Synthetic pathway activators

Abstract

Provided herein are novel synthetic pathway activators that include a multimerization region, a transmembrane domain, and an intracellular signaling domain.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 489,842, filed March 13, 2023, U.S. Provisional Application No. 63 / 495,865, filed April 13, 2023, and U.S. Provisional Application No. 63 / 613,713, filed December 21, 2023, each of which is incorporated by reference herein in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing, which is incorporated herein by reference in its entirety. The XML copy was created on February 27, 2024, is named ANB-219WO_SL, and is 252,803 bytes in size. [Background technology]

[0003] background Cancer is a disease characterized by the uncontrolled proliferation of cells. Many approaches to treating cancer have been attempted, including drug and radiation therapy. Recent cancer treatments attempt to use the body's own immune cells to attack cancer cells. One promising approach uses T cells taken from the patient and genetically engineered to produce chimeric antigen receptors (CARs), receptor proteins that give T cells new capabilities to target specific proteins. These receptors are chimeric because they combine antigen-binding and T-cell activation functions into a single receptor.

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

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

[0006] However, several concerns and limitations remain with CAR T cell-based immunotherapy. Clinically effective adoptive T cell therapy for the treatment of solid tumors relies on robust T cell proliferation, persistence, and efficacy. Therefore, additional therapies that increase T cell proliferation, persistence, and efficacy remain desirable. Summary of the Invention

[0007] overview In one aspect, i. optionally, an extracellular domain; and ii. a lipid anchor or transmembrane domain; iii. an intracellular signaling domain; and iv. Multimerization region and Provided herein are synthetic pathway activator (SPA) peptides, including chimeric polypeptides, comprising:

[0008] In some embodiments, multimerization of the chimeric polypeptide via the multimerization region results in constitutive activity of the intracellular signaling domain.

[0009] In some embodiments, the multimerization region comprises at least one of an unpaired cysteine ​​residue, a leucine zipper, a BCR domain, and a VASP domain.

[0010] In some embodiments, the multimerization region comprises at least an unpaired cysteine ​​residue.

[0011] In some embodiments, the multimerization region comprises at least an unpaired cysteine ​​residue and a leucine zipper.

[0012] In some embodiments, the multimerization domain is intracellular when expressed by a cell.

[0013] In some embodiments, the multimerization domain is extracellular when expressed by a cell.

[0014] In some embodiments, the intracellular signaling domain induces phosphorylation of STAT1, STAT3, or STAT5.

[0015] In some embodiments, the intracellular signaling domain comprises a type I cytokine receptor superfamily box 1 (IWPNVDP (SEQ ID NO: 106)) or box 2 (VSVVEIEANDKKP (SEQ ID NO: 107)) peptide motif.

[0016] In some embodiments, the intracellular signaling domain comprises a tyrosine phosphorylation motif including YXXQ or YXPQ.

[0017] In some embodiments, the intracellular signaling domain comprises a polypeptide sequence from an interleukin receptor.

[0018] In some embodiments, the interleukin receptor comprises a gp130 intracellular signaling domain.

[0019] In some embodiments, the intracellular signaling domain comprises a polypeptide sequence comprising amino acids 642 to 918 of gp130 (SEQ ID NO: 59).

[0020] In some embodiments, the intracellular signaling domain comprises a polypeptide sequence comprising the sequence set forth as SEQ ID NO:60.

[0021] In some embodiments, the interleukin receptor comprises a truncated gp130 intracellular signaling domain.

[0022] In some embodiments, the truncated gp130 intracellular signaling domain comprises a deletion of amino acids 771-811 of gp130 (SEQ ID NO: 59).

[0023] In some embodiments, the truncated gp130 intracellular signaling domain comprises a truncated gp130 intracellular domain of a sequence selected from the group set forth in SEQ ID NOs: 10-16 and 71-77.

[0024] In some embodiments, the gp130 intracellular signaling domain further comprises a Y759F mutation of gp130 (SEQ ID NO: 59).

[0025] In some embodiments, the intracellular signaling domain further comprises a prenylation motif at the C-terminus.

[0026] In some embodiments, the lipid anchor or transmembrane domain comprises a gp130 transmembrane domain, a CD8-alpha transmembrane domain, a prenylation motif, or a myristoylation domain derived from src, fyn, or lck.

[0027] In some embodiments, the transmembrane domain comprises a gp130 transmembrane domain.

[0028] In some embodiments, the transmembrane domain comprises a polypeptide sequence comprising amino acids 620 to 641 of gp130 (SEQ ID NO: 59).

[0029] In some embodiments, the transmembrane domain comprises a polypeptide sequence comprising the sequence set forth as SEQ ID NO:61.

[0030] In some embodiments, the SPA peptide further comprises the hinge domain of CD8-alpha.

[0031] In some embodiments, the extracellular domain comprises one or more of a CD34 epitope, a CD34 ectodomain, a BCR ectodomain, a thrombopoietin receptor (TpoR) ectodomain, or an erythropoietin receptor (EpoR) ectodomain.

[0032] In some embodiments, the thrombopoietin receptor (TpoR) ectodomain or the erythropoietin receptor (EpoR) ectodomain comprises an unpaired cysteine.

[0033] In some embodiments, the extracellular domain confers constitutive activity to the intracellular signaling domain.

[0034] In some embodiments, it comprises, from N-terminus to C-terminus, an extracellular domain comprising a CD34 epitope, a multimerization region comprising unpaired cysteine ​​residues, a gp130 transmembrane domain, and a gp130 intracellular signaling domain.

[0035] In some embodiments, the SPA peptide comprises a sequence selected from the sequences set forth in SEQ ID NOs: 1-58 or 63-104.

[0036] In some embodiments, the SPA peptide comprises the sequence set forth in SEQ ID NO:20.

[0037] In another aspect, provided herein are multimers of the SPA peptides disclosed herein.

[0038] In another aspect, provided herein are nucleic acids encoding the SPA peptides disclosed herein.

[0039] In another aspect, provided herein is a vector comprising a nucleic acid disclosed herein.

[0040] In another embodiment, i. a first chimeric polypeptide comprising a priming receptor; ii. a second chimeric polypeptide comprising a chimeric antigen receptor (CAR); and iii. an SPA peptide disclosed herein; Provided herein is a system comprising:

[0041] In some embodiments, the CAR induces expression of an SPA peptide.

[0042] In some embodiments, the SPA peptide is constitutively expressed.

[0043] In another aspect, provided herein is a cell or population of cells comprising an SPA peptide disclosed herein, a multimer disclosed herein, a nucleic acid disclosed herein, a vector disclosed herein, or a system disclosed herein.

[0044] In some embodiments, the cells are immune cells, and optionally, the immune cells are primary human immune cells.

[0045] In another aspect, provided herein is a pharmaceutical composition comprising a cell or population of cells disclosed herein and a pharmaceutically acceptable excipient.

[0046] In another aspect, provided herein is a pharmaceutical composition comprising a nucleic acid disclosed herein or a vector disclosed herein and a pharmaceutically acceptable excipient.

[0047] In another embodiment, i. providing a ribonucleoprotein complex (RNP)-nucleic acid complex, wherein the RNP comprises a nuclease domain and a guide RNA, and the nucleic acid comprises a nucleic acid disclosed herein, wherein the 5' and 3' ends of the nucleic acid comprise nucleotide sequences that are homologous to genomic sequences adjacent to an insertion site in the genome of an immune cell; ii. non-virally introducing an RNP-nucleic acid complex into an immune cell, wherein the guide RNA specifically hybridizes to a target region in the genome of the primary immune cell, and the nuclease domain cleaves the target region to create an insertion site in the genome of the immune cell; iii. editing an immune cell via insertion of a nucleic acid disclosed herein into an insertion site in the genome of the immune cell; Provided herein are methods for editing cells, including:

[0048] In another aspect, provided herein is a method of treating a disease in a subject, the method comprising administering to the subject a cell disclosed herein or a pharmaceutical composition disclosed herein.

[0049] In another aspect, provided herein is a method of inhibiting target cells in a subject, the method comprising administering to the subject a cell disclosed herein, wherein the cell inhibits the target cells.

[0050] In another embodiment, there is provided a method of modulating immune cell activity, comprising: i.1. An SPA peptide disclosed herein; 2. The system disclosed herein; 3. a nucleic acid disclosed herein, and / or 4. Vectors Disclosed Herein Obtaining cells comprising ii. contacting an immune cell with a target cell, wherein the synthetic pathway activator modulates the activity of the immune cell; Provided herein is a method comprising:

[0051] In another embodiment, there is provided a method of modulating immune cell activity, comprising: i.1. An SPA peptide disclosed herein; 2. The system disclosed herein; 3. a nucleic acid disclosed herein, and / or 4. Vectors Disclosed Herein Obtaining cells comprising ii. contacting an 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 a chimeric antigen receptor, binding of the chimeric antigen receptor to the CAR antigen on the target cell regulates the activity of the immune cell, and the synthetic pathway activator also regulates the activity of the immune cell; Provided herein is a method comprising:

[0052] In some aspects, provided herein are methods of treating a disease in a subject in need thereof, comprising determining or having determined expression of CD11c in a cell comprising a synthetic pathway activator (SPA) peptide disclosed herein or a nucleic acid disclosed herein, and administering or having administered the cell to the subject.

[0053] In some embodiments, provided herein is a method for determining the expression of SPA in a cell, the method comprising expressing one or more SPA peptides disclosed herein in the cell and determining CD11c expression in the cell.

[0054] In some embodiments, expression of CD11c in cells comprises the mRNA expression level of CD11c or the protein expression level of CD11c.

[0055] In some embodiments, the cell is an immune cell, a primary human immune cell, a natural killer (NK) cell, a T cell, a CD8+ T cell, a CD4+ T cell, a primary T cell, or a T cell precursor. [Brief explanation of the drawings]

[0056] These and other features, aspects, and advantages of the present disclosure will become better understood with regard to the following description and accompanying drawings.

[0057] [Figure 1] Logic gates 1-5 provide diagrams of various ICT transgene cassettes expressing ICT, shRNA, and SPA. [Figure 2A] 1A-1C show exemplary synthetic pathway activators and synthetic pathway activators increase potency and stem memory T phenotype. [Figure 2B] Additional exemplary synthetic pathway activators are shown. [Figure 3] All ICT cells constitutively expressed the PrimeR construct. [Figure 4] Figure 1 shows that ICT cells induced CAR expression when co-cultured with a cell line expressing the primeR antigen. [Figure 5] 1 shows that ICT cells expressing SPA exhibit approximately 2 logs higher pSTAT3 expression when compared to PrimeR- cells lacking SPA(EGFRt). [Figure 6] A shows cytotoxicity against parental K562 cells that do not express either the CAR or primeR antigens, B shows cytotoxicity against K562 cells that express only the CAR antigen, C shows cytotoxicity against K562 cells that express only the primeR antigen, and D shows cytotoxicity against K562 cells that express both the primeR and CAR antigens. [Figure 7] Shown is IFN-γ production from ICTs expressing logic gates 1–5 only in supernatants harvested from cocultures in which target cells expressed both primeR and CAR antigens. [Figure 8A]We show that ICTs expressing LG1-5 ICTs exhibited in vitro cytotoxicity against endogenous +CAR / +primeR antigen cell lines. [Figure 8B] Shows secretion of IFNγ, TNFα, GM-CSF, and IL-2 by ICT cells after co-culture with endogenous +CAR / +primeR antigen cells. [Figure 9] Figure 1 shows that co-culture with HUVEC-primeR antigen cells induced CAR protein expression in ICT cells and specific killing of CAR antigen+ cells. [Figure 10A] Figure 1 shows tumor volumes after tumor implantation in mice treated with ICTs expressing Logic Gates 1-5, RNP, or PBS generated from donor 1. [Figure 10B] Shown is the proliferation of total T cells and ICTs at day 12 post-inoculation, followed by a decline up to day 21. [Figure 10C] Total T cells expressing the priming receptor on days 12 and 21 are shown. [Figure 10D] Shown are tumor volumes after tumor implantation in mice treated with ICTs expressing Logic Gates 1-5, RNP, or PBS generated from donor 2. [Figure 10E] Shown is the proliferation of total T cells and ICTs at day 12 post-inoculation, followed by a decline up to day 21. [Figure 10F] Total T cells expressing the priming receptor on days 12 and 21 are shown. [Figure 11] A shows tumor growth inhibition (TGI) in single-positive CAR antigen-only flanks. B shows tumor growth inhibition (TGI) in double-positive primeR antigen / CAR antigen flanks. [Figure 12A] 1 shows the levels of pSTAT3 signaling induced by expression of the indicated SPAs in T cells after serum starvation. [Figure 12B] 1 shows the levels of pSTAT1 signaling induced by expression of the indicated SPAs in T cells after serum starvation. [Figure 13]A comparison of heat maps of pSTAT1 and pSTAT3 is provided for the indicated SPAs. [Figure 14-1] Granzyme B levels (upper panel) and IL-10 (lower panel) induced by the indicated SPAs are shown. [Figure 14-2] See description of Figure 14-1. [Figure 15] Tumor cell elimination (upper panel) and T cell proliferation (lower panel) of tumor cells expressing logic gate antigens in repeated stimulation assays are shown. [Figure 16] The percentage of ICT cells expressing the indicated SPA that expressed the indicated cell markers after repeated stimulation assays is shown. [Figure 17] In vivo tumor volumes after treatment with ICT expressing the indicated SPAs are shown. [Figure 18A] Figure 1 shows CD11c RNA and cell surface expression and SPA expression in both tumor and splenocytes on day 7 for both CD4+ and CD8+ cells. Cells with increased CD11c RNA and cell surface expression correlated with cells expressing SPA. [Figure 18B] Shown is CD11c (ITGAX) expression in splenocytes or tumor cells from mice treated with cells expressing SPA ICT (2 donors) compared to cells not expressing SPA ICT (1 donor), harvested on days 0 and 7 after treatment. DETAILED DESCRIPTION OF THE INVENTION

[0058] Detailed Description definition Terms used in the claims and specification, unless otherwise specified, are defined as set forth below.

[0059] As used herein, the term "gene" refers to the basic unit of heredity, consisting of a segment of DNA located along a chromosome that encodes a specific protein or segment of a protein. A gene typically includes a promoter, a 5' untranslated region, one or more coding sequences (exons), optionally introns, and a 3' untranslated region. A gene may further include a terminator, an enhancer, and / or a silencer.

[0060] As used herein, the term "locus" refers to a specific, fixed physical location on a chromosome where a gene or genetic marker is located.

[0061] The term "safe harbor locus" refers to a genetic locus at which a gene or genetic element may be integrated without disrupting the expression or regulation of adjacent genes. These safe harbor loci are also referred to as safe harbor sites (SHS). As used herein, safe harbor locus refers to an "integration site" or "knock-in site" at which a sequence encoding a transgene, as defined herein, may be inserted. In some embodiments, the insertion occurs with replacement of sequences located at the integration site. In some embodiments, the insertion occurs without replacement of sequences at the integration site. Examples of contemplated integration sites are provided in Table D.

[0062] As used herein, the term "insertion" refers to a nucleotide sequence that is integrated (inserted) into a target locus or a safe harbor site. An insertion can be used to refer to a gene or genetic element that is integrated into a target locus or a safe harbor site, for example, using homology-directed repair (HDR), CRISPR / Cas9 genome editing, or other methods for inserting nucleotide sequences into a genomic region known to those skilled in the art.

[0063] The term "insertion" refers to the manipulation of a nucleotide sequence to introduce a non-native sequence. This can be done, for example, by using restriction enzymes and ligases, whereby a DNA sequence of interest, usually encoding a gene of interest, can be incorporated into another nucleic acid molecule by digesting both molecules with the appropriate restriction enzyme to create compatible overlaps, and then joining the molecules together using ligase. Those skilled in the art are very familiar with such manipulations, and examples can be found in Sambrook et al. (Sambrook, Fritsch, & Maniatis, "Molecular Cloning: A Laboratory Manual", 2002). nd ed., Cold Spring Harbor Laboratory, 1989), which is incorporated herein by reference in its entirety, including any figures, drawings, and tables.

[0064] The "CRISPR / Cas" system refers to a broad class of bacterial systems for defense against foreign nucleic acids. CRISPR / Cas systems are found in a wide range of eubacterial and archaeal organisms. CRISPR / Cas systems include type I, type II, and type III subtypes. Wild-type type II CRISPR / Cas systems utilize an RNA-mediated nuclease, Cas9, in a complex with guide and activator RNAs to recognize and cleave foreign nucleic acids. Guide RNAs with both guide and activator RNA activity are also known in the art. In some cases, such dual-activity guide RNAs are referred to as small guide RNAs (sgRNAs).

[0065] Cas9 homologs are found in a wide variety of eubacteria, including, but not limited to, bacteria from the following taxa: Actinobacteria, Aquificae, Bacteroidetes-Chlorobi, Chlamydiae-Verrucomicrobia, Chloroflexi, Cyanobacteria, Firmicutes, Proteobacteria, Spirochaetes, and Thermotogae. An exemplary Cas9 protein is the Streptococcus pyogenes Cas9 protein. Additional Cas9 proteins and their homologs are described, for example, in 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 US 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 host cells.

[0066] As used herein, the term "Cas9" refers to an RNA-mediated nuclease (e.g., of bacterial or archaeal origin or derived therefrom). Exemplary RNA-mediated nucleases include the aforementioned Cas9 protein and homologs thereof, including, but 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, etc., refers to a complex between a Cas9 protein and a crRNA (e.g., a guide RNA or a small guide RNA), a Cas9 protein and a trans-activating crRNA (tracrRNA), a Cas9 protein and a small guide RNA, or a combination thereof (e.g., a complex containing a Cas9 protein, a tracrRNA, and a crRNA guide RNA).

[0067] As used herein, the phrase "immune cell" includes all cell types that can give rise to immune cells, including hematopoietic cells, such as hematopoietic stem cells, pluripotent stem cells, and induced pluripotent stem cells (iPSCs). In some embodiments, the immune cell is a B cell, a 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 precursor, or a dendritic cell. In some embodiments, the cell is an innate immune cell.

[0068] As used herein, the term "primary" in the context of primary cells or primary stem cells refers to cells that have not been transformed or immortalized. Such primary cells can be cultured, subcultured, or passaged a limited number of times (e.g., 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, primary cells are adapted to in vitro culture conditions. In some cases, primary cells are isolated from organisms, systems, organs, or tissues, optionally sorted, and used directly, for example, without culture or subculture. In some cases, 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-γ, or a combination thereof.

[0069] 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 specific foreign antigens in the body. These terms also refer to the major type of white blood cell that has various roles in the immune system, including activating and inactivating other immune cells. T cells can be any T cell, such as cultured T cells, e.g., primary T cells, or T cells from cultured T cell lines, e.g., JuRKAt, SuPT1, etc., or mammalian-derived T cells. T cells include, but are not limited to, naive T cells, stimulated T cells, primary T cells (e.g., not cultured), 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 subpopulations thereof. T cells can be CD3+ cells. T cells can be CD4+ cells. + , CD8 + , or CD4 + and CD8 +T cells can be any type of T cell, CD4+ / CD8+ double-positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral T cells, including, but not limited to, blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating lymphocytes (TILs), memory T cells, naive T cells, T cells, regulatory T cells, γδ T cells, and the like. It can be any T cell at any stage of development. Additional types of helper T cells include Th3 (Treg) cells, Th17 cells, Th9 cells, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (Tcm cells) and effector memory T cells (Tem cells and TEMRA cells). T cells can also refer to genetically modified T cells, such as T cells modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR). T cells can also be differentiated from stem or progenitor cells.

[0070] "CD4+ T cells" refer to a subset of T cells that express CD4 on their surface and are involved in cellular immune responses. CD4+ T cells are characterized by a post-stimulation secretory profile that can include secretion of cytokines such as IFN-γ, TNF-α, IL-2, IL-4, and IL-10. "CD4" is a 55 kD glycoprotein originally defined as a differentiation antigen on T lymphocytes, but has also been found on other cells, including monocytes / macrophages. The CD4 antigen is a member of the immunoglobulin superfamily and has been suggested as a binding recognition element in MHC (major histocompatibility complex) class II-restricted immune responses. On T lymphocytes, the CD4 antigen defines helper / inducer subsets.

