, or 33. In some embodiments, the full IL-2RP2 ICD sequence is provided in SEQ ID NO: 33. In some embodiments, the JAK / STAT signaling portion of the IL-2RP2 ICD sequence is provided in SEQ ID NO: 31. In some embodiments, the JAK signaling domain portion of the IL-2RP2 ICD sequence is provided in SEQ ID NO: 32.

[0211] In some embodiments, a SPA peptide can comprise a ligand agonist (e.g., a cytokine, e.g., an interleukin) that allows constitutive activation of the SPA. In some embodiments, the cytokine receptor and a soluble agonist are expressed simultaneously. In some embodiments, the cytokine receptor and a membrane-bound agonist are expressed simultaneously.

[0212] In some embodiments, the intracellular signaling domain comprises one or more JAK signaling motif(s) or JAK domains. Exemplary JAK domains for use in the SPA disclosed herein include, but are not limited to, JAK domains from gpl30 (IL6ST), IL-7Ra, G-CSFR, IL-12R, IL-9R, and IL-21R, or any combination thereof. In some embodiments, the SPA comprises a JAK domain comprising an amino acid sequence as set forth in SEQ ID NOs: 10, 13, 16, 19, 22, 25, 29, 32, 47, 48, 49, 50, 51, 52, 53, or 54. In some embodiments, the SPA comprises a JAK domain comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence as set forth in SEQ ID NOs: 10, 13, 16, 19, 22, 25, 29, 32, 47, 48, 49, 50, 51, 52, 53, or 54.

[0213] In some embodiments, the intracellular signaling domain comprises one or more co- stimulatory domains. An exemplary co- stimulatory domain is the CD28 co- stimulatory IPTS / 200097280.2 It,Attorney Ref: ANB-223WO domain, but the present invention is not limited to only the CD28 co- stimulatory domain. In some embodiments, the intracellular signaling domain comprises one or more CD28 costimulatory domain at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 46.

[0214] In some embodiments, the intracellular signaling domain comprises one or more JAK1 / 2 / 3 signaling motif(s) (e.g., any one or more JAK1, JAK2, and / or JAK3 signaling motif(s) domains). Exemplary JAK1 / 2 / 3 signaling motif domains include, but are not limited to, the JAK1 / 2 / 3 signaling motif(s) or domains present in the gpl30, IL-7Ra, G-CSFR, IL- 12R, IL9R, IL-21R, and / or IL-2RP proteins. In some embodiments, the intracellular signaling domain comprises one or more JAK1 / 2 / 3 signaling motif(s) or domains at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, or 54.

[0215] In some embodiments, the intracellular signaling domain comprises a signal adaptor comprising amino acids 339-379, 393-433, and 518-551 of IL-2RP (IL2RP (339- 379,393-433,518-551) (SEQ ID NO: 42)). In some embodiments, the intracellular signaling domain comprises a signal adaptor comprising amino acids 530-551 of IL-2RP (IL2Rp_530- 551 (SEQ ID NO: 43)). In some embodiments, the intracellular signaling domain comprises a signal adaptor comprising amino acids 373-520 of IL18Ra (IL18R_373-520 (SEQ ID NO: 44)). In some embodiments, the intracellular signaling domain comprises a signal adaptor comprising amino acids 369-541 of IL-18Ra (IL18R_369-541 (SEQ ID NO: 45)). In some embodiments, the intracellular signaling domain comprises one or more STAT and / or TIR adaptor sequences at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence as set forth in SEQ ID NOs: 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45.Lipid anchors and transmembrane domains

[0216] In various embodiments, SPA peptides are anchored to the cellular membrane via a lipid anchor. In other embodiments, SPA peptides comprise a transmembrane domain. In various embodiments, a SPA peptide comprises a lipid anchor or a transmembrane domain comprising a gpl30 transmembrane domain, an IL-7Ra transmembrane domain, a G-CFSR transmembrane domain, a G-CFSR transmembrane domain comprising a T640N mutation asIPTS / 200097280.2 37Attorney Ref: ANB-223WO compared to the sequence provided for UniProt Q99062, a CD8-alpha transmembrane domain, a prenylation motif, or a myristoylation domain derived from src, fyn, or lek.

[0217] In some embodiments, the SPA comprises a gpl30 transmembrane domain sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to the sequence set forth in SEQ ID NO: 6. In some embodiments, the SPA comprises a G-CFSR transmembrane domain sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to the sequence set forth in SEQ ID NO: 261. In some embodiments, the SPA comprises a G-CFSR T640N transmembrane domain at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to the sequence set forth in SEQ ID NO: 8. In some embodiments, the SPA comprises an IL-7Ra transmembrane domain sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to the sequence set forth in SEQ ID NO: 7 or 262.

[0218] In some embodiments, a SPA peptide comprises a sre-derived myristoylation domain, fyn-derived myristoylation domain, or Ick-derived myristoylation domain. In other embodiments, a SPA peptide comprises a prenylation motif. In some embodiments, a SPA peptide comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, a SPA peptide comprises comprise a transmembrane domain of an interleukin receptor.

[0219] In some embodiments, the transmembrane domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to the sequence set forth in SEQ ID NO: 6, 7, 8, 261, or 262. In some embodiments, the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 6, 7, 8, 261, or 262.Multimerization regions

[0220] In various embodiments, one or more structural alterations can be made to confer constitutive activity to a SPA or functional fragment thereof. In some embodiments, structures or mutations can be added to induce SPA multimerization (e.g, dimerization,IPTS / 200097280.2 38Attorney Ref: ANB-223WO trimerization, tetramerization, or higher order multimers). In some embodiments, one or more amino acids can be mutated to a cysteine to allow formation of one or more disulfide bond(s), e.g., between two receptor monomers. In some embodiments, one or more amino acids can be inserted into a wild-type receptor polypeptide to promote dimerization, e.g., through formation of one or more disulfide bond(s). In some embodiments, the multimerization domain comprises one or more unpaired cysteine residues. The one or more amino acids mutated to an unpaired cysteine can be in the extracellular domain, the transmembrane domain, a hinge domain, the intracellular signaling domain, or any linker used to link such domains. In some embodiments, the multimerization domain comprises one or more VASP domains. In some embodiments, the multimerization domain comprises one or more VASP tetramerization domains. In some embodiments, the multimerization domain comprises one or more leucine zippers. In some embodiments, the multimerization domain is intracellular or extracellular. In some embodiments, the multimerization domain is the transmembrane domain. An exemplary transmembrane domain that can multimerize is the IL7Ra-mutP2 TMD (SEQ ID NO: 7).

[0221] In some embodiments, an exogenous polypeptide is operatively linked to a cytokine receptor or functional fragment thereof to cause their multimerization or dimerization. In some embodiments, a leucine zipper polypeptide is operatively linked to a cytokine receptor or functional fragment thereof. In some embodiments, the leucine zipper polypeptide is a c-Jun leucine zipper. In some embodiments, an exogenous scaffold is operatively linked to a cytokine receptor or functional fragment thereof. In some embodiments, the exogenous scaffold is a CD34 ectodomain (e.g., SEQ ID NO: 242), erythropoietin receptor (EpoR) ectodomain, or thrombopoietin receptor (TpoR) ectodomain.

[0222] In some embodiments, multimerization of the chimeric polypeptide via the multimerization region results in constitutive activity of the intracellular signaling domain. In some embodiments, the SPA peptide comprises one or more multimerization region. In some embodiments, the SPA peptide comprises two or more multimerization regions. In some embodiments, the multimerization region comprises at least one of one or more unpaired cysteine residues, a leucine zipper, a BCR domain, and a VASP domain. In some embodiments, the multimerization region comprises at least one or more unpaired cysteine residues. In some embodiments, the multimerization region comprises at least one or more unpaired cysteine residue and a leucine zipper. In some embodiments, the SPA peptide comprises one or more VASP domain polypeptides to promote multimerization. In some embodiments, the VASP domain is the tetramerization domain of VASP. In someIPTS / 200097280.2 IQAttorney Ref: ANB-223WO embodiments, the BCR domain comprises a coiled coil tetramerization region. In such embodiments, the BCR ectodomain can result in multimerization through non-covalent interactions.

[0223] In some embodiments, the multimerization region is intracellular when expressed by a cell. In some embodiments, the multimerization region is extracellular when expressed by a cell. For example, the SPA peptide can comprise, in an N terminus to C terminus direction, i) one or more multimerization domains-an extracellular domain-a transmembrane domain-an intracellular signaling domain, ii) an extracellular domain- one or more multimerization domains -a transmembrane domain-an intracellular signaling domain, iii) one or more multimerization domains-a transmembrane domain-an intracellular signaling domain, iv) one or more multimerization domains-a lipid anchor-an intracellular signaling domain, v) an extracellular domain-a transmembrane domain-one or more multimerization domains-an intracellular signaling domain, vi) a lipid anchor-one or more multimerization domains-an intracellular signaling domain, vii) an extracellular domain-a transmembrane domain-an intracellular signaling domain-one or more multimerization domains, or viii) a lipid anchor-an intracellular signaling domain-one or more multimerization domains, or any combination thereof.

[0224] In some aspects, provided herein are dimers or multimers comprising one or more of the SPA peptide(s) disclosed herein. For example, a dimer is comprised of two of the SPA peptide(s) disclosed herein, while a multimer is comprised of three, four, five, six, or more of the SPA peptide(s) disclosed herein.Extracellular domain

[0225] A SPA peptide disclosed herein can also comprise an extracellular domain. In some embodiments, the extracellular domain conveys constitutive activity to the intracellular signaling domain. In some embodiments, the extracellular domain comprises a CD34 ectodomain or a truncated CD34 ectodomain (e.g., a CD34 extracellular domain, SEQ ID NO: 2, 3, 4, or 5). In some embodiments, the extracellular domain comprises a CD34 epitope In some embodiments, the extracellular domain comprises a CD34 epitope (e.g. a QB END 10 epitope, ELPTQGTFSNVSTNVS SEQ ID NO: 499) or a QBEND10 epitope with a cysteine linker as provided in SEQ ID NO: 1. In some embodiments, the extracellular domain comprises the sequence as provided in SEQ ID NO: 1 without the C terminal cysteine, the sequences provided in SEQ ID NOs: 475, 476, 477, or 478, or the sequences provided in SEQ ID NOs: 3, 4, or 5 without the C-terminal CGG amino acid residues. In some embodiments,IPTS / 200097280.2 40Attorney Ref: ANB-223WO the extracellular domain comprises a type I cytokine receptor extracellular domain (e.g., a thrombopoietin receptor (TpoR) ectodomain or erythropoietin receptor (EpoR) ectodomain). In some embodiments, the type I cytokine receptor extracellular domain (e.g., a thrombopoietin receptor (TpoR) ectodomain or erythropoietin receptor (EpoR) ectodomain) further comprises a type I cytokine receptor transmembrane domain (e.g., a thrombopoietin receptor (TpoR) transmembrane domain or erythropoietin receptor (EpoR) transmembrane domain). In some embodiments, the extracellular domain comprises one or more of a CD34 epitope (e.g. a QBEND10 epitope, SEQ ID NO: 499 or 1), a CD34 extracellular domain (SEQ ID NO: 475, 476, 477, 478, 2, 3, 4, or 5), optionally wherein the CD34 epitope or the CD34 extracellular domain lack the C terminal cysteine provided in SEQ ID NO: 1 or the C terminal GCC amino acids provided in SEQ ID NOs: 3, 4, or 5, a BCR ectodomain, a thrombopoietin receptor (TpoR) domain, or an erythropoietin receptor (EpoR) ectodomain. In various embodiments, the CD24 ectodomain or CD34 epitope further comprise one or more unpaired cysteines. In various embodiments, the thrombopoietin receptor (TpoR) ectodomain or erythropoietin receptor (EpoR) ectodomain further comprise one or more unpaired cysteines. In some embodiments, the BCR ectodomain comprises a coiled coil tetramerization region. In such embodiments, the BCR ectodomain can result in multimerization through non-covalent interactions.Exemplary SPAs

[0226] Diagrams of exemplary SPAs described herein are provided in FIG. 1A and IB with a figure legend provided in FIG. 1C.

[0227] In some embodiments, the transmembrane domain comprising an IL-7Ra transmembrane domain, a G-CSFR, transmembrane domain, or a gpl30 transmembrane domain. In some embodiments, the intracellular signaling domain comprises one or more Janus Kinase (JAK) signaling motif(s) or domain(s) comprising a gpl30 JAK signaling motif or domain, an IL-7Ra JAK signaling motif or domain, a G-CSFR JAK signaling motif or domain, an IL-12RP JAK signaling motif or domain, an IL-9R JAK signaling motif or domain, or an IL-21R JAK signaling motif or domain, or any combination thereof; and / or one or more Signal Transducer and Activator of Transcription (STAT)l, STAT3, STAT4, STAT5 and / or Toll-like receptor (TLR) / IL-1R (TIR) signaling motif(s) or domain(s), or any combination thereof. In some embodiments, the intracellular signaling domain comprises two or more STAT1, STAT3, STAT4, STAT5 and / or TIR signaling motif(s) or domains(s), or any combination thereof.IPTS / 200097280.2 41Attorney Ref: ANB-223WO

[0001] In some embodiments, the SPA comprises a truncated CD34 extracellular domain, an IL7Ra transmembrane domain, a gpl30 JAK domain, a gpl30 STAT1 and STAT3 signaling motif or domain, and an IL7Ra STAT5 signaling motif or domain; a truncated CD34 extracellular domain, an IL7Ra transmembrane domain, a gpl30 JAK signaling motif or domain, a gpl30 STAT1 and STAT3 signaling motif or domain, and an IL-2RP STAT5 signaling motif or domain; a truncated CD34 extracellular domain, an IL7Ra transmembrane domain, a gpl30 JAK signaling motif or domain, a gpl30 STAT1 and STAT3 signaling motif or domain, and an IL-12RP2 STAT4 signaling motif or domain; a CD34 epitope extracellular domain, a gpl30 transmembrane domain, a gpl30 JAK signaling motif or domain, a gpl30 STAT1 and STAT3 signaling motif or domain, and an IL7Ra STAT5 signaling motif or domain; a CD34 epitope extracellular domain, a gpl30 transmembrane domain, a gpl30 JAK signaling motif or domain, a gpl30 STAT1 and STAT3 signaling motif or domain, and an IL2RP STAT5 signaling motif or domain; a CD34 epitope extracellular domain, a gpl30 transmembrane domain, a gpl30 JAK signaling motif or domain, a gpl30 STAT1 and STAT3 signaling motif or domain, and an IL-12RP2 STAT4 signaling motif or domain; or a CD34 epitope extracellular domain, a gpl30 transmembrane domain, a G-CSFR JAK domain, a G-CSFR STAT1 and STAT3 signaling motif or domain, and an IL-12RP2 STAT4 signaling motif or domain.

[0228] In some embodiments, the SPA peptide comprises, from N terminus to C terminus, an extracellular domain comprising a CD34 epitope or CD34 extracellular domain; an optional multimerization region comprising one or more unpaired cysteine residues; a transmembrane selected from a GP130 (IL6ST) transmembrane domain, an IL7Ra transmembrane domain, a G-CFSR transmembrane domain, and a G-CFSR T640N transmembrane domain; and an intracellular domain comprising one or more JAK domains selected from gpl30, IL7Ra_JAK, G-CSFR, IL-12R, IL-9R, IL-21R, and IL-2RP, and a STAT motif or STAT signal adaptor selected from gpl30 (YXXQ)4 STAT adaptor (SEQ ID NO: 286 or 34), gpl30 STAT region with a Y759F mutation (SEQ ID NO: 35), a truncated gpl30( A 707-755) with a Y759F mutation (gpl30trunc7_Y759F (SEQ ID NO: 36)), IL9R_310-521 (YXXQ) (SEQ ID NO: 37), IL12Rp2_796-801 ((SEQ ID NO: 38), IL12RP2_714-862 (SEQ ID NO: 39), IL7Ra_316-459 (SEQ ID NO: 40), IL2Rp_333-551 (SEQ ID NO: 41), IL2RP (339-379,393-433,518-551) (SEQ ID NO: 42), IL2Rp_530-551 (SEQ ID NO: 43), IL18R_373-520 (SEQ ID NO: 44), and IL18R_369-541 (SEQ ID NO: 45). In some embodiments, the intracellular domain comprises a gpl30 ICD (SEQ ID NO:IPTS / 200097280.2 42Attorney Ref: ANB-223WO11), a IL-7Ra ICD (SEQ ID NO: 14), a G-CSFR ICD (SEQ ID NO: 17), a IL-12R ICD (SEQ ID NO: 20), a IL-9R ICD (SEQ ID NO: 23), a IL-21R ICD (SEQ ID NO: 26), or a IL-18R ICD (SEQ ID NO: 27). In some embodiments, the SPA peptide further comprises a leader sequence at the N terminus. In some embodiments, the leader sequence is a CD8a signal sequence, a GP130 (IL6ST) signal sequence, a CD34 signal sequence, or an Erythropoietin receptor (EpoR) signal sequence. In some embodiments, the leader sequence comprises MALPVTALLLPLALLLHAARP (SEQ ID NO: 255), MLVRRGARAGPRMPRGWTALCLLSLLPSGFM (SEQ ID NO: 256), MDHLGASLWPQVGSLCLLLAGAAW (SEQ ID NO: 257), or MLTLQTWLVQALFIFLTTESTG (SEQ ID NO: 258). In some embodiments, the SPA lacks a leader sequence of the sequence TALCLLSLLPSGFM (SEQ ID NO: 502), or as provided in SEQ ID NOs: 255, 256, 257, or 258.

[0229] In some embodiments, SPA peptide comprises a sequence selected from the group set forth in SEQ ID NOs: 57-254, 264-283, or 419-474. In some embodiments, the SPA peptide comprises a sequence with about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to a sequence as set forth in SEQ ID NOs: 57-254, 264-283, or 419-474. In some embodiments, the SPA peptide comprises the sequence set forth in SEQ ID NO: 421, 185, 114, 104, 79, 80, 81, 87, 103, 111, 116, 117, 143, 144, 145, 152, 153, 155, 156, 157, 159, 160, 182, 183, 184, 220, 234, 264, 268, 269, 270, 282, 419, 420, or 422. In some embodiments, the SPA peptide comprises a sequence with about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to a sequence as set forth in SEQ ID NOs: 421, 185, 114, 104, 79, 80, 81, 87, 103, 111, 116, 117, 143, 144, 145, 152, 153, 155, 156, 157, 159, 160, 182, 183, 184, 220, 234, 264, 268, 269, 270, 282, 419, 420, or 422. In some embodiments, the nucleic acid encodes a SPA peptide comprising a sequence selected from the group set forth in SEQ ID NOs: 57-254, 264-283, or 419-474. In some embodiments, the SPA peptide comprises a sequence with about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to a sequence as set forth in SEQ ID NOs: 57-254, 264-283, or 419-474. In some embodiments, the SPA peptide comprises the sequence set forth in SEQ ID NO: 421, 185, 114, 104, 79, 80, 81, 87, 103, 111, 116, 117, 143, 144, 145, 152, 153, 155, 156, 157, 159, 160, 182, 183, 184, 220, 234, 264, 268, 269, 270, 282, 419, 420, or 422. In some embodiments, the nucleic acid encodes a SPA peptide comprises a sequence with about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, IPTS / 200097280.2Attorney Ref: ANB-223WO90%, 95%, 99%, or 100% identity to a sequence as set forth in SEQ ID NOs: 421, 185, 114, 104, 79, 80, 81, 87, 103, 111, 116, 117, 143, 144, 145, 152, 153, 155, 156, 157, 159, 160, 182, 183, 184, 220, 234, 264, 268, 269, 270, 282, 419, 420, or 422.

[0230] In some embodiments, the nucleic acid encoding a SPA peptide comprises a sequence selected from the group set forth in SEQ ID NOs: 479-498. In some embodiments, the nucleic acid encoding a SPA peptide comprises a sequence with at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to a sequence as set forth in SEQ ID NOs: 479-498. In some embodiments, the nucleic acid does not encode a leader sequence, e.g., as provided in SEQ ID NOs: 480, 482, 484, 486, 488, 490, 492, 494, 496, or 498. In some embodiments, the nucleic acid does encode a leader sequence, e.g., as provided in SEQ ID NOs: 479, 481, 483, 495, 487, 489, 491, 493, 495, or 497.

[0231] Exemplary SPA structures are provided in Tables 1-12. In some embodiments, the SPA comprises any one of the SPAs provided in Tables 1-12.

[0232] Table 1: Provides SPAs with single STAT tiles with one of four ECDs (QbendlO- cys, CD34truncl, CD34truncl.l-cys, or CD34truncl.3-cys), one of three TMDS (gpl30_TMD, IL7RamutP2, or G-CFSR_T640N), and one of nine ICDs (gpl30, gpl30 trunc 7, IL-2R , IL-7Ra, G-CSFR, IL-12R, IL-9R, IL-21R, IL-18R) paired with the respective JAK domain. In some embodiments, the SPA comprises any one of the SPAs provided inTable 1.Attorney Ref: ANB-223WO

[0233] Table 2: Provides SPAs with combinatorial STAT tiles with two full length ICDs, ICD1 and ICD2, one of two ECDs (QbendlO-cys and CD34truncl), paired with a respective JAK from one of the two ICDs. In some embodiments, the SPA comprises any one of theSPAs provided in Table 2.IPTS / 200097280.2 45Attorney Ref: ANB-223WOIPTS / 200097280.2 46Attorney Ref: ANB-223WO

[0234] Table 3: Provides SPAs with combinatorial STAT tiles with a full length ICD with a paired JAK domain and one STAT / TIR adaptor. In some embodiments, the SPA comprises any one of the SPAs provided in Table 3.IPTS / 200097280.2 47Attorney Ref: ANB-223WOIPTS / 200097280.2 48Attorney Ref: ANB-223WO

[0235] Table 4: Provides SPAs with combinatorial STAT tiles with a JAK plus two STAT or TIR adaptors. In some embodiments, the SPA comprises any one of the SPAs provided in Table 4.IPTS / 200097280.2 49Attorney Ref: ANB-223WOIPTS / 200097280.2 50Attorney Ref: ANB-223WOIPTS / 200097280.2 51Attorney Ref: ANB-223WOIPTS / 200097280.2 52Attorney Ref: ANB-223WO

[0236] Table 5: Provides SPAs with a non-STAT signaling receptor (IL18R, e.g., IL-R18a or IL-18Rb) plus a JAK domain and a signaling adaptor comprising a STAT motif. In some embodiments, the SPA comprises any one of the SPAs provided in Table 5.

[0237] Table 6: Provides SPAs with one of three full length ICDs (STAT3, STAT4,STAT5) plus an IL18R (e.g., IL-R18a or IL-18Rb) TIR domain. In some embodiments, theSPA comprises any one of the SPAs provided in Table 6.IPTS / 200097280.2Attorney Ref: ANB-223WO

[0238] Table 7: Provides SPAs with a CD28 co- stimulatory domain along with a JAK signaling domain, an ICD, and a signaling adaptor (e.g., a STAT adaptor). In some embodiments, the SPA comprises any one of the SPAs provided in Table 7.

[0239] Table 8: Provides SPAs pairing the respective JAK domain for both ICDs. In some embodiments, the SPA comprises any one of the SPAs provided in Table 8.IPTS / 200097280.2 54Attorney Ref: ANB-223WO

[0240] Table 9: Provides SPAs pairing the respective JAK domain for both ICD and signal adaptor domains. In some embodiments, the SPA comprises any one of the SPAs provided in Table 9.IPTS / 200097280.2 55Attorney Ref: ANB-223WO

[0241] Table 10: Provides SPAs that include a CD28 co- stimulatory domain. In some embodiments, the SPA comprises any one of the SPAs provided in Table 10.

[0242] Table 11: Provides SPAs with a Y759F mutation that removes the ERK / MAPK activation potential by mutating the SHP2 site. In some embodiments, the SPA comprises a Y759F mutation in the SHP2 domain. In some embodiments, the SPA comprises any one of the SPAs provided in Table 11.IPTS / 200097280.2 56Attorney Ref: ANB-223WO

[0243] Table 12: Provides additional exemplary SPAs. In some embodiments, the SPA comprises any one of the SPAs provided in Table 12.Logic Gate Systems

[0244] As used herein, a “logic gate,” “circuit,” “circuit receptor,” “system” or “system receptor” refers to a two part protein expression system comprising a priming receptor and a chimeric antigen receptor. The system can be encoded on at least one nucleic acid inserted into a cell, where the priming receptor is expressed in the cell. The intracellular domain of theIPTS / 200097280.2 57Attorney Ref: ANB-223WO priming receptor is cleaved from the transmembrane domain upon binding of the priming receptor to its target antigen. The intracellular domain is then capable of translocating into a cell nucleus where it induces expression of the chimeric antigen receptor.

[0245] In one aspect, provided herein are systems comprising a priming receptor that binds to a target antigen and a chimeric antigen receptor that binds to a target antigen, wherein the transcription factor of the intracellular domain of the priming receptor is capable of inducing expression of the CAR and / or SPA. Such systems are alternatively termed “logic gates” or “circuits.” In some aspects, the system is encoded by nucleic acid transgenes inserted into an immune cell. The system can be encoded on a single nucleic acid insert or fragment that comprises both transgenes, or can be encoded on two nucleic acids that encode the system transgenes individually. The priming receptor and CAR of the system can be placed in any order on the single nucleic acid. For example, the priming receptor can be at the 5’ end and the CAR can be at the 3’ end, or the CAR can be at the 5’ end and the priming receptor can be at the 3’ end.

[0246] A constitutive promoter can be operably linked to the nucleotide sequence encoding the priming receptor and / or SPA. An inducible promoter can also be operably linked to the nucleotide sequence encoding the CAR. In some embodiments, when the system is encoded on a single nucleic acid insert or fragment that comprises both transgenes, the nucleic acid can comprise, in a 5’ to 3’ direction, the constitutive promoter; the nucleotide sequence encoding priming receptor; the inducible promoter; and the nucleotide sequence encoding chimeric antigen receptor. Alternatively, the nucleic acid can comprise, in a 5’ to 3’ direction, the inducible promoter; the nucleotide sequence encoding chimeric antigen receptor; the constitutive promoter; the nucleotide sequence encoding priming receptor. In some embodiments, the inducible promoter comprises one or more HNFla enhancer elements (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more HNFla enhancer elements). In some embodiments, the constitutive promoter comprises an EFla promoter.Priming Receptors

[0247] Provided herein are priming receptors comprising an extracellular antigen-binding domain that specifically binds target antigens and one or more intracellular domains from or derived from a transcriptional regulator and / or a DNA-binding domain.

[0248] In certain aspects of the present disclosure, the priming receptor is a synthetic receptor based on the Notch protein. Binding of a natural Notch receptor to a cognate ligand,IPTS / 200097280.2 58Attorney Ref: ANB-223WO such as those from the Delta family of proteins, causes intramembrane proteolysis that cleaves an intracellular fragment of the Notch protein. This intracellular fragment is a transcriptional regulator that only functions when cleaved from Notch. Cleavage may occur by sequential proteolysis by ADAM metalloprotease and the gamma-secretase complex. This intracellular fragment enters the nucleus of a cell and activates cell-cell signaling genes. In contrast to a natural Notch protein, a synthetic notch priming receptor replaces the natural Notch intracellular fragment with one that causes a gene encoding a protein of choice, such as a CAR, to be transcribed upon release of the intracellular fragment from the priming receptor.

[0249] Notch receptors have a modular domain organization. The ectodomains of Notch receptors consist of a series of N-terminal epidermal growth factor (EGF)-like repeats that are responsible for ligand binding. In synthetic Notch receptors or priming receptors, the Notch ligand-binding domain is replaced with a ligand binding domain that binds a selected target ligand or antigen. The EGF repeats are followed by three LIN -12 / Notch repeat (LNR) modules, which are unique to Notch receptors, and are widely reported to participate in preventing premature receptor activation. The heterodimerization (HD) domain of Notchl is divided by furin cleavage, so that its N-terminal part terminates the extracellular subunit, and its C -terminal half constitutes the beginning of the transmembrane subunit. Following the extracellular region, the receptor has a transmembrane segment and an intracellular domain (ICD), which includes a transcriptional regulator.

[0250] Multiple forms of priming receptors can be used in the methods, cells, and nucleic acids as described herein. One type of priming receptor contemplated for use in the methods and cells herein comprise a heterologous extracellular ligand binding domain, a linking polypeptide having substantial sequence identity with a Notch receptor including the NRR, a TMD, and an ICD. “Fn Notch” receptors comprise a heterologous extracellular ligand binding domain, a linking polypeptide having substantial sequence identity with a Robo receptor (such as a mammalian Robol, Robo2, Robo3, or Robo4), followed by 1, 2, or 3 fibronectin repeats (“Fn”), a TMD, and an ICD. “Mini Notch” receptors comprise a heterologous extracellular ligand binding domain, a linking polypeptide having substantial sequence identity with a Notch receptor (lacking the NRR), a TMD, and an ICD. “Minimal Linker Notch” receptors comprise a heterologous extracellular ligand binding domain, a linking polypeptide lacking substantial sequence identity with a Notch receptor (e.g., a synthetic (GGS)n polypeptide sequence), a TMD, and an ICD. “Hinge Notch” receptors comprise a heterologous extracellular ligand binding domain, a hinge sequence comprising an oligomerization domain (i.e., a domain that promotes dimerization, trimerization, or higher IPTS / 200097280.2 CQAttorney Ref: ANB-223WO order multimerization with a synthetic receptor and / or an existing host receptor), a TMD, and an ICD. All of these receptor classes are synthetic, recombinant, and do not occur in nature. In some embodiments, the non-naturally occurring receptors disclosed herein bind a target cell- surface displayed ligand, which triggers proteolytic cleavage of the receptors and release of a transcriptional regulator that modulates a custom transcriptional program in the cell. In some embodiments, the priming receptor does not include a LIN-12-Notch repeat (LNR) and / or a heterodimerization domain (HD) of a Notch receptor.Priming Receptor Extracellular Domain

[0251] In some embodiments, the extracellular domain includes the ligand-binding portion of a receptor. In some embodiments, the extracellular domain includes an antigen-binding moiety that binds to one or more target antigens. In some embodiments, the antigen-binding moiety includes one or more antigen-binding determinants of an antibody or a functional antigen-binding fragment thereof. In some embodiments, the antigen-binding moiety is selected from the group consisting of an antibody, a nanobody, a diabody, a triabody, or a minibody, a F(ab')2 fragment, a Fab fragment, a single chain variable fragment (scFv), and a single domain antibody (sdAb), or a functional fragment thereof. In some embodiments, the antigen-binding moiety comprises an scFv. The antigen-binding moiety can include naturally-occurring amino acid sequences or can be engineered, designed, or modified so as to provide desired and / or improved properties, e.g., increased binding affinity.

[0252] In various embodiments, a priming receptor comprises means for binding a target protein, optionally binding a human target protein. In some embodiments, the means binds a target protein. In some embodiments, the means binds a human target protein. In some embodiments, the means is an antibody or antigen-binding fragment or equivalent thereof (e.g., a full length antibody or a F(ab')2 fragment, a Fab fragment, a single chain variable fragment (scFv), and a single domain antibody (sdAb), or a functional fragment thereof).Transmembrane Domain

[0253] In some embodiments, the priming receptor comprises a hinge domain. In some embodiments, the hinge domain is a CD8 hinge.

[0254] As described above, the priming receptor comprises a transmembrane domain (TMD) comprising one or more ligand- inducible proteolytic cleavage sites.

[0255] In some embodiments, the TMD comprises a Notchl transmembrane domain.IPTS / 200097280.2 60Attorney Ref: ANB-223WO

[0256] Generally, the TMD suitable for the chimeric receptors disclosed herein can be any transmembrane domain of a Type 1 transmembrane receptor including at least one gamma- secretase cleavage site. Detailed description of the structure and function of the gamma- secretase complex as well as its substrate proteins, including amyloid precursor protein (APP) and Notch, can, for example, be found in a recent review by Zhang et al, Frontiers Cell Neurosci (2014). Non limiting suitable TMDs from Type 1 transmembrane receptors include those from CLSTN1, CLSTN2, APLP1, APLP2, LRP8, APP, BTC, TGBR3, SPN, CD44, CSF1R, CXCL16, CX3CL1, DCC, DLL1, DSG2, DAG1, CDH1, EPCAM, EPHA4, EPHB2, EFNB1, EFNB2, ErbB4, GHR, HLA- A, and IFNAR2, wherein the TMD includes at least one gamma secretase cleavage site. Additional TMDs suitable for the compositions and methods described herein include, but are not limited to, transmembrane domains from Type 1 transmembrane receptors IL1R1, IL1R2, IL6R, INSR, ERN1, ERN2, JAG2, KCNE1, KCNE2, KCNE3, KCNE4, KL, CHL1, PTPRF, SCN1B, SCN3B, NPR3, NGFR, PLXDC2, PAM, AGER, ROBO1, SORCS3, SORCS1, SORL1, SDC1, SDC2, SPN, TYR, TYRP1, DCT, YASN, FLT1, CDH5, PKHD1, NECTIN1, PCDHGC3, NRG1, LRP1B, CDH2, NRG2, PTPRK, SCN2B, Nradd, and PTPRM. In some embodiments, the TMD of the chimeric polypeptides or Notch receptors of the disclosure is a TMD derived from the TMD of a member of the calsyntenin family, such as, alcadein alpha and alcadein gamma. In some embodiments, the TMD of the chimeric polypeptides or Notch receptors of the disclosure is a TMD known for Notch receptors. In some embodiments, the TMD of the chimeric polypeptides or Notch receptors of the disclosure is a TMD derived from a different Notch receptor. For example, in a Mini Notch based on human Notchl, the Notchl TMD can be substituted with a Notch2 TMD, Notch3 TMD, Notch4 TMD, or a Notch TMD from a nonhuman animal such as Danio rerio, Drosophila melanogaster, Xenopus laevis, or Gallus gallus.

[0257] In some embodiments, the priming receptor comprises a Notch cleavage site, such as S2 or S3. Additional proteolytic cleavage sites suitable for the compositions and methods disclosed herein include, but are not limited to, ADAM10, a metalloproteinase cleavage site for a MMP selected from collagenase- 1, -2, and -3 (MMP-1, -8, and -13), gelatinase A and B (MMP-2 and -9), stromelysin 1, 2, and 3 (MMP-3, -10, and -11), matrilysin (MMP-7), and membrane metalloproteinases (MT 1 -MMP and MT2-MMP). Another example of a suitable protease cleavage site is a plasminogen activator cleavage site, e.g., a urokinase plasminogen activator (uPA) or a tissue plasminogen activator (tPA) cleavage site. Another example of a suitable protease cleavage site is a prolactin cleavage site. Specific examples of cleavage IPTS / 200097280.2 .1Attorney Ref: ANB-223WO sequences of uPA and tPA include sequences comprising Yal-Gly-Arg. Another example of a protease cleavage site that can be included in a proteolytically cleavable linker is a tobacco etch vims (TEV) protease cleavage site, e.g., Glu-Asn-Leu-Tyr-Thr-Gln-Ser (SEQ ID NO: 416), where the protease cleaves between the glutamine and the serine. Another example of a protease cleavage site that can be included in a proteolytically cleavable linker is an enterokinase cleavage site, e.g., Asp-Asp-Asp-Asp- Lys (SEQ ID NO: 417), where cleavage occurs after the lysine residue. Another example of a protease cleavage site that can be included in a proteolytically cleavable linker is a thrombin cleavage site, e.g., Leu-Val-Pro- Arg (SEQ ID NO: 418). Additional suitable linkers comprising protease cleavage sites include sequences cleavable by the following proteases: a PreScission™ protease (a fusion protein comprising human rhinovirus 3C protease and glutathione-S-transferase), a thrombin, cathepsin B, Epstein-Barr vims proteas, MMP-3 (stromelysin), MMP-7 (matrilysin), MMP-9; thermoly sin-like MMP, matrix metalloproteinase 2 (MMP-2), cathepsin L; cathepsin D, matrix metalloproteinase 1 (MMP-1), urokinase-type plasminogen activator, membrane type 1 matrixmetalloprotemase (MT- MMP), stromelysin 3 (or MMP-11), thermo lysin, fibroblast collagenase and stromelysin- 1, matrix metalloproteinase 13 (collagenase-3), tissue-type plasminogen activator(tPA), human prostate-specific antigen, kallikrein (hK3), neutrophil elastase, and calpain (calcium activated neutral protease). Proteases that are not native to the host cell in which the receptor is expressed (for example, TEV) can be used as a further regulatory mechanism, in which activation of the receptor is reduced until the protease is expressed or otherwise provided. Additionally, a protease may be tumor-associated or disease-associated (expressed to a significantly higher degree than in normal tissue), and serve as an independent regulatory mechanism. For example, some matrix metalloproteases are highly expressed in certain cancer types.

[0258] In some embodiments, the amino acid substitution(s) within the TMD includes one or more substitutions within a “GV” motif of the TMD. In some embodiments, at least one of such substitution(s) comprises a substitution to alanine. Additional sequences and substitutions are described in WO2021061872, hereby incorporated by reference in its entirety.Intracellular Domain

[0259] In some embodiments, the priming receptor comprises one or more intracellular domains from or derived from a transcriptional regulator and / or a DNA-binding domain. In some embodiments, the intracellular domain comprises means for modulating transcription ofIPTS / 200097280.2 62Attorney Ref: ANB-223WO one or more genes. In some embodiments, the means for modulating transcription of one or more genes comprises a transcriptional regulator, e.g., a transcriptional regulator provided herein or an equivalent thereof. In some embodiments, the priming receptor comprises one or more intracellular domains from or derived from a transcriptional regulator and / or a DNA- binding domain. In some embodiments, the intracellular domain comprises an HNFla / p65 domain or a Gal4 / VP64 domain.

[0260] Transcriptional regulators either activate or repress transcription from cognate promoters. Transcriptional activators typically bind nearby to transcriptional promoters and recruit RNA polymerase to directly initiate transcription. Transcriptional repressors bind to transcriptional promoters and sterically hinder transcriptional initiation by RNA polymerase. Other transcriptional regulators serve as either an activator or a repressor depending on where it binds and cellular conditions. Accordingly, as used herein, a “transcriptional activation domain” refers to the domain of a transcription factor that interacts with transcriptional control elements and / or transcriptional regulatory proteins (i.e., transcription factors, RNA polymerases, etc.) to increase and / or activate transcription of one or more genes. Nonlimiting examples of transcriptional activation domains include: a herpes simplex virus VP 16 activation domain, VP64 (which is a tetrameric derivative of VP16), HIV TAT, a NFkB p65 activation domain, p53 activation domains 1 and 2, a CREB (cAMP response element binding protein) activation domain, an E2A activation domain, NF AT (nuclear factor of activated T-cells) activation domain, yeast Gal4, yeast GCN4, yeast HAP1, MLL, RTG3, GLN3, OAF1, PIP2, PDR1, PDR3, PHO4, LEU3 glucocorticoid receptor transcription activation domain, B-cell POU homeodomain protein Oct2, plant Ap2, or any others known to one or ordinary skill in the art. In some embodiments, the transcriptional regulator is selected from Gal4-VP16, Gal4-VP64, tetR-VP64, ZFHD1-YP64, Gal4-KRAB, and HAP1- VP16. In some embodiments, the transcriptional regulator is Gal4-VP64. In some embodiments, the transcriptional regulator is a human or humanized transcriptional regulator. In some embodiments, the transcriptional regulator is HNFla-p65. In some embodiments, the human or humanized transcriptional regulator is HNFla-p65. A transcriptional activation domain can comprise a wild-type or naturally occurring sequence, or it can be a modified, mutant, or derivative version of the original transcriptional activation domain that has the desired ability to increase and / or activate transcription of one or more genes. In some embodiments, the transcriptional regulator can further include a nuclear localization signal.

[0261] In some embodiments, the priming receptor comprises one or more intracellular “DNA-binding domains” (or “DB domains”). Such “DNA-binding domains” refer toIPTS / 200097280.2 4.QAttorney Ref: ANB-223WO sequence- specific DNA binding domains that bind a particular DNA sequence element. Accordingly, as used herein, a “sequence- specific DNA-binding domain” refers to a protein domain portion that has the ability to selectively bind DNA having a specific, predetermined sequence. A sequence- specific DNA binding domain can comprise a wild-type or naturally occurring sequence, or it can be a modified, mutant, or derivative version of the original domain that has the desired ability to bind to a desired sequence. In some embodiments, the sequence- specific DNA binding domain is engineered to bind a desired sequence. Nonlimiting examples of proteins having sequence-specific DNA binding domains that can be used in synthetic proteins described herein include HNFla, Gal4, GCN4, reverse tetracycline receptor, THY1, SYN1, NSE / RU5', AGRP, CALB2, CAMK2A, CCK, CHAT, DLX6A, EMX1, zinc finger proteins or domains thereof, CRISPR / Cas proteins, such as Cas9, Cas3, Cas4, Cas5, Cas5e (or CasD), Cash, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, CaslO, CaslOd, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Cszl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul96, and TALES.

[0262] In those embodiments where a CRISPR / Cas-like protein is used, the CRISPR / Cas- like protein can be a wild type CRISPR / Cas protein, a modified CRISPR / Cas protein, or a fragment of a wild type or modified CRISPR / Cas protein. The CRISPR / Cas-like protein can be modified to increase nucleic acid binding affinity and / or specificity, alter an enzymatic activity, and / or change another property of the protein. For example, nuclease (i.e., DNase, RNase) domains of the CRISPR / Cas-like protein can be modified, deleted, or inactivated. Alternatively, the CRISPR / Cas-like protein can be truncated to remove domains that are not essential for the functions of the systems described herein. For example, a CRISPR enzyme that is used as a DNA binding protein or domain thereof can be mutated with respect to a corresponding wild-type enzyme such that the mutated CRISPR or domain thereof lacks the ability to cleave a nucleic acid sequence containing a DNA binding domain target site. For example, a D10A mutation can be combined with one or more of H840A, N854A, or N863A mutations to produce a Cas9 enzyme substantially lacking all DNA cleavage activity.Juxtamembrane Domain

[0263] The ECD and the TMD, or the TMD and the ICD, can be linked to each other with a linking polypeptide, such as a juxtamembrane domain. “SynNotch” or synthetic notch receptors comprise a heterologous extracellular ligand-binding domain, a linking polypeptideIPTS / 200097280.2 64Attorney Ref: ANB-223WO having substantial sequence identity with a Notch receptor JMD (including the NRR), a TMD, and an ICD. “Fn Notch” receptors comprise a heterologous extracellular ligand binding domain, a linking polypeptide having substantial sequence identity with a Robo receptor (such as a mammalian Robol, Robo2, Robo3, or Robo4), followed by 1, 2, or 3 fibronectin repeats (“Fn”), a TMD, and an ICD. “Mini Notch” receptors comprise a heterologous extracellular ligand binding domain, a linking polypeptide having substantial sequence identity with a Notch receptor JMD but lacking the NRR (the LIN-12-Notch repeat (LNR) modules, and the heterodimerization domain), a TMD, and an ICD. “Minimal Linker Notch” receptors comprise a heterologous extracellular ligand-binding domain, a linking polypeptide lacking substantial sequence identity with a Notch receptor (for example, without limitation, having a synthetic (GGS)n polypeptide sequence), a TMD, and an ICD. “Hinge Notch” receptors comprise a heterologous extracellular ligand-binding domain, a hinge sequence comprising an oligomerization domain (i.e., a domain that promotes dimerization, trimerization, or higher order multimerization with a synthetic receptor and / or an existing host receptor), a TMD, and an ICD.

[0264] In some embodiments, the priming receptor comprises a juxtamembrane domain (JMD) peptide in between the extracellular domain and the transmembrane domain. In some embodiments, the priming receptor comprises a juxtamembrane domain (JMD) peptide in between the transmembrane domain and the intracellular domain. In some embodiments, the JMD peptide comprises an LWF motif. The use of LWF motifs in receptor constructs is described in US Patent N. 10,858,443, hereby incorporated by reference in its entirety. In some embodiments, the JMD peptide has substantial sequence identity to the JMD of Notchl, Notch2, Notch3, and / or Notch4. In some embodiments, the JMD peptide has substantial sequence identity to the Notchl, Notch2, Notch3, and / or Notch4 JMD, but does not include a LIN-12-Notch repeat (LNR) and / or a heterodimerization domain (HD) of a Notch receptor. In some embodiments, the JMD peptide does not have substantial sequence identity to the Notchl, Notch2, Notch3, and / or Notch4 JMD. In some embodiments, the JMD peptide includes an oligimerization domain which promotes formation of dimers, trimers, or higher order assemblages of the receptor. Such JMD peptides are described in WO2021061872, hereby incorporated by reference in its entirety.

[0265] In the Mini Notch receptor, the linking polypeptide is derived from a Notch JMD sequence after deletion of the NRR and HD domain. The Notch JMD sequence may be the sequence from Notchl, Notch2, Notch3, or Notch4, and can be derived from a non-human homolog, such as those from Drosophila, Gallus, Danio, and the like. Four to 50 IPTS / 200097280.2 .CAttorney Ref: ANB-223WO amino acid residues of the remaining Notch sequence can be used as a polypeptide linker. In some embodiments, the length and amino acid composition of the linker polypeptide sequence are varied to alter the orientation and / or proximity of the ECD and the TMD relative to one another to achieve a desired activity of the chimeric polypeptide, such as the signal transduction level when ligand induced or in the absence of ligand.

[0266] In the Minimal Linker Notch receptor, the linking polypeptide does not have substantial sequence identity to a Notch JMD sequence, including the Notch JMD sequence from Notchl, Notch2, Notch3, or Notch4, or a non-human homolog thereof. Four to 50 amino acid residues can be used as a polypeptide linker. In some embodiments, the length and amino acid composition of the linker polypeptide sequence are varied to alter the orientation and / or proximity of the ECD and the TMD relative to one another to achieve a desired activity of the chimeric polypeptide of the disclosure. The Minimal Linker sequence can be designed to include or omit a protease cleavage site, and can include or omit a glycosylation site or sites for other types of post-translational modification. In some embodiments, the Minimal Linker does not comprise a protease cleavage site or a glysosylation site.

[0267] In some embodiments, the priming receptor further comprises a hinge. Hinge linkers that can be used in the priming receptor can include an oligomerization domain (e.g., a hinge domain) containing one or more polypeptide motifs that promote oligomer formation of the chimeric polypeptides via intermolecular disulfide bonding. In these instances, within the chimeric receptors disclosed herein, the hinge domain generally includes a flexible polypeptide connector region disposed between the ECD and the TMD. Thus, the hinge domain provides flexibility between the ECD and TMD and also provides sites for intermolecular disulfide bonding between two or more chimeric polypeptide monomers to form an oligomeric complex. In some embodiments, the hinge domain includes motifs that promote dimer formation of the chimeric polypeptides disclosed herein. In some embodiments, the hinge domain includes motifs that promote trimer formation of the chimeric polypeptides disclosed herein (e.g., a hinge domain derived from 0X40). Hinge polypeptide sequences suitable for the compositions and methods of the disclosure can be naturally-occurring hinge polypeptide sequences (e.g., those from naturally-occurring immunoglobulins) or can be engineered, designed, or modified so as to provide desired and / or improved properties, e.g., modulating transcription. Suitable hinge polypeptide sequences include, but are not limited to, those derived from IgA, IgD, and IgG subclasses, such as IgGl hinge domain, IgG2 hinge domain, IgG3 hinge domain, and IgG4 hinge domain, or a functional variant thereof. In some embodiments, the hinge polypeptide sequenceIPTS / 200097280.2 . .Attorney Ref: ANB-223WO contains one or more CXXC motifs. In some embodiments, the hinge polypeptide sequence contains one or more CPPC motifs (SEQ ID NO: 287).