[0071] "CD8+ T cells" refer to a subset of T cells that express CD8 on their surface, are MHC class I restricted, and function as cytotoxic T cells. The "CD8" molecule is a differentiation antigen present on thymocytes and on cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin superfamily and is the binding recognition element in major histocompatibility complex class I restricted interactions.

[0072] As used herein, the phrase "hematopoietic stem cells" refers to a type of stem cell that can give rise to blood cells. Hematopoietic stem cells can give rise to myeloid or lymphoid cells, or a combination thereof. Hematopoietic stem cells are primarily found in the bone marrow, but they can also be isolated from peripheral blood, or fractions thereof. Various cell surface markers can be used to identify, select, or purify hematopoietic stem cells. In some cases, hematopoietic stem cells express c-kit + and Lin - In some cases, human hematopoietic stem cells are identified as CD34 + , CD59 + , Thy1 / CD90 + , CD38 lo / - , C-kit / CD117 + , lin - In some cases, human hematopoietic stem cells are identified as CD34 - , CD59 + , Thy1 / CD90 + , CD38 lo / - , C-kit / CD117 + , lin - In some cases, human hematopoietic stem cells express CD133 + , CD59 + , Thy1 / CD90 + , CD38 lo / - , C-kit / CD117 + , lin - In some cases, mouse hematopoietic stem cells express CD34 lo / - , SCA-1 + , Thy1 + / lo , CD38+ , C-kit + , lin - In some cases, hematopoietic stem cells express CD150 + CD48 - CD244 - is.

[0073] As used herein, the phrase "hematopoietic cells" refers to cells derived from hematopoietic stem cells. Hematopoietic cells can be obtained or provided by isolation from an organism, system, organ, or tissue (e.g., blood, or a fraction thereof). Alternatively, hematopoietic cells can be obtained or provided by isolating hematopoietic stem cells and differentiating the stem cells. Hematopoietic cells include cells with limited potential to differentiate into additional cell types. Such hematopoietic cells include, but are not limited to, multipotent progenitor cells, lineage-restricted progenitor cells, common myeloid progenitor cells, granulocyte-macrophage progenitor cells, or megakaryocyte-erythroid progenitor cells. Hematopoietic cells include lymphoid and myeloid cells, such as lymphocytes, erythrocytes, granulocytes, monocytes, and platelets.

[0074] As used herein, the term "construct" refers to a macromolecule or complex of molecules that includes a polynucleotide.

[0075] As used herein, the term "integration" refers to the process of stably inserting one or more nucleotides of a construct into a cell genome, i.e., covalently linking them to a nucleic acid sequence in the chromosomal DNA of the cell. It can also refer to a nucleotide deletion at the site of integration. If there is a deletion at the insertion site, "integration" can further include the replacement of the deleted endogenous sequence or nucleotides with one or more inserted nucleotides.

[0076] As used herein, the term "exogenous" refers to a molecule or activity that is introduced into a host cell and is not native to that cell. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material, e.g., by integration into a host chromosome, or as non-chromosomal genetic material such as a plasmid. Thus, when used in connection with expression of an encoding nucleic acid, the term refers to introducing 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, when used in connection with expression of an encoding nucleic acid, the term refers to expression of an encoding nucleic acid that is contained within the cell and not exogenously introduced.

[0077] The term "heterologous" refers to a nucleic acid or polypeptide sequence or domain that is not native to the flanking sequence, e.g., a heterologous sequence is not found in nature coupled to a nucleic acid or polypeptide sequence that occurs at one or both termini.

[0078] The term "homologous" refers to a nucleic acid or polypeptide sequence or domain that is natural to the adjacent sequence, e.g., a homologous sequence is found in nature coupled to a nucleic acid or polypeptide sequence occurring at one or both termini.

[0079] As used herein, a "polynucleotide donor construct" refers to a nucleotide sequence (e.g., a DNA sequence) that is genetically inserted into a polynucleotide and is exogenous to that polynucleotide. The polynucleotide donor construct is transcribed into RNA and optionally translated into a polypeptide. The polynucleotide donor construct can include prokaryotic sequences, cDNA derived from eukaryotic mRNA, genomic DNA sequences derived from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. For example, the polynucleotide donor construct can be an miRNA, an shRNA, a native polypeptide (i.e., a naturally occurring polypeptide) or a fragment thereof, or a variant polypeptide (e.g., a naturally occurring polypeptide having less than 100% sequence identity to the native polypeptide) or a fragment thereof.

[0080] As used herein, the terms "complementary" or "complementarity" refer 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 include a DNA target sequence that is fully complementary or substantially complementary (e.g., has 1-4 mismatches) to a sequence, e.g., a genomic sequence in a cell.

[0081] As used herein, the term "transgene" refers to a polynucleotide that is transferred from one organism to another, either naturally or by any of several genetic engineering techniques, and that is optionally translated into a polypeptide. As used, transgene can refer to a polynucleotide that encodes a polypeptide.

[0082] "Protein," "polypeptide," and "peptide" are used interchangeably herein.

[0083] As used herein, the terms "operably linked" or "operably linked" refer to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the function of the other. For example, a promoter is operably linked to a coding sequence or functional RNA if it is capable of affecting the expression of the coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter). A coding sequence can be operably linked to a regulatory sequence in both sense and antisense orientation.

[0084] As used herein, the term "developmental cell state" refers, for example, to when a cell is inactive, actively developing, differentiating, senescent, etc. A developmental cell state can also refer to a cell in a precursor state (e.g., a T cell precursor).

[0085] As used, the term "encoding" refers to a nucleic acid sequence that encodes a protein or polypeptide of interest. The nucleic acid sequence can be either a DNA or an RNA molecule. In a preferred embodiment, the molecule is a DNA molecule. In another preferred embodiment, the molecule is an RNA molecule. When present as an RNA molecule, it contains a sequence that instructs the host cell's ribosomes to begin translation (e.g., a start codon, ATG) and a sequence that instructs the ribosomes to terminate translation (e.g., a stop codon). Between the start codon and the stop codon is an open reading frame (ORF). Such terms are known to those of skill in the art.

[0086] 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 the engineered cells or populations thereof provided herein. In some aspects, the disease or condition is cancer.

[0087] As used herein, the term "promoter" refers to a nucleotide sequence (e.g., a DNA sequence) capable of controlling the expression of a coding sequence or functional RNA. A promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. A promoter can be derived entirely from a native gene, can be composed of different elements from different promoters found in nature, and / or can include synthetic DNA segments. A promoter, as contemplated herein, can be endogenous to the cell of interest or exogenous to the cell of interest. It will be understood by those skilled in the art that different promoters can induce gene expression in different tissues or cell types, or at different developmental stages, or in response to different environmental conditions. As is known in the art, promoters can be selected according to the strength of the promoter and / or the conditions under which the promoter is active, e.g., constitutive promoters, strong promoters, weak promoters, inducible / repressible promoters, tissue-specific or developmentally regulated promoters, cell cycle-dependent promoters, etc.

[0088] The promoter may be an inducible promoter (e.g., a heat shock promoter, a tetracycline-regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, an estrogen receptor-regulated promoter, an HNF1α promoter, etc.). The promoter may be a constitutive promoter (e.g., a CMV promoter, a UBC promoter). In some embodiments, the promoter may be a spatially restricted and / or temporally restricted promoter (e.g., a tissue-specific promoter, a cell-type specific promoter, etc.). See, e.g., U.S. Publication No. 20180127786, the disclosure of which is incorporated herein by reference in its entirety.

[0089] As contemplated herein, gene editing can involve knocking in or knocking out a gene (or nucleotide sequence). As used herein, the term "knock-in" refers to the addition of a DNA sequence or a fragment thereof to a genome. Such a knocked-in DNA sequence may include an entire gene or multiple genes, and may include regulatory sequences associated with a gene or any portion or fragment thereof. For example, a polynucleotide donor construct encoding a protein can be inserted into the genome of a cell harboring a mutant gene. In some embodiments, the knock-in strategy involves replacing an existing sequence with a provided sequence, e.g., replacing a mutant allele with a wild-type copy. On the other hand, the term "knock-out" refers to the removal of a gene or the expression of a gene. For example, a gene can be knocked out by either deleting or adding a nucleotide sequence, which leads to a disruption of the reading frame. As another example, a gene can be knocked out by replacing a portion of the gene with an unrelated (e.g., non-coding) sequence.

[0090] As used herein, the term "non-homologous end joining" or NHEJ refers to a cellular process in which broken or nicked ends of a DNA strand are directly ligated without the need for a homologous template nucleic acid. NHEJ can result in the addition, deletion, substitution, or combination of one or more nucleotides at the repair site.

[0091] As used herein, "homology-directed repair" or HDR refers to the cellular process in which the broken or nick end of a DNA strand is 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 a homologous sequence (sister chromatid, homologous chromosome, or repeat region on the same or different chromosome) located elsewhere in the genome. Alternatively, exogenous template nucleic acid can be introduced to obtain specific HDR-induced changes in the sequence at the target site. In this way, specific mutations can be introduced at the break site.

[0092] As used herein, single-stranded DNA template or double-stranded DNA template refers to the DNA oligonucleotide that can be used by cell as template for HDR.Generally, single-stranded DNA template or double-stranded DNA template has at least one region of homology with target site.In some cases, single-stranded DNA template or double-stranded DNA template has two homologous regions adjacent to the region that contains the heterologous sequence that is inserted into target cleavage site.

[0093] The terms "vector" and "plasmid" are used interchangeably and, as used herein, refer to a polynucleotide vehicle useful for introducing genetic material into a cell. A vector can be linear or circular. A vector can integrate into a target genome of a host cell or replicate independently within the host cell. A vector can include, for example, an origin of replication, a multicloning site, and / or a selectable marker. An expression vector typically includes an expression cassette. Vectors and plasmids include, but are not limited to, integrative vectors, prokaryotic plasmids, eukaryotic plasmids, plant synthetic chromosomes, episomes, cosmids, and artificial chromosomes.

[0094] As used herein, the term "introducing" in the context of introducing a nucleic acid or a complex containing a nucleic acid, such as an RNP-DNA template complex, refers to the translocation of the nucleic acid sequence or RNP-DNA template complex from outside the cell to inside the cell. In some cases, introducing refers to the translocation of the nucleic acid or complex from outside the cell to 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, etc.

[0095] As used herein, the term "expression cassette" refers to a recombinantly or synthetically produced polynucleotide construct that includes 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 within the host cell, or transcription and translation of the selected polynucleotide within the host cell. The expression cassette can, for example, be integrated into the genome of the host cell or can be present in an expression vector.

[0096] As used herein, the phrase "subject in need thereof" refers to a subject who exhibits and / or is diagnosed with one or more symptoms or signs of a disease or disorder described herein.

[0097] "Chemotherapeutic agents" refer to chemical compounds useful in the treatment of cancer. Chemotherapeutic agents include "antihormonal agents" or "endocrine therapy agents" that act to regulate, reduce, block, or inhibit the effects of hormones that can promote cancer growth.

[0098] The term "composition" refers to a mixture containing, for example, engineered cells or proteins contemplated herein. In some embodiments, a composition may contain additional components, such as adjuvants, stabilizers, excipients, etc. The term "composition" or "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredients contained therein to be effective in treating a subject, and that does not contain additional ingredients that are unacceptably toxic to a subject in the amounts provided in the pharmaceutical composition.

[0099] The term "in situ" refers to processes that occur within living cells grown isolated from an organism, for example, grown in tissue culture.

[0100] The term "in vivo" refers to a process that occurs within a living organism.

[0101] As used herein, the term "ex vivo" generally includes experiments or measurements performed in or on living tissue, preferably in an artificial environment outside the organism, preferably with minimal variation from natural conditions.

[0102] As used herein, the term "mammal" includes both humans and non-humans, including, but not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0103] The term "percent identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have the same specified percentage of nucleotide or amino acid residues when compared and aligned for maximum correspondence, as determined using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those of skill in the art) or by visual inspection. Depending on the application, the percent "identity" can exist over a region of the sequences being compared, e.g., over a functional domain, or alternatively, over the entire length of the two sequences being compared.

[0104] For sequence comparison, typically, one sequence serves 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 as necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence(s) relative to the reference sequence based on the designated program parameters.

[0105] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc' Nat'l. Acad. Sci. USA 85:2444 (1988), by computer 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).

[0106] 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 / ).

[0107] The term "sufficient amount" means an amount sufficient to produce a desired effect, for example, an amount sufficient to modulate protein aggregation in a cell.

[0108] The term "therapeutically effective amount" is an amount effective to ameliorate symptoms of the disease.

[0109] The term "ameliorate" refers to any therapeutically beneficial outcome in the treatment of a disease state, for example, a cancer disease state, a reduction in its severity or progression, a remission thereof, or a cure thereof.

[0110] As used herein, the term "effective amount" refers to a sufficient amount of a compound (e.g., a composition described herein, a cell described herein) to produce a beneficial or desired result. 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 route of administration.

[0111] As used herein, the term "treating" includes any effect that results in an improvement of the condition, disease, disorder, etc. or improves the symptoms thereof, e.g., alleviating, reducing, modulating, ameliorating, or eliminating.

[0112] The terms "modulate" and "modulation" refer to decreasing or inhibiting, or alternatively activating or increasing, the recited variable.

[0113] The terms "increase" and "activate" refer to a 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 increase in the recited variable.

[0114] The terms "reduce" and "inhibit" refer to a 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 decrease in the recited variable.

[0115] With respect to antibody binding to a target molecule, the terms "binds to," "specific binding to," "specifically binds to," "specific for," "selectively binds to," and "selective for" a particular antigen (e.g., a polypeptide target) or epitope on a particular antigen refer to binding that is measurably different from nonspecific or nonselective interactions (e.g., with non-target molecules). For example, an antibody that "selectively binds to" or "specifically binds to" an antigen is an antigen-binding moiety that binds to the antigen with high affinity and does not significantly bind to other, unrelated antigens. Specific binding can be measured, for example, by measuring binding to the target molecule and comparing it to binding to non-target molecules. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. In this case, specific binding is indicated if the binding of the antibody to the target molecule is competitively inhibited by the control molecule.

[0116] "Affinity" refers to the strength of the sum 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 otherwise indicated, as used herein, "affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen or epitope). The affinity of a molecule X for its partner Y is determined by the dissociation equilibrium constant (K D ) 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®).

[0117] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops ("hypervariable loops"). Typically, a naturally occurring four-chain antibody contains six HVRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs typically contain amino acid residues from the hypervariable loops and / or from the complementarity-determining regions (CDRs), the latter being those with the highest sequence variability and / or involved in antigen recognition. With the exception of CDR1 in VH, CDRs typically contain 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 interchangeably herein with respect to the portions of the variable regions that form the antigen-binding region. This particular region is described by Kabat et al., US Department of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and Chothia et al., J Mol Biol 196:901-917 (1987), and the definitions include overlapping or subsets of amino acid residues when compared with each other. However, application of either definition to refer to a CDR of an antibody or variant thereof is intended to be within the scope of the term as defined and used herein. The exact residue numbers encompassing a particular CDR will vary depending on the sequence and size of the CDR. One of ordinary skill in the art can routinely determine which residues comprise a particular CDR, given the amino acid sequence of the variable region of an antibody.

[0118] The amino acid sequence boundaries of the CDRs can be determined by one of skill in the art using any of several known numbering schemes, including those described by Kabat et al. supra (the "Kabat" numbering scheme), Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (the "Chothia" numbering scheme), MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (the "Contact" numbering scheme), Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (the "IMGT" numbering scheme), and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 (the "AHo" numbering scheme), each of which is incorporated by reference in its entirety.

[0119] 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.

[0120] CDRs can 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, which is incorporated by reference in its entirety.

[0121] The "EU numbering scheme" is generally used when referring to residues in antibody heavy chain constant regions (e.g., as reported in Kabat et al., supra). Unless otherwise stated, the EU numbering scheme is used to refer to residues in antibody heavy chain constant regions described herein.

[0122] As used herein, the term "single chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In certain such embodiments, 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 a light chain (VL), which is connected from its C-terminus to the N-terminus of the variable domain of a heavy chain (VH) by a polypeptide chain. Alternatively, an scFv comprises a polypeptide chain, and the C-terminus of the VH is connected to the N-terminus of the VL by a polypeptide chain.

[0123] A "Fab fragment" (also called fragment antigen binding) contains the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1), as well as the variable domains VL and VH on the light and heavy chains, respectively. The variable domains contain the complementarity-determining loops (CDRs, also called hypervariable regions) 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 CH1 domain, including one or more cysteines from the antibody hinge region.

[0124] An "F(ab')2" fragment contains two Fab' fragments linked by a disulfide bond near the hinge region. F(ab')2 fragments can be produced, for example, by recombinant methods or by pepsin digestion of an intact antibody. F(ab')2 fragments can be dissociated, for example, by treatment with β-mercaptoethanol.

[0125] An "Fv" fragment comprises a non-covalently associated dimer of one heavy- and one light-chain variable domain.

[0126] A "single-chain Fv" or "scFv" comprises 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 enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Plückthun 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 WO 93 / 16185, U.S. Patent No. 5,571,894, and U.S. Patent No. 5,587,458.

[0127] 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 other variable domains. Single domain antibodies and fragments thereof are described in Arabi Ghahroudi et al., FEBS Letters, 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 easily expressed as fusion partners with the Fc chain of an antibody (Harmsen MM, De Haard HJ (2007) "Properties, production, and applications of camelid single-domain antibody fragments", Appl. Microbiol. Biotechnol. 77(1):13-22).

[0128] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0129] Synthetic pathway activators In various embodiments, the systems disclosed herein employ one or more "synthetic pathway activator" (SPA) peptides. CAR-expressing immune cells can be limited by the need for in vivo expansion after infusion. To achieve robust expansion, T cells use three signals: antigen stimulation, costimulation, and cytokine-induced stimulation. CAR activation is sufficient to induce the first two signals but cannot recapitulate cytokine signaling. Furthermore, the tumor microenvironment is often immunosuppressive and lacks pro-inflammatory cytokines. Thus, for example, SPA peptides can be used to stimulate robust in vivo expansion and enhance desirable properties (i.e., survival, persistence, and efficacy) of T cells expressing the priming receptors and / or CARs described herein.

[0130] In one aspect, provided herein is a synthetic pathway activator (SPA) peptide comprising a chimeric polypeptide comprising a lipid anchor or transmembrane domain; an intracellular signaling domain; and a multimerization region. In another aspect, provided herein is a synthetic pathway activator (SPA) peptide comprising a chimeric polypeptide comprising an extracellular domain, a lipid anchor or transmembrane domain; an intracellular signaling domain; and a multimerization region. In some embodiments, the SPA peptide further comprises the hinge domain of CD8-alpha. In some embodiments, the SPA peptide is constitutively expressed in cells. In some embodiments, expression of the SPA peptide in cells is induced, for example, by T cell activation. For example, after engagement of a CAR T cell with a CAR cognate ligand on a target cell, inducible SPA can be expressed. In some embodiments, expression of the SPA peptide in cells is induced by priming receptor and / or CAR signaling.

[0131] SPA Structure In various embodiments, the SPA peptides mimic the activation of interleukin signaling. Interleukin receptors are cytokine receptors that signal through signal transducers and activators of transcription (STAT) transcription factors (e.g., STAT1, STAT3, and STAT5). Interleukin receptors typically function by dimerization in response to ligand binding. Upon dimerization, the receptor can bind to Janus-associated kinases (JAKs) to induce JAK cross-phosphorylation and downstream "JAK / STAT" signaling. Thus, induction of receptor agonism or ligand-independent receptor dimerization can be used to allow synthetic pathway activators to induce constitutive receptor activity and, therefore, constitutive cytokine signaling.

[0132] In various embodiments, the SPA peptide comprises an interleukin receptor or a functional fragment thereof. In some embodiments, the SPA comprises or is derived from an interleukin receptor intracellular signaling domain or a functional fragment thereof. In some embodiments, the SPA peptide comprises or is derived from an interleukin-6 signal transduction substrate (IL6ST) polypeptide or a functional fragment thereof. Interleukin-6 signal transduction substrate (IL6ST) is also known as glycoprotein 130 (gp130). The SPA can comprise a multimerized SPA peptide, e.g., two dimerized, e.g., homodimerized, SPA peptides. Multimerization encompasses dimerization, trimerization, tetramerization, or higher order combinations of interacting SPA peptides.

[0133] Intracellular signaling domains In some embodiments, the intracellular signaling domain induces phosphorylation of STAT1, STAT3, and / or STAT5. In some embodiments, the functional SPA induces phosphorylation of STAT1, STAT3, and / or STAT5. In some embodiments, the intracellular signaling domain comprises a type I cytokine receptor superfamily box 1 (IWPNVDP (SEQ ID NO: 106)) or box 2 (VSVVEIEANDKKP (SEQ ID NO: 107)) peptide motif. In some embodiments, the intracellular signaling domain comprises a tyrosine phosphorylation motif comprising YXXQ or YXPQ. In some embodiments, the intracellular signaling domain comprises one or more minimal STAT binding motifs from STAT1, STAT3, and / or STAT5 proteins. In some embodiments, the intracellular signaling domain comprises one or more minimal STAT binding motifs from STAT1 and STAT3 proteins. In some embodiments, the intracellular signaling domain comprises one or more minimal STAT binding motifs from STAT1 and STAT5 proteins. In some embodiments, the intracellular signaling domain comprises one or more minimal STAT binding motifs from STAT3 and STAT5 proteins.

[0134] In some embodiments, the intracellular signaling domain comprises a polypeptide sequence from an interleukin receptor.

[0135] In some embodiments, the interleukin receptor comprises a gp130 intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a polypeptide sequence comprising amino acids 642 to 918 of gp130 (SEQ ID NO: 59).

[0136] In some embodiments, the interleukin receptor comprises a truncated gp130 intracellular signaling domain. In some embodiments, the SPA peptide comprises a truncated gp130 intracellular domain. In some embodiments, the truncated gp130 intracellular signaling domain comprises a truncated gp130 intracellular domain of a sequence selected from the group set forth in SEQ ID NOs: 10-16 and 71-77. In some embodiments, the truncated gp130 intracellular signaling domain comprises a sequence that is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the gp130 intracellular signaling domain provided in the sequence set forth in SEQ ID NO: 60.

[0137] In some embodiments, the truncated gp130 intracellular domain comprises the GP130Δ771-811 fragment. In some embodiments, the truncated gp130 intracellular domain comprises the GP130Δ707-755 fragment. In some embodiments, the truncated gp130 intracellular domain comprises the GP130Δ818-901 fragment. In some embodiments, the truncated gp130 intracellular domain comprises one or more truncations selected from the group consisting of GP130Δ707-755, GP130Δ771-811, and GP130Δ818-901. In some embodiments, the truncated gp130 intracellular signaling domain comprises a deletion of amino acids 771-811 of gp130 (SEQ ID NO:59). In some embodiments, the truncated gp130 intracellular signaling domain comprises a deletion of amino acids 707-755 of gp130 (SEQ ID NO:59). In some embodiments, the truncated gp130 intracellular signaling domain comprises a deletion of amino acids 818-901 of gp130 (SEQ ID NO:59). In some embodiments, the truncated gp130 intracellular signaling domain comprises a deletion of one or more (e.g., one, two, or three) amino acids selected from the group consisting of amino acids 707-755, 771-811, and 818-901 of gp130 (SEQ ID NO:59). In other embodiments, the gp130 intracellular domain comprises a Y759F mutation (resulting in a SOCS-proof mutant). In some embodiments, the gp130 intracellular signaling domain further comprises a Y759F mutation of gp130 (SEQ ID NO:59). In other embodiments, the gp130 intracellular domain comprises a Y759F mutation of gp130 (SEQ ID NO:59) and a Δ771-811 truncation (gp130Y759FΔ771-811). In another embodiment, the gp130 intracellular domain comprises a Y759F mutation and a Δ707-755 truncation of gp130 (SEQ ID NO: 59) (gp130Y759FΔ707-755). In another embodiment, the gp130 intracellular domain comprises a Y759F mutation and a Δ818-901 truncation of gp130 (SEQ ID NO: 59) (gp130Y759FΔ818-901).

[0138] In some embodiments, the gp130 intracellular signaling domain comprises an amino acid sequence that is 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 set forth in SEQ ID NO: 60. In some embodiments, the gp130 intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO:60.

[0139] In some embodiments, the SPA peptide can include a ligand agonist (e.g., a cytokine, e.g., an interleukin) that allows for constitutive activation of the SPA. In some embodiments, the cytokine receptor and soluble agonist are co-expressed. In some embodiments, the cytokine receptor and membrane-bound agonist are co-expressed.

[0140] Lipid anchor and transmembrane domain In various embodiments, the SPA peptide is anchored to the cell membrane via a lipid anchor. In other embodiments, the SPA peptide comprises a transmembrane domain. In various embodiments, the SPA peptide comprises a lipid anchor or a transmembrane domain comprising a gp130 transmembrane domain, a CD8-alpha transmembrane domain, a prenylation motif, or a myristoylation domain derived from src, fyn, or lck.

[0141] In some embodiments, the SPA peptide comprises a src-derived myristoylation domain, a fyn-derived myristoylation domain, or a lck-derived myristoylation domain. In other embodiments, the SPA peptide comprises a prenylation motif. In some embodiments, the SPA peptide comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the SPA peptide comprises the transmembrane domain of an interleukin receptor.

[0142] In some embodiments, the transmembrane domain comprises an amino acid sequence that is 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 set forth in SEQ ID NO: 61. In some embodiments, the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 61.

[0143] Multimerization region In various embodiments, one or more structural modifications can be made to confer constitutive activity to an SPA or functional fragment thereof. In some embodiments, structures or mutations can be added to induce multimerization of an SPA (e.g., dimerization, trimerization, tetramerization, or higher multimers). In some embodiments, one or more amino acids can be mutated to cysteine ​​to form one or more disulfide bond(s), for example, between two receptor monomers. In some embodiments, one or more amino acids can be inserted into a wild-type receptor polypeptide to promote dimerization, for example, by forming 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 unpaired cysteines can be in the extracellular domain, transmembrane domain, hinge domain, 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.

[0144] In some embodiments, the exogenous polypeptide is operably linked to a cytokine receptor or functional fragment thereof to cause their multimerization or dimerization. In some embodiments, a leucine zipper polypeptide is operably 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 operably 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), an erythropoietin receptor (EpoR) ectodomain, or a thrombopoietin receptor (TpoR) ectodomain.

[0145] 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 regions. 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. An exemplary BCR domain is provided in SEQ ID NO: 239. An exemplary VASP domain is provided in SEQ ID NO: 240. 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 ​​residues and a leucine zipper. In some embodiments, the SPA peptide comprises one or more VASP domain polypeptides (e.g., SEQ ID NO: 240) to promote multimerization. In some embodiments, the VASP domain is the tetramerization domain of VASP. In some embodiments, the BCR domain comprises a coiled-coil tetramerization region. In such embodiments, the BCR ectodomain can mediate multimerization through non-covalent interactions.

[0146] In some embodiments, the multimerization domain is intracellular when expressed by a cell. In some embodiments, the multimerization domain is extracellular when expressed by a cell. For example, an SPA peptide can include, from N-terminus to C-terminus, i) one or more multimerization domains-extracellular domain-transmembrane domain-intracellular signaling domain, ii) extracellular domain-one or more multimerization domains-transmembrane domain-intracellular signaling domain, iii) one or more multimerization domains-transmembrane domain-intracellular signaling domain, iv) one or more multimerization domains-lipid anchor-intracellular signaling domain, v) extracellular domain-transmembrane domain-one or more multimerization domains-intracellular signaling domain, vi) lipid anchor-one or more multimerization domains-intracellular signaling domain, vii) extracellular domain-transmembrane domain-intracellular signaling domain-one or more multimerization domains, or viii) lipid anchor-intracellular signaling domain-one or more multimerization domains, or any combination thereof.

[0147] Extracellular domain The SPA peptides disclosed herein can also comprise an extracellular domain. In some embodiments, the extracellular domain confers constitutive activity to the intracellular signaling domain. In some embodiments, the extracellular domain comprises a CD34 ectodomain (e.g., a CD34 ectodomain, SEQ ID NO: 238). In some embodiments, the extracellular domain comprises a CD34 epitope (e.g., a QBEND10 epitope, SEQ ID NO: 238). In some embodiments, the extracellular domain comprises a type I cytokine receptor ectodomain (e.g., a thrombopoietin receptor (TpoR) ectodomain or an erythropoietin receptor (EpoR) ectodomain). In some embodiments, the type I cytokine receptor ectodomain (e.g., a thrombopoietin receptor (TpoR) ectodomain or an erythropoietin receptor (EpoR) ectodomain) further comprises a type I cytokine receptor transmembrane domain (e.g., a thrombopoietin receptor (TpoR) transmembrane domain or an erythropoietin receptor (EpoR) transmembrane domain). In some embodiments, the extracellular domain comprises one or more of a CD34 epitope (e.g., the QBEND10 epitope, SEQ ID NO: 238), a CD34 ectodomain (SEQ ID NO: 242), a BCR ectodomain (SEQ ID NO: 239), a thrombopoietin receptor (TpoR) domain (SEQ ID NO: 243), or an erythropoietin receptor (EpoR) ectodomain (SEQ ID NO: 241). In various embodiments, the thrombopoietin receptor (TpoR) ectodomain or the erythropoietin receptor (EpoR) ectodomain further comprises one or more unpaired cysteines. In some embodiments, the BCR ectodomain comprises a coiled-coil tetramerization region. In such embodiments, the BCR ectodomain can mediate multimerization through non-covalent interactions.

[0148] Exemplary SPA In some embodiments, the SPA peptide comprises leucine zipper-gp130 (interchangeably referred to herein as "L-gp130" or "gp130") or the L-gp130 intracellular signaling domain. L-gp130 is a homodimer in which each monomer comprises (a) an extracellular domain and a c-Jun leucine zipper that contains an inserted cysteine ​​residue that forms a disulfide bond with another monomer, and (b) an IL6ST (GP130) transmembrane domain and an intracellular signaling domain. The cysteine ​​residues and leucine zipper on each polypeptide can induce the formation of a stable homodimer that mimics constitutive IL-6R activation. Additional details regarding the construction of L-gp130 are described in Stuhlmann-Laeisz et al. Mol Biol Cell. 2006 Jul;17(7):2986-95 and WO2020200325, which are incorporated by reference in their entireties. L-gp130 and other exemplary SPAs described herein are provided in Figure 2B.

[0149] In some embodiments, the SPA peptide comprises, from N- to C-terminus, an extracellular domain comprising a CD34 epitope or a CD34 extracellular domain, a multimerization region comprising one or more unpaired cysteine ​​residues, a GP130 (IL6ST) transmembrane domain, and a GP130 (IL6ST) intracellular signaling domain. In some embodiments, the SPA peptide further comprises a leader sequence at the N-terminus. In some embodiments, the leader sequence is a CD8α 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: 108), MLVRRGARAGPRMPRGWTALCLLSLLPSGFM (SEQ ID NO: 109), MDHLGASLWPQVGSLCLLLAGAAW (SEQ ID NO: 110), or MLTLQTWLVQALFIFLTTESTG (SEQ ID NO: 111).

[0150] In some embodiments, the SPA peptide comprises a sequence selected from the group set forth in SEQ ID NOS: 1-58 or 63-104. SPAs with N-terminal leader sequences are provided in SEQ ID NOS: 1-58. SPAs without N-terminal leader sequences are provided in SEQ ID NOS: 63-104. In some embodiments, the SPA peptide comprises a sequence selected from the group set forth in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102 , 51, 52, 53, 54, 55, 56, 57, 58, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, or 104. In some embodiments, the SPA peptide comprises a sequence selected from the group set forth in SEQ ID NOs: 1-58 or 63-104. In some embodiments, the SPA peptide is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, or 104. In some embodiments, the SPA peptide comprises a sequence set forth in SEQ ID NO:20.In some embodiments, the SPA peptide comprises the sequence set forth in SEQ ID NO: 81. In some embodiments, the SPA peptide comprises a sequence that is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the sequence set forth in SEQ ID NO: 20 or 81.

[0151] Logic Gate System As used herein, "logic gate," "circuit," "circuit receptor," "system," or "system receptor" refers to a bipartite 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, and the priming receptor is expressed in the cell. The intracellular domain of the priming receptor is cleaved from the transmembrane domain when the priming receptor binds to its target antigen. The intracellular domain then translocates into the cell nucleus, where it can induce expression of the chimeric antigen receptor.

[0152] In one aspect, a system is provided herein that includes a priming receptor that binds to a target antigen and a chimeric antigen receptor that binds to the target antigen, wherein a transcription factor in the intracellular domain of the priming receptor can induce the expression of a CAR and / or an SPA. Such a system is alternatively referred to as a "logic gate" or "circuit." In some aspects, the system is encoded by a nucleic acid transgene inserted into an immune cell. The system may be encoded on a single nucleic acid insert or fragment that includes both transgenes, or on two nucleic acids that individually encode the system transgenes. The priming receptor and CAR of the system can be arranged in any order on a single nucleic acid. For example, the priming receptor can be at the 5' end and the CAR at the 3' end, or the CAR can be at the 5' end and the priming receptor at the 3' end.

[0153] A constitutive promoter can be operably linked to a nucleotide sequence encoding a priming receptor and / or an SPA. An inducible promoter can also be operably linked to a nucleotide sequence encoding a CAR. In some embodiments, when the system is encoded by a single nucleic acid insert or fragment containing both transgenes, the nucleic acid can comprise, from 5' to 3', a constitutive promoter; a nucleotide sequence encoding a priming receptor; an inducible promoter; and a nucleotide sequence encoding a chimeric antigen receptor. Alternatively, the nucleic acid can comprise, from 5' to 3', an inducible promoter; a nucleotide sequence encoding a chimeric antigen receptor; a constitutive promoter; and a nucleotide sequence encoding a priming receptor. In some embodiments, the inducible promoter comprises one or more HNF1α 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 HNF1α enhancer elements). In some embodiments, the constitutive promoter comprises an EF1α promoter.

[0154] Priming Receptors Provided herein are priming receptors that include an extracellular antigen-binding domain that specifically binds to a target antigen, and one or more intracellular domains from or derived from a transcriptional regulator, and / or a DNA-binding domain.

[0155] In certain embodiments of the present disclosure, the priming receptor is a synthetic receptor based on the Notch protein. Binding of a native Notch receptor to its cognate ligand, such as a ligand from the Delta family of proteins, triggers intramembrane proteolysis, cleaving an intracellular fragment of the Notch protein. This intracellular fragment is a transcriptional regulator that functions only when cleaved from Notch. Cleavage can occur through sequential proteolysis by ADAM metalloproteases and the gamma-secretase complex. This intracellular fragment enters the cell nucleus and activates intercellular signaling genes. In contrast to the native Notch protein, the synthetic Notch priming receptor replaces the native Notch intracellular fragment with one that transcribes a gene encoding a selected protein, such as a CAR, upon release of the intracellular fragment from the priming receptor.

[0156] Notch receptors have a modular domain organization. The ectodomain of Notch receptors consists of a series of N-terminal epidermal growth factor (EGF)-like repeats involved in 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 that are unique to Notch receptors and have been widely reported to be involved in preventing premature receptor activation. The heterodimerization (HD) domain of Notch1 is split by furin cleavage, resulting in its N-terminal portion terminating in the extracellular subunit and its C-terminal half forming the beginning of the transmembrane subunit. Following the extracellular region, the receptor contains a transmembrane segment and an intracellular domain (ICD) containing transcriptional regulators.

[0157] Multiple forms of priming receptors can be used in the methods, cells, and nucleic acids described herein. One type of priming receptor contemplated for use in the methods and cells herein comprises a heterologous extracellular ligand-binding domain, a linked polypeptide having substantial sequence identity with a Notch receptor containing an NRR, a TMD, and an ICD. An "Fn Notch" receptor comprises a heterologous extracellular ligand-binding domain, a linked polypeptide having substantial sequence identity with a Robo receptor (such as mammalian Robo1, Robo2, Robo3, or Robo4), followed by one, two, or three fibronectin repeats ("Fn"), a TMD, and an ICD. A "mini-Notch" receptor comprises a heterologous extracellular ligand-binding domain, a linked polypeptide having substantial sequence identity with a Notch receptor (lacking an NRR), a TMD, and an ICD. A "minimal linker Notch" receptor comprises a heterologous extracellular ligand-binding domain, a linking polypeptide lacking substantial sequence identity to a Notch receptor (e.g., a synthetic (GGS)N polypeptide sequence), a TMD, and an ICD. A "hinge Notch" receptor comprises 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 synthetic receptors and / or pre-existing host receptors), 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 to a ligand displayed on the target cell surface, which triggers proteolytic cleavage of the receptor, releasing a transcriptional regulator that regulates a custom transcriptional program in the cell. In some embodiments, the priming receptor does not comprise the LIN-12-Notch repeat (LNR) and / or heterodimerization domain (HD) of a Notch receptor.

[0158] Priming receptor extracellular domain In some embodiments, the extracellular domain comprises a ligand-binding portion of a receptor. In some embodiments, the extracellular domain comprises an antigen-binding portion that binds to one or more target antigens. In some embodiments, the antigen-binding portion comprises one or more antigen-binding determinants of an antibody or functional antigen-binding fragment thereof. In some embodiments, the antigen-binding portion is selected from the group consisting of an antibody, nanobody, diabody, triabody, or 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 portion comprises an scFv. The antigen-binding portion may comprise a naturally occurring amino acid sequence or may be engineered, designed, or modified to provide desired and / or improved properties, e.g., increased binding affinity.

[0159] In various embodiments, the priming receptor comprises a means for binding to a target protein, optionally a human target protein. In some embodiments, the means binds to the target protein. In some embodiments, the means binds to a human target protein. In some embodiments, the means is an antibody or antigen-binding fragment or equivalent (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 functional fragments thereof).

[0160] Transmembrane domain In some embodiments, the priming receptor comprises a hinge domain. In some embodiments, the hinge domain is a CD8 hinge.

[0161] As noted above, priming receptors contain a transmembrane domain (TMD) that contains one or more ligand-inducible proteolytic cleavage sites.

[0162] In some embodiments, the TMD comprises a Notch1 transmembrane domain.

[0163] Generally, a suitable TMD for the chimeric receptors disclosed herein can be any transmembrane domain of a type 1 transmembrane receptor that contains at least one gamma-secretase cleavage site. A detailed description of the structure and function of the gamma-secretase complex and its substrate proteins, including amyloid precursor protein (APP) and Notch, can be found, for example, 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 comprises 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 a chimeric polypeptide or Notch receptor of the present 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 a chimeric polypeptide or Notch receptor of the present disclosure is a known TMD for a Notch receptor. In some embodiments, the TMD of a chimeric polypeptide or Notch receptor of the present disclosure is a TMD derived from a different Notch receptor.For example, in a mini-Notch based on human Notch1, the Notch1 TMD can be replaced with a Notch2 TMD, a Notch3 TMD, a Notch4 TMD, or a Notch TMD from a non-human animal such as Danio rerio, Drosophila melanogaster, Xenopus laevis, or Gallus gallus.