[0268] Hinge polypeptide sequences can also be derived from a CD8a hinge domain, a CD28 hinge domain, a CD 152 hinge domain, a PD-1 hinge domain, a CTLA4 hinge domain, an 0X40 hinge domain, and functional variants thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from a CD8 a hinge domain or a functional variant thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from a CD28 hinge domain or a functional variant thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from an 0X40 hinge domain or a functional variant thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from an IgG4 hinge domain or a functional variant thereof.

[0269] The Fn Notch linking polypeptide is derived from the Robol JMD, which contains a fibronectin repeat (Fn) domain, with a short polypeptide sequence between the Fn repeats and the TMD. The Fn Notch linking polypeptide does not contain a Notch negative regulatory region (NRR), or the Notch HD domain. The Fn linking polypeptide can contain 1, 2, 3, 4, or 5 Fn repeats. In some embodiments, the chimeric receptor comprises a Fn linking polypeptide having about 1 to about 5 Fn repeats, about 1 to about 3 Fn repeats, or about 2 to about 3 Fn repeats. The short polypeptide sequence between the Fn repeats and the TMD can be from about 2 to about 30 amino acid residues. In some embodiments, the short polypeptide sequence can be between about 5 and about 20 amino acids, of any sequence. In some embodiments, the short polypeptide sequence can be between about 5 and about 20 naturally- occurring amino acids, of any sequence. In some embodiments, the short polypeptide sequence can be between about 5 and about 20 amino acids, of any sequence but having no more than one proline. In some embodiments, the short polypeptide sequence can be between about 5 and about 20 amino acids, and about 50% or more of the amino acids are glycine. In some embodiments, the short polypeptide sequence can be between about 5 and about 20 amino acids, where the amino acids are selected from glycine, serine, threonine, and alanine. In some embodiments, the length and amino acid composition of the Fn linking polypeptide sequence can be varied to alter the orientation and / or proximity of the ECD and the TMD relative to one another to achieve a desired activity of the chimeric polypeptide of the disclosure.IPTS / 200097280.2 67Attorney Ref: ANB-223WOStop-Transfer Sequence

[0270] In some embodiments, the priming receptor further comprises a stop-transfer sequence (STS) in between the transmembrane domain and the intracellular domains. The STS comprises a charged, lipophobic sequence. Without being bound by any theory, the STS serves as a membrane anchor, and is believed to prevent passage of the intracellular domain into the plasma membrane. The use of STS domains in priming receptors is described in WO202 1061872, hereby incorporated by reference in its entirety. Non-limiting exemplary STS sequences include APLP1, APLP2, APP, TGBR3, CSF1R, CXCL16, CX3CL1, DAG1, DCC, DNER, DSG2, CDH1, GHR, HLA-A, IFNAR2, IGF1R, IE1R1, ERN2, KCNE1, KCNE2, CHE1, ERP1, ERP2, ERP18, PTPRF, SCN1B, SCN3B, NPR3, NGFR, PEXDC2, PAM, AGER, ROBO1, SORCS3, SORCS1, SORL1, SDC1, SDC2, SPN, TYR, TYRP1, DCT, VASN, FLT1, CDH5, PKTFD1, NECTIN1, KL, IL6R, EFNB1, CD44, CLSTN1, LRP8, PCDHGC3, NRG1, LRP1B, JAG2, EFNB2, DLL1, CLSTN2, EPCAM, ErbB4, KCNE3, CDH2, NRG2, PTPRK, BTC, EPHA4, IL1R2, KCNE4, SCN2B, Nradd, PTPRM, Notchl, Notch2, Notch3, and Notch4 STS sequences. In some embodiments, the STS is heterologous to the transmembrane domain. In some embodiments, the STS is homologous to the transmembrane domain. STS sequences are described in WO2021061872, hereby incorporated by reference in its entirety.Chimeric Antigen Receptors

[0271] In another aspect, provided herein are chimeric antigen receptors comprising an extracellular antigen-binding domain that specifically binds to a target antigen or ligand.

[0272] In some embodiments, the chimeric antigen receptor includes an extracellular portion comprising an antigen binding domain. The antigen recognition domain of a receptor such as a CAR can be linked to one or more intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as a TCR complex, in the case of a CAR, and / or signal via another cell surface receptor. Thus, in some embodiments, the extracellular binding component (e.g., ligand-binding or antigenbinding domain) is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain that naturally is associated with one of the domains in the receptor, e.g., CAR, is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembraneIPTS / 200097280.2 68Attorney Ref: ANB-223WO domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0273] In some aspects, the chimeric antigen receptor includes an extracellular portion comprising an antigen binding domain described herein and an intracellular signaling domain. In some embodiments, an antibody or fragment includes an scFv, a VH, or a single-domain VH antibody and the intracellular domain contains an ITAM. In some aspects, the intracellular signaling domain includes a signaling domain of a zeta chain of a CD3-zeta (CD3) chain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain.

[0274] In some aspects, the transmembrane domain contains a transmembrane portion of CD8a or CD28. The extracellular domain and transmembrane can be linked directly or indirectly. In some embodiments, the extracellular domain and transmembrane are linked by a spacer, such as any described herein. In some embodiments, the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule, such as between the transmembrane domain and intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 41BB.Chimeric Antigen Receptor Extracellular Domain

[0275] In some embodiments, the extracellular domain includes the ligand-binding portion of a receptor. In some embodiments, the extracellular domain includes an antigen-binding moiety that binds to one or more target antigens. In some embodiments, the antigen-binding moiety includes one or more antigen-binding determinants of an antibody or a functional antigen-binding fragment thereof. In some embodiments, the antigen-binding moiety is selected from the group consisting of an antibody, a nanobody, a diabody, a triabody, or a minibody, a F(ab')2 fragment, a Fab fragment, a single chain variable fragment (scFv), and a single domain antibody (sdAb), or a functional fragment thereof. In some embodiments, the antigen-binding moiety comprises an scFv. The antigen-binding moiety can include naturally-occurring amino acid sequences or can be engineered, designed, or modified so as to provide desired and / or improved properties, e.g., increased binding affinity.

[0276] In various embodiments, a CAR comprises means for binding a target protein. In some embodiments, the means binds a target protein. In some embodiments, the means binds a human target protein. In some embodiments, the means is an antibody or antigen-binding fragment or equivalent thereof (e.g., a full length antibody or a F(ab')2 fragment, a FabIPTS / 200097280.2 69Attorney Ref: ANB-223WO fragment, a single chain variable fragment (scFv), and a single domain antibody (sdAb), or a functional fragment thereof) means for binding a target protein.CAR Transmembrane Domain

[0277] The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane -bound or transmembrane protein. Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T- cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, and / or CD 154. Alternatively the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. In some embodiments, the linkage is by linkers, spacers, and / or transmembrane domain(s).

[0278] In some embodiments, the transmembrane domain of the receptor, e.g., the CAR, is a transmembrane domain of human CD28 or variant thereof, e.g., a 27-amino acid transmembrane domain of a human CD28 (Accession No.: P10747.1).

[0279] In some embodiments, the CAR comprises a CD8a or CD28 TMD.CAR Hinge

[0280] In some embodiments, the CAR further includes a spacer, which may be or include at least a portion of an immunoglobulin constant region or variant or modified version thereof, such as a hinge region, e.g., a CD8a hinge, an IgG4 hinge region, and / or a CH1 / CL and / or Fc region. In some embodiments, the constant region or portion is of a human IgG, such as IgG4 or IgGl. In some aspects, the portion of the constant region serves as a spacer region between the antigenrecognition component, e.g., scFv, and transmembrane domain. The spacer can be of a length that provides for increased responsiveness of the cell following antigen binding, as compared to in the absence of the spacer. In some examples, the spacer is at or about 12 amino acids in length or is no more than 12 amino acids in length. Exemplary spacers include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and including any integer between theIPTS / 200097280.2 70Attorney Ref: ANB-223WO endpoints of any of the listed ranges. In some embodiments, a spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include CD8a hinge, IgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains, or IgG4 hinge linked to the CH3 domain. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153 or international patent application publication number WO2014031687. In some embodiments, the CAR hinge comprises a CD8a CD8a, truncated CD8a, or CD28 hinge domain.

[0281] Among the intracellular signaling domains are those that mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and / or a signal through a costimulatory receptor alone. In some embodiments, a short oligo- or polypeptide linker, for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-serine doublet, is present and forms a linkage between the transmembrane domain and the cytoplasmic signaling domain of the receptor.CAR Intracellular Domain

[0282] In some embodiments, upon ligation of the CAR, the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the receptor. In some embodiments, the CAR comprises means for activating at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the receptor. For example, in some contexts, the receptor induces a function of a T cell such as cytolytic activity or T-helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of an intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immuno stimulatory chain, for example, if it transduces the effector function signal. In some embodiments, the intracellular signaling domain or domains include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptor to initiate signal transduction following antigen receptor engagement, and / or any derivative or variant of such molecules, and / or any synthetic sequence that has the same functional capability. In some embodiments, the means for at least one of the normal effector functions or responses of the immune cell comprises an CAR intracellular activation domain, e.g., an intracellular activation domain provided herein or an equivalent thereof. In some embodiments, the means for at least one of the normal effector functions or responses of the immune cell comprises an CARIPTS / 200097280.2 71Attorney Ref: ANB-223WO intracellular activation domain and a CAR co-stimulatory domain, e.g., a co- stimulatory domain provided herein or an equivalent thereof.

[0283] In some aspects, the receptor includes a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM containing primary cytoplasmic signaling sequences include those derived from TCR or CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and CD66d. In some embodiments, cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta. In some embodiments, the intracellular activation domain comprises a CD3(^ domain.

[0284] In some embodiments, the intracellular signaling domain comprises a human CD3 zeta stimulatory signaling domain or functional variant thereof, such as a 112 AA cytoplasmic domain of isoform 3 of human CD3.zeta. (Accession No.: P20963.2) or a CD3 zeta signaling domain as described in U.S. Pat. No. 7,446,190 or U.S. Pat. No. 8,911,993.

[0285] The receptor, e.g., the CAR, can include at least one intracellular signaling component or components. In some embodiments, the receptor includes an intracellular component of a TCR complex, such as a TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain. Thus, in some aspects, the extracellular domain is linked to one or more cell signaling modules. In some embodiments, cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and / or other CD transmembrane domains. In some embodiments, the receptor, e.g., CAR, further includes a portion of one or more additional molecules such as Fc receptor-gamma, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR includes a chimeric molecule between CD3-zeta or Fc receptor-gamma and CD8, CD4, CD25 or CD16.

[0286] In some embodiments, the intracellular domain comprises an intracellular costimulatory signaling domain of 41BB or functional variant or portion thereof, such as a 42-amino acid cytoplasmic domain of a human 4- IBB (Accession No. Q07011.1) or functional variant or portion thereof.

[0287] In some embodiments, the receptor encompasses one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., primary activation domain, in the cytoplasmic portion. Exemplary receptors include intracellular components of CD3-zeta, CD28, and 4-1BB. In some embodiments, the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule. In some aspects, the T cell costimulatory molecule is 4- IBB.IPTS / 200097280.2 77Attorney Ref: ANB-223WO

[0288] In some embodiments, the receptor includes a signaling domain and / or transmembrane portion of a costimulatory receptor, such as CD28, 4- IBB, 0X40, DAP10, and ICOS. In some aspects, the same receptor includes both the activating and costimulatory components.

[0289] In certain embodiments, the intracellular signaling domain comprises a CD8a transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain. In some embodiments, the intracellular signaling domain comprises a 4-1BB (CD137, TNFRSF9) co- stimulatory domains, linked to a CD3 zeta intracellular domain. In some embodiments, the CAR comprises a 4- IBB co- stimulatory domain.

[0290] In some embodiments, the CAR or other antigen receptor further includes a marker, such as a cell surface marker, which may be used to confirm transduction or engineering of the cell to express the receptor, such as a truncated version of a cell surface receptor, such as truncated EGFR (tEGFR). In some aspects, the marker includes all or part (e.g., truncated form) of CD34, a nerve growth factor receptor (NGFR), or epidermal growth factor receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding for a linker sequence, such as a cleavable linker sequence or a ribosomal skip sequence, e.g., T2A. See WO2014031687. In some embodiments, introduction of a construct encoding the CAR and EGFRt separated by a T2A ribosome switch can express two proteins from the same construct, such that the EGFRt can be used as a marker to detect cells expressing such construct. In some embodiments, a marker, and optionally a linker sequence, can be any as disclosed in published patent application No. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR) that is, optionally, linked to a linker sequence, such as a T2A ribosomal skip sequence.

[0291] In some embodiments, the marker is a molecule, e.g., cell surface protein, not naturally found on T cells or not naturally found on the surface of T cells, or a portion thereof.

[0292] In some embodiments, the molecule is a non-self molecule, e.g., non-self protein, i.e., one that is not recognized as "self" by the immune system of the host into which the cells will be adoptively transferred.

[0293] In some embodiments, the marker serves no therapeutic function and / or produces no effect other than to be used as a marker for genetic engineering, e.g., for selecting cells successfully engineered. In other embodiments, the marker may be a therapeutic molecule or molecule otherwise exerting some desired effect, such as a ligand for a cell to be encountered in vivo, such as a costimulatory or immune checkpoint molecule to enhance and / or dampen responses of the cells upon adoptive transfer and encounter with ligand.IPTS / 200097280.2 73Attorney Ref: ANB-223WO

[0294] The CAR may comprise one or modified synthetic amino acids in place of one or more naturally-occurring amino acids. Exemplary modified amino acids include, but are not limited to, aminocyclohexane carboxylic acid, norleucine, a-amino n-decanoic acid, homoserine, S- acetylaminomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4- nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, (3-phenylserine (3- hydroxyphenylalanine, phenylglycine, a-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N' -benzyl-N'-methyl-lysine, N',N' -dibenzyl-lysine, 6- hydroxylysine, ornithine, a- aminocyclopentane carboxylic acid, a- aminocyclohexane carboxylic acid, a-aminocycloheptane carboxylic acid, a-(2-amino-2-norbomane )-carboxylic acid, a,y - diaminobutyric acid, a,y -diaminopropionic acid, homophenylalanine, and a-tertbutylglycine.

[0295] For example, in some embodiments, the CAR includes an antibody or fragment thereof, including single chain antibodies (sdAbs, e.g. containing only the VH region), VH domains, and scFvs, described herein, a spacer such as a CD8a hinge, a CD8a transmembrane domain, a 4- 1BB intracellular signaling domain, and a CD3 zeta signaling domain. In some embodiments, the CAR includes an antibody or fragment, including sdAbs and scFvs described herein, a spacer such as a CD8a hinge, a CD8a transmembrane domain, a 4- IBB intracellular signaling domain, and a CD3 zeta signaling domain.

[0296] Transgenes expressing the priming receptor and CAR system may be introduced into cells, such as a T cell, using, for example, a site-specific technique. With site specific integration of the transgenes (e.g. priming receptor and CAR), the transgenes may be targeted to a safe harbor locus or TRAC. Examples of site-specific techniques for integration into the safe harbor loci include, without limitation, homology-dependent engineering using nucleases and homology independent targeted insertion using Cas9.

[0297] The engineered cells have applications to immune-oncology. The priming receptor and CAR, for example, can be selected to target different specific tumor antigens. Examples of cancers that can be effectively targeted using such cells are blood cancers or solid cancers. In some embodiments, immune cell therapy can be used to treat solid tumors.Nucleic Acids and Vectors

[0298] In another aspect, provided herein are one or more nucleic acids, wherein the one or more nucleic acids encode a synthetic pathway activator described herein. In another aspect, provided herein are one or more nucleic acids, wherein the one or more nucleic acids encode a sequence selected from the group consisting of SEQ ID NOS: 1-58 or 63-104.IPTS / 200097280.2 74Attorney Ref: ANB-223WO

[0299] In some embodiments, the one or more nucleic acid(s) further comprises a 5’ homology directed repair arm and / or a 3’ homology directed repair arm complementary to an insertion site in a host cell chromosome. In some embodiments, the one or more nucleic acid(s) comprises the 5’ homology directed repair arm and the 3’ homology directed repair arm. In some embodiments, the one or more nucleic acid(s) is incorporated into an expression cassette or an expression vector. In some embodiments, the expression cassette or the expression vector further comprises a constitutive promoter upstream of the one or more nucleic acid(s).

[0300] In some embodiments, the priming receptor, CAR, and synthetic pathway activator are incorporated into a single expression cassette or a single expression vector. In some embodiments, the priming receptor, CAR, and synthetic pathway activator are incorporated into two or more expression cassettes or expression vectors. In some embodiments, the expression vector(s) is a non- viral vector.

[0301] In some embodiments, the present disclosure contemplates nucleic acid DNA template inserts that comprise one or more transgenes encoding the synthetic pathway activators as described herein. In some embodiments, the DNA template insert encodes a synthetic pathway activator. In some embodiments, the nucleic acid DNA template further comprises a priming receptors and / or CAR. In some embodiments, the DNA template insert encodes a priming receptor transgene. In some embodiments, the DNA template insert encodes a chimeric antigen receptor transgene. In some embodiments, the DNA template insert comprises a synthetic pathway activator, a priming receptor transgene, and a chimeric antigen receptor transgene.

[0302] In some embodiments, the one or more nucleic acid(s) are encoded on a single DNA template insert. In some embodiments, the one or more nucleic acid(s) are encoded on multiple DNA template inserts. For example, the one or more nucleic acid(s) can be encoded on two, three, or four DNA template inserts.

[0303] The DNA template insert can also comprise a self-cleaving peptide. Examples of self-cleaving peptides include, but are not limited to, self-cleaving viral 2A peptides, for example, a porcine teschovirus-1 (P2A) peptide, a Thosea asigna virus (T2A) peptide, an equine rhinitis A virus (E2A) peptide, or a foot-and-mouth disease virus (F2A) peptide. Selfcleaving 2A peptides allow expression of multiple gene products from a single construct. (See, for example, Chang et al. “Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells,” MAbs 7(2): 403-412 (2015)).IPTS / 200097280.2 75Attorney Ref: ANB-223WO

[0304] The DNA template insert can also comprise a WPRE element. WPRE elements are generally described in Higashimoto, T., et al. Gene Ther 14, 1298-1304 (2007); and Zufferey, R., et al. J Virol. 1999 Apr;73(4):2886-92., both of which are hereby incorporated by reference.

[0305] The DNA template insert can also comprise an SV40 polyA tail.Cells

[0306] Also provided herein are cells or immune cells comprising at least one DNA template non-virally inserted into a target region of the genome of the cell, wherein DNA template encodes one or more of the synthetic pathway activators as described herein. In some embodiments, the DNA template further encodes a priming receptor and CAR system as described herein.

[0307] A cell comprising a DNA template insert at a target locus or safe harbor site as described in the present disclosure can be referred to as an engineered cell. In some embodiments, the cell or immune cell is any cell that can give rise to a pluripotent immune cell. In some embodiments, the immune cell is a primary immune cell. In some embodiments, the immune cell can be an induced pluripotent stem cell (iPSC) or a human pluripotent stem cell (HSPC). In some embodiments, the immune cell comprises primary hematopoietic cells or primary hematopoietic stem cells. In some embodiments, that engineered cell is a stem cell, a human cell, a primary cell, an hematopoietic cell, an adaptive immune cell, an innate immune cell, a natural killer (NK) cell, a T cell, a CD8+ cell, a CD4+ cell, or a T cell progenitor cell. In some embodiments, the immune cells are T cells. In some embodiments, the T cells are regulatory T cells, effector T cells, or naive T cells. In some embodiments, the T cells are CD8+T cells. In some embodiments, the T cells are CD4+T cells. In some embodiments, the T cells are CD4+CD8+T cells.

[0308] In some embodiments, the engineered cell is a stem cell, a human cell, a primary cell, an hematopoietic cell, an hematopoietic stem cell, an adaptive immune cell, an innate immune cell, a T cell or a T cell progenitor. Non-limiting examples of immune cells that are contemplated in the present disclosure include T cell, B cell, natural killer (NK) cell, NKT / iNKT cell, macrophage, myeloid cell, and dendritic cells. Non-limiting examples of stem cells that are contemplated in the present disclosure include pluripotent stem cells (PSCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), embryo- derived embryonic stem cells obtained by nuclear transfer (ntES; nuclear transfer ES), male germline stem cells (GS cells), embryonic germ cells (EG cells), hematopoieticIPTS / 200097280.2 76Attorney Ref: ANB-223WO stem / progenitor stem cells (HSPCs), somatic stem cells (adult stem cells), hemangioblasts, neural stem cells, mesenchymal stem cells and stem cells of other cells (including osteocyte, chondrocyte, myocyte, cardiac myocyte, neuron, tendon cell, adipocyte, pancreocyte, hepatocyte, nephrocyte and follicle cells and so on). In some embodiments, the engineered cells is a T cell, NK cells, iPSC, and HSPC. In some embodiments, the engineered cells used in the present disclosure are human cell lines grown in vitro (e.g. deliberately immortalized cell lines, cancer cell lines, etc.).

[0309] Also provided herein are populations of cells comprising a plurality of the cells or immune cell. In some embodiments, the genome of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or greater of the cells comprises the priming receptor and CAR system as described herein.Method of Treating Immune-Related Condition of Disease

[0310] In another aspect, the disclosure provides methods of treating an immune -related condition (e.g., cancer) in an individual comprising administering to the individual an effective amount of a composition comprising a synthetic pathway activator described herein, such as a cell comprising a synthetic pathway activator described herein. In some embodiments, the composition further comprises a priming receptor that specifically binds to a target antigen and a chimeric antigen receptor that specifically binds to a target antigen. In another aspect, the disclosure provides methods of enhancing an immune response in an individual comprising administering to the individual an effective amount of a composition comprising a synthetic pathway activator described herein, such as such as a cell comprising a synthetic pathway activator described herein. In some embodiments, the composition further comprises a priming receptor that specifically binds to a target antigen and a chimeric antigen receptor that specifically binds to a target antigen.

[0311] In some embodiments, the methods provided herein are useful for the treatment of an immune -related condition in an individual. In one embodiment, the individual is a human.

[0312] In some embodiments, the methods provided herein (such as methods of increasing or enhancing an immune response) are useful for the treatment of cancer and as such an individual receiving the synthetic pathway activator described herein has cancer. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is immunoevasive. In some embodiments, the cancer is immunoresponsive. In some embodiments, the cancer is immunoresponsive. In particular embodiments, the cancer isIPTS / 200097280.2 77Attorney Ref: ANB-223WO kidney cancer, renal cell carcinoma, clear cell renal cell carcinoma (ccRcc), colorectal cancer, or lung cancer. In some embodiments, the cancer is mesothelioma.

[0313] In another aspect, the disclosure provides methods of inhibiting (e.g., killing, disabling, or preventing growth or expansion) a target cell that expresses both of the CAR antigen and the priming receptor antigen. In another aspect, the invention provides methods of killing a target cell that expresses both of the CAR antigen and the priming receptor antigen. In some embodiments, the target cell is a cancer cell.

[0314] In some embodiments, the treatment results in a decrease in the cancer volume or size. In some embodiments, the treatment is effective at reducing a cancer volume as compared to the cancer volume prior to administration of the antibody. In some embodiments, the treatment results in a decrease in the cancer growth rate. In some embodiments, the treatment is effective at reducing a cancer growth rate as compared to the cancer growth rate prior to administration of the antibody. In some embodiments, the treatment is effective at eliminating the cancer.

[0315] In some embodiments, the treatment results in an increase in immune cell potency (e.g., cytotoxic activity) and / or persistence (e.g., viability or cell number) of an immune cell comprising one or more SPA proteins or nucleic acids as described herein. The increase in potency and / or persistence can be as compared to a cell lacking the SPA proteins or nucleic acids as described herein.