[0164] 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, metalloproteinase cleavage sites of ADAM10, i.e., MMPs 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 (MT1-MMP and MT2-MMP). Another example of a suitable protease cleavage site is a plasminogen activator cleavage site, such as a urokinase plasminogen activator (uPA) or tissue plasminogen activator (tPA) cleavage site. Another example of a suitable protease cleavage site is a prolactin cleavage site. Specific examples of cleavage sequences for uPA and tPA include sequences comprising Yal-Gly-Arg. Another example of a protease cleavage site that may be included in a proteolytically cleavable linker is a tobacco etch virus (TEV) protease cleavage site, e.g., GLu-AsN-LEu-Thr-GLN-S. Another example of a protease cleavage site that may be included in a proteolytically cleavable linker is a tobacco etch virus (TEV) protease cleavage site, e.g., Glu-Asn-Leu-Thr-Gln-Ser (SEQ ID NO: 112), where the protease cleaves between glutamine and serine. Another example of a protease cleavage site that may be included in a proteolytically cleavable linker is an enterokinase cleavage site, e.g., Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 113), where cleavage occurs after the lysine residue. Another example of a protease cleavage site that may be included in a proteolytically cleavable linker is a thrombin cleavage site, e.g., Leu-Val-Pro-Arg (SEQ ID NO: 114).Additional suitable linkers that contain a protease cleavage site include sequences cleavable by the following proteases: PreScission™ protease (a fusion protein comprising human rhinovirus 3C protease and glutathione-S-transferase), thrombin, cathepsin B, Epstein-Barr virus protease, MMP-3 (stromelysin), MMP-7 (matrilysin), MMP-9; thermolysin-like MMPs, matrix metalloproteinase 2 (MMP-2), cathepsin L; Examples of receptor-specific proteins include tepsin D, matrix metalloproteinase 1 (MMP-1), urokinase-type plasminogen activator, membrane type 1 matrix metalloproteinase (MT-MMP), stromelysin 3 (or MMP-11), thermolysin, 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 not native to the host cells in which the receptor is expressed (e.g., TEV) can be used as an additional regulatory mechanism, reducing receptor activation until the protease is expressed or otherwise provided. Additionally, proteases can be tumor- or disease-associated (expressed to a significantly higher degree than normal tissue) and serve as an independent regulatory mechanism. For example, several matrix metalloproteinases are highly expressed in certain cancer types.

[0165] In some embodiments, the amino acid substitution(s) within the TMD comprise one or more substitutions within the "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, which is incorporated herein by reference in its entirety.

[0166] Intracellular domain In some embodiments, the priming receptor comprises one or more intracellular domains from or derived from a transcription regulator and / or a DNA binding domain. In some embodiments, the intracellular domain comprises a means for regulating transcription of one or more genes. In some embodiments, the means for regulating transcription of one or more genes comprises a transcription regulator, such as a transcription regulator provided herein or its equivalent. In some embodiments, the priming receptor comprises one or more intracellular domains from or derived from a transcription regulator and / or a DNA binding domain. In some embodiments, the intracellular domain comprises an HNF1α / p65 domain or a Gal4 / VP64 domain.

[0167] Transcriptional regulators activate or repress transcription from their cognate promoters. Transcriptional activators typically bind to nearby transcriptional promoters and recruit RNA polymerase to directly initiate transcription. Transcriptional repressors bind to transcriptional promoters and sterically inhibit transcription initiation by RNA polymerase. Other transcriptional regulators function as either activators or repressors, depending on where they bind and the cellular conditions. Thus, as used herein, "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. Non-limiting examples of transcriptional activation domains include herpes simplex virus VP16 activation domain, VP64 (a tetrameric derivative of VP16), HIV TAT, NFkB p65 activation domain, p53 activation domains 1 and 2, CREB (cAMP response element binding protein) activation domain, E2A activation domain, NFAT (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 transcriptional activation domain, B-cell POU homeodomain protein Oct2, plant Ap2, or any others known to those of 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. The transcriptional activation domain can comprise a wild-type or naturally occurring sequence, or can be a modified, mutated, 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 comprise a nuclear localization signal.

[0168] In some embodiments, the priming receptor comprises one or more intracellular "DNA-binding domains" (or "DB domains"). Such "DNA-binding domains" refer to sequence-specific DNA-binding domains that bind to specific DNA sequence elements. Thus, as used herein, a "sequence-specific DNA-binding domain" refers to a protein domain portion that has the ability to selectively bind to DNA having a specific, predetermined sequence. The sequence-specific DNA-binding domain can comprise a wild-type or naturally occurring sequence, or can be a modified, mutant, or derivative version of the original domain that has the desired ability to bind to the desired sequence. In some embodiments, the sequence-specific DNA-binding domain is engineered to bind to the desired sequence. Non-limiting examples of proteins having sequence-specific DNA binding domains that can be used in the synthetic proteins described herein include HNF1a, 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, Cas8a1, Cas8a2, Cas8b, Cas8c, and Ca. Examples include s10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu196, as well as TALES.

[0169] In embodiments in which 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 enzymatic activity, and / or change another property of the protein. For example, the nuclease (i.e., DNase, RNase) domain 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 function of the systems described herein. For example, a CRISPR enzyme used as a DNA-binding protein or domain thereof can be mutated relative to the corresponding wild-type enzyme such that the mutated CRISPR or domain thereof lacks the ability to cleave a nucleic acid sequence containing the DNA-binding domain target site. For example, the D10A mutation can be combined with one or more of the H840A, N854A, or N863A mutations to produce a Cas9 enzyme that lacks substantially all DNA cleavage activity.

[0170] Juxtamembrane domain The ECD and TMD, or the TMD and ICD, can be linked to each other with a linking polypeptide such as a juxtamembrane domain. A "SynNotch" or synthetic Notch receptor comprises a heterologous extracellular ligand-binding domain, a linking polypeptide having substantial sequence identity with a Notch receptor JMD (including an NRR), a TMD, and an ICD. An "Fn Notch" receptor comprises a heterologous extracellular ligand-binding domain, a linking polypeptide having substantial sequence identity with a Robo receptor (such as a mammalian Robo1, Robo2, Robo3, or Robo4), followed by one, two, or three fibronectin repeats ("Fn"), a TMD, and an ICD. A "mini-Notch" receptor comprises a heterologous extracellular ligand-binding domain, a linking polypeptide having substantial sequence identity with a Notch receptor JMD but lacking an NRR (a LIN-12-Notch repeat (LNR) module and a heterodimerization domain), a TMD, and an ICD. A "minimal linker Notch" receptor comprises a heterologous extracellular ligand-binding domain, a linking polypeptide (such as, but not limited to, a synthetic (GGS)) lacking substantial sequence identity with the Notch receptor. n "Hinge Notch" receptors comprise a heterologous extracellular ligand-binding domain, a hinge sequence containing an oligomerization domain (i.e., a domain that promotes dimerization, trimerization, or higher order multimerization with synthetic receptors and / or pre-existing host receptors), a TMD, and an ICD.

[0171] In some embodiments, the priming receptor comprises a juxtamembrane domain (JMD) peptide between the extracellular domain and the transmembrane domain. In some embodiments, the priming receptor comprises a juxtamembrane domain (JMD) peptide between the transmembrane domain and the intracellular domain. In some embodiments, the JMD peptide comprises an LWF motif. The use of an LWF motif in receptor constructs is described in U.S. Patent No. 10,858,443, which is incorporated herein by reference in its entirety. In some embodiments, the JMD peptide has substantial sequence identity to the JMD of Notch1, Notch2, Notch3, and / or Notch4. In some embodiments, the JMD peptide has substantial sequence identity to the Notch1, Notch2, Notch3, and / or Notch4 JMD but does not include the LIN-12-Notch repeat (LNR) and / or heterodimerization domain (HD) of the Notch receptor. In some embodiments, the JMD peptide does not have substantial sequence identity to the Notch1, Notch2, Notch3, and / or Notch4 JMD. In some embodiments, the JMD peptide comprises an oligomerization domain that promotes the formation of receptor dimers, trimers, or higher order aggregates. Such JMD peptides are described in WO2021061872, the entire contents of which are incorporated herein by reference.

[0172] In mini-Notch receptors, the linking polypeptide is derived from the Notch JMD sequence after deletion of the NRR and HD domains. The Notch JMD sequence may be from Notch1, Notch2, Notch3, or Notch4, or may be derived from a non-human homologue, such as from Drosophila, Gallus, or Danio. The remaining 4-50 amino acid residues of the 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 TMD relative to each other to achieve a desired activity of the chimeric polypeptide, such as the level of signal transduction upon ligand induction or in the absence of ligand.

[0173] In minimal linker Notch receptors, the linking polypeptide does not have substantial sequence identity to a Notch JMD sequence (including a Notch JMD sequence from Notch1, Notch2, Notch3, or Notch4, or a non-human homolog thereof). Between 4 and 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 TMD relative to one another to achieve the desired activity of the chimeric polypeptides of the present disclosure. The minimal linker sequence can be designed to include or omit a protease cleavage site and can include or omit multiple sites for glycosylation or other types of post-translational modification. In some embodiments, the minimal linker does not include a protease cleavage site or a glycosylation site.

[0174] In some embodiments, the priming receptor further comprises a hinge. Hinge linkers that can be used in priming receptors can include an oligomerization domain (e.g., a hinge domain) containing one or more polypeptide motifs that promote oligomerization of chimeric polypeptides through intermolecular disulfide bonds. In these cases, within the chimeric receptors disclosed herein, the hinge domain generally comprises a flexible polypeptide connector region located between the ECD and the TMD. Thus, the hinge domain provides flexibility between the ECD and the TMD and also provides a site for intermolecular disulfide bonding between two or more chimeric polypeptide monomers to form an oligomeric complex. In some embodiments, the hinge domain comprises a motif that promotes dimerization of the chimeric polypeptides disclosed herein. In some embodiments, the hinge domain comprises a motif that promotes trimerization of the chimeric polypeptides disclosed herein (e.g., a hinge domain from OX40). Hinge polypeptide sequences suitable for the compositions and methods of the present disclosure can be naturally occurring hinge polypeptide sequences (e.g., derived from naturally occurring immunoglobulins) or can be engineered, designed, or modified to provide desired and / or improved properties, such as transcriptional regulation. Suitable hinge polypeptide sequences include, but are not limited to, those derived from IgA, IgD, and IgG subclasses, such as IgG1 hinge domain, IgG2 hinge domain, IgG3 hinge domain, and IgG4 hinge domain, or functional variants thereof. In some embodiments, the hinge polypeptide sequence contains one or more CXXC motifs. In some embodiments, the hinge polypeptide sequence includes one or more CPPC motifs (SEQ ID NO: 115).

[0175] The hinge polypeptide sequence may also be derived from the CD8α hinge domain, CD28 hinge domain, CD152 hinge domain, PD-1 hinge domain, CTLA4 hinge domain, OX40 hinge domain, and functional variants thereof. In some embodiments, the hinge domain comprises a hinge polypeptide sequence derived from the CD8α hinge domain or a functional variant thereof. In some embodiments, the hinge domain comprises a hinge polypeptide sequence derived from the CD28 hinge domain or a functional variant thereof. In some embodiments, the hinge domain comprises a hinge polypeptide sequence derived from the OX40 hinge domain or a functional variant thereof. In some embodiments, the hinge domain comprises a hinge polypeptide sequence derived from the IgG4 hinge domain or a functional variant thereof.

[0176] The Fn-Notch-linked polypeptide is derived from the Robo1 JMD, which contains a fibronectin repeat (Fn) domain, with a short polypeptide sequence between the Fn repeat and the TMD. The Fn-Notch-linked polypeptide does not contain a Notch negative regulatory region (NRR) or a Notch HD domain. The Fn-linked polypeptide can contain 1, 2, 3, 4, or 5 Fn repeats. In some embodiments, the chimeric receptor comprises an Fn-linked 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 repeat and the TMD can be about 2 to about 30 amino acid residues. In some embodiments, the short polypeptide sequence can be about 5 to about 20 amino acids of any sequence. In some embodiments, the short polypeptide sequence can be about 5 to about 20 naturally occurring amino acids of any sequence. In some embodiments, the short polypeptide sequence can be about 5 to about 20 amino acids of any sequence, but with no more than one proline. In some embodiments, the short polypeptide sequence can be from about 5 to about 20 amino acids, with about 50% or more of the amino acids being glycine. In some embodiments, the short polypeptide sequence can be from about 5 to about 20 amino acids, with the amino acids selected from glycine, serine, threonine, and alanine. In some embodiments, the length and amino acid composition of the FN-linked polypeptide sequence is varied to alter the orientation and / or proximity of the ECD and TMD relative to one another to achieve a desired activity of the chimeric polypeptide of the present disclosure.

[0177] Stop transfer sequence In some embodiments, the priming receptor further comprises a stop transfer sequence (STS) between the transmembrane domain and the intracellular domain. The STS comprises a charged lipophobic sequence. Without being bound by any theory, it is believed that the STS functions as a membrane anchor and prevents the intracellular domain from passing through to the plasma membrane. The use of the STS domain in the priming receptor is described in WO2021061872, the entire contents of which are incorporated herein by reference. Non-limiting exemplary STS sequences include APLP1, APLP2, APP, TGBR3, CSF1R, CXCL16, CX3CL1, DAG1, DCC, DNER, DSG2, CDH1, GHR, HLA-A, IFNAR2, IGF1R, IL1R1, ERN2, KCNE1, KCNE2, CHL1, LRPL, LRP2, LRP18, PTPRF, SCN1B, SCN3B, NPR3, NGFR, PLXDC2, PAM, AGER, ROBOL, SORCS3, SORCS1, SORL1, SDC1, S Examples of STS sequences include DC2, SPN, TYR, TYRP1, DCT, VASN, FLT1, CDH5, PKTFD1, NECTINL, 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, Notch1, Notch2, Notch3, and Notch4. 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, incorporated herein by reference in its entirety.

[0178] Chimeric Antigen Receptor In another aspect, provided herein is a chimeric antigen receptor comprising an extracellular antigen-binding domain that specifically binds to a target antigen or ligand.

[0179] In some embodiments, a chimeric antigen receptor comprises 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 a signaling component that mimics activation via an antigen receptor complex, e.g., a TCR complex, in the case of a CAR, and / or to a signal via another cell surface receptor. Thus, in some embodiments, the extracellular binding component (e.g., a ligand-binding or antigen-binding 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, one of the domains within the receptor, e.g., the transmembrane domain naturally associated with the CAR, is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins and minimize interaction with other members of the receptor complex.

[0180] In some embodiments, the chimeric antigen receptor comprises an extracellular portion comprising an antigen-binding domain described herein and an intracellular signaling domain. In some embodiments, the antibody or fragment comprises an scFv, VH, or single-domain VH antibody, and the intracellular domain comprises an ITAM. In some embodiments, the intracellular signaling domain comprises the signaling domain of the zeta chain of the CD3-zeta (CD3) chain. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain connecting the extracellular domain and the intracellular signaling domain.

[0181] In some embodiments, the transmembrane domain comprises the transmembrane portion of CD8A or CD28. The extracellular domain and the transmembrane can be directly or indirectly linked. In some embodiments, the extracellular domain and the transmembrane are linked by a spacer, such as any of those described herein. In some embodiments, the chimeric antigen receptor contains, for example, an intracellular domain of a T cell costimulatory molecule between the transmembrane domain and the intracellular signaling domain. In some embodiments, the T cell costimulatory molecule is CD28 or 41BB.

[0182] Chimeric antigen receptor extracellular domain In some embodiments, the extracellular domain comprises a ligand-binding portion of a receptor. In some embodiments, the extracellular domain comprises an antigen-binding portion that binds to one or more target antigens. In some embodiments, the antigen-binding portion comprises one or more antigen-binding determinants of an antibody or functional antigen-binding fragment thereof. In some embodiments, the antigen-binding portion is selected from the group consisting of an antibody, nanobody, diabody, triabody, or 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 portion comprises an scFv. The antigen-binding portion may comprise a naturally occurring amino acid sequence or may be engineered, designed, or modified to provide desired and / or improved properties, e.g., increased binding affinity.

[0183] In various embodiments, the CAR comprises a means for binding to a target protein. In some embodiments, the means binds to the target protein. In some embodiments, the means binds to a human target protein. In some embodiments, the means is an antibody or antigen-binding fragment or equivalent (e.g., a full-length antibody or F(ab')2 fragment, Fab fragment, single-chain variable fragment (scFv), and single-domain antibody (sdAb), or functional fragment thereof) means for binding to the target protein.

[0184] CAR transmembrane domain In some embodiments, the transmembrane domain is derived from either a natural or synthetic source. If the source is natural, in some aspects, the domain is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e., comprising 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, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and / or CD154. Alternatively, in some embodiments, the transmembrane domain is synthetic. In some aspects, synthetic transmembrane domains comprise primarily hydrophobic residues such as leucine and valine. In some aspects, triplets of phenylalanine, tryptophan, and valine are found at each end of the synthetic transmembrane domain. In some embodiments, the linkage is by a linker, spacer, and / or transmembrane domain(s).

[0185] In some embodiments, the transmembrane domain of a receptor, e.g., a CAR, is the transmembrane domain of human CD28 or a variant thereof, e.g., the 27 amino acid transmembrane domain of human CD28 (Accession Number: P10747.1).

[0186] In some embodiments, the CAR comprises a CD8a or CD28 TMD.

[0187] CAR hinge In some embodiments, the CAR further comprises a spacer, which may be or include at least a portion of an immunoglobulin constant region, or a 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 that of a human IgG, such as IgG4 or IGG1. In some aspects, the portion of the constant region functions as a spacer region between the antigen-recognition component, e.g., an scFv, and the transmembrane domain. The spacer may be of a length that results in increased cellular responsiveness after antigen binding compared to the absence of the spacer. In some examples, the spacer is about 12 amino acids in length, or 12 amino acids or less in length. Exemplary spacers include those having at least about 10-229 amino acids, about 10-200 amino acids, about 10-175 amino acids, about 10-150 amino acids, about 10-125 amino acids, about 10-100 amino acids, about 10-75 amino acids, about 10-50 amino acids, about 10-40 amino acids, about 10-30 amino acids, about 10-20 amino acids, or about 10-15 amino acids, including any integer between any of the endpoints of the recited ranges. In some embodiments, the 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 a CD8a hinge, an IgG4 hinge alone, an IgG4 hinge linked to CH2 and CH3 domains, or an IgG4 hinge linked to a 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 No. WO2014031687. In some embodiments, the CAR hinge comprises a CD8a CD8α, a truncated CD8α, or a CD28 hinge domain.

[0188] Some intracellular signaling domains mimic or approximate signaling through natural antigen receptors, signaling through such receptors in combination with costimulatory receptors, and / or signaling through costimulatory receptors alone. In some embodiments, a short oligo- or polypeptide linker, e.g., a linker 2-10 amino acids in length, containing glycine and serine, e.g., a glycine-serine doublet, is present to form the link between the transmembrane domain and the cytoplasmic signaling domain of the receptor.

[0189] CAR intracellular domain 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 an immune cell, e.g., a T cell engineered to express the receptor. In some embodiments, the CAR comprises a means for activating at least one of the normal effector functions or responses of an immune cell, e.g., a T cell engineered to express the receptor. For example, in some contexts, the receptor induces a T cell function, such as cytolytic activity or T helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of the intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, e.g., when it transduces an effector function signal. In some embodiments, the intracellular signaling domain or domains comprise the cytoplasmic sequence of a T cell receptor (TCR), and in some aspects, any derivative or variant of such a molecule that acts in concert with such a receptor in its natural context to signal following antigen receptor engagement, and / or any derivative or variant of such a molecule, and / or any synthetic sequence having the same functional capability. In some embodiments, the means for at least one of a normal effector function or response of an immune cell comprises a 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 a normal effector function or response of an immune cell comprises a CAR intracellular activation domain and a CAR costimulatory domain, e.g., a costimulatory domain provided herein or an equivalent thereof.

[0190] In some embodiments, the receptor comprises a primary cytoplasmic signaling sequence that regulates the primary activation of the TCR complex. The stimulatory primary cytoplasmic signaling sequence may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAMs 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, the cytoplasmic signaling molecule(s) in the CAR contain a cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3 zeta. In some embodiments, the intracellular activation domain comprises a CD3 zeta domain.

[0191] In some embodiments, the intracellular signaling domain comprises a human CD3 zeta stimulatory signaling domain or a functional variant thereof, such as the 112 AA cytoplasmic domain of human CD3 zeta isoform 3 (Accession Number: P20963.2) or a CD3 zeta signaling domain described in U.S. Pat. No. 7,446,190 or U.S. Pat. No. 8,911,993.

[0192] A receptor, e.g., a CAR, can comprise at least one intracellular signaling component(s). In some embodiments, the receptor comprises an intracellular component of the TCR complex, such as a TCR CD3 chain, e.g., a CD3-zeta chain, which mediates T cell activation and cytotoxicity. Thus, in some embodiments, the extracellular domain is linked to one or more cell signaling modules. In some embodiments, the cell signaling module comprises a CD3 transmembrane domain, a CD3 intracellular signaling domain, and / or other CD transmembrane domains. In some embodiments, the receptor, e.g., a CAR, further comprises a portion of one or more additional molecules, such as Fc receptor gamma, CD8, CD4, CD25, or CD16. For example, in some embodiments, the CAR comprises a chimeric molecule between CD3-zeta or Fc receptor-gamma and CD8, CD4, CD25, or CD16.