[0316] In some embodiments, the CAR antigen and / or the priming receptor antigen are expressed at a higher level in the cancer as compared to a non-cancer cell. Levels of CAR antigen and / or the priming receptor antigen can be assessed by any technique known in the field, including, but not limited to, protein assays or nucleic assays such as FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blotting, immunodetection methods, HPLC, surface plasmon resonance, optical spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY technique, and FISH, and combinations thereof.Method of Immune Modulation

[0317] Methods of administration of a cell comprising a synthetic pathway activator described herein can result in modulation of the activity of a cell. In some embodiments, the activity is an immune response. Modulation can be an increase or decrease in an immune response. In some embodiments, modulation is an increase in an immune response. In someIPTS / 200097280.2 78Attorney Ref: ANB-223WO embodiments, the activity is increased cytotoxicity, immune cell persistence, and / or tumor infiltration as compared to a cell that does not comprise the SPA nucleic acids or peptide disclosed herein.

[0318] In one aspect, administration of a cell comprising a synthetic pathway activator described herein can result in induction of pro-inflammatory molecules, such as cytokines or chemokines. Generally, induced pro-inflammatory molecules are present at levels greater than that achieved with isotype control. Such pro-inflammatory molecules in turn result in activation of anti-tumor immunity, including, but not limited to, T cell activation, T cell proliferation, T cell differentiation, Ml -like macrophage activation, and NK cell activation. Thus, the administration of a cell comprising a synthetic pathway activator described herein can induce multiple anti-tumor immune mechanisms that lead to tumor destruction. In some embodiments, the immune activity of the cell is cytolytic activity.

[0319] In another aspect, provided herein are methods of increasing an immune response in an individual comprising administering to the individual an effective amount of a cell comprising a synthetic pathway activator described herein. In some embodiments, the method of increasing an immune response in a subject comprises administering to the subject a cell comprising a synthetic pathway activator described herein..

[0320] In some embodiments, the cell is present in a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

[0321] In any and all aspects of increasing an immune response as described herein, any increase or decrease or alteration of an aspect of characteristic(s) or function(s) is as compared to a cell not comprising a composition comprising a synthetic pathway activator described herein.

[0322] Increasing an immune response can be both enhancing an immune response or inducing an immune response. For instance, increasing an immune response encompasses both the start or initiation of an immune response, or ramping up or amplifying an on-going or existing immune response. In some embodiments, the treatment induces an immune response. In some embodiments, the induced immune response is an adaptive immune response. In some embodiments, the induced immune response is an innate immune response. In some embodiments, the treatment enhances an immune response. In some embodiments, the enhanced immune response is an adaptive immune response. In some embodiments, the enhanced immune response is an innate immune response. In some embodiments, the treatment increases an immune response. In some embodiments, the increased immune response is an adaptive immune response. In some embodiments, the increased immuneIPTS / 200097280.2 -Attorney Ref: ANB-223WO response is an innate immune response. In some embodiments, the immune response is started or initiated by administration of a cell comprising a synthetic pathway activator described herein. In some embodiments, the immune response is enhanced by administration of cell comprising a synthetic pathway activator described herein. In some embodiments, the immune response is enhanced by administration of cell comprising a synthetic pathway activator and a priming receptor and CAR system described herein.

[0323] In some embodiments, the increased immune response is an increase in one or more STAT1 / 3 / 4 / 5 and / or TIR signaling pathways, e.g., an increase in one of more of the STAT1, STAT3, STAT4, STAT5, and / or the TIR signaling pathways in a cell comprising the SPA proteins or nucleic acids as described herein. An increase in the one or more STAT and / or TIR signaling pathways in a cell can be determined by an increase in the phosphorylation of one or more of STAT1, STAT3, STAT4, STAT5, and / or the TIR or MyD88 proteins in the cell comprising the SPA as compared to a cell that does not comprise the SPA. Alternatively, an increase in the one or more STAT1 / 3 / 4 / 5 and / or TIR signaling pathways in a cell can be determined by changes in gene expression (e.g., increase or decreases) of genes whose expression is controlled by one or more of STAT1, STAT3, STAT4, STAT5, and / or the TIR or MyD88 proteins. Exemplary genes regulated by STAT1 include, but are not limited to, Interferon-Stimulated Genes (ISGs, such as APOL1, APOL2, HIRA, and USP18), MHC class I and II genes, inflammatory and cytokine genes such as IL- 15, and IL-33.

[0324] Exemplary genes regulated by STAT3 include, but are not limited to, genes involved in the cell growth, differentiation (e.g., of Thl7 cells), and apoptosis regulation, such as CL-xL, Cyclin DI, c-Myc, Survivin, SOCS3, IL-10, IL-6, NTRK3, DUSP10, CASP1, CXCR5, IGEBP5, BECN1 and PIK3C3.

[0325] Exemplary genes regulated by STAT4 include, but are not limited to, perforin, IFNy, TNEa, IL-21, IL-18R1, IL-18r|32, IL-18RAP, TBX21, GATA3, KLRK1, and MYD88.

[0326] Exemplary genes regulated by STAT5 include, but are not limited to, CIS (Cytokine-Inducible SH2-Containing Protein), DOCK8, c-Myc, BCL-2, IL-4Ra, Blimp- 1, SOCS1, Cyclin DI, Spi2.1 (Serine / cysteine proteinase inhibitor), Ryk (Receptor- like tyrosine kinase), and LOXP3.

[0327] Exemplary genes regulated by TIR include, but are not limited to, NF-KB, IKBO., AP-1 (Activator protein 1), IRFs (Interferon Regulatory Factors), IL-ip, IL-6, TNFa, IL-12, IFN-a, IFN-P, MCP-1 / CCL2, IL-8 / CXCL8, and COX-2. Additional description of the TIR signaling pathway is provided in Loiarro M, el al. Targeting TLR / IL-1R signaling in human IPTS / 200097280.2 onAttorney Ref: ANB-223WO diseases. Mediators Inflamm. 2010;2010:674363. doi: 10.1155 / 2010 / 674363, which is hereby incorporated by reference in its entirety.

[0328] In some embodiments, the increased immune response is an increase in immune cell potency (e.g., cytotoxic activity) and / or persistence (e.g., viability or cell number) of an immune cell comprising one or more SPA proteins or nucleic acids as described herein. The increase in potency and / or persistence can be as compared to a cell lacking the SPA proteins or nucleic acids as described herein.

[0329] In some embodiments, the increased activity and / or immune response is a 0.25- fold, 0.50-fold, 0.75-fold, 1.00-fold, 1.25-fold, 1.50-fold, 1.75-fold, 2.00-fold, 2.25-fold, 2.50-fold, 2.75-fold, 3.00-fold, 3.25-fold, 3.50-fold, 3.75-fold, 4.00-fold, 4.25-fold, 4.50-fold,4.75-fold, or 5.00-fold increase in STAT1, STAT3, STAT4, STAT5, and / or TIR phosphorylation in a cell comprising the SPA proteins or nucleic acids as described herein as compared to a cell lacking the SPA proteins or nucleic acids as described herein. In some embodiments, the increased activity and / or immune response is between a 0.25-0.5-fold, 0.5- 0.75-fold, 0.75-1.0-fold, 1.0-1.25-fold, 1.25-1.5-fold, 1.5-1.75-fold, 1.75-2.0-fold, 2.0-2.25- fold, 2.25-2.5-fold, 2.5-2.75-fold, 2.75-3.0-fold, 3.0-3.25-fold, 3.25-3.5-fold, 3.5-3.75-fold,3.75-4.0-fold, 4.0-4.25-fold, 4.25-4.5-fold, 4.5-4.75-fold, or 4.75-5.0-fold increase in STAT1, STAT3, STAT4, STAT5, and / or TIR phosphorylation in a cell comprising the SPA proteins or nucleic acids as described herein as compared to a cell lacking the SPA proteins or nucleic acids as described herein.

[0330] In some embodiments, the increased activity and / or immune response is a 0.25- fold, 0.50-fold, 0.75-fold, 1.00-fold, 1.25-fold, 1.50-fold, 1.75-fold, 2.00-fold, 2.25-fold, 2.50-fold, 2.75-fold, 3.00-fold, 3.25-fold, 3.50-fold, 3.75-fold, 4.00-fold, 4.25-fold, 4.50-fold,4.75-fold, or 5.00-fold increase in STAT1, STAT3, STAT4, and STAT5 phosphorylation as compared to a cell that does not comprise the SPA proteins or nucleic acids as described herein. In some embodiments, the increased activity and / or immune response is a 0.25-fold, 0.50-fold, 0.75-fold, 1.00-fold, 1.25-fold, 1.50-fold, 1.75-fold, 2.00-fold, 2.25-fold, 2.50-fold,2.75-fold, 3.00-fold, 3.25-fold, 3.50-fold, 3.75-fold, 4.00-fold, 4.25-fold, 4.50-fold, 4.75-fold, or 5.00-fold increase in STAT1, STAT3, and STAT4 phosphorylation as compared to a cell that does not comprise the SPA proteins or nucleic acids as described herein.

[0331] In another aspect, the present application provides methods of genetically editing a cell with a synthetic pathway activator described herein, which results in the modulation of the immune function of the cell. The modulation can be increasing an immune response. In some embodiments, the modulation is an increase in immune function. In some IPTS / 200097280.2 01Attorney Ref: ANB-223WO embodiments, the modulation of function leads to the expression of cytokine or interleukin. In some embodiments, the modulation of function leads to the activation of an immune cell.

[0332] In some embodiments, the cell is a natural killer (NK) cell, a T cell, a CD8+ T cell, a CD4+ T cell, a primary T cell, or a T cell progenitor.

[0333] In some embodiments, the modulation of function of the cells comprising a synthetic pathway activator as described herein leads to an increase in the cells’ abilities to stimulate both native and activated T-cells, for example, by increasing cytokine or chemokine secretion by the cells expressing the synthetic pathway activator described herein. In some embodiments, the modulation of function enhances or increases the cells’ ability to produce cytokines, chemokines, CARs, or costimulatory or activating receptors. In some embodiments, the modulation increases the T-cell stimulatory function of the cells expressing a synthetic pathway activator described herein, including, for example, the cells’ abilities to trigger T-cell receptor (TCR) signaling, T-cell proliferation, or T-cell cytokine production.

[0334] In some embodiments, the increased immune response is secretion of cytokines and chemokines. In some embodiments, a synthetic pathway activator described herein induces increased expression of at least one cytokine or chemokine in a cell as compared to an isotype control cell. In some embodiments, the at least one cytokine or chemokine is selected from the group consisting of: IL-2 and IFNy. In some embodiments, the cytokine or chemokine is IL-2. In some embodiments, the cytokine or chemokine is IFNy. In some embodiments, the cytokine or chemokine secretion is increased a between bout 1-100-fold 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70- 80, 80-90, or 90-100 fold as compared to an untreated cell or a cell treated with an isotype control antibody. In some embodiments, the chemokine is IL-2 and the secretion is increased between about 1-100-fold, 1-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 1-10-fold, 10-20-fold, 20-30-fold, 30-40-fold, 40-50-fold, 50-60-fold, 60-70-fold, 70-80-fold, 80-90-fold, or 90-100-fold as compared to an untreated cell or a cell treated with an isotype control antibody. In some embodiments, the cytokine is IFNy and the secretion is increased between about 1-100-fold, 1-fold, 5-fold, 10-fold, 20- fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 1-10-fold, 10-20- fold, 20-30-fold, 30-40-fold, 40-50-fold, 50-60-fold, 60-70-fold, 70-80-fold, 80-90-fold, or 90-100-fold as compared to an untreated cell or a cell treated with an isotype control antibody.

[0335] In some embodiments, the enhanced immune response is anti-tumor immune cell recruitment and activation.IPTS / 200097280.2 82Attorney Ref: ANB-223WO

[0336] In some embodiments, the cell expressing a synthetic pathway activator described herein induces a memory immune response as compared to an isotype control cell. In general, a memory immune response is a protective immune response upon a subsequent exposure to pathogens or antigens that the immune system encountered previously. Exemplary memory immune responses include the immune response after infection or vaccination with an antigen. In general, memory immune responses are mediated by lymphocytes such as T cells or B cells. In some embodiments, the memory immune response is a protective immune response to cancer, including cancer cell growth, proliferation, or metastasis. In some embodiments, the memory immune response inhibits, prevents, or reduces cancer cell growth, proliferation, or metastasis.Methods of Editing Cells

[0337] The terms “gene editing” or “genome editing”, as used herein, refer to a type of genetic manipulation in which DNA is inserted, replaced, or removed from the genome using artificially manipulated nucleases or “molecular scissors”. It is a useful tool for elucidating the function and effect of sequence- specific genes or proteins or altering cell behavior (e.g. for therapeutic purposes).

[0338] Currently available genome editing tools include zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs) to incorporate genes at safe harbor loci (.e.g. the adeno-associated virus integration site 1 (AAVS1) safe harbor locus). The DICE (dual integrase cassette exchange) system utilizing phiC31 integrase and Bxbl integrase is a tool for target integration. Additionally, clustered regularly interspaced short palindromic repeat / Cas9 (CRISPR / Cas9) techniques can be used for targeted gene insertion.

[0339] Site specific gene editing approaches can include homology dependent mechanisms or homology independent mechanisms.

[0340] All methods known in the art for targeted insertion of gene sequences are contemplated in the methods described herein to insert constructs at gene targets or safe harbor loci.

[0341] Provided herein are methods of inserting nucleotide sequences greater than about 5 kilobases in length into the genome of a cell, in the absence of a viral vector. In some embodiments, the nucleotide sequence greater than about 5 kilobase in length can be inserted into the genome of a primary immune cell, in the absence of a viral vector

[0342] Integration of large nucleic acids, for example nucleic acids greater than 5 kilobase in size, into cells, can be limited by low efficiency of integration, off-target effects and / or lossIPTS / 200097280.2 83Attorney Ref: ANB-223WO of cell viability. Described herein are methods and compositions for achieving integration of a nucleotide sequence, for example, a nucleotide sequence greater than about 5 kilobases in size, into the genome of a cell. In some methods the efficiency of integration is increased, off-target effects are reduced and / or loss of cell viability is reduced.

[0343] The plasmid can be introduced into an immune cell with a nuclease, such as a CRISPR-associated system (Cas). The nuclease can be introduced in a ribonucleoprotein format with a guide RNA (gRNA) that targets a specific site on the genome of the immune cell. The nuclease cuts the genomic DNA at this specific site. The specific site may be a portion of the genome that encodes an endogenous immune cell receptor. Thus, cutting the genome at this site will cause the immune cell to no longer express an endogenous immune cell receptor.

[0344] The plasmid may include 5’ and 3’ homology-directed repair arms complementary to sequences at a specific site on the genome of the immune cell. The complementary sequences are on either side of the site cut by the nuclease, which allows the plasmid to be incorporated at a specified insertion site on the immune cell’s genome. Once the plasmid is incorporated, the cell will express the SPA peptide. In examples where the SPA peptide is also co-expressed with a system comprising a priming receptor and CAR, the priming receptor is also expressed by the cell. However, as explained, the design of the transgene cassette ensures that non-virally delivered circuit system receptors do not express CAR until the priming receptor binds to its cognate ligand and releases the cleavable transcription factor.

[0345] Initially, a T cell is activated. The T cell may be obtained from a patient. Thus, the present disclosure provides methods in which immune cells, such as T cells, are harvested from a patient. Then, the plasmid that encodes the CAR and priming receptor are introduced into a T cell. Advantageously, the plasmids of the present disclosure can be introduced using electroporation. When introducing the plasmid via electroporation, the nuclease may also be introduced. By using electroporation, methods of the present disclosure avoid the use of viral vectors for introducing transgenes, which is a known bottleneck in immune cell engineering. The T cells are then expanded and co-cultured to create a sufficient quantity of engineered immune cells to be used as a therapeutic treatment.

[0346] Methods for editing the genome of a cell can include a) providing a Cas 9 ribonucleoprotein complex (RNP)-DNA template complex comprising: (i) the RNP, wherein the RNP comprises a Cas9 nuclease domain and a guide RNA, wherein the guide RNA specifically hybridizes to a target region of the genome of the cell, and wherein the Cas9 IPTS / 200097280.2 O / 1Attorney Ref: ANB-223WO nuclease domain cleaves the target region to create an insertion site in the genome of the cell; and (ii) a double- stranded or single- stranded DNA template, wherein the size of the DNA template is greater than about 200 nucleotides, wherein the 5’ and 3’ ends of the DNA template comprise nucleotide sequences that are homologous to genomic sequences flanking the insertion site, and wherein the molar ratio of RNP to DNA template in the complex is from about 3:1 to about 100:1; and b) introducing the RNP-DNA template complex into the cell.

[0347] In some embodiments, the methods described herein provide an efficiency of delivery of the RNP-DNA template complex of at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, 99.5%, 99%, or higher. In some cases, the efficiency is determined with respect to cells that are viable after introducing the RNP-DNA template into the cell. In some cases, the efficiency is determined with respect to the total number of cells (viable or non-viable) in which the RNP-DNA template is introduced into the cell.

[0348] As another example, the efficiency of delivery can be determined by quantifying the number of genome edited cells in a population of cells (as compared to total cells or total viable cells obtained after the introducing step). Various methods for quantifying genome editing can be utilized. These methods include, but are not limited to, the use of a mismatchspecific nuclease, such as T7 endonuclease I; sequencing of one or more target loci (e.g., by sanger sequencing of cloned target locus amplification fragments); and high-throughput deep sequencing.

[0349] In some embodiments, loss of cell viability is reduced as compared to loss of cell viability after introduction of naked DNA into a cell or introduction of DNA into a cell using a viral vector. The reduction can be a reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any percentage in between these percentages. In some embodiments, off-target effects of integration are reduced as compared to off-target integration after introduction of naked DNA into a cell or introduction of DNA into a cell using a viral vector. The reduction can be a reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any percentage in between these percentages.

[0350] In some cases, the methods described herein provide for high cell viability of cells to which the RNP-DNA template has been introduced. In some cases, the viability of the cells to which the RNP-DNA template has been introduced is at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, 99.5%, 99%, or higher. In some cases, the viability of the cells to which the RNP-DNA template has IPTS / 200097280.2 OCAttorney Ref: ANB-223WO been introduced is from about 20% to about 99%, from about 30% to about 90%, from about 35% to about 85% or 90% or higher, from about 40% to about 85% or 90% or higher, from about 50% to about 85% or 90% or higher, from about 50% to about 85% or 90% or higher, from about 60% to about 85% or 90% or higher, or from about 70% to about 85% or 90% or higher.

[0351] In the methods provided herein, the molar ratio of RNP to nucleic acid (e.g., DNA template) can be from about 3:1 to about 100:1. For example, the molar ratio can be from about 3:1 to 10:1, from about 3:1 to about 15:1, 3:1 to about 20:1; 3:1 to about 25:1; from about 3:1 to 50:1, from about 3:1 to 75:1, from about 3:1 to 100:1; from about 5:1 to 10:1, from about 5:1 to about 15:1, 5:1 to about 20:1; 5:1 to about 25:1; from about 5:1 to 50:1, from about 5:1 to 75:1, from about 5:1 to 100:1; from about 8:1 to about 12:1; from about 8:1 to about 15:1, from about 8:1 to about 20:1, from about 8:1 to about 25:1, from about 8:1 to 50:1, from about 8:1 to 75:1, from about 8:1 to 100:1; from about 10:1 to about 15:1, 10:1 to about 20:1, 10:1 to about 25:1; from about 10:1 to 50:1, from about 10:1 to 75:1, or from about 10:1 to 100:1.

[0352] In some embodiments, the DNA template is at a concentration of about 2.5 pM to about 25 pM. For example, the concentration of DNA template can be about 2.5, 3, 3.5, 4,4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15,15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25 pM or any concentration in between these concentrations.

[0353] In some embodiments, the size or length of the nucleic acid (e.g., DNA template) is greater than about 4.5 kb, 5.0 kb, 5.1 kb, 5.2 kb, 5.3 kb, 5.4 kb, 5.5 kb, 5.6 kb, 5.7 kb, 5.8 kb,5.9 kb, 6.0 kb, 6.1 kb, 6.2 kb, 6.3 kb, 6.4 kb, 6.5 kb, 6.6 kb, 6.7 kb, 6.8 kb, 6.9 kb, 7.0 kb, 7.1 kb, 7.2 kb, 7.3 kb, 7.4 kb, 7.5 kb, 7.6 kb, 7.7 kb, 7.8 kb, 7.9 kb, 8.0 kb, 8.1 kb, 8.2 kb, 8.3 kb,8.4 kb, 8.5 kb, 8.6 kb, 8.7 kb, 8.8 kb, 8.9 kb, 9.0 kb, 9.1 kb, 9.2 kb, 9.3 kb, 9.4 kb, 9.5 kb, 9.6 kb, 9.7 kb, 9.8 kb, 9.9 kb, 10 kb, 11 kb, 12 kb, 13kb, 14 kb, 15kb, or 16kb or any size of nucleic acid (e.g., DNA template) in between these sizes. For example, the size of the DNA template can be about 4.5 kb to about 15 kb, about 4.5 kb to about 14 kb, about 4.5 kb to about 10 kb, about 5 kb to about 15 kb, about 5 kb to about 14 kb, about 5 kb to about 10 kb, about 5 kb to about 9 kb, about 5 kb to about 8 kb, about 5 kb to about 7 kb, about 5 kb to about 6 kb, about kb 6 to about 15 kb, about kb 6 to about 14 kb, about kb 6 to about 10 kb, about 6 kb to about 9 kb, about 6 kb to about 8 kb, about 6 kb to about 7 kb, about 7 kb to about 15 kb, about 7 kb to about 14 kb, about 7 kb to about 10 kb, about 7 kb to about 9 kb, about 7 kb to about 8 kb, about 8 kb to about 15 kb, about 8 kb to about 14 kb, about 8 kb to IPTS / 200097280.2 Of.Attorney Ref: ANB-223WO about 10 kb, about 8 kb to about 9 kb, about 9 kb to about 15 kb, about 9 kb to about 14 kb, about 9 kb to about 13 kb, about 9 kb to about 12 kb, about 9 kb to about 11 kb, about 9 kb to about 10 kb, about 10 kb to about 15 kb, about 10 kb to about 14 kb, about 10 kb to about 13 kb, about 10 kb to about 12 kb, or about 10 kb to about 11 kb.

[0354] In some embodiments, the amount of DNA template is about 1 pg to about 10 pg. For example, the amount of DNA template can be about 1 pg to about 2 pg, about 1 pg to about 3 pg, about 1 pg to about 4 pg, about 1 pg to about 5 pg, about 1 pg to about 6 pg, about 1 pg to about 7 pg, about 1 pg to about 8 pg, about 1 pg to about 9 pg, about 1 pg to about 10 pg. In some embodiments the amount of DNA template is about 2 pg to about 3 pg, about 2 pg to about 4 pg, about 2 pg to about 5 pg, about 2 pg to about 6 pg, about 2 pg to about 7 pg, about 2 pg to about 8 pg, about 2 pg to about 9 pg, or 2 pg to about 10 pg. In some embodiments the amount of DNA template is about 3 pg to about 4 pg, about 3 pg to about 5 pg, about 3 pg to about 6 pg, about 3 pg to about 7 pg, about 3 pg to about 8 pg, about 3 pg to about 9 pg, or about 3 pg to about 10 pg. In some embodiments, the amount of DNA template is about 4 pg to about 5 pg, about 4 pg to about 6 pg, about 4 pg to about 7 pg, about 4 pg to about 8 pg, about 4 pg to about 9 pg, or about 4 pg to about 10 pg. In some embodiments, the amount of DNA template is about 5 pg to about 6 pg, about 5 pg to about 7 pg, about 5 pg to about 8 pg, about 5 pg to about 9 pg, or about 5 pg to about 10 pg. In some embodiments, the amount of DNA template is about 6 pg to about 7 pg, about 6 pg to about 8 pg, about 6 pg to about 9 pg, or about 6 pg to about 10 pg. In some embodiments, the amount of DNA template is about 7 pg to about 8 pg, about 7 pg to about 9 pg, or about 7 pg to about 10 pg. In some embodiments, the amount of DNA template is about 8 pg to about 9 pg, or about 8 pg to about 10 pg. In some embodiments, the amount of DNA template is about 9 pg to about 10 pg.