[0193] In some embodiments, the intracellular domain comprises the intracellular costimulatory signaling domain of 41BB, or a functional variant or portion thereof, e.g., the 42 amino acid cytoplasmic domain of human 4-1BB (Accession No. Q07011.1), or a functional variant or portion thereof.

[0194] In some embodiments, the receptor includes one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., a primary activation domain, in the cytoplasmic portion. Exemplary receptors include CD3-zeta, CD28, and the intracellular component of 4-1BB. In some embodiments, the chimeric antigen receptor contains the intracellular domain of a T cell costimulatory molecule. In some aspects, the T cell costimulatory molecule is 4-1BB.

[0195] In some embodiments, the receptor comprises the signaling domain and / or transmembrane portion of a costimulatory receptor such as CD28, 4-1BB, OX40, DAP10, and ICOS. In some aspects, the same receptor comprises both an activating component and a costimulatory component.

[0196] 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) costimulatory domain linked to a CD3 zeta intracellular domain. In some embodiments, the CAR comprises a 4-1BB costimulatory domain.

[0197] In some embodiments, the CAR or other antigen receptor further comprises a marker, such as a cell surface marker, which can be used to confirm the transduction or engineering of cells to express a receptor, such as a truncated version of a cell surface receptor, such as truncated EGFR (tEGFR). In some embodiments, the marker comprises all or a portion (e.g., a truncated form) of CD34, 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 linker sequence, e.g., a cleavable linker sequence or a ribosomal skip sequence, e.g., a polynucleotide encoding T2A. See WO2014031687. In some embodiments, introduction of a construct encoding CAR and EGFRt separated by a T2A ribosomal switch allows expression of the two proteins from the same construct, such that EGFRt can be used as a marker to detect cells expressing such a construct. In some embodiments, the marker, and optionally the linker sequence, can be any of the sequences disclosed in published patent application WO2014031687. For example, the marker can be a truncated EGFR (tEGFR), optionally linked to a linker sequence such as a T2A ribosomal skip sequence.

[0198] In some embodiments, the marker is a molecule that is not naturally found on T cells or that is not naturally found on the surface of T cells, for example, a cell surface protein, or portion thereof.

[0199] In some embodiments, the molecule is a non-self molecule, eg, a non-self protein, ie, a molecule that is not recognized as "self" by the immune system of the host into which the cells are adoptively transferred.

[0200] In some embodiments, the marker does not serve a therapeutic function and / or produce any effect other than being used as a marker for genetic manipulation, e.g., to select successfully engineered cells. In other embodiments, the marker may be a therapeutic molecule or a molecule that exerts some desired effect in other ways, e.g., a ligand for cells encountered in vivo, e.g., a costimulatory or immune checkpoint molecule to enhance and / or attenuate the response of cells upon adoptive transfer and encounter with the ligand.

[0201] A CAR may contain one or more modified synthetic amino acids in place of one or more naturally occurring amino acids. Exemplary modified amino acids include aminocyclohexanecarboxylic acid, norleucine, α-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, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2, These include, but are not limited to, 3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,γ-diaminopropionic acid, homophenylalanine, and α-tertbutylglycine.

[0202] For example, in some embodiments, a CAR comprises a single chain antibody (sdAb, e.g., containing only a VH region), an antibody or fragment thereof comprising a VH domain and an scFv described herein, and a spacer, e.g., a CD8A hinge, a CD8A transmembrane domain, a 4-1BB intracellular signaling domain, and a CD3 zeta signaling domain. In some embodiments, a CAR comprises an antibody or fragment thereof comprising a sdAb and an scFv described herein, and a spacer, e.g., a CD8A hinge, a CD8A transmembrane domain, a 4-1BB intracellular signaling domain, and a CD3 zeta signaling domain.

[0203] The transgenes expressing the priming receptor and CAR system can be introduced into cells, e.g., T cells, using, for example, site-specific techniques. For site-specific integration of the transgene (e.g., the priming receptor and CAR), the transgene can be targeted to a safe harbor locus or TRAC. Examples of site-specific techniques for integration into a safe harbor locus include, but are not limited to, homology-dependent engineering using nucleases and homology-independent targeted insertion using Cas9.

[0204] The engineered cells have applications in immuno-oncology. For example, priming receptors and CARs can be selected to target different specific tumor antigens. Examples of cancers that can be effectively targeted using such cells include blood cancers or solid cancers. In some embodiments, immune cell therapy can be used to treat solid tumors.

[0205] Nucleic acids and vectors In another aspect, provided herein is one or more nucleic acids, wherein the one or more nucleic acids encode a synthetic pathway activator described herein. In another aspect, provided herein is 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.

[0206] In some embodiments, the one or more nucleic acids further comprise a 5' homology-directed repair arm and / or a 3' homology-directed repair arm complementary to the insertion site in the host cell chromosome. In some embodiments, the one or more nucleic acids further comprise a 5' homology-directed repair arm and a 3' homology-directed repair arm. In some embodiments, the one or more nucleic acids are incorporated into an expression cassette or expression vector. In some embodiments, the expression cassette or expression vector further comprises a constitutive promoter upstream of the one or more nucleic acids.

[0207] In some embodiments, the priming receptor, CAR, and synthetic pathway activator are combined into a single expression cassette or a single expression vector. In some embodiments, the priming receptor, CAR, and synthetic pathway activator are combined into two or more expression cassettes or expression vectors. In some embodiments, the expression vector(s) are non-viral vectors.

[0208] In some embodiments, the present disclosure contemplates nucleic acid DNA template inserts comprising one or more transgenes encoding synthetic pathway activators 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 receptor and / or a 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.

[0209] In some embodiments, one or more nucleic acid(s) are encoded on a single DNA template insert. In some embodiments, one or more nucleic acid(s) are encoded on multiple DNA template inserts. For example, one or more nucleic acid(s) can be encoded on two, three, or four DNA template inserts.

[0210] The DNA template insert can also include a self-cleaving peptide. Examples of self-cleaving peptides include, but are not limited to, self-cleaving viral 2A peptides, such as porcine teschovirus-1 (P2A) peptide, Thosea asigna virus (T2A) peptide, equine rhinitis A virus (E2A) peptide, or foot-and-mouth disease virus (F2A) peptide. Self-cleaving 2A peptides enable the 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)).

[0211] The DNA template insert can also contain a WPRE element, which is reviewed 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 incorporated herein by reference.

[0212] The DNA template insert can also include an SV40 polyA tail.

[0213] cell Also provided herein are cells or immune cells comprising at least one DNA template non-virally inserted into a target region of the cell's genome, wherein the DNA template encodes one or more of the synthetic pathway activators described herein. In some embodiments, the DNA template further encodes a priming receptor and a CAR system described herein.

[0214] A cell containing a DNA template insert at a target locus or safe harbor site, as described in this disclosure, may 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 a primary hematopoietic cell or a primary hematopoietic stem cell. In some embodiments, the engineered cell is a stem cell, a human cell, a primary cell, 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 precursor. 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 CD8+ T cell. In some embodiments, the T cell is a CD4+ T cell. + In some embodiments, the T cells are CD4 + CD8+ T cells.

[0215] In some embodiments, the engineered cells are stem cells, human cells, primary cells, hematopoietic cells, hematopoietic stem cells, adaptive immune cells, innate immune cells, T cells, or T cell precursors. Non-limiting examples of immune cells contemplated by the present disclosure include T cells, B cells, natural killer (NK) cells, NKT / iNKT cells, macrophages, myeloid cells, and dendritic cells. Non-limiting examples of stem cells contemplated by the present disclosure include pluripotent stem cells (PSCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), embryo-derived embryonic stem cells (ntES; nuclear transfer ES), male germ cells (GS cells), embryonic germ cells (EG cells), hematopoietic stem / progenitor stem cells (HSPCs), somatic cells (adult stem cells), hemangioblasts, neural stem cells, mesenchymal stem cells, and other stem cells, including bone cells, chondrocytes, myocytes, cardiomyocytes, neurons, tenocytes, adipocytes, pancreatic cells, hepatocytes, kidney cells, and follicular cells. In some embodiments, the engineered cells are T cells, NK cells, iPSCs, and HSPCs. In some embodiments, the engineered cells used in the present disclosure are human cell lines (e.g., intentionally immortalized cell lines, cancer cell lines, etc.) grown in vitro.

[0216] Also provided herein are populations of cells comprising a plurality of cells or immune cells, in some embodiments, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more of the genomes of the cells comprise the priming receptor and CAR system described herein.

[0217] Methods of Treating Immune-Related Conditions of Disease In another aspect, the present disclosure provides a method of treating an immune-related condition (e.g., cancer) in an individual, the method comprising administering to the individual an effective amount of a composition comprising a synthetic pathway activator described herein, e.g., cells 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 the target antigen. In another aspect, the present disclosure provides a method of enhancing an immune response in an individual, the method comprising administering to the individual an effective amount of a composition comprising a synthetic pathway activator described herein, e.g., cells 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 the target antigen.

[0218] In some embodiments, the methods provided herein are useful for treating an immune-related condition in an individual. In certain embodiments, the individual is a human.

[0219] In some embodiments, the methods provided herein (e.g., methods of enhancing an immune response) are useful for treating cancer, and thus, an individual who receives a 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 certain embodiments, the cancer is kidney cancer, renal cell carcinoma, clear cell renal cell carcinoma (ccRcc), colon cancer, or lung cancer. In some embodiments, the cancer is mesothelioma.

[0220] In another aspect, the present disclosure provides methods of inhibiting (e.g., killing, disabling, or preventing the growth or proliferation of) target cells that express both a CAR antigen and a priming receptor antigen. In another aspect, the present invention provides methods of killing target cells that express both a CAR antigen and a priming receptor antigen. In some embodiments, the target cells are cancer cells.

[0221] In some embodiments, the treatment results in a decrease in cancer volume or size. In some embodiments, the treatment is effective in reducing cancer volume compared to the cancer volume before administration of the antibody. In some embodiments, the treatment results in a decrease in cancer growth rate. In some embodiments, the treatment is effective in reducing cancer growth rate compared to the cancer growth rate before administration of the antibody. In some embodiments, the treatment is effective in eliminating the cancer.

[0222] In some embodiments, the CAR antigen and / or priming receptor antigen is expressed at a higher level in cancer compared to non-cancer cells. The level of the CAR antigen and / or priming receptor antigen can be assessed by any technique known in the art, including, but not limited to, protein or nuclear 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, multiplexed qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY techniques, and FISH, and combinations thereof.

[0223] Immunomodulatory methods The methods of administration of cells comprising synthetic pathway activators described herein can result in modulation of the immune response. The modulation can be an increase or a decrease in the immune response. In some embodiments, the modulation is an increase in the immune response.

[0224] In one aspect, administration of cells containing a synthetic pathway activator described herein can result in the induction of pro-inflammatory molecules, such as cytokines or chemokines. Generally, the induced pro-inflammatory molecules are present at levels greater than those achieved with isotype controls. Such pro-inflammatory molecules then lead to the activation of anti-tumor immunity, including, but not limited to, T cell activation, T cell proliferation, T cell differentiation, M1-like macrophage activation, and NK cell activation. Thus, administration of cells containing a synthetic pathway activator described herein can induce multiple anti-tumor immune mechanisms that result in tumor destruction. In some embodiments, the immune activity of the cells is cytolytic activity.

[0225] In another aspect, provided herein is a method of increasing an immune response in an individual, the method comprising administering to the individual an effective amount of cells, wherein the cells comprise cells 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 cells comprising a synthetic pathway activator described herein.

[0226] In some embodiments, the cells are present in a pharmaceutical composition that further comprises a pharmaceutically acceptable excipient.

[0227] In any of the embodiments described herein for increasing an immune response, the increase or decrease or alteration of any aspect of characteristic(s) or function(s) is compared to cells that do not include a composition comprising a synthetic pathway activator described herein.

[0228] Increasing an immune response can be both enhancing an immune response or inducing an immune response. For example, increasing an immune response encompasses both initiating or initiating an immune response or increasing or amplifying an ongoing 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 immune response is an innate immune response. In some embodiments, the immune response is started or initiated by administration of cells comprising a synthetic pathway activator described herein. In some embodiments, the immune response is enhanced by administration of cells comprising a synthetic pathway activator described herein. In some embodiments, the immune response is enhanced by administration of cells comprising a synthetic pathway activator and a priming receptor CAR system described herein.

[0229] In another aspect, the application provides a method of gene editing a cell with a synthetic pathway activator described herein, wherein the gene editing results in modulation of the immune function of the cell. The modulation can be an increased immune response. In some embodiments, the modulation is an increase in immune function. In some embodiments, the modulation of function results in expression of a cytokine or interleukin. In some embodiments, the modulation of function results in activation of an immune cell.

[0230] 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 precursor.

[0231] In some embodiments, modulating the function of a cell comprising a synthetic pathway activator described herein results in an increase in the cell's ability to stimulate both naive and activated T cells, for example, by increasing cytokine or chemokine secretion by cells expressing a synthetic pathway activator described herein. In some embodiments, modulating the function enhances or increases the cell's ability to produce cytokines, chemokines, CARs, or costimulatory or activating receptors. In some embodiments, modulating increases the T cell stimulatory function of a cell expressing a synthetic pathway activator described herein, where T cell stimulatory function includes, for example, the cell's ability to induce T cell receptor (TCR) signaling, T cell proliferation, or T cell cytokine production.

[0232] In some embodiments, the increased immune response is cytokine and chemokine secretion. In some embodiments, the synthetic pathway activators described herein induce increased expression of at least one cytokine or chemokine in cells compared to isotype control cells. In some embodiments, the at least one cytokine or chemokine is selected from the group consisting of IL-2 and IFNγ. In some embodiments, the cytokine or chemokine is IL-2. In some embodiments, the cytokine or chemokine is IFNγ. In some embodiments, cytokine or chemokine secretion is increased by about 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 compared to untreated cells or cells treated with an isotype control antibody. In some embodiments, the chemokine is IL-2 and secretion is increased by 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 compared to untreated cells or cells treated with an isotype control antibody. In some embodiments, the cytokine is IFNγ and secretion is increased by 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 compared to untreated cells or cells treated with an isotype control antibody.

[0233] In some embodiments, the enhanced immune response is anti-tumor immune cell recruitment and activation.

[0234] In some embodiments, cells expressing a synthetic pathway activator described herein induce a memory immune response compared to isotype control cells. Generally, a memory immune response is a protective immune response upon subsequent exposure to a pathogen or antigen previously encountered by the immune system. Exemplary memory immune responses include immune responses following infection or vaccination with an antigen. Generally, memory immune responses are mediated by lymphocytes, e.g., 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.

[0235] How to edit cells The term "gene editing" or "genome editing," as used herein, refers to a type of genetic manipulation in which DNA is inserted into, replaced, or removed from a genome using engineered nucleases or "molecular scissors." This is a useful tool for elucidating the function and effects of sequence-specific genes or proteins or for modifying cellular behavior (e.g., for therapeutic purposes).

[0236] Currently available genome editing tools include zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) for integrating genes into safe harbor loci (e.g., the adeno-associated virus integration site 1 (AAVS1) safe harbor locus). The DICE (dual integrase cassette exchange) system, which utilizes phiC31 integrase and Bxb1 integrase, is a tool for targeted integration. Additionally, clustered regularly interspaced short palindromic repeats / Cas9 (CRISPR / Cas9) technology can be used for targeted gene insertion.

[0237] Site-specific gene editing approaches can include homology-dependent or homology-independent mechanisms.

[0238] All methods known in the art for targeted insertion of gene sequences are contemplated in the methods described herein for inserting constructs into gene target or safe harbor loci.

[0239] Provided herein is a method for inserting a nucleotide sequence of more than about 5 kilobases in length into the genome of a cell in the absence of a viral vector. In some embodiments, a nucleotide sequence of more than about 5 bases in length can be inserted into the genome of a primary immune cell in the absence of a viral vector.

[0240] The integration of large nucleic acids, e.g., nucleic acids greater than 5 kilobases in size, into cells can be limited by low integration efficiency, off-target effects, and / or loss of cell viability. Methods and compositions for achieving integration of nucleotide sequences, e.g., nucleotide sequences greater than about 5 bases in size, into the genome of a cell are described herein. Some methods improve integration efficiency, reduce off-target effects, and / or reduce loss of cell viability.

[0241] The plasmid can be introduced into immune cells using 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 cleaves the genomic DNA at this specific site. The specific site can be a portion of the genome that encodes an endogenous immune cell receptor. Therefore, by cleaving the genome at this site, the immune cell no longer expresses the endogenous immune cell receptor.

[0242] The plasmid may contain 5' and 3' homology-directed repair arms complementary to sequences at specific sites on the genome of the immune cell. The complementary sequences are on either side of the site cleaved by the nuclease, allowing the plasmid to be integrated into the designated insertion site on the genome of the immune cell. Once the plasmid is integrated, the cell expresses the SPA peptide. In examples where the SPA peptide is co-expressed with a system comprising a priming receptor and a CAR, the priming receptor is also expressed by the cell. However, as described, the design of the transgene cassette ensures that the non-viral delivery circuitry receptor does not express the CAR until the priming receptor binds to its cognate ligand and releases the cleavable transcription factor.

[0243] First, T cells are activated. The T cells can be obtained from a patient. Thus, the present disclosure provides a method in which immune cells such as T cells are harvested from a patient. Then, a plasmid encoding a CAR and a priming receptor is introduced into the T cells. Advantageously, the plasmid of the present disclosure can be introduced using electroporation. When introducing the plasmid via electroporation, a nuclease can also be introduced. By using electroporation, the method of the present disclosure avoids the use of viral vectors to introduce transgenes, which is a known bottleneck in immune cell engineering. The T cells are then expanded and co-cultured to generate a sufficient amount of engineered immune cells for use as a therapeutic treatment.

[0244] A method for editing the genome of a cell can include: a) providing a Cas9 ribonucleoprotein complex (RNP)-DNA template complex comprising: (i) an 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 the Cas9 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 DNA template is greater than about 200 nucleotides in size, the 5' and 3' ends of the DNA template comprise nucleotide sequences that are homologous to genomic sequences adjacent to the insertion site, and the molar ratio of RNP to DNA template in the complex is between about 3:1 and about 100:1; and b) introducing the RNP-DNA template complex into the cell.

[0245] In some embodiments, the methods described herein provide an efficiency of delivery of RNP-DNA template complexes 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 more. In some cases, the efficiency is determined in terms of cells that are viable after introducing the RNP-DNA template into cells. In some cases, the efficiency is determined in terms of the total number of cells (viable or nonviable) into which the RNP-DNA template is introduced.

[0246] As another example, the efficiency of delivery can be determined by quantifying the number of genome-edited cells in a cell population (compared to the total cells or total viable cells obtained after the introduction step). Various methods for quantifying genome editing are available. These methods include, but are not limited to, the use of mismatch-specific nucleases such as T7 endonuclease I, sequencing of one or more target loci (e.g., by Sanger sequencing of cloned target locus amplified fragments), and high-throughput large-scale sequencing.

[0247] In some embodiments, the loss of cell viability is reduced compared to the loss of cell viability after naked DNA is introduced into cells or after DNA is introduced into cells using a viral vector. The reduction can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage between these. In some embodiments, the off-target effects of integration are reduced compared to off-target integration after naked DNA is introduced into cells or after DNA is introduced into cells using a viral vector. The reduction can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage between these.

[0248] In some cases, the methods described herein provide high cell viability of cells into which the RNP-DNA template has been introduced, in some cases, the viability of cells into 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 more. In some cases, the viability of cells into which the RNP-DNA template has been introduced is about 20% to about 99%, about 30% to about 90%, about 35% to about 85% or 90% or more, about 40% to about 85% or 90% or more, about 50% to about 85% or 90% or more, about 50% to about 85% or 90% or more, about 60% to about 85% or 90% or more, or about 70% to about 85% or 90% or more.

[0249] In the methods provided herein, the molar ratio of RNP to nucleic acid (e.g., DNA template) can be about 3:1 to about 100:1. For example, the molar ratio can be about 3:1 to 10:1, about 3:1 to about 15:1, 3:1 to about 20:1, 3:1 to about 25:1, about 3:1 to about 50:1, about 3:1 to 75:1, about 3:1 to 100:1, about 5:1 to 10:1, about 5:1 to about 15:1, 5:1 to about 20:1, 5:1 to about 25:1, about 5:1 to 50:1, about 5:1 to 75:1, or about 5:1 to 100:1. , about 8:1 to about 12:1, about 8:1 to about 15:1, about 8:1 to about 20:1, about 8:1 to about 25:1, about 8:1 to about 50:1, about 8:1 to 75:1, about 8:1 to 100:1, about 10:1 to about 15:1, 10:1 to about 20:1, 10:1 to about 25:1, about 10:1 to 50:1, about 10:1 to 75:1, or about 10:1 to 100:1.

[0250] In some embodiments, the DNA template has a concentration of about 2.5 PM to about 25 PM. For example, the DNA template concentration 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 therebetween.

[0251] In some embodiments, the size or length of a nucleic acid (e.g., a DNA template) is 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, The nucleic acid (e.g., DNA template) may be 7.8KB, 7.9KB, 8.0KB, 8.1KB, 8.2KB, 8.3KB, 8.4KB, 8.5KB, 8.6KB, 8.7KB, 8.8KB, 8.9KB, 9.0KB, 9.1KB, 9.2KB, 9.3KB, 9.4KB, 9.5KB, 9.6KB, 9.7KB, 9.8KB, 9.9KB, or 10KB, 11kb, 12kb, 13kb, 14kb, 15kb, or 16kb, or any size in between these sizes. For example, the size of the DNA template may 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 6 kb to about 15 kb, about 6 kb to about 14 kb, about 6 kb 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 1 It may be 4 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 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.

[0252] In some embodiments, the amount of DNA template can be about 1 μg to about 10 μg. For example, the amount of DNA template can be about 1 μg to about 2 μg, about 1 μg to about 3 μg, about 1 μg to about 4 μg, about 1 μg to about 5 μg, about 1 μg to about 6 μg, about 1 μg to about 7 μg, about 1 μg to about 8 μg, about 1 μg to about 9 μg, or about 1 μg to about 10 μg. In some embodiments, the amount of DNA template is about 2 μg to about 3 μg, about 2 μg to about 4 μg, about 2 μg to about 5 μg, about 2 μg to about 6 μg, about 2 μg to about 7 μg, about 2 μg to about 8 μg, about 2 μg to about 9 μg, or 2 μg to about 10 μg. In some embodiments, the amount of DNA template is about 3 μg to about 4 μg, about 3 μg to about 5 μg, about 3 μg to about 6 μg, about 3 μg to about 7 μg, about 3 μg to about 8 μg, about 3 μg to about 9 μg, or about 3 μg to about 10 μg. In some embodiments, the amount of DNA template is about 4 μg to about 5 μg, about 4 μg to about 6 μg, about 4 μg to about 7 μg, about 4 μg to about 8 μg, about 4 μg to about 9 μg, or about 4 μg to about 10 μg. In some embodiments, the amount of DNA template is about 5 μg to about 6 μg, about 5 μg to about 7 μg, about 5 μg to about 8 μg, about 5 μg to about 9 μg, or about 5 μg to about 10 μg. In some embodiments, the amount of DNA template is about 6 μg to about 7 μg, about 6 μg to about 8 μg, about 6 μg to about 9 μg, or about 6 μg to about 10 μg. In some embodiments, the amount of DNA template is about 7 μg to about 8 μg, about 7 μg to about 9 μg, or about 7 μg to about 10 μg. In some embodiments, the amount of DNA template is about 8 μg to about 9 μg, or about 8 μg to about 10 μg. In some embodiments, the amount of DNA template is about 9 μg to about 10 μg.

[0253] In some cases, the size of the DNA template is large enough and sufficient in 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 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more genes.

[0254] In some embodiments, the DNA template comprises regulatory sequences, such as promoter and / or enhancer sequences, for regulating expression of the heterologous protein or fragment thereof after insertion into the genome of the cell.

[0255] 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, "pure single-stranded DNA" refers to single-stranded DNA that is substantially devoid of other strands or opposite strands of DNA. "Substantially devoid" means that the pure single-stranded DNA is devoid of one DNA strand at least 100 times more than another DNA strand.

[0256] In some cases, the RNP-DNA template complex is formed by incubating the RNP with the DNA template for less than about 1 minute to about 30 minutes at a temperature of about 20° C. to about 25° C. For example, the RNP can be incubated with the DNA template at a temperature of about 20° C., 21° C., 22° C., 23° C., 24° C., or 25° C. 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, 30 minutes, or any amount of time in between these times. In another example, the RNP can be incubated with the DNA template at a temperature of about 20° C. to about 25° C. for about less than 1 minute to about 1 minute, about less than 1 minute to about 5 minutes, about less than 1 minute to about 10 minutes, about 5 to 10 minutes, about 5 to 15 minutes, about 10 to about 15 minutes, about 10 to about 20 minutes, or about 10 to about 30 minutes. In some embodiments, the RNP-DNA template complex and cells are mixed before introducing the RNP-DNA template complex into the cells.

[0257] In some embodiments, introducing the RNP-DNA template complex comprises electroporation. Methods, compositions, and devices for electroporating cells to introduce the RNP-DNA template complex can include those described in the Examples herein. Additional or alternative methods, compositions, and devices for electroporating cells to introduce the RNP-DNA template complex can include those described in WO / 2006 / 001614 or Kim, JA et al. Biosens. Bioelectron. 23, 1353-1360 (2008). Additional or alternative methods, compositions, and devices for electroporating cells to introduce the RNP-DNA template complex can include those described in U.S. Patent Application Publication Nos. 2006 / 0094095, 2005 / 0064596, or 2006 / 0087522. Additional or alternative methods, compositions, and devices for electroporating cells to introduce RNP-DNA template complexes can include those described in Li, L. Het al. Cancer Res. Treat. 1, 341-350 (2002), U.S. Patent Nos. 6,773,669, 7,186,559, 7,771,984, 7,991,559, 6485961, 7029916, and U.S. Patent Application Publication Nos. 2014 / 0017213 and 2012 / 0088842, all of which are incorporated herein by reference. Additional or alternative methods, compositions, and devices for electroporating cells to introduce RNP-DNA template complexes 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 incorporated herein by reference.

[0258] In some embodiments, the Cas9 protein can be in an active endonuclease form so that when it binds to a target nucleic acid as part of a complex with a guide RNA or 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 by HDR to introduce specific mutations. Various Cas9 nucleases can be used 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 used. Such a Cas9 nuclease can target any region of the genome that contains an NGG sequence. As another example, a Cas9 protein with an orthogonal PAM motif requirement can be used to target a sequence that does not have an adjacent NGG PAM sequence. Exemplary Cas9 proteins with orthogonal PAM sequence specificity 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 incorporated herein by reference).

[0259] In some cases, Cas9 protein is a nickase, so that when it binds to target nucleic acid as part of a complex with guide RNA, it introduces single-strand breaks or nicks into target nucleic acid.A pair of Cas9 nickases, each of which is bound to structurally different guide RNAs, can target two adjacent sites in the target genome region, and thus introduce a pair of adjacent single-strand breaks into the target genome region.Nickase pairs can increase specificity because off-target effects are more likely to result in a single nick, which is generally repaired without lesions by base excision repair mechanisms.Exemplary Cas9 nickases include Cas9 nucleases with D10A or H840A mutations.

[0260] 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 distinct RNP complexes. In some embodiments, the at least two structurally distinct RNP complexes contain structurally distinct Cas9 nuclease domains. In some embodiments, the at least two structurally distinct RNP complexes contain structurally distinct guide RNAs. In some embodiments, where the at least two structurally distinct RNP complexes contain structurally distinct guide RNAs, each of the structurally distinct RNP complexes comprises a Cas9 nickase, and the structurally distinct guide RNAs hybridize to opposite strands of the target region.

[0261] In some cases, multiple RNP-DNA templates containing structurally distinct ribonucleoprotein complexes are introduced into cells. For example, a Cas9 protein can be complexed with multiple (e.g., 2, 3, 4, 5, or more, e.g., 2-10, 5-100, 20-100) structurally distinct guide RNAs to target insertion of the DNA templates at multiple structurally distinct target genomic regions.

[0262] In the methods and compositions provided herein, cells include, but are not limited to, eukaryotic cells, prokaryotic cells, animal cells, plant cells, fungal cells, etc. Optionally, the cells are mammalian cells, e.g., human cells. The cells can be in vitro, ex vivo, or in vivo. The cells can also be primary cells, germ cells, stem cells, or progenitor cells. Progenitor cells can be, for example, pluripotent stem cells or hematopoietic stem cells. In some embodiments, the cells are primary hematopoietic cells or primary hematopoietic stem cells. In some embodiments, the primary hematopoietic cells are immune cells. 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 CD4 + In some embodiments, the T cells are CD8 + In some embodiments, the T cells are CD4 + CD8 + In some embodiments, the T cells are CD4 - CD8 - The modified cells are T cells. Also provided are any populations of cells modified by any of the methods described herein. In some embodiments, the method further comprises expanding the population of modified cells.

[0263] In some cases, 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 are delivered to the patient in vivo. See, e.g., U.S. Patent No. 9,737,604 and Zhang et al. "Lipid nanoparticle-mediated efficient delivery of CRISPR / Cas9 for tumor therapy," NPG Asia Materials Volume 9, page e441 (2017) (both incorporated herein by reference).

[0264] In some embodiments, the RNP-DNA template complex is about 1×10 5 ~Approx. 2×10 6 For example, the RNP-DNA template complex is introduced into approximately 1 × 10 cells. 5 ~Approx. 5×10 5 cells, approximately 1 x 10 5 ~Approx. 1×10 6 , 1×10 5 ~Approx. 1.5×10 6 , 1×10 5 ~Approx. 2×10 6 , about 1×10 6 ~Approx. 1.5×10 6 cells, or approximately 1 x 10 6 ~Approx. 2×10 6 can be introduced into

[0265] In some cases, the methods and compositions described herein can be used to generate, modify, use, or control 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, infectious disease, or autoimmune disease in a subject. For example, in some embodiments, one or more gene products are inserted or knocked into T cells to express a heterologous protein (e.g., a chimeric antigen receptor (CAR) or a priming receptor).

[0266] Insertion site Methods for editing the genome of a T cell specifically include methods for editing the genome of a human T cell comprising inserting a nucleic acid sequence or construct into a target region in exon 1 of the TCR-α subunit (TRAC) in a human T cell. In some embodiments, the target region is in exon 1 of the constant domain of the 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-α transmembrane domain. In some embodiments, the target region is the GS94 genomic safe harbor.

[0267] Methods for editing the genome of a T cell also include methods for editing the genome of a human T cell, comprising inserting a nucleic acid sequence or construct into a target region in exon 1 of a TCR-β subunit (TRBC) in a human T cell. In some embodiments, the target region is in exon 1 of the TRBC1 or TRBC2 gene.

[0268] Methods for editing the genome of T cells specifically include methods for editing the genome of human T cells, comprising inserting a nucleic acid sequence or construct into a target region of a genomic safe harbor (GSH).

[0269] Methods for editing the genome of a T cell also include methods of editing the genome of a human T cell, comprising inserting a nucleic acid sequence or construct into the GS94 target region (locus chr11:128340000-128350000).

[0270] In some embodiments, the target region is the GS94 locus.

[0271] 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 because it reduces the risk 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 low knock-in efficiency, risk of insertional oncogenesis, unstable and / or abnormal expression of adjacent genes or transgenes, and low accessibility (e.g., within 20 KB of adjacent genes). These challenges can be addressed, in part, by identifying and using safe harbor loci or safe harbor sites (SHSs), which are sites where genes or genetic elements can be integrated without disrupting the expression or regulation of adjacent genes.

[0272] The most widely used of the putative human safe harbor sites is the AAVS1 site on chromosome 19Q, which was originally identified as a site for recurrent adeno-associated virus insertion. Other potential SHSs have been identified based on homology to sites originally identified in other species (e.g., the human homolog of the permissive mouse Rosa26 locus) or in an increasing number of human genes that appear non-essential under some circumstances. One putative SHS of this type is the CCR5 chemokine receptor gene, whose disruption confers resistance to human immunodeficiency virus infection. Additional potential genomic SHSs have been identified in humans and other cell types based on viral integration site mapping or gene trap analysis, similar to the original mouse Rosa26 locus. The top three SHSs, AAVS1, CCR5, and Rosa26, are located near many protein-coding genes and regulatory elements. (See SADELAiN, M., Et AL. (2012). Safe harbors for the integration of new DNA in the human genome. Nature reviews Cancer, 12(1), 51-58, the relevant disclosure of which is incorporated herein by reference in its entirety).

[0273] AAVS1 (also known as the PPP1R12C locus) on human chromosome 19 is a known SHS for hosting transgenes (e.g., DNA transgenes) with expected functions. It is located at position 19q13.42. It has an open chromatin structure and is transcriptionally competent. The canonical SHS locus for AAVS1 is chr19:55, 625, 241 to 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 disclosure of which is incorporated herein by reference. Exemplary AAVS1-targeting gRNAs and target sequences are provided below. AAVS1-gRNA sequence: ggggccactagggacaggatGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT (SEQ ID NO: 116) AAVS1 target sequence: ggggccactagggacaggat (SEQ ID NO: 117)

[0274] CCR5, located on chromosome 3 at position 3p21.31, encodes the primary co-receptor for HIV-1. Disruption of this site in the CCR5 gene is beneficial in HIV / AIDS treatment and has prompted the development of zinc finger nucleases targeting its third exon. The canonical SHS locus for CCR5 is chr3:46, 414, 443 to 46, 414, 942. 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 disclosure of which is incorporated herein by reference).

[0275] The mouse Rosa26 locus is particularly useful for genetic modification because 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 is incorporated herein by reference, identified the human homolog, human ROSA26, on 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 disclosure of which is incorporated herein by reference.

[0276] 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 disclosure of which is incorporated herein by reference. Additional examples of integration sites are provided in Table D.

[0277] In some embodiments, safe harbor sites allow for high transgene expression (sufficient to enable transgene functionality or treatment of a disease of interest) and stable expression of the transgene over days, weeks, or months. In some embodiments, knockout of a gene at a safe harbor locus confers a benefit to cellular function, or the gene at a safe harbor locus has no known function in the cell. In some embodiments, safe harbor loci result in stable transgene expression in vitro with or without CD3 / CD28 stimulation, negligible off-target cleavage detected by iGuide-Seq or CRISPR-Seq, less off-target cleavage compared to other loci 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 are located outside of cancer-associated genes.

[0278] When used, "neighboring genes" can refer to genes that are within about 100 KB, about 125 KB, about 150 KB, about 175 KB, about 200 KB, about 225 KB, about 250 KB, about 275 KB, about 300 KB, about 325 KB, about 350 KB, about 375 KB, about 400 KB, about 425 KB, about 450 KB, about 475 KB, about 500 KB, about 525 KB, or about 550 KB of the safe harbor locus (integration site).

[0279] In some embodiments, the present disclosure contemplates inserts containing one or more transgenes. The transgenes can encode therapeutic proteins, antibodies, peptides, or any other gene of interest. Transgene integration can result in, for example, improved therapeutic properties. These enhanced therapeutic properties, as used herein, refer to enhanced therapeutic properties of cells compared to typical immune cells of the same normal cell type. For example, T cells with "enhanced therapeutic properties" have enhanced, improved, and / or increased therapeutic outcomes compared to typical, unmodified, and / or naturally occurring T cells. Therapeutic properties of immune cells can include, but are not limited to, cell engraftment, trafficking, homing, viability, self-renewal, persistence, immune response control and modulation, survival, and cytotoxicity. Therapeutic properties of immune cells can also be manifested by expression of antigen-targeting receptors, HLA presentation or lack thereof, tolerance to the tumor microenvironment, induction and immunomodulation of bystander immune cells, improved target specificity through reduction, and resistance to treatments such as chemotherapy.

[0280] As used herein, "insert size" refers to the length of the nucleotide sequence to be integrated (inserted) into a target locus or safe harbor site. In some embodiments, the insert size comprises at least about 4.5 kilobase pairs (KB) to about 10 kilobase pairs (KB). In some embodiments, the insert size comprises about 5,000 or more nucleotide base pairs. 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 (kilobase pairs), or any size therebetween. 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 any size therebetween. In some embodiments, the insert size is within the range of 4.5 to 15 KBP, or any number within that range. In some embodiments, the insert size is in the range of 4.8-8.3 KBP, or any number within that range. In some embodiments, the insert size is in the range of 5-8.3 KBP, or any number within that range. In some embodiments, the insert size is in the range of 5-15 KBP, or any number within that range. In some embodiments, the insert size is in the range of 4.5-20 KBP, or any number within 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 to 17, 6 to 17, 7 to 17, 8 to 17, 9 to 17, 10 to 17, 11 to 17, 12 to 17, 13 to 17, or 14 to 17, 15 to 17, or 16 to 17 KBP. In some embodiments, the insert size is 4.5 to 18, 6 to 18, 7 to 18, 8 to 18, 9 to 18, 10 to 18, 11 to 18, 12 to 18, 13 to 18, 14 to 18, 15 to 18, 16 to 18, or 17 to 18 KBP. In some embodiments, the insert size is 4.5 to 19, 6 to 19, 7 to 19, 8 to 19, 9 to 19, 10 to 19, 11 to 19, 12 to 19, 13 to 19, 14 to 19, 15 to 19, 16 to 19, 17 to 19, or 18 to 19 KBP. In some embodiments, the insert size is 4.5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, 10 to 20, 11 to 20, 12 to 20, 13 to 20, 14 to 20, 15 to 20, 16 to 20, 17 to 20, 18 to 20, or 19 to 20 KBP.

[0281] An insert, as used herein, refers to a nucleic acid molecule or polynucleotide inserted into a target locus or safe harbor site. In some embodiments, the nucleotide sequence is a DNA molecule, e.g., genomic DNA, or comprises deoxyribonucleotides. In some embodiments, the insert comprises smaller fragments of DNA, such as plastid DNA, mitochondrial DNA, or DNA isolated in the form of a plasmid, fosmid, cosmid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), and / or any other subgenomic segment of DNA. In some embodiments, the insert is an RNA molecule or comprises ribonucleotides. Nucleotides in the insert are contemplated as naturally occurring nucleotides, non-naturally occurring nucleotides, and modified nucleotides. Nucleotides may be chemically or biochemically modified or contain non-natural or derivatized nucleotide bases, as will be readily understood by those skilled in the art. Such modifications include, for example, labeling, methylation, substitution of one or more naturally occurring nucleotides with an analog, and internucleotide modifications. Polynucleotides can be in any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hairpinned, circular conformations, and other three-dimensional conformations contemplated in the art.

[0282] An insert can have coding and / or non-coding regions. An insert can include non-coding sequences (e.g., regulatory elements, e.g., promoter sequences). In some embodiments, an insert encodes a transcription factor. In some embodiments, an insert encodes an antigen-binding receptor, e.g., a single receptor, a T cell receptor (TCR), a priming receptor, a CAR, a mAb, etc. In some embodiments, an insert is a human sequence. In some embodiments, an insert is chimeric. In some embodiments, an insert is a multigene / multimodule therapeutic cassette. A multigene / multimodule therapeutic cassette refers to an insert or cassette having one or more receptors (e.g., a synthetic receptor such as a CAR or priming receptor), other exogenous protein-coding sequences, non-coding RNA, transcriptional regulatory elements, and / or insulator sequences, etc.

[0283] In some embodiments, nucleic acid sequences are inserted into the genome of cells such as immune cells or T cells via non-viral delivery.In non-viral delivery methods, nucleic acids can be naked DNA or can be in non-viral plasmids or vectors.Non-viral delivery techniques can be site-specific integration techniques described herein or known to those skilled in the art.Examples of site-specific techniques for integration into safe harbor loci include, but are not limited to, homology-dependent manipulation using nucleases and homology-independent targeted insertion using Cas9 or other CRISPR endonucleases.