[0355] In some cases, the size of the DNA template is large enough and in sufficient quantity to be lethal as naked DNA. In some embodiments, the DNA template encodes a heterologous protein or a fragment thereof. In some embodiments, the DNA template encodes at least one gene. In some embodiments, the DNA template encodes at least two genes. In some embodiments, the DNA template encodes one, two, three, four, five, six, seven, eight, nine, ten, or more genes.

[0356] In some embodiments, the DNA template includes regulatory sequences, for example, a promoter sequence and / or an enhancer sequence to regulate expression of the heterologous protein or fragment thereof after insertion into the genome of a cell.IPTS / 200097280.2 87Attorney Ref: ANB-223WO

[0357] In some cases, the DNA template is a linear DNA template. In some cases, the DNA template is a single- stranded DNA template. In some cases, the single- stranded DNA template is a pure single- stranded DNA template. As used herein, by “pure single-stranded DNA” is meant single- stranded DNA that substantially lacks the other or opposite strand of DNA. By “substantially lacks” is meant that the pure single- stranded DNA lacks at least 100- fold more of one strand than another strand of DNA.

[0358] In some cases, the RNP-DNA template complex is formed by incubating the RNP with the DNA template for less than about one minute to about thirty minutes, at a temperature of about 20° C to about 25° C. For example, the RNP can be incubated with the DNA template for about 5 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes or 30 minutes or any amount of time in between these times, at a temperature of about 20° C, 21° C, 22° C, 23° C, 24° C4or 25° C. In another example, the RNP can be incubated with the DNA template for less than about one minute to about one minute, for less than about one minute to about 5 minutes, for less than about 1 minute to about 10 minutes, for about 5 minutes to 10 minutes, for about 5 minutes to 15 minutes, for about 10 to about 15 minutes, for about 10 minutes to about 20 minutes, or for about 10 minutes to about 30 minutes, at a temperature of about 20° C to about 25° C. In some embodiments, the RNP-DNA template complex and the cell are mixed prior to introducing the RNP-DNA template complex into the cell.

[0359] In some embodiments introducing the RNP-DNA template complex comprises electroporation. Methods, compositions, and devices for electroporating cells to introduce a RNP-DNA template complex can include those described in the examples herein. Additional or alternative methods, compositions, and devices for electroporating cells to introduce a RNP-DNA template complex can include those described in WO / 2006 / 001614 or Kim, J.A. et al. Biosens. Bioelectron. 23, 1353-1360 (2008). Additional or alternative methods, compositions, and devices for electroporating cells to introduce a RNP-DNA template complex can include those described in U.S. Patent Appl. Pub. Nos. 2006 / 0094095; 2005 / 0064596; or 2006 / 0087522. Additional or alternative methods, compositions, and devices for electroporating cells to introduce a RNP-DNA template complex can include those described in Li, L.H. et al. Cancer Res. Treat. 1, 341-350 (2002); U.S. Patent Nos.: IPTS / 200097280.2 OOAttorney Ref: ANB-223WO6,773,669; 7,186,559; 7,771,984; 7,991,559; 6485961; 7029916; and U.S. Patent Appl. Pub. Nos: 2014 / 0017213; and 2012 / 0088842, all of which are hereby incorporated by reference. Additional or alternative methods, compositions, and devices for electroporating cells to introduce a RNP-DNA template complex can include those described in Geng, T. et al.. J. Control Release 144, 91-100 (2010); and Wang, J., et al. Lab. Chip 10, 2057-2061 (2010), all of which are hereby incorporated by reference.

[0360] In some embodiments, the Cas9 protein can be in an active endonuclease form, such that when bound to target nucleic acid as part of a complex with a guide RNA or part of a complex with a DNA template, a double strand break is introduced into the target nucleic acid. The double strand break can be repaired by NHEJ to introduce random mutations, or HDR to introduce specific mutations. Various Cas9 nucleases can be utilized in the methods described herein. For example, a Cas9 nuclease that requires an NGG protospacer adjacent motif (PAM) immediately 3’ of the region targeted by the guide RNA can be utilized. Such Cas9 nucleases can be targeted to any region of a genome that contains an NGG sequence. As another example, Cas9 proteins with orthogonal PAM motif requirements can be utilized to target sequences that do not have an adjacent NGG PAM sequence. Exemplary Cas9 proteins with orthogonal PAM sequence specificities include, but are not limited to, CFP1, those described in Nature Methods 10, 1116-1121 (2013), and those described in Zetsche et al., Cell, Volume 163, Issue 3, p759-771, 22 October 2015, both of which are hereby incorporated by reference.

[0361] In some cases, the Cas9 protein is a nickase, such that when bound to target nucleic acid as part of a complex with a guide RNA, a single strand break or nick is introduced into the target nucleic acid. A pair of Cas9 nickases, each bound to a structurally different guide RNA, can be targeted to two proximal sites of a target genomic region and thus introduce a pair of proximal single stranded breaks into the target genomic region. Nickase pairs can provide enhanced specificity because off-target effects are likely to result in single nicks, which are generally repaired without lesion by base-excision repair mechanisms. Exemplary Cas9 nickases include Cas9 nucleases having a D10A or H840A mutation.

[0362] In some embodiments, the RNP comprises a Cas9 nuclease. In some embodiments, the RNP comprises a Cas9 nickase. In some embodiments, the RNP-DNA template complex comprises at least two structurally different RNP complexes. In some embodiments, the at least two structurally different RNP complexes contain structurally different Cas9 nuclease domains In some embodiments, the at least two structurally different RNP complexes contain structurally different guide RNAs. In some embodiments, wherein the at least two IPTS / 200097280.2 OQAttorney Ref: ANB-223WO structurally different RNP complexes contain structurally different guide RNAs, each of the structurally different RNP complexes comprises a Cas9 nickase, and the structurally different guide RNAs hybridize to opposite strands of the target region.

[0363] In some cases, a plurality of RNP-DNA templates comprising structurally different ribonucleoprotein complexes is introduced into the cell. For example a Cas9 protein can be complexed with a plurality (e.g., 2, 3, 4, 5, or more, e.g., 2-10, 5-100, 20-100) of structurally different guide RNAs to target insertion of a DNA template at a plurality of structurally different target genomic regions.

[0364] In the methods and compositions provided herein, cells include, but are not limited to, eukaryotic cells, prokaryotic cells, animal cells, plant cells, fungal cells and the like. Optionally, the cell is a mammalian cell, for example, a human cell. The cell can be in vitro, ex vivo or in vivo. The cell can also be a primary cell, a germ cell, a stem cell or a precursor cell. The precursor cell can be, for example, a pluripotent stem cell, or a hematopoietic stem cell. In some embodiments, the cell is a primary hematopoietic cell or a primary hematopoietic stem cell. In some embodiments, the primary hematopoietic cell is an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a regulatory T cell, an effector T cell, or a naive T cell. In some embodiments, the T cell is a CD4+T cell. In some embodiments, the T cell is a CD8+T cell. In some embodiments, the T cell is a CD4+CD8+T cell. In some embodiments, the T cell is a CD4 CD8' T cell.Populations of any of the cells modified by any of the methods described herein are also provided. In some embodiments, the methods further comprise expanding the population of modified cells.

[0365] In some cases, the cells are removed from a subject, modified using any of the methods described herein and administered to the patient. In other cases, any of the constructs described herein is delivered to the patient in vivo. See, for example, U.S. Patent No. 9737604 and Zhang et al. “Lipid nanoparticle-mediated efficient delivery of CRISPR / Cas9 for tumor therapy,” NPGAsia Materials Volume 9, page e441 (2017), both of which are hereby incorporated by reference.

[0366] In some embodiments, the RNP- DNA template complex is introduced into about 1 x 105to about 2 x 106cells. For example, the RNP- DNA template complex can be introduced into about 1 x 105to about 5 x 105cells, about 1 x 105to about 1 x 106, 1 x 105to about 1.5 x 106, 1 x 105to about 2 x 106, about 1 x 106to about 1.5 x 106cells or about 1 x 106to about 2 x 106.IPTS / 200097280.2 90Attorney Ref: ANB-223WO

[0367] In some cases, the methods and compositions described herein can be used for generation, modification, use, or control of recombinant T cells, such as chimeric antigen receptor T cells (CAR T cells). Such CAR T cells can be used to treat or prevent cancer, an infectious disease, or autoimmune disease in a subject. For example, in some embodiments, one or more gene products are inserted or knocked-in to a T cell to express a heterologous protein (e.g., a chimeric antigen receptor (CAR) or a priming receptor).In vivo editins

[0368] In one aspect, provided herein are compositions and methods for in vivo immune cell editing, by contacting an immune cell in vivo with one or more encapsulated nucleic acid(s) encoding a SPA disclosed herein, and optionally a priming receptor and / or CAR. In such embodiments, once the immune cell (e.g., a T cell) has been contacted by a particle encapsulating the nucleic acid, the nucleic acid is delivered to the cytoplasm where it can be expressed to produce the gene product encoded therein (e.g., a protein or a gene modulating molecule such as an RNAi molecule). Any particle described herein can be used to contact the immune cell in vivo, e.g., a viral vector or viral particle, a bioscaffold, a lipid nanoparticle (LNP), a liposome, a polymeric nanoparticle, a biodegradable microparticle, a nanovesicle, a microvesicle, or microcapsule. Methods of in vivo immune cell engineering are disclosed in Li, YR et al. In vivo CAR engineering for immunotherapy. Nat Rev Immunol (2025). doi.org / 10.1038 / s41577-025-01174-l, hereby incorporated by reference in its entirety.Viral Particles

[0001] Nucleic acids of the disclosure can be delivered using a variety of delivery methods including viral particles. Exemplary viral particles include, but are not limited to, adeno- associated virus (AAV) and lentivirus vectors. Such viral particles can be pseudotyped.

[0002] In some aspects, provided herein are compositions comprising a viral particle encapsulating a nucleic acid encoding a synthetic pathway activator comprising a chimeric polypeptide comprising optionally, an extracellular domain; a lipid anchor or a transmembrane domain; an intracellular signaling domain; and a multimerization region.

[0003] The term “pseudotyped” or “pseudotyped viral particle”, as used herein, refers to a viral particle bearing capsid or membrane proteins (e.g., envelope glycoproteins or capsid proteins) derived from a virus that is different from the parental virus. The host range of the vector particles can thus be expanded or altered depending on the type of cell surface receptor used by the glycoprotein. For example, a HIV lentiviral vector can have the HIV envelopeIPTS / 200097280.2 91Attorney Ref: ANB-223WO glycoprotein be replaced with the VSV envelope glycoprotein, or an AAV particle can have the capsid protein replaced with the capsid of another AAV serotype.

[0004] A “lentivirus” as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.

[0005] In certain embodiments, the virus particle is a lentiviral particle. Lentiviral particles are derived from lentiviruses, which are retroviruses that, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function (see, e.g., U.S. Pat. Nos. 6,013,516 and 5,994,136). Some examples of lentiviruses include the Human Immunodeficiency Viruses (HIV-1, HIV-2) and the Simian Immunodeficiency Virus (SIV). Lentiviral particles have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe. Lentiviral particles are capable of infecting non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression, e.g., of a nucleic acid encoding a transgene.

[0006] Methods for producing and / or modifying lentiviral vectors are disclosed in US Patent Nos. 5,994,136, US 9,481,728; US 10,226,538; US 11,617,760, and US 12,163,119, each of which are hereby incorporated by reference.. Exemplary lentiviral vectors for use in making the lentiviral particles for use with the present disclosure include, but are not limited to, selfinactivating recombinant vectors that include at least a lentiviral gag, pol and rev genes, that is, those genes required for virus production, which permit their manufacture in reasonable quantities using available producer cell lines. In general, lentiviral genes and backbone (i.e., long terminal repeats or LTRs) used in preparing lentivectors in accordance with the present disclosure will be one that is human immunodeficiency virus (HIV) derived, and more particularly, HIV-1 derived. Thus, the gag, pol and rev genes will preferably be HIV genes and more preferably HIV-1 genes. However, the gag, pol and rev genes and LTR regions from other lentiviruses may be employed for certain applications in accordance with the present invention, including the genes and LTRs of HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus, bovine immunodeficiency virus, equine infectious anemia virus, caprine arthritis encephalitis virus and the like. Such constructs could be useful, for example, where one desires to modify certain cells of non-human origin.IPTS / 200097280.2 C)2Attorney Ref: ANB-223WO

[0007] An “adeno-associated virus” or “AAV” as used herein refers to a genus of the Parvoviridae family. Adeno-associated virus (AAV) is a dependent parvovirus twenty nanometers in size which requires co-infection with another virus (either adenovirus or certain members of the herpes virus group) to undergo a productive infection in cultured cells. In the absence of co-infection with helper virus, the AAV virion binds to a cellular receptor and enters the cell, migrating to the nucleus, and delivers a single- stranded DNA genome that can establish latency by integration into the host chromosome.

[0008] Methods for producing and / or modifying AAV particles such as pseudotyped AAV particles are disclosed in International Patent Publications W0200028004, W0200123001, W02004112727, W02005005610, W02005072364, and W02019028306, the contents of each of which are incorporated herein by reference in their entirety). In some embodiments, the AAV serotype may be, or have, a sequence as described in U.S. Patent Application Publication No. US20030138772, the contents of which are herein incorporated by reference in their entirety.Bio Scaffolds

[0009] Nucleic acids of the disclosure can be delivered using a variety of delivery methods including bioinstructive scaffolds. Exemplary bioinstructive scaffolds include, but are not limited to alginate scaffolds and collagen scaffolds. Such scaffolds can be seeded with the particles disclosed herein and provide a localized and controlled microenvironment that enhances interactions between the target immune cell and the particles disclosed herein.

[0010] In some aspects, provided herein are compositions comprising a bioinstructive scaffold comprising a lipid or viral particle encapsulating a nucleic acid encoding a synthetic pathway activator comprising a chimeric polypeptide comprising optionally, an extracellular domain; a lipid anchor or a transmembrane domain; an intracellular signaling domain; and a multimerization region.

[0011] Alginate and collagen bioscaffolds are described in US Patent No. 8,303,972, and Li, YR et al. In vivo CAR engineering for immunotherapy. Nat Rev Immunol (2025). doi.org / 10.1038 / s41577-025-01174-l, each of which are hereby incorporated by reference in thier entirety.Nanoparticles

[0012] Nucleic acids of the disclosure can be delivered using a variety of membranous molecular assembly delivery methods including lipid nanoparticles (LNPs), liposomes,IPTS / 200097280.2 93Attorney Ref: ANB-223WO polymeric, biodegradable microparticles, nanovesicles, or microcapsule delivery devices known in the art. In some embodiments, the one or more nucleic acids disclosed herein is encapsulated in a membranous nanoparticle.

[0013] For example, a colloidal dispersion system may be used for targeted delivery of a nucleic acid described herein. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in- water emulsions, micelles, mixed micelles, and liposomes.

[0014] In some aspects, provided herein are compositions comprising a membranous particle encapsulating a nucleic acid encoding a synthetic pathway activator comprising a chimeric polypeptide comprising optionally, an extracellular domain; a lipid anchor or a transmembrane domain; an intracellular signaling domain; and a multimerization region.

[0015] The term “lipid nanoparticle” (LNP) refers to particles which include one or more lipids that can self-assemble into structured particles. LNP membranes may be lamellar or non-lamellar and may be comprised of 1, 2, 3, 4, 5 or more layers. In some embodiments, LNPs may comprise a cargo or a payload into their interior space, into the inter membrane space, onto their exterior surface, or any combination thereof. Some examples of LNPs include but are not limited to liposomes, nonoemulsions, solid lipid nanoparticles, nanostructured lipid carriers, and lipid polymer hybrid nanoparticles. Examples lipids can be found in WO2021113777, hereby incorporated by reference.

[0016] Nucleic acids of the disclosure may be fully encapsulated in a lipid formulation, e.g., a lipid nanoparticle (LNP), or other nucleic acid-lipid particle. LNPs are useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). LNPs include "pSPLP," which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00 / 03683. The particles typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present disclosure are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Publication No. 2010 / 0324120 and PCT Publication No. WO 1996 / 40964, each of which is hereby incorporated by reference. Additional lipid compositions for nucleic acid delivery areIPTS / 200097280.2 94Attorney Ref: ANB-223WO disclosed in PCT Publication Nos. W02012 / 170930A1, WO2013 / 149141A1, and WO2014 / 152211A1, each of which is hereby incorporated by reference.

[0017] Liposomes can be neutral (cholesterol) or bipolar and include phospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyelin (SM) and other type of bipolar lipids including dioleoyl phosphatidylethanolamine (DOPE), with a hydrocarbon chain length in the range of 14-22, and saturated or with one or more double C=C bonds. Additional compositions that can be used to construct liposomes are disclosed in WO2019213308A1, hereby incorporated by reference in its entirety.

[0018] Lipid nanoparticles comprising nucleic acids and their method of preparation are disclosed in, e.g., US Patent No. 9,889,210; US Patent No. 9,856,477, U.S. Patent Publication Nos. 2004 / 0142025, 2007 / 0042031, PCT Pub. Nos. WO 2013 / 016058, WO 2013 / 086373, W02020101740, and Dahlman, et al., In vivo endothelial siRNA delivery using polymeric nanoparticles with low molecular weight, Nat Nanotechnol. 2014 Aug; 9(8): 648-655; the full disclosures of which are herein incorporated by reference in their entirety for all purposes.

[0019] Methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide / cationic lipid complex as a structural component of the delivery vehicle, are further described in, e.g., WO 1996 / 37194, the entire contents of which are incorporated herein by reference. Liposome formation can also include one or more aspects of exemplary compositions or methods described in Feigner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417; U.S. Pat. No. 4,897,355; U.S. Pat. No. 5,171,678; Bangham et al., (1965) M. Mol. Biol. 23:238; Olson et al., (1979) Biochim. Biophys. Acta 557:9; Szoka et al., (1978) Proc. Natl. Acad. Sci. 75: 4194; Mayhew et al., (1984) Biochim. Biophys. Acta 775:169; Kim et al., (1983) Biochim. Biophys. Acta 728:339; and Fukunaga et al., (1984) Endocrinol. 115:757. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer et al., (1986) Biochim. Biophys. Acta 858:161. Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew et al., (1984) Biochim. Biophys. Acta 775:169).

[0020] LNPs can be polymeric nanoparticles composition comprising a positively-charged carrier matrix comprising a positively charged lipid or polymer. In some embodiments, the positively-charged carrier matrix comprises a positively charged lipid or polymer. In some embodiments, the positively charged lipid or polymer comprises PBAE, poly(L-lysine), poly(ethylene imine) (PEI), poly-(amidoamine) dendrimers (PAMAMs), poly(amine-co- IPTS / 200097280.2 OAttorney Ref: ANB-223WO esters), poly(dimethylaminoethyl methacrylate) (PDMAEMA), chitosan, poly- (L-lactide-co- L-lysine), poly[a-(4-aminobutyl)-L-glycolic acid] (PAGA), or poly(4-hydroxy-L- proline ester) (PHP).

[0021] A positively charged synthetic cationic lipid, N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylamrnonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of oligonucleotide (see, e.g., Feigner, P. L. el al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417, and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with nucleic acids). A DOTMA analogue, l,2-bis(oleoyloxy)-3- (trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form nucleic acid-complexing vesicles. Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide ("DOGS") (TRANSFECTAM™, Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5- carboxyspermyl-amide ("DPPES") (see, e.g., U.S. Pat. No. 5,171,678). Other cationic lipids suitable for the delivery of nucleic acids are described in WO 1998 / 39359 and WO 1996 / 37194, each of which are hereby incorporated by reference in their entirety. Other formulations amenable to the present disclosure are described in PCT Publication Nos. WO 2009 / 088891, WO 2009 / 132131, and WO 2008 / 042973, which are hereby incorporated by reference in their entirety.

[0022] The ionizable / non-cationic lipid can be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1- carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearoyl-2-oleoyl- phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid can be, for example, from about 5 mol % to about 90 mol %, about 10 mol %, or about 60 mol % if cholesterol is included, of the total lipid present in the particle.IPTS / 200097280.2 QAttorney Ref: ANB-223WO

[0023] In some embodiments, the LNP is comprised of poly P-amino ester (PBAE) polymer (e.g, PBAE 447 and / or with l-(3- aminopropyl)pyrrolidine end caps). In some embodiments, the LNP further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 50 mol % of the total lipid present in the particle.Insertion sites

[0024] Methods for editing the genome of a T cell, specifically, include a method of editing the genome of a human T cell comprise inserting a nucleic acid sequence or construct into a target region in exon 1 of the TCR-a subunit (TRAC) gene in the human T cell. In some embodiments, the target region is in exon 1 of the constant domain of TRAC gene. In other embodiments, the target region is in exon 1, exon 2 or exon 3, prior to the start of the sequence encoding the TCR-a transmembrane domain. In some embodiments, the target region is the GS94 genomic safe harbor.

[0025] Methods for editing the genome of a T cell also include a method of editing the genome of a human T cell comprise inserting a nucleic acid sequence or construct into a target region in exon 1 of a TCR-P subunit (TRBC) gene in the human T cell. In some embodiments, the target region is in exon 1 of the TRBC1 or TRBC2 gene.

[0026] Methods for editing the genome of a T cell, specifically, include a method of editing the genome of a human T cell comprise inserting a nucleic acid sequence or construct into a target region of a genomic safe harbor (GSH).

[0027] Methods for editing the genome of a T cell also include a method of editing the genome of a human T cell comprise inserting a nucleic acid sequence or construct into a GS94 target region (locus chrl 1:128340000- 128350000).

[0028] In some embodiments, the target region is the GS94 locus or GS102 locus as provided in Table D.

[0001] Gene editing therapies include, for example, vector integration and site specific integration. Site-specific integration is a promising alternative to random integration of viral vectors, as it mitigates the risks of insertional mutagenesis or insertional oncogenesis (Kolb et al. Trends Biotechnol. 2005 23:399-406; Porteus et al. Nat Biotechnol. 2005 23:967-973; Paques et al. Curr Gen Ther. 2007 7:49-66). However, site specific integration continues to face challenges such as poor knock-in efficiency, risk of insertional oncogenesis, unstable and / or anomalous expression of adjacent genes or the transgene, low accessibility (e.g. within 20 kB of adjacent genes), etc.. These challenges can be addressed, in part, through the identification and use of safe harbor loci or safe harbor sites (SHS), which are sites in whichIPTS / 200097280.2 97Attorney Ref: ANB-223WO genes or genetic elements can be incorporated without disruption to expression or regulation of adjacent genes.

[0029] The most widely used of the putative human safe harbor sites is the AAVS1 site on chromosome 19q, which was initially identified as a site for recurrent adenoassociated virus insertion. Other potential SHS have been identified on the basis of homology, with sites first identified in other species (e.g., the human homolog of the permissive murine Rosa26 locus) or among the growing number of human genes that appear non-essential under some circumstances. One putative SHS of this type is the CCR5 chemokine receptor gene, which, when disrupted, confers resistance to human immunodeficiency virus infection. Additional potential genomic SHS have been identified in human and other cell types on the basis of viral integration site mapping or gene-trap analyses, as was the original murine Rosa26 locus. The three top SHS, AAVS1, CCR5, and Rosa26, are in close proximity to many protein coding genes and regulatory elements. (See Sadelain, M., et al. (2012). Safe harbours for the integration of new DNA in the human genome. Nature reviews Cancer, 12(1), 51-58, the relevant disclosures of which are herein incorporated by reference in their entirety).

[0030] The AAVS1 (also known as the PPP1R12C locus) on human chromosome 19 is a known SHS for hosting transgenes (e.g. DNA transgenes) with expected function. It is at position 19ql3.42. It has an open chromatin structure and is transcription-competent. The canonical SHS locus for AAVS1 is chrl9: 55,625,241-55,629,351. See Pellenz et al. “New Human Chromosomal Sites with "Safe Harbor" Potential for Targeted Transgene Insertion.” Human gene therapy vol. 30,7 (2019): 814-828, the relevant disclosures of which are herein incorporated by reference. An exemplary AAVS1 target gRNA and target sequence are provided below:• AAVS1 -gRNA sequence: ggggccactagggacaggatGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTA GTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT (SEQ ID NO: 294)• AAVS1 target sequence: ggggccactagggacaggat (SEQ ID NO: 295) CCR5, which is located on chromosome 3 at position 3p21.31, encodes the major co-receptor for HIV-1. Disruption at this site in the CCR5 gene has been beneficial in HIV / AIDS therapy and prompted the development of zinc-finger nucleases that target its third exon. The canonical SHS locus for CCR5 is chr3: 46,414,443-46,414,942. See Pellenz et al. “New Human Chromosomal Sites with "Safe Harbor" Potential forIPTS / 200097280.2 98Attorney Ref: ANB-223WOTargeted Transgene Insertion.” Human gene therapy vol. 30,7 (2019): 814-828, the relevant disclosures of which are herein incorporated by reference.

[0031] The mouse Rosa26 locus is particularly useful for genetic modification as it can be targeted with high efficiency and is expressed in most cell types tested. Irion et al. 2007 ("Identification and targeting of the ROSA26 locus in human embryonic stem cells." Nature biotechnology 25.12 (2007): 1477-1482, the relevant disclosure of which are herein incorporated by reference) identified the human homolog, human ROSA26, in chromosome 3 (position 3p25.3).The canonical SHS locus for human Rosa26 (hRosa26) is chr3: 9,415,082- 9,414,043. See Pellenz et al. “New Human Chromosomal Sites with "Safe Harbor" Potential for Targeted Transgene Insertion.” Human gene therapy vol. 30,7 (2019): 814-828, the relevant disclosures of which are herein incorporated by reference.

[0032] Additional examples of safe harbor sites are provided in Pellenz et al. “New Human Chromosomal Sites with "Safe Harbor" Potential for Targeted Transgene Insertion.” Human gene therapy vol. 30,7 (2019): 814-828, the relevant disclosures of which are herein incorporated by reference. Examples of additional integration sites are provided in Table D.

[0033] In some embodiments, the safe harbor sites allow for high transgene expression (sufficient to allow for transgene functionality or treatment of a disease of interest) and stable expression of the transgene over several days, weeks or months. In some embodiments, knockout of the gene at the safe harbor locus confers benefit to the function of the cell, or the gene at the safe harbor locus has no known function within the cell. In some embodiments the safe harbor locus results in stable transgene expression in vitro with or without CD3 / CD28 stimulation, negligible off-target cleavage as detected by iGuide-Seq or CRISPR-Seq, less off-target cleavage relative to other loci as detected by iGuide-Seq or CRISPR-Seq, negligible transgene-independent cytotoxicity, negligible transgene-independent cytokine expression, negligible transgene-independent chimeric antigen receptor expression, negligible deregulation or silencing of nearby genes, and positioned outside of a cancer-related gene.

[0034] As used, a “nearby gene” can refer to a gene that is within about lOOkB, about 125kB, about 150kB, about 175kB, about 200kB, about 225kB, about 250kB, about 275kB, about 300kB, about 325kB, about 350kB, about 375kB, about 400kB, about 425kB, about 450kB, about 475kB, about 500kB, about 525kB, about 550kB away from the safe harbor locus (integration site).

[0035] In some embodiments, the present disclosure contemplates inserts that comprise one or more transgenes. The transgene can encode a therapeutic protein, an antibody, a peptide, or any other gene of interest. The transgene integration can result in, for example, enhanced IPTS / 200097280.2 QQAttorney Ref: ANB-223WO therapeutic properties. These enhanced therapeutic properties, as used herein, refer to an enhanced therapeutic property of a cell when compared to a typical immune cell of the same normal cell type. For example, a T cell having “enhanced therapeutic properties” has an enhanced, improved, and / or increased treatment outcome when compared to a typical, unmodified and / or naturally occurring T cell. The therapeutic properties of immune cells can include, but are not limited to, cell transplantation, transport, homing, viability, self-renewal, persistence, immune response control and regulation, survival, and cytotoxicity. The therapeutic properties of immune cells are also manifested by: antigen-targeted receptor expression; HLA presentation or lack thereof; tolerance to the intratumoral microenvironment; induction of bystander immune cells and immune regulation; improved target specificity with reduction; resistance to treatments such as chemotherapy.

[0036] As used herein, the term “insert size” refers to the length of the nucleotide sequence being integrated (inserted) at the target locus or safe harbor site. In some embodiments, the insert size comprises at least about 4.5 kilobasepairs (kb) to about 10 kilobasepairs (kb). In some embodiments, the insert size comprises about 5000 nucleotides or more basepairs. In some embodiments, the insert size comprises up to 4.5, 4.8, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 kbp (kilo basepairs) or the sizes in between. In some embodiments, the insert size is greater than 4.5, 4.8, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 kbp or the sizes in between. In some embodiments, the insert size is within the range of 4.5-15 kbp or is any number in that range. In some embodiments, the insert size is within the range of4.8-8.3 kbp or is any number in that range. In some embodiments, the insert size is within the range of 5-8.3 kbp or is any number in that range. In some embodiments, the insert size is within the range of 5-15 kbp or is any number in that range. In some embodiments, the insert size is within the range of 4.5-20 kbp or is any number in that range. In some embodiments, the insert size is 5-10 kbp. In some embodiments, the insert size is 4.5-10, 5-10, 6-10, 7-10,8-10, 9-10 kbp. In some embodiments, the insert size is 4.5-11, 6-11, 7-11, 8-11, 9-11, or 10-11 kbp. In some embodiments, the insert size is 4.5-12, 6-12, 7-12, 8-12, 9-12, 10-12, or 11-12 kbp. In some embodiments, the insert size is 4.5-13, 6-13, 7-13, 8-13, 9-13, 10-13, 11-13, or 12-13 kbp. In some embodiments, the insert size is 4.5-14, 6-14, 7-14, 8-14, 9-14, 10-14, 11-14, 12-14 or 13-14 kbp. In some embodiments, the insert size is 4.5-15, 6-15, 7-15, 8-15,9-15, 10-15, 11-15, 12-15, 13-15, or 14-15 kbp. In some embodiments, the insert size is 4.5-16, 6-16, 7-16, 8-16, 9-16, 10-16, 11-16, 12-16, 13-16, 14-16 or 15-16 kbp. In some embodiments, the insert size is 4.5-17, 6-17, 7-17, 8-17, 9-17, 10-17, 11-17, 12-17, 13-17, or 14-17, 15-17 or 16-17 kbp. In some embodiments, the insert size is 4.5-18, 6-18, 7-18, 8-18,IPTS / 200097280.2 | QQAttorney Ref: ANB-223WO9-18, 10-18, 11-18, 12-18, 13-18, 14-18, 15-18, 16-18 or 17-18 kbp. In some embodiments, the insert size is 4.5-19, 6-19, 7-19, 8-19, 9-19, 10-19, 11-19, 12-19, 13-19, 14-19, 15-19, 16-19, 17-19, or 18-19 kbp. In some embodiments, the insert size is 4.5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 11-20, 12-20, 13-20, 14-20, 15-20, 16-20, 17-20, 18-20, or 19-20 kbp.

[0037] The inserts of the present disclosure refer to nucleic acid molecules or polynucleotide inserted at a target locus or safe harbor site. In some embodiments, the nucleotide sequence is a DNA molecule, e.g., genomic DNA, or comprises deoxy -ribonucleotides. In some embodiments, the insert comprises a smaller fragment of DNA, such as a plastid DNA, mitochondrial DNA, or DNA isolated in the form of a plasmid, a fosmid, a cosmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), and / or any other sub-genome segment of DNA. In some embodiments, the insert is an RNA molecule or comprises ribonucleotides. The nucleotides in the insert are contemplated as naturally occuring nucleotides, non-naturally occuring, and modified nucleotides. Nucleotides may be modified chemically or biochemically, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications. The polynucleotides can be in any topological conformation, including single- stranded, double-stranded, partially duplexed, triplexed, hairpinned, circular conformations, and other three-dimension conformations contemplated in the art.

[0038] The inserts can have coding and / or non-coding regions. The insert can comprises a non-coding sequence (e.g., control elements, e.g., a promoter sequence). In some embodiments, the insert encodes transcription factors. In some embodiments, the insert encodes an antigen binding receptors such as single receptors, T-cell receptors (TCRs), priming receptors, CARs, mAbs, etc. In some embodiments, the the insert is a human sequence. In some embodiments, the insert is chimeric. In some embodiments, the insert is a multi-gene / multi-module therapeutic cassette. A multi-gene / multi-module therapeutic cassette refers to an insert or cassette having one or more than one receptor (e.g., synthetic receptors such as a CAR or a priming receptor), other exogenous protein coding sequences, non-coding RNAs, transcriptional regulatory elements, and / or insulator sequences, etc.

[0039] In some embodiments, the nucleic acid sequence is inserted into the genome of the cell such as an immune cell or T cell via non- viral delivery. In non-viral delivery methods, the nucleic acid can be naked DNA, or in a non-viral plasmid or vector. Non-viral delivery techniques can be site-specific integration techniques, as described herein or known to those IPTS / 200097280.2 1 A1Attorney Ref: ANB-223WO of ordinary skill in the art. Examples of site-specific techniques for integration into the safe harbor loci include, without limitation, homology-dependent engineering using nucleases and homology independent targeted insertion using Cas9 or other CRISPR endonucleases.

[0040] In some embodiments, the insert is integrated at a safe harbor site by introducing into the engineered cell, (a) a targeted nuclease that cleaves a target region in the safe harbor site to create the insertion site; and (b) the nucleic acid sequence (insert), wherein the insert is incorporated at the insertion site by, e.g., HDR. Examples of non-viral delivery techniques that can be used in the methods of the present disclosure are provided in US Patent Nos.US11033584B2 and US11814624B2, the relevant disclosures of which are herein incorporated by reference in their entirety.

[0041] Examples of integration sites contemplated are provided in Table D.Table D: sgRNA sequencesIPTS / 200097280.2 102Attorney Ref: ANB-223WOIPTS / 200097280.2 103Attorney Ref: ANB-223WOIPTS / 200097280.2 104Attorney Ref: ANB-223WOCRISPR-Cas Editins

[0042] One effective example of gene editing is the CRISPR-Cas approach (e.g. CRISPR- Cas9). This approach incorporates the use of a guide polynucleotide (e.g. guide ribonucleic acid or gRNA) and a cas endonuclease (e.g. Cas9 endonuclease).

[0043] As used herein, a polypeptide referred to as a “Cas endonuclease” or having “Cas endonuclease activity” refers to a CRISPR-related (Cas) polypeptide encoded by a Cas gene, wherein a Cas polypeptide is a target DNA sequence that can be cleaved when operably linked to one or more guide polynucleotides (see, e.g., US Pat. No. 8,697,359). Also included in this definition are variants of Cas endonuclease that retain guide polynucleotide-dependent endonuclease activity. The Cas endonuclease used in the donor DNA insertion method detailed herein is an endonuclease that introduces double- strand breaks into DNA at the target site (e.g., within the target locus or at the safe harbor site).

[0044] As used herein, the term “guide polynucleotide” relates to a polynucleotide sequence capable of complexing with a Cas endonuclease and allowing the Cas endonuclease to recognize and cleave a DNA target site. The guide polynucleotide can be a single molecule or a double molecule. The guide polynucleotide sequence can be an RNA sequence, a DNA sequence, or a combination thereof (RNA-DNA combination sequence). A guide polynucleotide comprising only ribonucleic acid is also referred to as “guide RNA”. In some embodiments, a polynucleotide donor construct is inserted at a safe harbor locus using a guide RNA (gRNA) in combination with a cas endonuclease (e.g. Cas9 endonuclease).

[0045] The guide polynucleotide includes a first nucleotide sequence domain (also referred to as a variable targeting domain or VT domain) that is complementary to a nucleotide sequence in the target DNA, and a second nucleotide that interacts with a Cas endonucleaseIPTS / 200097280.2 105Attorney Ref: ANB-223WO polypeptide. It can be a double molecule (also referred to as a double- stranded guide polynucleotide) comprising a sequence domain (referred to as a Cas endonuclease recognition domain or CER domain). The CER domain of this double molecule guide polynucleotide comprises two separate molecules that hybridize along the complementary region. The two separate molecules can be RNA sequences, DNA sequences and / or RNA- DNA combination sequences.

[0046] Genome editing using CRISPR-Cas approaches relies on the repair of site- specific DNA double-strand breaks (DSBs) induced by the RNA-guided Cas endonuclease (e.g. Cas 9 endonuclease). Homology-directed repair (HDR) of these DSBs enables precise editing of the genome by introducing defined genomic changes, including base substitutions, sequence insertions, and deletions. Conventional HDR-based CRISPR / Cas9 genome-editing involves transfecting cells with Cas9, gRNA and donor DNA containing homologous arms matching the genomic locus of interest.

[0047] HITI (homology independent targeted insertion) uses a non-homologous end joining (NHEJ)-based homology-independent strategy and the method can be more efficient than HDR. Guide RNAs (gRNAs) target the insertion site. For HITI, donor plasmids lack homology arms and DSB repair does not occur through the HDR pathway. The donor polynucleotide construct can be engineered to include Cas9 cleavage site(s) flanking the gene or sequence to be inserted. This results in Cas9 cleavage at both the donor plasmid and the genomic target sequence. Both target and donor have blunt ends and the linearized donor DNA plasmid is used by the NHEJ pathway resulting integration into the genomic DSB site. (See, for example, Suzuki, K., et al. (2016). In vivo genome editing via CRISPR / Cas9 mediated homology-independent targeted integration. Nature, 540(7631), 144-149, the relevant disclosures of which are herein incorporated in their entirety).

[0048] Methods for conducing gene editing using CRISPR-Cas approaches are known to those of ordinary skill in the art. (See, for example, US Application Nos. US 16 / 312,676, US 15 / 303,722, and US 15 / 628,533, the disclosures of which are herein incorporated by reference in their entirety). Additionally, uses of endonucleases for inserting transgenes into safe harbor loci are described, for example, in US Application No. 13 / 036,343, the disclosures of which are herein incorporated by reference in their entirety.

[0049] The guide RNAs and / or mRNA (or DNA) encoding an endonuclease can be chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Non-limiting examples of such moieties include lipid moieties such as a cholesterol moiety, cholic acid, a thioether, a IPTS / 200097280.2 1 n .Attorney Ref: ANB-223WO thiocholesterol, an aliphatic chain (e.g., dodecandiol or undecyl residues), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1 ,2-di-O-hexadecyl- rac-glycero-3-H- phosphonate, a polyamine or a polyethylene glycol chain, adamantane acetic acid, a palmityl moiety and an octadecylamine or hexylamino-carbonyl-t oxy cholesterol moiety. See for example US Patent Publication No. 20180127786, the disclosure of which is herein incorporated by reference in its entirety.Therapeutic Applications

[0050] For therapeutic applications, the engineered cells, populations thereof, or compositions thereof are administered to a subject, generally a mammal, generally a human, in an effective amount. The engineered cells may be administered to a subject by infusion (e.g., continuous infusion over a period of time) or other modes of administration known to those of ordinary skill in the art.

[0051] The engineered cells provided herein not only find use in gene therapy but also in non-pharmaceutical uses such as, e.g., production of animal models and production of recombinant cell lines expressing a protein of interest.

[0052] The engineered cells of the present disclosure can be any cell, generally a mammalian cell, generally a human cell that has been modified by integrating a transgene at a safe harbor locus described herein. Exemplary cells are provided in the Recombinant Cells section.

[0053] The engineered cells, compositions and methods of the present disclosure are useful for therapeutic applications such as CAR T cell therapy and TCR T cell therapy. In some embodiments, the insertion of a sequence encoding a transgene within a safe harbor locus maintains the TCR expression relative to instances when there is no insertion and enables transgene expression while maintaining TCR function.

[0054] In some embodiments, the present disclosure provides methods of treating a subject in need of treatment by administering to the subject a composition comprising any of the engineered cells described herein. In some embodiments, administration of the engineered cell composition results in a desired pharmacological and / or physiological effect. That effect can be partial or complete cure of the disease and / or adverse effects resulting from the disease. In some embodiments, treatment encompasses any treatment of a disease in a subject (e.g., mammal, e.g., human). Further, treatment may stabilize or reduce undesirable clinical symptoms in subjects (e.g., patients). The cells provided herein populations thereof, or compositions thereof may be administered during or after the occurrence of the disease.IPTS / 200097280.2 107Attorney Ref: ANB-223WO

[0055] In certain embodiments, the subject has a disease, condition, and / or injury that can be treated and / or ameliorated by cell therapy. In some embodiments, the subject in need of cell therapy is a subject having an injury, disease, or condition, thereby causing cell therapy (e.g., therapy in which cellular material is administered to the subject). However, it is contemplated that it is possible to treat, ameliorate and / or reduce the severity of at least one symptom associated with the injury, disease or condition.Method of Administration

[0056] An effective amount of the immune cell comprising the SPA peptide comprises may be administered for the treatment of cancer. The appropriate dosage of the immune cell comprising the SPA peptide may be determined based on the type of cancer to be treated, the type of the immune cell comprising the SPA peptide, the severity and course of the cancer, the clinical condition of the individual, the individual’s clinical history and response to the treatment, and the discretion of the attending physician.Determining Expression of CD11c

[0057] Also provided herein are methods of treating a cancer in a subject in need thereof comprising: determining or having determined the expression of CD11c in a cell comprising a synthetic pathway activator (SPA) peptide disclosed herein, optionally wherein the SPA is inserted into a target region of the genome of the cell; and administering or having administered to the subject the cell comprising the SPA.

[0058] CD11c is also known as Integrin Subunit Alpha X, Integrin, Alpha X (Complement Component 3 Receptor 4 Subunit), or ITGAX (HGNC: 6152, NCBI Gene: 3687, UniProtKB / Swiss-Prot: P20702).

[0059] In some aspects, provided herein are methods of determining an expression level of CD11c protein in a sample from a subject comprising contacting the sample with an antiCD 11c antibody and performing a FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, monoplex immunohistochemistry, multiplex immunohistochemistry, immunofluorescence, radioimmunoassay, dot blotting, immunodetection methods, surface plasmon resonance, optical spectroscopy, mass spectrometry assay, or any combination thereof. In some aspects, provided herein are methods of determining an expression level of CD11c mRNA in a sample from a subject comprising performing qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY technique, Luminex, MSD, or FISH, and combinations thereof.IPTS / 200097280.2 108Attorney Ref: ANB-223WO

[0060] In some aspects, provided herein are methods of producing a CD1 Ic-positive(CD1 lc+) cell comprising inserting a nucleic acid encoding for any SPA peptide disclosed herein and expressing the SPA peptide in the cell. In other aspects, provided herein are methods of detecting a SPA, optionally a functional SPA, in a cell comprising a SPA peptide disclosed herein, optionally wherein the SPA is inserted into the genome of the cell.

[0061] In some aspects, provided herein are methods of screening a cell for expression of a SPA, optionally wherein the SPA is a functional SPA, comprising expressing one or more SPAs in a cell and detecting CD11c expression in the cell, wherein the detection of CD11c indicates a functional SPA peptide. In such embodiments, a functional SPA is one that has functional signaling (e.g., can stimulate the cell, such as a T cell, via phosphorylation of STAT1, STAT3, and / or STAT5).

[0062] In some aspects, provided herein are assays to detect cells, such as primary cells and / or immune cells, engineered to express a SPA comprising: determining or having determined CD11c expression in the primary cell, wherein CD11c expression indicates that the cell expresses the SPA.

[0063] In some aspects, provided herein are methods of treating a patient with an engineered CDl lc-expressing T cell comprising: administering a T cell comprising a SPA disclosed herein to the patient.

[0064] In some embodiments, the CD11c expression is determined in the T cell, from a biological sample from a patient administered the cell expressing the SPA peptide, or in the tumor of a patient administered the cell expressing the SPA peptide. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a primary immune cell. In some embodiments, the immune cell is a hematopoietic cell, an adaptive immune cell, an innate immune cell, a natural killer (NK) cell, a T cell, a CD8+ cell, a CD4+ cell, or a T cell progenitor cell. In some embodiments, the immune cells are T cells. In some embodiments, the T cells are regulatory T cells, effector T cells, or naive T cells. In some embodiments, the T cells are CD8+T cells. In some embodiments, the T cells are CD4+T cells. In some embodiments, the T cells are CD4+CD8+T cells.

[0065] In some embodiments, the expression level of CD11c comprises the mRNA expression level of CD11c. In some embodiments, the expression level of CD11c comprises the protein expression level of CD11c. In some embodiments the expression level of CD11c is detected in the sample using a method selected from the group consisting of FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, monoplex immunohistochemistry, multiplex immunohistochemistry, immunofluorescence, IPTS / 200097280.2 109Attorney Ref: ANB-223WO radioimmunoassay, dot blotting, immunodetection methods, HPLC, surface plasmon resonance, optical spectroscopy, mass spectrometry, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY technique, Luminex, MSD, and FISH, and combinations thereof.Pharmaceutical compositions

[0066] The engineered recombinant cells provided herein can be administered as part of a pharmaceutical compositions. These compositions can comprise, in addition to one or more of the recombinant cells, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material can depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes. The pharmaceutical composition may comprise one or more pharmaceutical excipients. Any suitable pharmaceutical excipient may be used, and one of ordinary skill in the art is capable of selecting suitable pharmaceutical excipients. Accordingly, the pharmaceutical excipients provided below are intended to be illustrative, and not limiting. Additional pharmaceutical excipients include, for example, those described in the Handbook of Pharmaceutical Excipients, Rowe el al. (Eds.) 6th Ed. (2009), incorporated by reference in its entirety.

[0067] Various modes of administering the additional therapeutic agents are contemplated herein. In some embodiments, the additional therapeutic agent is administered by any suitable mode of administration.

[0068] A composition can be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.Kits and Articles of Manufacture

[0069] The present application provides kits comprising any one or more of the SPA peptides or cell compositions described herein along with instructions for use. The instructions for use can be present in the kits as a package insert, in the labeling of the container of the kit or components thereof, or can be in digital form (e.g. on a CD-ROM, via a link on the internet). A kit can include one or more of a genome-targeting nucleic acid, a polynucleotide encoding a genome-targeting nucleic acid, a site-directed polypeptide, and / or a polynucleotide encoding a site-directed polypeptide. Additional components within the kits are also contemplated, for example, buffer (such as reconstituting buffer, stabilizing buffer, diluting buffer), and / or one or more control vectors.IPTS / 200097280.2 110Attorney Ref: ANB-223WO

[0070] In some embodiments, the kits further contain a component selected from any of secondary antibodies, reagents for immunohistochemistry analysis, pharmaceutically acceptable excipient and instruction manual and any combination thereof. In one specific embodiment, the kit comprises a pharmaceutical composition comprising any one or more of the antibody compositions described herein, with one or more pharmaceutically acceptable excipients.

[0071] The present application also provides articles of manufacture comprising any one of the antibody compositions or kits described herein. Examples of an article of manufacture include vials (including sealed vials).EXAMPLES

[0072] Below are examples of specific embodiments for carrying out the present disclosure. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present disclosure in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0073] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989);Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rdEd. (Plenum Press) Vols A and B( 1992).Example 1: Synthesis and in vitro Characterization of Synthetic Pathway Activators

[0074] Synthetic pathway activators comprising intracellular domains that induce constitutive STAT signaling were designed and characterized. Without wishing to be bound by theory, the use of intracellular domains that induce STAT signaling can increase the anti-tumor potency, antigen sensitivity, and exhaustion resistance of immune cells such as T cells.

[0075] Materials and Methods

[0076] The SPA architecture is composed of up to four modular components: an extracellular scaffold domain, co-stim domain, JAK juxtamembrane signaling domain, and tiled STATIPTS / 200097280.2 111Attorney Ref: ANB-223WO intracellular signaling domain. The tiled STAT signaling domains were adapted from cytokine receptor intracellular domains- either full length or a truncated portion. The combination of domains derived from distinct cytokine receptors results in transduction of distinct signals through the phosphorylation of different STAT proteins. The STAT tile designs were adapted from the endogenous sequence of GP130, IL21R, GCSFR, IL9R, IL12RP, IL2Ra, IL7Ra, IL15R, and IL18R. Tables 1-12 provide the structure of the SPAs constructed and tested.