[0284] In some embodiments, the insert is integrated into the safe harbor site by introducing into the engineered cell (a) a target nuclease that cleaves the target region of the safe harbor site to create the insertion site, and (v) a nucleic acid sequence (insert), where the insert is integrated into the insertion site, for example, by HDR. Examples of non-viral delivery techniques that can be used in the methods of the present disclosure are provided in U.S. Patent Nos. US11033584B2 and US11814624B2, the relevant disclosures of which are incorporated herein by reference in their entireties.

[0285] Examples of contemplated integration sites are provided in Table D.

[0286] Table D. sgRNA sequences TIFF2026508611000002.tif221165TIFF2026508611000003.tif234165TIFF2026508611000004.tif217165TIFF2026508611000005.tif217165 TIFF2026508611000006.tif207165TIFF2026508611000007.tif201165TIFF2026508611000008.tif178165TIFF2026508611000009.tif141165

[0287] CRISPR-Cas editing One effective example of gene editing is the CRISPR-Cas approach (e.g., CRISPR-Cas9), which incorporates the use of a guide polynucleotide (e.g., a guide ribonucleic acid or gRNA) and a cas endonuclease (e.g., Cas9 endonuclease).

[0288] As used herein, a polypeptide referred to as a "Cas endonuclease" or having "Cas endonuclease activity" refers to a CRISPR-associated (CAS) polypeptide encoded by a Cas gene, where the Cas polypeptide is a target DNA sequence that can be cleaved when operably linked to one or more guide polynucleotides (see, e.g., U.S. Patent No. 8,697,359). This definition also includes variants of Cas endonucleases that retain guide polynucleotide-dependent endonuclease activity. The Cas endonucleases used in the donor DNA insertion methods detailed herein are endonucleases that introduce double-strand breaks in DNA at target sites (e.g., within a target locus or at a safe harbor site).

[0289] As used herein, the term "guide polynucleotide" refers to a polynucleotide sequence that can complex with a Cas endonuclease and enable the Cas endonuclease to recognize and cleave a DNA target site. A guide polynucleotide can be a single molecule or a double molecule. A guide polynucleotide sequence can be an RNA sequence, a DNA sequence, or a combination thereof (RNA-DNA combination sequence). A guide polynucleotide that contains only ribonucleic acid is also referred to as a "guide RNA." In some embodiments, a polynucleotide donor construct is inserted into a safe harbor locus using a guide RNA (gRNA) in combination with a Cas endonuclease (e.g., Cas9 endonuclease).

[0290] The guide polynucleotide comprises 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 sequence that interacts with a Cas endonuclease polypeptide. The guide polynucleotide may be a duplex molecule (also referred to as a double-stranded guide polynucleotide) that comprises a sequence domain (also referred to as a Cas endonuclease recognition domain or CER domain). The CER domain of this duplex guide polynucleotide comprises two separate molecules that hybridize along complementary regions. The two separate molecules may be RNA sequences, DNA sequences, and / or RNA-DNA combination sequences.

[0291] Genome editing using the CRISPR-Cas approach relies on the repair of site-specific DNA double-strand breaks (DSBs) induced by RNA-guided Cas endonucleases (e.g., Cas9 endonuclease). Homology-directed repair (HDR) of these DSBs allows for 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.

[0292] HITI (homology-independent targeted insertion) uses a homology-independent strategy based on non-homologous end joining (NHEJ), which can be more efficient than HDR. A guide RNA (gRNA) targets the insertion site. For HITI, the donor plasmid lacks homology arms, and DSB repair does not occur via the HDR pathway. The donor polynucleotide construct can be engineered to contain Cas9 cleavage site(s) adjacent to the gene or sequence to be inserted. This results in Cas9 cleavage in both the donor plasmid and the genomic target sequence. Both the target and donor have blunt ends, and the linearized donor DNA plasmid is used by the NHEJ pathway, which leads to integration into the genomic DSB site. (See, e.g., Suzuki, K., et al. (2016). In vivo genome editing via CRISPR / Cas9 mediated homology-independent targeted integration. Nature, 540(7631), 144-149, the relevant disclosure of which is incorporated herein in its entirety.)

[0293] Methods for gene editing using CRISPR-Cas approaches are known to those skilled in the art. (See, e.g., U.S. Application Nos. US16 / 312,676, US15 / 303,722, and US15 / 628,533, the disclosures of which are incorporated herein by reference in their entireties.) Additionally, the use of endonucleases to insert transgenes into safe harbor loci is described, for example, in U.S. Application No. 13 / 036,343, the disclosure of which is incorporated herein by reference in its entirety.

[0294] The guide RNA and / or mRNA (or DNA) encoding the endonuclease can be chemically conjugated 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 cholesterol moieties, cholic acid, thioethers, thiocholesterol, aliphatic chains (e.g., dodecanediol or undecyl residues), phospholipids such as dihexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-3-H-phosphonate, polyamine or polyethylene glycol chains, adamantane acetic acid, palmityl moieties, and lipid moieties such as octadecylamine or hexylamino-carbonyl-t-oxycholesterol moieties. See, e.g., U.S. Patent Publication No. 20180127786, the disclosure of which is incorporated herein by reference in its entirety.

[0295] therapeutic use 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 skilled in the art.

[0296] The engineered cells provided herein find use in gene therapy as well as non-pharmaceutical uses, such as, for example, the production of animal models and the production of recombinant cell lines expressing a protein of interest.

[0297] The engineered cells of the present disclosure can be any cell, generally a mammalian cell, generally a human cell, that has been modified by incorporating a transgene into a safe harbor locus as described herein. Exemplary cells are provided in the recombinant cell section.

[0298] 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, insertion of a sequence encoding a transgene within a safe harbor locus maintains TCR expression relative to the absence of the insertion, allowing transgene expression while maintaining TCR function.

[0299] 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. This effect can be a partial or complete cure of the disease and / or adverse effects resulting from the disease. In some embodiments, treatment includes any treatment of a disease in a subject (e.g., a mammal, e.g., a human). Additionally, treatment can stabilize or reduce undesirable clinical symptoms in a subject (e.g., a patient). The cells, populations thereof, or compositions thereof provided herein can be administered during or after the onset of a disease.

[0300] In certain embodiments, a subject has a disease, condition, and / or injury that can be treated and / or ameliorated by cell therapy. In some embodiments, a subject in need of cell therapy is a subject with an injury, disease, or condition that triggers cell therapy (e.g., therapy in which cellular material is administered to the subject). However, it is contemplated that the severity of at least one symptom associated with the injury, disease, or condition can be treated, ameliorated, and / or reduced.

[0301] Method of administration An effective amount of immune cells comprising an SPA peptide can be administered to treat cancer. The appropriate dosage of immune cells comprising an SPA peptide can be determined based on the type of cancer being treated, the type of immune cells comprising the SPA peptide, the severity and course of the cancer, the individual's clinical condition, the individual's clinical history and response to treatment, and the judgment of the attending physician.

[0302] Determine CD11c expression Also provided herein is a method of treating cancer in a subject in need thereof, comprising determining or having determined expression of CD11c in cells containing a synthetic pathway activator (SPA) peptide disclosed herein, optionally wherein the SPA is inserted into a target region of the cell's genome, and administering or having administered to the subject the cells containing the SPA.

[0303] 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).

[0304] In some embodiments, provided herein is a method for determining the expression level of CD11c protein in a sample from a subject, the method comprising contacting the sample with an anti-CD11c antibody and performing 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 embodiments, provided herein is a method for determining the expression level of CD11c mRNA in a sample from a subject, the method comprising performing qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY technology, Luminex, MSD, or FISH, and combinations thereof.

[0305] In some embodiments, provided herein are methods of generating CD11c-positive (CD11c+) cells, the methods comprising inserting a nucleic acid encoding any of the SPA peptides disclosed herein and expressing the SPA peptide in the cells. In other embodiments, provided herein are methods of detecting SPA, optionally functional SPA, in cells containing an SPA peptide disclosed herein, where optionally the SPA is inserted into the genome of the cell.

[0306] In some aspects, provided herein are methods of screening cells for expression of SPAs, optionally functional SPAs, comprising expressing one or more SPAs in the cells and detecting CD11c expression in the cells, wherein detection of CD11c indicates a functional SPA peptide. In such embodiments, a functional SPA is one that has functional signaling (e.g., one that can stimulate a cell, e.g., a T cell, via phosphorylation of STAT1, STAT3, and / or STAT5).

[0307] In some embodiments, provided herein is an assay for detecting cells, e.g., primary cells and / or immune cells, that have been engineered to express SPA, comprising determining or having determined CD11c expression in the primary cells, wherein CD11c expression indicates that the cells express SPA.

[0308] In some embodiments, provided herein are methods of treating a patient with engineered CD11c-expressing T cells, the methods comprising administering to the patient T cells comprising a SPA disclosed herein.

[0309] In some embodiments, CD11c expression is determined in T cells, from a biological sample from a patient administered cells expressing the SPA peptide, or in a tumor from a patient administered cells 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 precursor. 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 CD8+ T cell. In some embodiments, the T cell is a CD4 + In some embodiments, the T cells are CD4 + CD8+ T cells.

[0310] In some embodiments, the expression level of CD11c comprises an mRNA expression level. In some embodiments, the expression level of CD11c comprises a protein expression level of CD11c. In some embodiments, the expression level of CD11c is detected in a sample using a method selected from the group consisting of FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, monoplex immunohistochemistry, multiplex immunohistochemistry, immunofluorescence, 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 technology, Luminex, MSD, and FISH, and combinations thereof.

[0311] Pharmaceutical Compositions The engineered recombinant cells provided herein can be administered as part of a pharmaceutical composition. These compositions can contain, in addition to one or more of the recombinant cells, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials known to those of skill 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 may depend on the route of administration, e.g., oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, or intraperitoneal. Pharmaceutical compositions can contain one or more pharmaceutical excipients. Any suitable pharmaceutical excipient may be used, and those of skill in the art can select a suitable pharmaceutical excipient. Therefore, the pharmaceutical excipients provided below are intended to be exemplary and not limiting. Additional pharmaceutical excipients include, for example, those described in Handbook of Pharmaceutical Excipients, Rowe et al. (Eds.) 6th Ed. (2009), incorporated by reference in its entirety.

[0312] Various modes of administering the additional therapeutic agent are contemplated herein. In some embodiments, the additional therapeutic agent is administered by any suitable mode of administration.

[0313] The compositions can be administered alone or in combination with other treatments, either simultaneously or sequentially, depending on the condition to be treated.

[0314] Kits and Products The present application provides kits comprising any one or more of the SPA peptides or cell compositions described herein and instructions for use. The instructions may be present in the kit as a package insert, on a label on the container of the kit or its components, or in digital form (e.g., on a CD-ROM or via an internet link). The kit may include one or more of a genome-targeting nucleic acid, a polynucleotide encoding the genome-targeting nucleic acid, a site-directed polypeptide, and / or a polynucleotide encoding the site-directed polypeptide. Additional components in the kit, such as buffers (reconstitution buffer, stabilization buffer, dilution buffer, etc.), and / or one or more control vectors, are also contemplated.

[0315] In some embodiments, the kit further contains a component selected from any of a secondary antibody, a reagent for immunohistochemistry, a pharmaceutically acceptable excipient, and instructions, and any combination thereof. In a specific embodiment, the kit comprises a pharmaceutical composition comprising any one or more of the antibody compositions described herein and one or more pharmaceutically acceptable excipients.

[0316] The present application also provides an article of manufacture comprising any one of the antibody compositions or kits described herein. An example of an article of manufacture is a vial (including a sealed vial). [Example]

[0317] Below are examples of specific embodiments for carrying out the present disclosure. The examples are provided 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.

[0318] 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 fully explained in the literature, 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 3 rd Ed. (Plenum Press) Vols A and B (1992).

[0319] Example 1: Synthesis and in vitro characterization of logic gate circuits containing synthetic pathway activators Materials and Methods Expression of ICT constructs in T cells Integrated circuit T (ICT) cells were generated by site-specific CRISPR-mediated knock-in (KI). T cells were activated using CD3-CD28 beads for two days. On day 2, the beads were removed, and then the ICT transgene was delivered to the GS94 site in the T cell genome. Transgene integration was achieved using a CRISPR-mediated process and electroporation step that combined activated T cells, CRISPR / Cas9 RNPs targeting the GS94 non-coding autosomal integration site, and plasmid DNA that constituted a repair template to drive insertion of the transgene cassette via cellular DNA repair mechanisms.

[0320] The GS94 CRISPR / Cas9 RNP used was generated by complexing a single guide RNA (sgRNA) with recombinant Streptococcus pyogenes Cas9 (SpCas9). The sgRNA contained a protospacer sequence that directed the CRISPR / Cas9 RNP to the GS94 transgene integration site. The plasmid DNA repair template contained an ICT transgene cassette flanked by 450 base pairs (bp) of sequence homology to regions flanking the integration site to effect repair-mediated insertion.

[0321] A schematic diagram of the five ICT transgene cassettes generated is provided in Figure 1. ICT constructs 1, 2, 3, and 4 contained a constitutively expressed priming receptor, an inducible CAR (collectively forming a logic gate or "LG"), a constitutively expressed shRNA, and a synthetic pathway activator (SPA). ICT5 contained LNGFR in place of SPA.

[0322] After electroporation, cells were harvested and grown in T cell medium for 7 days. Optionally, negative control T cells were generated using a mock electroporation process to edit T cells with ribonucleoprotein (RNP) in the absence of donor plasmid, referred to as "RNP control."

[0323] ICT cells were evaluated for expression of the transgene KI and PrimeR and CAR using flow-based staining. The constructs contained a signal peptide followed by the tags myc and FLAG on the distal extracellular portion of PrimeR and CAR, respectively. ICT cells 7 days after activation were stained with myc, FLAG, and CD3 antibodies at 4°C for 30 minutes. After activation, cells were washed in FAC buffer and run by flow cytometry. After gating each sample on live CD3+ cells, ICTs were analyzed for PrimeR and CAR expression.

[0324] CAR induction by ICT ICTs were generated as described above from T cells from two donors. 11 days after activation, ICTs were measured for CAR and PRiMER expression by FLAG and MyC staining. KI% was quantified by summing the % of T cells in a sample that were PrimeR+ or CAR+. Prior to co-culture setup, ICTs were normalized to the same KI% using the addition of donor-matched RNP-only cells. 1 x 10 7 ICT, 1 x 10 7 The cells were co-cultured with target cells or medium for 72 hours and stained using flag staining to calculate the % of CAR+ cells. Basal CAR expression was measured during the assay setup.

[0325] Synthetic pathway activators Synthetic pathway activators (SPAs) constitutively induce STAT signaling without the need for exogenous cytokine input. Through rational design, SPAs can be engineered to harness the activity of multiple STAT family transcription factors at variable levels. Exemplary class I SPAs primarily increase pSTAT3 activity, while exemplary class II SPAs primarily increase pSTAT5 activity. Figure 2 shows the structures of exemplary synthetic pathway activators.

[0326] A gp130-based synthetic pathway activator (SPA) was constructed as shown in SEQ ID NOs: 20 and 81. SEQ ID NO: 20 includes the leader sequence. SPA comprises the transmembrane and intracellular domains of gp130 linked to an ectodomain derived from the cell adhesion protein CD34. An unpaired cysteine ​​residue was introduced into the receptor ectodomain to allow for covalent bond formation and subsequent dimerization of individual synthetic gp130 monomers. SPA induces constitutive recruitment and phosphorylation of STAT1 and STAT3 transcription factors (Figure 5, data not shown).

[0327] To demonstrate the ability of the SPA module to induce constitutive STAT3 phosphorylation, ICTs expressing the SPA module under unstimulated conditions were fixed, permeabilized, and stained for pSTAT3 and the myc epitope tag to distinguish edited from nonedited cells (data not shown).

[0328] Cytotoxic, manipulated K562 cells ICT cells expressing integrated circuits containing Logic Gate 1 IC, Logic Gate 2 IC, Logic Gate 3 IC, Logic Gate 4 IC, or Logic Gate 5 IC (LG-15 IC) with shRNA and SPA were co-cultured with K562_EFG, K562_EFG_CAR, K562_EFG_primeR, or K562_EFG_CAR_primeR target antigens at various E:T ratios at 37°C for 72 hours. After incubation, cytotoxicity was measured using a luciferase reporter assay. Data are shown as the mean ± standard deviation of four donors.

[0329] Cytokine secretion To further evaluate the specificity and function of ICT cells expressing logic gates 1–5, supernatants were collected from K562-target cytotoxicity co-cultures (1:1 effector:target ratio, 72 h co-culture). After incubation, supernatants were collected at endpoint, and cytokine release levels were measured using the LuMiNEx assay. Data from four donors are shown.

[0330] Cytotoxicity in endogenous CAR cells ICT cells expressing logic gates 1-5 were co-cultured with cells endogenously expressing the CAR target and engineered to express the primeR target at various E:T ratios for 72 hours at 37°C. After incubation, cytotoxicity was measured using a luciferase reporter assay. Data are shown as the mean ± standard deviation of four donors. Supernatants were collected at endpoint, prior to luciferase readout as described above, and the cytokines IFN-γ, TNFα, GM-CSF, and IL-2 were measured using the LuMiNEx assay. Data from four donors are shown.

[0331] Cytotoxicity of mixed co-cultures ICT cells expressing LG1-5 IC were co-cultured with primeR target+ / CAR target- HUVECs and luciferase-expressing primeR target- / CAR target+ cells (K562-EFG-CAR) at various E:T ratios for 72 hours at 37°C. After incubation, cytotoxicity was measured using a luciferase reporter assay. Data are from one normal donor. ICT-mediated killing of CAR+ target cells was assessed using a luciferase reporter assay against a negative control electroporated with RNP.

[0332] result All ICT cells constitutively expressed the PrimeR construct, as indicated by myc expression (Figure 3). Inducible CAR was not expressed basally in ICT cells, as indicated by the lack of FLAG expression (Figure 3), indicating that the priming receptor did not induce CAR expression.

[0333] As shown in Figure 4, ICT cells induce CAR expression when co-cultured with cell lines expressing the primeR target antigen. The numbers shown in Figure 4 are calculated as (CAR%) / (KI% normalized to the start of the assay). *100. Thus, the logic gate circuit functioned correctly by preventing CAR expression in the absence of binding of primeR to its target antigen (Figure 3) and inducing CAR expression upon binding of primeR to its cognate ligand on target cells (Figure 4).

[0334] As shown in Figure 5, flow cytometry analysis revealed that ICT cells expressing SPA (LG1-4 IC) exhibit approximately 1 log higher pSTAT3 expression when compared to PrimeR- cells lacking SPA (EGFRt). T cells edited with the EGFRt (non-signaling) module instead of SPA do not exhibit increased pSTAT3 staining when compared to PrimeR- cells. Overall, the results indicate that ICT expressing the SPA module exhibit increased STAT3 phosphorylation.

[0335] ICT expressing the LG1-5 IC showed cytotoxicity only against cells expressing both the CAR and primeR antigens, compared to unedited control cells (RNP). Figure 6A shows cytotoxicity against parental K562 cells expressing neither target antigen. Figure 6B shows cytotoxicity against K562 cells expressing only the CAR target antigen. Figure 6C shows cytotoxicity against K562 cells expressing only the primeR target antigen. Figure 6D shows cytotoxicity against K562 cells expressing both the primeR and CAR target antigens. As shown in Figure 6D, ICT showed cytotoxicity only against cells expressing both the primeR and CAR target antigens, compared to unedited control cells (RNP).

[0336] IFN-γ production from ICTs expressing LG1-5 ICTs was observed only in supernatants collected from cocultures in which target cells expressed both the primeR and CAR target antigens (Figure 7). The cytokine analysis results were consistent with the cytotoxicity data. Collectively, these data further demonstrate that ICT activity is induced by coexpression of the primeR and CAR target antigens.

[0337] ICTs expressing the LG1-5 IC demonstrated in vitro cytotoxicity against cell lines expressing the endogenous CAR target antigen and the engineered primeR target antigen (Figure 8A). ICTs also secrete cytokines after coculture with CAR / primeR target antigen cell lines. Figure 8B shows the secretion of IFNγ, TNFα, GM-CSF, and IL-2 by ICT cells after coculture with +CAR / +primeR target antigen cells. Thus, ICTs expressing the LG1-5 IC secreted cytokines and killed ccRCC cell lines expressing the endogenous CAR antigen in the presence of the primeR target antigen.