[0077] Programmable Circuit (PC) T cells (PCT cells) (formerly called Integrated Circuit (IC) T cells (PCT cells)) were electroporated with a gene cassette that integrated into a genomic safe harbor locus (GS94) through homology-directed repair (HDR). The transgene encoded a programmable circuit (PC) expressing a constitutive cistron driven by the EFla promoter. The SPA-containing cistron encoded an exemplary priming receptor (PrimeR) followed by a 2A cleavage site, followed by the SPA gene sequence, and a PrimeR-inducible promoter that induced expression of an exemplary chimeric antigen receptor (CAR). T cells were activated for 48 hours. Ribonucleoprotein complexes (RNPs) containing Cas9 and guide RNAs targeting the genomic safe harbor locus and plasmid DNA encoding the PC were mixed with activated T cells and electroporated using a Lonza EP system. Cells were recovered with IL-7 and IL- 15 for 7 days, after which time co-culture experiments were initiated.

[0078] Target Cell Survival Assay (Potency)

[0079] On day 7 post electroporation, PCT cells (engineered T cells expressing a SPA) were cocultured at a 1:50 E:T with RPMI cells expressing high levels of prime and cytolytic antigens. Every 2-3 days a media change was performed by aspirating half of coculture and replenishing with RPMI + Glutamax + 10% PBS + 15ng / mL gentamicin. On day 6, a rechallenge was performed by adding 25,000 RPMI cells expressing high levels of prime and cytolytic antigens to coculture. On day 8 and 12, a split and rechallenge was performed by transferring !4 of the coculture into a new plate containing 25,000 RPMI cells expressing high levels of prime and cytolytic antigens. Tumor control was measured through GPP counts on the Incucyte over 372 hours.

[0080] pSTAT profiling

[0081] On Day 7 post activation (5 days post electroporation), PCT cells were starved overnight in media free of cytokine. The next day, T cells were fixed and permeabilized for intracellular pSTAT staining. PCT cells were incubated with pSTAT antibodies for 30 mins before flow cytometry readout via Attune (FIG. 3).IPTS / 200097280.2 1 1Attorney Ref: ANB-223WO

[0082] pSTAT5 profiling

[0083] On day 5 post gene insertion via electroporation, PCT cells were starved overnight in media free of any cytokine in addition. The next day, T cells were fixed and permeabilized for intracellular pSTAT staining. PCT cells were incubated with pSTAT antibodies for 30 mins before flow cytometry readout via Attune. PCT survival was measured by seeding 50,000 T cells in a U-Bottom plate with TexMACS + 3% human serum + 15ng / mL gentamicin and placed in the incubator. After 9 days, PCT cells were measured and counted by flow cytometry. Fold change was measured by dividing PCT count obtained on Day 9 by initial Day 0 PCT count. Simple linear regression was performed to evaluate pSTAT correlation with T cell survival (FIG. 4A-E).

[0084] T cell survival assay

[0085] Long term PCT survival was measured by seeding 50,000 T cells on day 5 post gene insertion via electroporation in a U-Bottom plate with TexMACS + 3% human serum + 15ng / mL gentamicin without cytokine supplementation and placed in the incubator to be read out weekly. T cells expressing the 30 candidate SPAs were used. Every 2-3 days, a media change was performed by centrifuging the U-bottom plate containing PCT cells at 400 x g for 5 minutes and resuspending. Every week (Day 7, Day 14, Day 21, Day 28, and Day 33) PCT cells were measured and counted by flow cytometry. Fold change was measured by dividing PCT count obtained on Day 7, 14, 21, 28, and 33 by initial Day 0 PCT count (FIG. 5A-B).

[0086] Extended survival assay

[0087] Long term PCT survival was measured by seeding 50,000 T cells on day 5 post gene insertion via electroporation in a U-Bottom plate with TexMACS + 3% human serum + 15ng / mL gentamicin without cytokine supplementation and placed in the incubator to be read out weekly. T cells expressing the 30 candidate SPAs were used. Every 2-3 days, a media change was performed by centrifuging the U-bottom plate containing PCT cells at 400 x g for 5 minutes and resuspending. At Day 14, PCT cells were measured and counted by flow cytometry. Fold change was measured by dividing PCT cell count obtained on Day 14 by initial Day 0 PCT cell count.

[0088] On day 7 post gene insertion, PCT cells were cocultured at a 1:50 E:T with RPMI cells expressing high levels of prime and cytolytic antigens. Every 2-3 days a media change was performed by aspirating half of coculture and replenishing with RPMI + Glutamax + 10% FBS + 15ng / mL gentamicin. On day 6 a rechallenge was performed by adding 25,000 RPMI cells expressing high levels of prime and cytolytic antigens to coculture. On day 8 and 12, a split and rechallenge was performed by transferring !4 of the coculture into a new plate IPTS / 200097280.2 1 1Attorney Ref: ANB-223WO containing 25,000 RPMI cells expressing high levels of prime and cytolytic antigens. Tumor control was measured through GFP counts on the Incucyte. Cell survival CSA index was calculated by integration of both the area under curve of tumor growth and terminal stage tumor cell count. Higher CSA index values reflect higher cumulative tumor control. Simple linear regression was performed to compare potency (measured by CSA index) with cytokine free survival. (FIG 5A-B).

[0089] Results

[0090] As shown in FIG. 2, multiple newly constructed SPA peptides outperformed the SPA comprising SEQ ID NO: 259 or in a cell survival assy.

[0091] The left panel of FIG. 3 shows the pSTATl, pSTAT3, pSTAT4, pSTAT5, pSTAT6, and pERK expressions profile of the entire SPA library. Raw MFI data was re-normalized to Z-score system on the scale from -5 to 5. The right panel of FIG. 2 shows the 30 top candidates with highest potency were highlighted with their pSTAT expression profile from the entire library in a ranked manner from top to bottom. Candidates were further categorized based on their STAT tile designs. The candidates comprised SPAs with the sequences set forth in SEQ ID NOs: 79, 80, 81, 87, 103, 104, 111, 116, 117, 143, 144, 145, 152, 153, 155, 156, 157, 159, 160, 182, 183, 184, 185, 220, 234, 264, 268, 269, 270, and 282.

[0092] As shown in FIG. 4A-4E, pSTAT5 showed the most correlation with day-9 cytokine independent outgrowth out of all the pSTAT pathways assessed. FIG. 4A shows pSTAT5 z- score correlated to T cell survival, FIG. 4B shows pSTATl z-score correlated to T cell survival, FIG. 4C shows pSTAT3 z-score correlated to T cell survival, FIG. 4D shows pSTAT4 z-score correlated to T cell survival, FIG. 4E shows pSTAT6 z-score correlated to T cell survival.

[0093] As shown in FIG. 5A and 5B, many of the top 30 SPA expressing PCT cells contracted after 14 days of cytokine withdrawal. SPA expressing PCT cells with the best survival in T cells from two donors are indicated in FIG. 5A and 5B (SEQ ID NOs: 184, 183, 159, 264, and 144). Receptor tethered IL2rb-IL15 was used as a control.

[0094] As shown in FIG. 6A and 6B, 22 of the 30 SPAs tested demonstrated increased potency with limited cytokine-free survival (e.g., high potency, but limited proliferation of the T cells). In particular, SPAs of SEQ ID NOs: 182, 152, 185, 153, 117 and 269 had high potency and low cytokine free survival.IPTS / 200097280.2 114Attorney Ref: ANB-223WOExample 2: Additional in vitro characterization of Synthetic Pathway Activators

[0095] As described in Example 1, the novel SPAs described herein incorporate additional JAK / STAT signaling motifs that provide further signaling pathway activation beyond the STAT1 signaling pathway. These additional signaling motifs provide further enhanced antitumor killing over certain existing SPAs (e.g., a SPA of SEQ ID NO: 259 provided in W02024192100), as well as improved anti-tumor function without lymphodepletion, such as cytotoxicity, persistence and tumor-infiltration.

[0096] Materials and Methods

[0097] Cells expressing selected SPAs as shown in SEQ ID NOs: 185, 184, 182, 117, 152, 104, 79, 114, 156, or 268 were further characterized for STAT activation (pSTAT profiling), long term immune cell potency, cytokine secretion, cell persistence in the absence of cytokine media, and immune cell expansion. The SPA of SEQ ID NO: 185 is also provided in SEQ ID NO: 421 without the CD34 leader peptide. The SPA of SEQ ID NO: 184 is also provided in SEQ ID NO: 420 without the CD34 leader peptide. The SPA of SEQ ID NO: 182 is also provided in SEQ ID NO: 419 without the CD34 leader peptide. The SPA of SEQ ID NO: 152 is also provided in SEQ ID NO: 422 without the CD34 leader peptide.

[0098] Integrated Circuit (IC) T cells expressing the selected SPA as well as an exemplary priming receptor and CAR that bound to exemplary priming and cytolytic antigens were engineered as described in Example 1. PCT cells expressing a constitutively expressed CAR that bound to the cytolytic target were used as a positive control, PCT cells expressing a SPA of SEQ ID NO: 259 were used as a baseline comparison, PCT cells expressing NGFR instead of a SPA or edited using RNP only were used as a negative control and for data normalization.

[0099] pSTAT profiling

[0100] pSTAT profiling for pSTATl, pSTA3, pSTAT4, and pSTAT5 was performed as described in Example 1. Briefly, PCTs were thawed and cultured in 12.5 ng / mL of IL7 and IL 15 for 24 hours. After 24 hours, PCT cells were starved overnight in media free of cytokine. The following day, T cells were fixed and permeabilized for intracellular pSTAT staining. PCTs were incubated with pSTAT antibodies for 30 mins before flow cytometry readout via Attune. pSTAT fold change was measured relative to NGFR PCTs.

[0101] Target Cell Survival Assay (Potency)

[0102] On day 7 post electroporation, PCT cells (engineered T cells expressing a SPA and an exemplary priming receptor and CAR) were cocultured at a 1:50 E:T with RPMI cells expressing high levels of prime and cytolytic antigens. Every 2-3 days a media change was IPTS / 200097280.2 1 1 <Attorney Ref: ANB-223WO performed by aspirating half of coculture and replenishing with RPMI + Glutamax + 10% FBS + 15ng / mL gentamicin. On day 6, a rechallenge was performed by adding 25,000 RPMI cells expressing high levels of prime and cytolytic antigens to coculture. On day 8 and 12, a split and rechallenge was performed by transferring !4 of the coculture into a new plate containing 25,000 RPMI cells expressing high levels of prime and cytolytic antigens. Tumor control was measured through GFP counts on the Incucyte.

[0103] Cytokine Secretion

[0104] To further assess the specificity and function of PCT cells expressing the indicated SPA and an exemplary priming receptor and CAR, supernatants were collected from PCTs co-cultured with 786-0 cells expressing both priming and cytolytic antigens (Effector: Target ratio of 1:1, 48 hour co-culture). Following incubation, supernatants were collected at endpoint and cytokine release levels were measured using a Luminex assay. Data from 3 donors are shown.

[0105] T cell survival

[0106] Long term PCT cell survival was measured by seeding 50,000 T cells on day 5 post gene insertion via electroporation in a U-Bottom plate with TexMACS + 3% human serum + 15ng / mL gentamicin without cytokine supplementation and placed in the incubator to be read out weekly. T cells expressing the selected SPAs were used. Every 2-3 days, a media change was performed by centrifuging the U-bottom plate containing PCT cells at 400 x g for 5 minutes and resuspending. Every week (Day 7, Day 14, Day 21, and Day 28) PCT cells were measured and counted by flow cytometry. Fold change was measured by dividing PCT count obtained on Day 7, 14, 21, 28, and 33 by initial Day 0 PCT count.

[0107] T cell persistence in cytokine-free media

[0108] Long term PCT cell persistence was measured by seeding 50,000 frozen PCT cells the selected SPAs in a U-Bottom plate with TexMACS + 3% human serum + 15ng / mL gentamicin on Day -1. On day 0, cells were cultured without cytokine supplement, with a 125 pg / ml IL-7 and IL- 15 gradient, or with 12.5 ng / ml IL-7 and IL- 15 and placed in the incubator to be read out weekly. Media was refreshed twice a week before day 20 centrifuging the LJ- bottom plate containing PCT cells at 400 x g for 5 minutes and resuspending. After day 20, media was refreshed once per week. On Days 5, 11, 18, 40, and 57 PCTs were measured and counted by flow cytometry. The percent of edited cell number change over time was measured by dividing PCT cell count obtained on Days 5, 11, 18, 40, and 57 by initial Day 0 PCT cell count.

[0109] Expansion and Repetitive Stimulation AssayIPTS / 200097280.2 116Attorney Ref: ANB-223WO

[0110] Engineered PCT-cells expressing the SPAs of SEQ ID NOs: 185 / 421, 104, 114, and 259 (baseline) were co-cultured with dual-antigen expressing tumor cells at an E:T of 1:1 (one PCT cell for every 1 tumor cell) at 37°C in cell media lacking IL-2. Every 3 days, the supernatant containing the T-cells was collected and half of it was cocultured with fresh tumor cells (volumetric split), for up to 12 days. T cell expansion was tracked via flow cytometry staining of PrimeR protein. On Days 5 and 12 cell cultures were re-normalized to 1:1 E:T ratios.

[0111] Results

[0112] pSTAT profiling

[0113] The pSTAT profiling results for the selected SPAs is provided in Table 13. Fold change is relative to negative control PCT cells expressing NGFR.

[0114] As shown in Table 13, the SPA STAT activation profiles in PCT cells expressing the SPAs separated into three distinct categories: strong multi-STAT signaling, mild multi- STAT signaling, and STAT4-centric signaling.

[0115] The SPAs provided in SEQ ID NOs: 185 / 421, 184 / 420 and 182 / 419 grouped together as strong multi-STAT signaling proteins that induced at least a 3-fold increase in STAT1, STAT3, STAT4 and STAT5 phosphorylation. The SPAs provided in SEQ ID NOs: 117, 152 / 422, and 104 grouped together as mild multi-STAT signaling proteins that induced at least a 3-fold increase in STAT3 phosphorylation and at least a 1.5-fold increase in STAT1, STAT4 and STAT5 phosphorylation. The SPAs provided in SEQ ID NOs: 79, 114, and 268 grouped together as STAT4-centric signaling proteins that induced at least a 3-fold increase in STAT3 and STAT4 phosphorylation and at least a 1.5-fold increase in STAT1,IPTS / 200097280.2 117Attorney Ref: ANB-223WO phosphorylation. In particular, SPAs comprising the (YXXQ)4 STAT adaptor (SEQ ID NO: 34 or 286) and a second STAT signaling domain from IL-2RP or IL-7Ra showed strong multi-STAT signaling, while SPAs comprising a longer gpl30 intracellular domain fragment in combination with and a second STAT signaling domain from IL-2RP or IL-7Ra showed mild multi-STAT signaling. The SPAs comprising an IL-12RP2 intracellular domain induced STAT4 centric signaling.

[0116] Without wishing to be bound by theory, the phosphorylation of specific STAT proteins can be less selective, especially when minimal phosphorylation motifs are synthetically staggered together from truncations of endogenous receptor proteins. In addition, the JAK kinases are also well known to be less selective. As observed in the data, the synthetic pathway activators are expressed highly in PC T cells, generating stronger and active JAK signaling and thus resulting in potentially hypersensitivity in multiple phosphorylation signals, including pSTAT4. Thus, SPAs that lack canonical STAT4 signaling domains may still induce STAT4 signaling due to the synthetic nature of combining the minimal STAT and JAK signaling domains.

[0117] Furthermore, without wishing to be bound by theory, the dimerization of the SPA may contribute to the amount and type of STAT phosphorylation and signaling. For example, dimerization driven by the cysteine linker versus the IE7Ra transmembrane domain can contribute to different pSTAT levels. In some instances, SPAs containing the IE7RamutP2 TMD induced higher pSTAT levels than SPAs comprising the cysteine linker despite both containing a smaller gpl30 ICD (e.g., SPAs of SEQ ID NOs: 185 / 421, 184 / 420, and 182 / 419 as compared to the SPA of SEQ ID NO: 268). In addition, the length of the gpl30 intracellular domain may play a role in the strength of the pSTAT signaling. For example, the SPA of SEQ ID NO: 104 comprises a gpl30 ICD with the Y759 residue and which is longer than the (YXXQ)4 adaptor used in the SPAs of SEQ ID NOs: 185 / 421, 184 / 420, and 182 / 419. The Y759 residue is known to recruit negative regulators such as SHP-2 and SOCS- 3 that prevent excessive STAT activation. Thus, without wishing to be bound by theory, the effect and characterization of the SPAs as strong and mild multi-STAT signaling inducers may be a combination of dimerization force and the ICD sequence.

[0118] The STAT signaling axis for the new SPAs based on signaling group (e.g., strong multi-STAT, mild multi-STAT, and STAT4-centric) for an exemplary SPA within each of those groups as compared to the control SPA provided in SEQ ID NO: 259 is shown in FIG. 8. The exemplary SPA for the strong multi-STAT group is the SPA of SEQ ID NO: 185 / 421,IPTS / 200097280.2 118Attorney Ref: ANB-223WO the exemplary SPA for the mild multi-STAT group is the SPA of SEQ ID NO: 104, and the exemplary SPA for the STAT4-centric group is the SPA of SEQ ID NO: 114.

[0119] Target Cell Survival Assay (Potency)

[0120] PCT cells expressing the selected SPAs also demonstrated superior long term potency as compared to the baseline SPA of SEQ ID NO: 259 (FIG. 8A and 8B). As shown in FIG. 8A, PCT cells expressing the selected SPAs demonstrated increased target cell killing as compared to the PCT cells expressing the exemplary CAR, NGFR, or the baseline SPA of SEQ ID: 259. Increased target cell killing resulted in lower target cells counts. Thus, the lower lines at the end of the elapsed time in FIG. 8A indicate increased PCT cell potency. The fold benefit for T cell potency of the selected SPAs as compared to the SPA of SEQ ID NO: 259 was also calculated by dividing the two donor average endpoint target cell counts of SEQ ID NO: 259 with each SPA (FIG. 8B). Paired T test relative to SPA of SEQ ID NO: 259. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. As shown in FIG. 8B, the SPAs of SEQ ID NOs: 185 / 421, 184 / 420, 117, 152 / 422, 104, 79, and 114 demonstrated statistically significant increase potency as compared to the SPA of SEQ ID NO: 259.

[0121] Cytokine Secretion

[0122] PCT cells expressing the selected SPAs demonstrate elevated cytokine secretion relative to the SPA of SEQ ID NO: 259. As shown in FIG. 9A-9D, dual antigen stimulation of the PCT cells expressing the selected SPAs with cells expressing both the priming receptor and CAR antigens resulted in greater secretion of GM-CSF, IFNy, TNFa, and IL- 10 as compared to PCT cells expressing the SPA of SEQ ID NO: 259. FIG. 9A shows secretion of GM-CSF by PCT cells expressing the indicated SPA, a constitutively expressed CAR, or cells engineered with RNP. FIG. 9B shows secretion of IFNy. FIG. 9C shows secretion of TNFa. FIG. 9D shows secretion of IL- 10. Paired T test for cytokine concentration relative to SPA of SEQ ID NO: 259. * P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001.

[0123] T cell survival

[0124] PCT cells expressing the selected SPAs exhibited extended survival and persistence without cytokine support for up to 28 days. SPAs incorporating IL2RP and IL- 7Ra domains in particular demonstrated this phenotype (e.g., the SPAs of SEQ ID NOs: 185, 184, 182, 152, 104) (FIG. 10A and 10B). FIG. 10A provides the PCT cell survival for cells from donor 1. FIG. 10B provides the PCT cell survival for cells from donor 2. LLOD indicates the lower limit of detection. Jurkat T cells and primary PCT cells expressing NGFR were used as positive and negative controls.

[0125] T cell persistence in cytokine-free media IPTS / 200097280.2 1 19Attorney Ref: ANB-223WO

[0126] PCT cells expressing all of the selected SPA proteins, with the exception of the SPA provided in SEQ ID NO: 184 demonstrated T cell population contraction when incubated for up to 60 days without cytokine support (FIG. 11A), with only serum-level cytokine support at 125 pg / ml (FIG. 11B), or with supraphysiological cytokine support at 12.5 ng / ml (e.g., the concentration of cytokines used in T cell expansion media, FIG. 11C). Thus, the SPAs of SEQ ID NOs: 185 / 421, 182 / 419, 117, 152 / 422, 104, 79, 114, 156, and 268, had reduced cytokine independent growth risk.

[0127] Expansion and Repetitive Stimulation Assay

[0128] PCT cells expressing all of the selected SPA proteins showed higher expansion compared to the SPA of SEQ ID NO: 259 after a repeated antigen stimulation assay (RSA) (FIG. 12A and 12B). FIG. 12A provides the data from donor 1, FIG. 12B provides that data form donor 2. Consistent with the other cytolytic assays, in a RSA without exogenous IL-2 support, all of the selected SPA compositions showed higher PCT cell expansion as compared to the SPA of SEQ ID NO: 259.

[0129] A summary diagram of the STAT signaling axis for standard T cells, PCT cells expressing the baseline SPA of SEQ ID NO: 259, and PCT cells expressing the new SPAs disclosed herein is provided in FIG. 13A. As shown in FIG. 13A, the new SPAs have increased STAT signaling on all of the STAT axis (STAT1, STAT3, STAT4, and STAT5) as compared to standard T cells, and increased STAT signaling on the STAT1, STAT4, and STAT5 axis as compared to the baseline SPA of SEQ ID NO: 259. In addition, PCT cells expressing the new SPAs demonstrated increased T cell potency and persistence as compared to standard T cells and T cells expressing the baseline SPA of SEQ ID NO: 259 (FIG. 13B). Without wishing to be bound by theory, the increased STAT signaling results in more powerful T cells with increased potency (cytotoxicity), expansion, and persistence. Thus, the new SPAs disclosed herein provide superior technical effects and functionality as compared to the baseline SPA of SEQ ID NO: 259.

[0130] While the disclosure has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the disclosure.

[0131] All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.IPTS / 200097280.2 120Attorney Ref: ANB-223WOINFORMAL SEQUENCE LISTINGIPTS / 200097280.2 121Attorney Ref: ANB-223WOIPTS / 200097280.2 122Attorney Ref: ANB-223WOIPTS / 200097280.2 123Attorney Ref: ANB-223WOIPTS / 200097280.2 124Attorney Ref: ANB-223WOIPTS / 200097280.2 125Attorney Ref: ANB-223WOIPTS / 200097280.2 126Attorney Ref: ANB-223WOIPTS / 200097280.2 127Attorney Ref: ANB-223WOIPTS / 200097280.2 128Attorney Ref: ANB-223WOIPTS / 200097280.2 129Attorney Ref: ANB-223WOIPTS / 200097280.2 130Attorney Ref: ANB-223WOIPTS / 200097280.2 131Attorney Ref: ANB-223WOIPTS / 200097280.2 132Attorney Ref: ANB-223WOIPTS / 200097280.2 133Attorney Ref: ANB-223WOIPTS / 200097280.2 134Attorney Ref: ANB-223WOIPTS / 200097280.2 135Attorney Ref: ANB-223WOIPTS / 200097280.2 136Attorney Ref: ANB-223WOIPTS / 200097280.2 137Attorney Ref: ANB-223WOIPTS / 200097280.2 138Attorney Ref: ANB-223WOIPTS / 200097280.2 139Attorney Ref: ANB-223WOIPTS / 200097280.2 140Attorney Ref: ANB-223WOIPTS / 200097280.2 141Attorney Ref: ANB-223WOIPTS / 200097280.2 142Attorney Ref: ANB-223WOIPTS / 200097280.2 143Attorney Ref: ANB-223WOIPTS / 200097280.2 144Attorney Ref: ANB-223WOIPTS / 200097280.2 145Attorney Ref: ANB-223WOIPTS / 200097280.2 146Attorney Ref: ANB-223WOIPTS / 200097280.2 147Attorney Ref: ANB-223WOIPTS / 200097280.2 148Attorney Ref: ANB-223WOIPTS / 200097280.2 149Attorney Ref: ANB-223WOIPTS / 200097280.2 150Attorney Ref: ANB-223WOIPTS / 200097280.2 151Attorney Ref: ANB-223WOIPTS / 200097280.2 152Attorney Ref: ANB-223WOIPTS / 200097280.2 153Attorney Ref: ANB-223WOIPTS / 200097280.2 154Attorney Ref: ANB-223WOIPTS / 200097280.2 155Attorney Ref: ANB-223WOIPTS / 200097280.2 156Attorney Ref: ANB-223WOIPTS / 200097280.2 157Attorney Ref: ANB-223WOIPTS / 200097280.2 158Attorney Ref: ANB-223WOIPTS / 200097280.2 159Attorney Ref: ANB-223WOIPTS / 200097280.2 160Attorney Ref: ANB-223WOIPTS / 200097280.2 161Attorney Ref: ANB-223WOIPTS / 200097280.2 162Attorney Ref: ANB-223WOIPTS / 200097280.2 163Attorney Ref: ANB-223WOIPTS / 200097280.2 164Attorney Ref: ANB-223WOIPTS / 200097280.2 165

Claims

Attorney Ref: ANB-223WOCLAIMS1. One or more nucleic acids encoding a synthetic pathway activator (SPA) peptide comprising a chimeric polypeptide comprising: a. optionally, an extracellular domain; b. a transmembrane domain; c. optionally, a costimulatory domain; and d. an intracellular signaling domain comprising: i. one or more Janus Kinase (JAK) signaling domains; and ii. a combinatorial signaling domain comprising two or more Signal Transducer and Activator of Transcription (STAT)l, STAT3, STAT4, STAT5 and / or Toll-like receptor (TLR) / IL-1R (TIR) signaling domains(s), or any combination thereof.

2. One or more nucleic acids encoding a synthetic pathway activator (SPA) peptide comprising a chimeric polypeptide comprising: a. optionally, an extracellular domain; b. a transmembrane domain comprising an IL-7Ra transmembrane domain, a G- CSFR, transmembrane domain, or a gpl3O transmembrane domain; c. optionally, a costimulatory domain; and d. an intracellular signaling domain comprising: i. one or more Janus Kinase (JAK) signaling domain(s) comprising a gpl3O JAK signaling domain, an IL-7Ra JAK signaling domain, a G- CSFR JAK signaling domain, an IL-12RP2 JAK signaling domain, an IL-9R JAK signaling domain, or an IL-21R JAK signaling domain, or any combination thereof; and ii. one or more Signal Transducer and Activator of Transcription (STAT)l, STAT3, STAT4, STAT5 and / or Toll-like receptor (TLR) / IL- 1R (TIR) signaling domains(s), or any combination thereof.IPTS / 200097280.2 166Attorney Ref: ANB-223WO3. The nucleic acid of claim 2, wherein the intracellular signaling domain comprises two or more STAT1, STAT3, STAT4, STAT5 and / or TIR signaling domain(s), or any combination thereof.

4. The nucleic acid of claim 1-3, wherein the STAT1, STAT3, STAT4, and / or STAT5 signaling domain(s) comprises one or more tyrosine phosphorylation motifs comprising YXXQ or YXPQ.

5. The nucleic acid of claims 1-4, wherein the STAT1, STAT3, STAT4, STAT5 and / or TIR signaling domain(s) induces phosphorylation of one or more of STAT1, STAT3, STAT4, STAT5 and / or TIR upon multimerization of the SPA in a cell.

6. The nucleic acid of claims 1-4, wherein the intracellular signaling domain induces phosphorylation of at least STAT1, STAT3, STAT4 and / or STAT5 upon multimerization of the SPA in a cell.

7. The nucleic acid of claims 1-4, wherein the intracellular signaling domain induces phosphorylation of at least STAT4 upon multimerization of the SPA in a cell.

8. The nucleic acid of any one of claims 1-5, wherein the intracellular signaling domain comprises at least one polypeptide sequence from an interleukin receptor or a cytokine receptor.

9. The nucleic acid of any one of claims 1-8 wherein the STAT1, STAT3, STAT4, STAT5 and / or (TIR) signaling domain(s)comprises at least one STAT1 and / or STAT3 signaling domain from at least one of gpl30, IL-21R, G-CSFR, or IL-9R, or any combination thereof.

10. The nucleic acid of any one of claims 1-9 wherein the STAT1, STAT3, STAT4, STAT5 and / or (TIR) signaling domain(s) comprises at least one STAT4 signaling domain from IL-12RP2.

11. The nucleic acid of any one of claims 1-10 wherein the STAT1, STAT3, STAT4, STAT5 and / or (TIR) signaling domain(s)comprises at least one STAT5 signaling domain from at least one of IL-2RP , IL-7Ra, or IL-15R, or any combination thereof.

12. The nucleic acid of any one of claims 1-11 wherein the STAT1, STAT3, STAT4, STAT5 and / or (TIR) signaling domain(s) comprises at least one TIR domain from IL-18R.

13. The nucleic acid of any one of claims 1-12, wherein the STAT1, STAT3, STAT4, STAT5 and / or Toll-like receptor (TLR) / IL-1R (TIR) signaling domain(s) comprises at leastIPTS / 200097280.2 | gyAttorney Ref: ANB-223WO one signal adaptor comprising one or more amino acid sequences at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence of: a gpl30 (YXXQ)4 STAT adaptor (SEQ ID NO: 34 or 286), a gpl30_Y759F STAT adaptor (SEQ ID NO: 35), a truncated gpl30(A707-755) with a Y759F mutation (gpl30trunc7_Y759F (SEQ ID NO: 36)), IL9R_310-521 (SEQ ID NO: 37), IL12Rp2_796- 801 ((SEQ ID NO: 38), IL 12Rp2_714-862 (SEQ ID NO: 39), IL7Ra_316-459 (SEQ ID NO: 40), IL2Rp_333-551 (SEQ ID NO: 41), IL2RP (339-379,393-433,518-551) (SEQ ID NO: 42), IL2Rp_530-551 (SEQ ID NO: 43), IL18R_373-520 (SEQ ID NO: 44), or IL18R_369- 541 (SEQ ID NO: 45).

14. The nucleic acid of any one of claims 1-13, wherein the intracellular signaling domain comprises one or more amino acid sequences at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from the sequences provided as SEQ ID NOs: 9, 11, 12, 14, 15, 17, 28, 20, 21, 23, 24, 26, 27, 28, 30, 31, 33, 55, or 56.

15. The nucleic acid of any one of claims 1-14, wherein the intracellular signaling domain induces phosphorylation of at least one of JAK1, JAK2, and / or JAK3 upon multimerization of the SPA.

16. The nucleic acid of any one of claims 1, or 4-15, wherein the one or more JAK signaling domains comprises at least one JAK signaling domain from at least one of gpl30, IL-7Ra, G-CSFR, IL-12RP2, IL-9R, and IL-21R, or any combination thereof.

17. The nucleic acid of any one of claims 1-16, wherein the JAK signaling domain comprises one or more amino acid sequences at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence as set forth in SEQ ID NOs: 10, 13, 16, 19, 22, 25, 29, 32, 47, 48, 49, 50, 51, 52, 53, or 54.

18. The nucleic acid of any one of claims 1-17, wherein the co- stimulatory domain comprises a CD28 co- stimulatory domain.

19. The nucleic acid of claim 18, wherein the CD28 co-stimulatory domain comprises a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 46.IPTS / 200097280.2Attorney Ref: ANB-223WO20. The nucleic acid of any one of claims 1 or 4-19, wherein the transmembrane domain comprises a gpl30 transmembrane domain, an IL-7Ra transmembrane domain, a G-CFSR transmembrane domain, or a G-CFSR transmembrane domain, or a portion thereof.

21. The nucleic acid of claim 20, wherein the G-CFSR transmembrane domain comprises a T640N mutation as compared to the full length G-CFSR protein (SEQ ID NO: 263).

22. The nucleic acid of any one of claims 1-21, wherein the transmembrane domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NOs: 6, 7, 8, 261, or 262.

23. The nucleic acid of any one of claims 1-22, wherein the extracellular domain comprises a CD34 epitope, a QB END 10 epitope, a CD34 extracellular domain, or a truncated CD34 extracellular domain.

24. The nucleic acid of claim 23, wherein the extracellular domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NOs: 499, 475-478, 1, 2, 3, 4, or 5.

25. The nucleic acid of any one of claims 1-24, wherein the extracellular domain conveys constitutive activity to the intracellular signaling domain upon multimerization of the SPA.

26. The nucleic acid of any one of claims 1-25, wherein the extracellular domain comprises an unpaired cysteine.

27. The nucleic acid of any one of claims 1-26, wherein the SPA peptide comprises a. a truncated CD34 extracellular domain, an IL7Ra transmembrane domain, a gpl30 JAK domain, a gpl30 STAT1 and STAT3 signaling domain, and an IL7Ra STAT5 signaling domain; b. a truncated CD34 extracellular domain, an IL7Ra transmembrane domain, a gpl30 JAK domain, a gpl30 STAT1 and STAT3 signaling domain, and an IL- 2RP STAT5 signaling domain; c. a truncated CD34 extracellular domain, an IL7Ra transmembrane domain, a gpl30 JAK domain, a gpl30 STAT1 and STAT3 signaling domain, and an IL- 12RP2 STAT4 signaling domain;IPTS / 200097280.2 169Attorney Ref: ANB-223WO d. a CD34 epitope extracellular domain, a gpl3O transmembrane domain, a gpl3O JAK domain, a gpl3O STAT1 and STAT3 signaling domain, and an IL7Ra STAT5 signaling domain; e. a CD34 epitope extracellular domain, a gpl3O transmembrane domain, a gpl3O JAK domain, a gpl3O STAT1 and STAT3 signaling domain, and an IL- 2RP STAT5 signaling domain; f. a CD34 epitope extracellular domain, a gpl3O transmembrane domain, a gpl3O JAK domain, a gpl3O STAT1 and STAT3 signaling domain, and an IL- 12RP STAT4 signaling domain; or g. a CD34 epitope extracellular domain, a gpl3O transmembrane domain, a G- CSFR JAK domain, a G-CSFR STAT1 and STAT3 signaling domain, and an IL-12RP STAT4 signaling domain.

28. The nucleic acid of any one of claims 1-27, wherein the SPA peptide comprises a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from the sequences set forth in SEQ ID NOs: 57-254, 264- 283, or 419-474.

29. The nucleic acid of any one of claims 1-28, wherein the SPA peptide comprises a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from the sequences set forth in SEQ ID NOs: 421, 185, 114, 104, 79, 80, 81, 87, 103, 111, 116, 117, 143, 144, 145, 152, 153, 155, 156, 157, 159, 160, 182, 183, 184, 220, 234, 264, 268, 269, 270, 282, 419, 420, or 422.

30. The nucleic acid of any one of claims 1-29, wherein the SPA peptide induces at least 1.5-fold, 2-fold, or 3-fold more phosphorylation of at least STAT1, STAT3, STAT4 and / or STAT5 upon multimerization of the SPA in a cell as compared to a cell that lacks the SPA peptide.

31. The nucleic acid of any one of claims 1-29, wherein the SPA peptide induces at least 1.5-fold, 2-fold, or 3-fold more phosphorylation of at least STAT1, STAT3, and / or STAT4 upon multimerization of the SPA in a cell as compared to a cell that lacks the SPA peptide.IPTS / 200097280.2 170Attorney Ref: ANB-223WO32. The nucleic acid of any one of claims 1-29, wherein the SPA peptide induces at least 1.5-fold, 2-fold, or 3-fold more phosphorylation of at least STAT4 upon multimerization of the SPA in a cell as compared to a cell that lacks the SPA peptide.

33. The nucleic acid of any one of claims 1-29, wherein the SPA peptide induces at least 1.5-fold, 2-fold, or 3-fold more phosphorylation of at least STAT5 upon multimerization of the SPA in a cell as compared to a cell that lacks the SPA peptide.

34. The nucleic acid of any one of claims 1-33, wherein the nucleic acid comprises a sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to a sequence as set forth in SEQ ID NOs: 479-498.

35. The nucleic acid of claim 34, wherein the nucleic acid comprises a sequence as set forth in any one of SEQ ID NOs: 479-498.

36. One or more nucleic acids encoding a synthetic pathway activator (SPA) peptide at least 90% identical to any one of the sequences as set forth in SEQ ID NOs: 479-498.

37. The nucleic acid of claim 36, wherein the nucleic acid comprises a sequence at least 95% identical to a sequence as set forth in SEQ ID NOs: 479-498.

38. The nucleic acid of claim 36 or 37, wherein the nucleic acid comprises a sequence at least 90% identical to a sequence as set forth in SEQ ID NOs: 479, 480, 489, 490, 493, or 494.

39. The nucleic acid of claim 36 or 37, wherein the nucleic acid comprises a sequence at least 95% identical to a sequence as set forth in SEQ ID NOs: 479, 480, 489, 490, 493, or 494.

40. The nucleic acid of claim 36 or 37, wherein the nucleic acid comprises a sequence at as set forth in SEQ ID NOs: 479, 480, 489, 490, 493, or 494.

41. One or more nucleic acids encoding a synthetic pathway activator (SPA) peptide at least 90% identical to any one of the sequences as set forth in SEQ ID NOs: 421, 420, 422,419, 185, 184, 182, 104, 114, 117, 152, 79, 156, 268, 57-78, 80-103, 105-113, 115-116, 118- 151, 153-155, 157-181, 183, 186-254, 264-267, 269-283, or 423-474.

42. The nucleic acid of claim 41, wherein the nucleic acid encodes a synthetic pathway activator (SPA) peptide at least 95% identical to a sequence as set forth in SEQ ID NOs: 421,420, 422, 419, 185, 184, 182, 104, 114, 117, 152, 79, 156, 268, 57-78, 80-103, 105-113, 115- 116, 118-151, 153-155, 157-181, 183, 186-254, 264-267, 269-283, or 423-474.IPTS / 200097280.2 171Attorney Ref: ANB-223WO43. The nucleic acid of claim 41 or 42, wherein the nucleic acid encodes a synthetic pathway activator (SPA) peptide at least 90% identical to a sequence as set forth in SEQ ID NOs: 421, 185, 104, or 114.

44. The nucleic acid of claim 41 or 42, wherein the nucleic acid encodes a synthetic pathway activator (SPA) peptide at least 95% identical to a sequence as set forth in SEQ ID NOs: 421, 185, 104, or 114.

45. The nucleic acid of claim 41 or 42, wherein the nucleic acid encodes a synthetic pathway activator (SPA) peptide comprising a sequence at as set forth in SEQ ID NOs: 421, 185, 104, or 114.

46. One or more SPA peptides encoded by the one or more nucleic acid(s) of any one of claims 1-45.

47. A dimer or multimer comprising the one or more SPA peptides of claim 46.

48. At least one vector comprising the at least one nucleic acid of any one of claims 1-45.

49. A system comprising: a. one or more nucleic acid(s) encoding a first chimeric polypeptide comprising a priming receptor; b. one or more nucleic acid(s) encoding a second chimeric polypeptide comprising a chimeric antigen receptor (CAR); and c. the one or more nucleic acids encoding a SPA peptide of any one of claims 1- 45.

50. A system comprising: a. a first chimeric polypeptide comprising a priming receptor; b. a second chimeric polypeptide comprising a chimeric antigen receptor (CAR); and c. the one or more SPA peptides of claim 46.

51. The system of claim 49 or 50, wherein binding by the CAR or priming receptor to a target induces expression of the one or more SPA peptides.

52. The system of claim 49 or 50, wherein the one or more SPA peptides is constitutively or inducibly expressed.IPTS / 200097280.2 172Attorney Ref: ANB-223WO53. A cell or population of cells comprising the one or more nucleic acids of any one of claims 1-45, the one or more SPA peptides of claim 46, the dimer or multimer of claim 47, the vector of claim 48, or the system of any one of claims 49-52.

54. The cell of claim 53, wherein the cell is an immune cell or a stem cell, optionally wherein the immune cell is a primary human immune cell.

55. A pharmaceutical composition comprising the cell or population of cells of claims 53 or 54, and a pharmaceutically acceptable excipient.

56. A pharmaceutical composition comprising the nucleic acid of any one of claims 1-45 or the vector of claim 48, and a pharmaceutically acceptable excipient.

57. A method of engineering a cell, comprising introducing the nucleic acid of any one of claims 1-45 into the cell.

58. The method of claims 57, wherein the nucleic acid is introduced into an insertion site in the genome of the cell.

59. The method of claims 57 or 58, wherein the nucleic acid is introduced to the cell non- virally.

60. The method of claim 57-59, wherein the cell is an immune cell, optionally a primary immune cell.

61. A method of editing a cell, comprising: a. providing a nuclease domain and a guide RNA and the nucleic acid of any one of claims 1-45, and wherein the 5’ and 3’ ends of the nucleic acid comprise nucleotide sequences that are homologous to genomic sequences flanking an insertion site in the genome of the cell; b. introducing the nuclease domain and nucleic acid into the cell, wherein the guide RNA specifically hybridizes to a target region of the genome of the cell, and wherein the nuclease domain cleaves the target region to create an insertion site in the genome of the cell; and c. editing the cell via insertion of the nucleic acid into the insertion site in the genome of the cell.

62. The method of claim 61, wherein the nuclease domain and nucleic acid are introduced to the cell non-virally.IPTS / 200097280.2 173Attorney Ref: ANB-223WO63. A method of editing an immune cell, comprising: a. providing a ribonucleoprotein complex (RNP) complex wherein the RNP comprises a nuclease domain and a guide RNA, and the nucleic acid of any one of claims 1-45, wherein the 5’ and 3’ ends of the nucleic acid comprise nucleotide sequences that are homologous to genomic sequences flanking an insertion site in the genome of the immune cell; b. non-virally introducing the RNP-nucleic acid complex into the immune cell, wherein the guide RNA specifically hybridizes to a target region of the genome of the primary immune cell, and wherein the nuclease domain cleaves the target region to create the insertion site in the genome of the immune cell; and c. editing the immune cell via insertion of the nucleic acid of claim 1 into the insertion site in the genome of the immune cell.

64. The method of claims 59, 62, or 63, wherein non-virally introducing comprises electroporation.

65. The method of any one of claims 61-64, wherein the nuclease domain comprises a CRISPR-associated endonuclease (Cas), optionally a Cas9 nuclease.

66. The method of any one of claims 61 to 65, wherein the target region of the genome of the cell is a genomic safe harbor (GSH) locus or a T Cell Receptor Alpha Constant (TRAC) locus.

67. The method of any one of claims 61 to 66, wherein the target region is the GS94 locus (locus chr 11 : 128340000-128350000).

68. The method of any one of claims 57 to 67, wherein the nucleic acid is a doublestranded nucleic acid or a single- stranded nucleic acid.

69. The method of any one of claims 57 to 68, wherein the nucleic acid is a linear nucleic acid or a circular nucleic acid, optionally wherein the circular nucleic acid is a plasmid.

70. The method of any one of claims 63 to 69, wherein the cell is an immune cell or a stem cell, optionally a primary human immune cell.

71. The method of any one of claims 63 to 70, wherein the immune cell is an autologous immune cell.IPTS / 200097280.2 174Attorney Ref: ANB-223WO72. The method of any one of claims 63 to 70, wherein the immune cell is an allogeneic immune cell.

73. The method of any one of claims 63 to 72, wherein the immune cell is a natural killer (NK) cell, a T cell, a CD8+ T cell, a CD4+ T cell, a primary T cell, or a T cell progenitor cell.

74. The method of any one of claims 63 to 73, wherein the immune cell is a primary T cell.

75. The method of any one of claims 63 to 74, wherein the immune cell is a primary human T cell.

76. The method of any one of claims 57 to 75, wherein the cell is virus-free.

77. The method of any one of claims 57 to 76, further comprising determining CD11c expression in the cell.

78. The method of any one of claims 57 to 77, further comprising obtaining the cell from a patient and introducing the nucleic acid in vitro.

79. A method of treating a disease in a subject comprising administering the nucleic acid of any one of claims 1-45, the vector of claim 48, the cell or population of cells of claims 53 or 54, or the pharmaceutical composition of claims 55 or 56 to the subject.

80. The method of claim 79, wherein the disease is cancer.

81. The method of claim 80, wherein the cancer is a solid cancer or a liquid cancer.

82. The method of claim 80 or 81, wherein the cancer is kidney cancer, clear cell renal cell carcinoma (ccRcc), colorectal cancer, or lung cancer.

83. A method of inhibiting a target cell in a subject comprising administering the one or more nucleic acids of any one of claims 1-45, the one or more SPA peptides of claim 46, the dimer or multimer of claim 47, the vector of claim 48, the cell or population of cells of claims 53 or 54, or the pharmaceutical composition of claims 55 or 56 to the subject, wherein the cell inhibits the target cell.

84. A method of modulating the activity of a cell or an immune cell comprising: a. obtaining a cell or an immune cell comprising i. the one or more nucleic acids of any one of claims 1-45;IPTS / 200097280.2 175Attorney Ref: ANB-223WO ii. the SPA peptide of claim 46; iii. the dimer or multimer of claim 47 ; iv. the system of any one of claims 49-52; and / or v. the vector claim 48; and b. contacting the cell or immune cell with a target cell, wherein the synthetic pathway activator modulates the activity of the cell or immune cell.

85. A method of modulating the activity of an cell or immune cell comprising: a. obtaining a cell or immune cell comprising i. the one or more nucleic acids of any one of claims 1-45; ii. the SPA peptide of claim 46; iii. the dimer or multimer of claim 47 ; iv. the system of any one of claims 49-52; and / or v. the vector claim 48; and b. contacting the cell or immune cell with a target cell expressing a priming receptor antigen and a CAR antigen, wherein binding of the priming receptor to the priming receptor antigen on the target cell induces activation of the priming receptor and expression of the chimeric antigen receptor, wherein binding of the chimeric antigen receptor to CAR antigen on the target cell modulates the activity of the cell or immune cell, and wherein the synthetic pathway activator also modulates the activity of the cell or immune cell.

86. The method of claims 84 or 85, wherein the activity comprises inducing the JAK- STAT signaling pathway.

87. The methods of claims 84 or 85, wherein the activity comprises an enhanced immune activity as compared to a cell that does not comprise the SPA88. The methods of claims 87, wherein the immune activity comprises increased cytotoxicity, immune cell persistence, and / or tumor infiltration as compared to a cell that does not comprise the SPA.IPTS / 200097280.2 176Attorney Ref: ANB-223WO89. The method of any one of claims 84-88, wherein the SPA induces phosphorylation of at least STAT1, STAT3, STAT4, STAT5, and / or TIR upon multimerization of the SPA in the cell.

90. The method of claim 89, wherein the SPA peptide induces at least 1.5-fold, 2-fold, or 3-fold more phosphorylation of at least STAT1, STAT3, STAT4 and STAT5 upon multimerization of the SPA in a cell as compared to a cell that lacks the SPA peptide.

91. The method of claim 89, wherein the SPA peptide induces at least 1.5-fold, 2-fold, or 3-fold more phosphorylation of at least STAT1, STAT3, and STAT4 upon multimerization of the SPA in a cell as compared to a cell that lacks the SPA peptide.

92. The method of claim 89, wherein the SPA peptide induces at least 1.5-fold, 2-fold, or 3-fold more phosphorylation of STAT4 upon multimerization of the SPA in a cell as compared to a cell that lacks the SPA peptide.

93. The method of claim 89, wherein the SPA peptide induces at least 1.5-fold, 2-fold, or 3-fold more phosphorylation of STAT5 upon multimerization of the SPA in a cell as compared to a cell that lacks the SPA peptide.IPTS / 200097280.2 177

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