[0338] Co-culture with HUVEC-primeR antigen+ cells induced CAR protein expression in ICT cells, confirming specific killing of CAR antigen-positive cells (Figure 9). Therefore, ICTs expressing logic gates 1-5 were able to induce CAR expression through interaction with primeR target antigen-positive endothelial cells and then specifically bind to and kill CAR target antigen-positive tumor cells. Therefore, without wishing to be bound by theory, ICTs may be primed by binding to endothelial cells expressing the primeR target antigen to express CAR and then kill CAR-targeted tumor cells.

[0339] Therefore, we developed a logic-gated ICT cell line containing a signaling pathway activator that exploits the presence of two antigens to induce tumor cell killing and enhance tumor specificity to improve the therapeutic index of CAR T cells. CAR induction was gated to the expression of the primeR target antigen found in the neovascular network of ccRCC tumors. When the priming receptor (PrimeR) binds to the primeR target antigen, PrimeR binding triggers the proteolytic release of a transcription factor that induces CAR expression. We confirmed the feasibility of vascular priming using a transwell assay, in which ICT cells were primed by an endothelial cell line expressing the primeR target antigen, then migrated through the transwell membrane and killed CAR antigen-expressing RCC cells.

[0340] When constitutively expressed in ICT cells, SPA significantly enhanced T cell potency and proliferation. Repeated stimulation assays, in which T cells were stimulated with tumor cells every 2 days, demonstrated that class I SPA resulted in a greater than 6-log improvement in tumor cell elimination over the 2-week assay period (data not shown). Across various mouse xenograft models (Figure 2, Figure 10A, Figure 10D, Figure 11B, and data not shown), SPA-expressing ICT achieved at least a 6-fold improvement in tumor growth inhibition. RNA-seq and ATAC-seq analyses demonstrated altered gene expression profiles in T cells expressing class I SPA, maintaining a T cell stem-like phenotype and restricting access to various exhaustion marker genes (Figure 2 and data not shown). Importantly, despite significantly increased proliferation levels, SPA-equipped ICT did not become immortal or exhibit signs of cytokine-independent growth (data not shown). Furthermore, SPA-expressing ICT cells rapidly shrink after tumor clearance in vivo (Figures 10B, 10C, 10E, 10F).

[0341] Collectively, these results demonstrate that SPA-loaded primeR / CAR ICT cells (i) can selectively target antigens that generally cannot be safely targeted by conventional CARs and (ii) can overcome multiple suppressive mechanisms in the tumor microenvironment.

[0342] Example 2: In vivo efficacy of primeR and CAR logic-gated T cells expressing synthetic pathway activators Materials and Methods RCC Efficacy Model Human ccRCC cells express endogenous levels of CAR target antigen, and they were engineered to express physiological levels of primeR target antigen. 6 PrimeR target antigen cells were inoculated into the right dorsal flank of 5-6 week-old female NSG MHC I / II DKO mice. 35 days after tumor inoculation, the average tumor volume was 150 mm. 3The tumor-bearing animals were randomized into various treatment groups so that the mean tumor volume per group was within 10% of the overall mean. Seven mice per group were injected with 0.15 x 10 PrimeR+ICT cells expressing one of the five LG ICTs (LG1 IC, LG2 IC, LG3 IC, LG4 IC, or LG5 IC) described in Example 1, RNP, or PBS. 6 The test was repeated using ICT generated from two different normal donors. Tumor volume and body weight were recorded every two weeks. Tumor volume was calculated using the formula 1 / 2 * L * W 2 The calculation was performed according to the formula: where L is the tumor length and W is the tumor width.

[0343] Blood pharmacokinetics showed proliferation of ICT on day 14 after T cell injection, followed by a complete decline by day 42 after injection. PrimeR+ ICT was quantified in the blood of mice using flow cytometry with counting bright beads for T cell quantification / volume, and ICT proliferation was tracked. Mean and SEM were plotted.

[0344] Double flank model Human ccRCC 786-O cells were engineered to express either the CAR target antigen and the primeR target antigen, or the CAR target antigen alone. 6 786-O-CAR+ cells and 786-O-CAR antigen+-primeR antigen+ cells were inoculated into the left and right dorsal flanks, respectively, of 5- to 6-week-old female NSG MHC I / II DKO mice. At 35 days after tumor inoculation, the mean tumor volume in each flank was 150-200 mm. 3 The tumor-bearing animals were randomized into various treatment groups so that the mean tumor volume per group on the right flank was within 10% of the overall mean. Seven mice per group received 0.25 x 10 PrimeR+ICT cells, constitutive CAR T cells, RNP, or PBS control. 6 or 1×10 6 The tumor volume and body weight were recorded every two weeks. The tumor volume was calculated using the formula 1 / 2 * L * W2 (where L is tumor length and W is tumor width). (B) Tumor volume in the 786-O CAR antigen-only flank (left) and (C) tumor volume in the 786-O-CAR+ / primeR antigen+ flank (right). Data represent a single-donor study with 7 mice per group, and the mean and SEM are plotted.

[0345] result A498 RCC Efficacy Model ICTs expressing LG1-5 ICTs demonstrated tumor elimination in a ccRCC model. Figures 10A and 10D show tumor volume after tumor implantation in mice treated with ICTs expressing Logic Gates 1-5, RNP, or PBS generated from T cells from either donor 1 (Figures 10A-C) or donor 2 (Figures 10D-F). Figures 10B and 10E show the proliferation of total T cells and ICTs on day 12 postinoculation, followed by a decline by day 21. Figures 10C and 10F show the total T cells expressing the priming receptors on days 12 and 21. In both replicates, ICT cells demonstrated significant tumor growth suppression in mice (P<0.05).

[0346] Double flank model ICTs expressing LG1-5 ICs demonstrated specificity in the double-flank model (Figures 11A and 11B). Greater tumor growth inhibition (TGI) was observed in double-positive primeR / CAR flanks (Figure 11B) than in single-positive CAR-only flanks (Figure 11A). Thus, the double-flank xenograft model demonstrates that the logic gated circuit (ICT) more selectively killed tumors expressing both CAR and primeR target antigens, rather than tumors expressing CAR alone.

[0347] Example 3: Generation and characterization of novel synthetic pathway activators method Production and Synthesis A library of novel gp130-based STAT1 / 3 synthetic pathway activators (SPAs) was generated by diversifying extracellular and intracellular domains, multimerization modalities, membrane anchor modalities, and epitope tags / signaling peptides. SPAs were generated to explore optimization and improvement of SPA performance, including improved potency, reduced immunogenicity, detectability, and reduced gene size. Various forms of the gp130 intracellular domain (ICD) were used, including the full-length gp130 ICD and truncated forms containing gp130 ICD Δ707-755, Δ771-811, Δ818-901, and combinations thereof. The gp130 Y759F mutation was also included in some ICD constructs. The multimerization modalities used were unpaired cysteine, leucine zipper, BCR ectodomain (SEQ ID NO: 239), and VASP tetramerization domain (SEQ ID NO: 240). The extracellular domains used were the full-length CD34 ectodomain (SEQ ID NO: 242), CD34 epitope (SEQ ID NO: 238), BCR ectodomain (SEQ ID NO: 239), thrombopoietin receptor domain (SEQ ID NO: 243), and erythropoietin receptor (EpoR) ectodomain (SEQ ID NO: 241). The membrane anchor modalities used were prenylation and myristoylation domains derived from src, fyn, or lck. Prenylation modifications were also used at the C-terminus of some SPAs. Some SPAs also contained the CD8 alpha hinge domain (FACD). In some SPAs, SPA expression was inducible upon T cell activation. Other SPAs were constitutively expressed. The sequences of the novel SPAs are provided in SEQ ID NOs: 1-58 and 63-104. The SPAs were screened first by pSTAT activation screening, then by in vitro functional assessment, and finally by in vivo efficacy assessment.

[0348] The activity of novel pathway activators was assessed by intracellular pSTAT staining after 24 hours of serum starvation. ICTs expressing the SPA construct modules were starved for 24 hours without antigen stimulation or cytokine support, then fixed, permeabilized, and stained for pSTAT protein. MFI values ​​for pSTAT3 and pSTAT1 were measured for two independent donors, and the average values ​​were plotted on a heatmap.

[0349] Cytokine induction Various ICTs expressing novel STAT1 / STAT3 SPAs were cultured with K562 cells at a 1:1 ratio. After 72 hours of coculture, supernatants were isolated and analyzed by Luminex for granzyme B and IL-10 production. Representative cytokines showing different expression levels are shown (plotted by group according to pSTAT1 levels).

[0350] Repetitive stimulation and memory phenotypes T cells expressing logic gate (ICT) and various novel class I STAT1 / STAT3 SPAs were stimulated with K562 cells expressing primeR and CAR antigens supplemented with IL-2 in a 14-day repeated stimulation assay. ICTs and tumor cells were renormalized to a fixed number every other day to maintain a 1:1 E:T ratio. Total tumor cell proliferation and T cell proliferation over the experimental period were normalized to EGFRt controls and plotted between groups according to pSTAT1 levels. L-gp130 was used as a control SPAs. Additional controls included expression of cJun, EGFRt, mbIL-15, and IL7Ra-IL7 in ICTs.

[0351] Memory phenotype was measured by flow cytometry for CD45RA and CD27 expression at day 0 and at the endpoint of the repeated stimulation assay (day 14) and plotted between groups according to pSTAT1 profile.

[0352] result Approximately 60% of the novel SPAs showed elevated levels of pSTAT1 (Figure 12B) and / or pSTAT3 (Figure 12A) compared to logic-gated ICTs expressing inactive truncated EGFR molecules instead of SPAs. The pSTAT1 vs. pSTAT3 heat map highlights the combinatorial diversity achieved by modifying the SPA structure (Figure 13). Thus, without wishing to be bound by theory, STAT-induced cell surface receptors exhibit remarkable flexibility in their structure, allowing for diversification of STAT profiles.

[0353] Diversification of pSTAT1 and pSTAT3 signaling resulted in the secretion of various cytokines, including granzyme B (Figure 14, upper panel) and IL-10 (Figure 14, lower panel). Thus, the divergence of pSTAT1 / 3 signaling achieved via the novel SPA induced cytokine responses.

[0354] All of the novel STAT1 / STAT3 SPAs demonstrated dramatic levels of tumor clearance in repeated stimulation assays. Figure 15 (upper panel) shows tumor growth compared to control ICT under long-term antigen stimulation. ICTs expressing the novel SPAs significantly reduced tumor growth and increased tumor clearance compared to control ICT. Figure 15 (lower panel) shows T cell proliferation. Without wishing to be bound by theory, the novel class I SPAs thus demonstrated superior antitumor activity compared to L-gp130 (SEQ ID NO: 62) and also demonstrated a favorable safety profile due to rapid tumor shrinkage after tumor clearance.

[0355] The novel SPAs induced diverse memory and functional phenotypes based on pSTAT profiles (Figure 16). Figure 16 shows the percentage of ICT cells expressing novel SPAs that expressed CD45RA and / or CD27 and the corresponding T cell types (e.g., Tscm, Tcm, Teff) after repeated stimulation assays.

[0356] Without wishing to be bound by theory, the novel SPA thus represents a novel, improved, and tunable T cell-intrinsic approach to manipulate cell fate that results in potent anti-tumor properties compared to, for example, L-gp130.

[0357] Example 4: In vivo efficacy of second primeR and CAR logic-gated T cells expressing synthetic pathway activators Materials and Methods In the mesothelioma (MSTO) solid tumor model, mice were implanted with MSTO tumors expressing different primeR and CAR antigens compared to Example 2. Tumors were 100 mm 3 Upon reaching 100% T cell mass, 300,000 ICT cells expressing an exemplary primeR and CAR logic gate and a novel class I SPA (SPA of SEQ ID NO: 20, 30, or 16), or expressing an exemplary primeR and CAR logic gate but no additional SPA, were intravenously administered. T cells expressing an exemplary primeR and CAR logic gate and L-gp130 (SPA001, SEQ ID NO: 62) were used as an additional control. For pharmacokinetic (PK) studies, blood was collected weekly on days 7, 14, 21, and 28 after T cell injection. Tumor growth inhibition (TGI) was measured three times a week for 45 days after T cell injection.

[0358] result ICT cells expressing logic gate and novel class I SPA variants of SEQ ID NOs: 20, 30, and 16 showed improved antitumor efficacy compared to ICT cells expressing logic gate and L-gp130 (SPA01, FIG. 17). The novel class I SPA variants also showed improved T cell proliferation compared to the base SPA01 molecule. Thus, the novel SPAs were superior to L-gp130 in in vivo studies assessing anticancer efficacy.

[0359] Example 5: SPA expression in T cells induces CD11c expression Materials and Methods Five- to six-week-old female NSG MHC DKO mice were implanted with 786-O B2M KO tumors expressing the primeR and CAR antigens from Example 2. On day 36 after tumor implantation, mice were intravenously injected with LG1 IC T cells or non-SPA IC T cells (LG 5 ICT). Tumor and spleen samples were collected from 15 mice treated with either LG1 IC T cells (2 donors) or non-SPA ICT (1 donor) on day 7 after ICT injection. Tumors were lysed in DMEM containing Dnase / collagenase / hyaluronidase. Tumor dissociation was performed on a gentleMacs (Octo) using a custom-built program. Spleens were mechanically digested using a "handle-end" syringe plunger with a 70 μm filter. After digestion, the cell suspension was filtered, subjected to RBC lysis, and stained with live dead Zombie NIR dye for 15 minutes at room temperature in the dark. After live-dead staining, cells were centrifuged, washed, and stained for 30 minutes at room temperature in the dark with a cell surface antibody cocktail containing mouse CD45, mouse GR-1, human CD3, CAR idiotype (CAR receptor), and prime idiotype (prime receptor) in the presence of human and mouse Fc blocks. Cells were then centrifuged at 400 g for 10 minutes, washed, and suspended in flow staining buffer (BD Biosciences). Cells were then sorted using a BD FACS Aria. Receptor-positive cells were sorted and subjected to RNA-seq and CITE-seq.

[0360] CITE labeling protocol TotalSeq-C Human Universal Cocktail, V1.0 (Biolegend catalog no. 399905) antibody mix was reconstituted in 26 μl of 1% BSA in PBS. Cells were blocked with Fc Block, and 13 μl of the antibody mix was added to 500,000 cells. After a 30-minute incubation on ice, cells were washed, resuspended, and transferred for GEM generation and barcoding using the 10x Genomics Chromium Next Gem 5' Single Cell Kit (catalog no. 1000263). Cells were combined with the GEM Master Mix and loaded onto a 10x K chip in a Chromium X controller for GEM generation, followed by incubation in a thermal cycler for reverse transcription. After GEM cleanup and cDNA amplification, gene expression sequencing libraries were generated according to the 10x Genomics published protocol. Using the supernatant fraction from the cDNA amplification cleanup step, CITE-Seq libraries were constructed using 8 cycles of PCR amplification using a 10x Genomics Dual Index Plate TN Set A (Cat. No. 1000250). After library preparation, samples were sequenced on an Illumina NovaSeq 6000.

[0361] Bioinformatics analysis Single-cell RNA-seq and CITE-seq data were first analyzed using Cellranger 7.0 to generate a cell matrix of genes. Cells were filtered to exclude low-UMI cells, cells with high mitochondria content, and non-T cells. The filtered matrix was then analyzed using ScVi 0.20 to generate low-dimensional visualizations of individual cells. The filtered matrix was also analyzed using Seurat 5.0 to define differentially expressed genes. These genes were significantly up- or down-regulated in SPA-positive cells compared with SPA-negative cells (SPA vs. no SPA). Genes up-regulated in SPA compared with no SPA across all D0, D7 tumor, and D7 spleen samples were intersected to generate a shared list. The same analysis was performed for both RNA-seq and CITE-seq to generate two shared lists. The two shared lists were then further intersected to nominate a final list of cell surface protein genes most associated with SPA expression. CD11c was nominated by manual review of the final nominated genes.

[0362] Detection of CD11c by flow cytometry Peripheral blood samples were collected in EDTA-coated tubes. Red blood cells were lysed with ammonium chloride, and the remaining cell fraction was stained with a fixable amine-reactive viability dye and Fc receptors were blocked using an anti-CD16 / CD32 monoclonal antibody. After washing, cell surface antigens were stained using fluorochrome-conjugated monoclonal antibodies against human CD45, mouse CD45, mouse Gr-1, Flag tag (chimeric antigen receptor), and Myc tag (priming receptor). Cells were then fixed with Cytofix fixation buffer (BD Biosciences) and permeabilized with Phosflow Perm buffer III (BD Biosciences) according to the manufacturer's recommendations. After permeabilization, cells were stained with fluorochrome-conjugated monoclonal antibodies against CD11c (CD11c is also known as integrin, alpha X, or ITGAX) and intracellular pSTAT3. Samples were analyzed using an Attune NxT flow cytometer (Thermo Fisher Scientific).

[0363] result In both tumor and spleen samples, at D7, there was a strong correlation between CD11c RNA and cell surface expression and SPA expression in both CD4+ and CD8+ cells (Figure 18A). Increased CD11c expression was observed in SPA-expressing CD8 and CD4 cells compared with non-SPA-expressing CD8 and CD4 cells. Figure 18B shows CD11c (ITGAX) expression in splenocytes or tumor cells from mice treated with SPA ICT-expressing cells (donor 2) compared with non-SPA ICT-expressing cells (donor 1), collected on days 0 and 7 after treatment. CD11c (i.e., ITGAX) was expressed at higher levels in SPA-containing T cells isolated from tumors and spleens compared with non-SPA-containing T cells (Figure 18B). Therefore, quantification of CD11c (mRNA or protein) expression can be used as a marker of SPA expression in both CD4+ and CD8+ cells.

[0364] While the present disclosure has been particularly shown and described with reference to preferred and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes in form and detail can be made therein without departing from the spirit and scope of the present disclosure.

[0365] All references, issued patents, and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes.

[0366] Unofficial sequence listing TIFF2026508611000010.tif216165TIFF2026508611000011.tif208165TIFF2026508611000012.tif202165TIFF2026508611000013.tif227165TIFF2026508611000014.tif214165TIFF2026508611000015.tif193165TIFF2026508611000016.tif208165TIFF2026508611000017.tif214165TIFF2026508611000018.tif227165TIFF2026508611000019.tif209165TIFF2026508611000020.tif226165TIFF2026508611000021.tif233165TIFF2026508611000022.tif233165TIFF2026508611000023.tif225165TIFF2026508611000024.tif204165TIFF2026508611000025.tif214165TIFF2026508611000026.tif204165TIFF2026508611000027.tif221165TIFF2026508611000028.tif214165TIFF2026508611000029.tif173165TIFF2026508611000030.tif197165TIFF2026508611000031.tif226165TIFF2026508611000032.tif203165TIFF2026508611000033.tif190165TIFF2026508611000034.tif226165TIFF2026508611000035.tif206165

Claims

1. 1. A synthetic pathway activator (SPA) peptide comprising a chimeric polypeptide, comprising: a. optionally, an extracellular domain; and b. a lipid anchor or transmembrane domain; c. an intracellular signaling domain; d. a multimerization region; A synthetic pathway activator (SPA) peptide comprising said chimeric polypeptide, comprising:

2. the SPA peptide comprises, from N-terminus to C-terminus, an extracellular domain comprising a CD34 epitope, a multimerization region comprising unpaired cysteine ​​residues, a gp130 transmembrane domain, and a gp130 intracellular signaling domain; a. the SPA peptide comprises the sequence set forth in SEQ ID NO: 81 or 20, or b. multimerization of the chimeric polypeptide via the multimerization region results in constitutive activity of the intracellular signaling domain; The SPA peptide of claim 1.

3. 3. The SPA peptide of claim 1 or 2, wherein the multimerization region comprises at least one of an unpaired cysteine ​​residue, a leucine zipper, a BCR domain, and a VASP domain.

4. The SPA peptide of claim 3 , wherein the multimerization region comprises at least one unpaired cysteine ​​residue.

5. 5. The SPA peptide of claim 3 or 4, wherein the multimerization domain comprises at least an unpaired cysteine ​​residue and a leucine zipper.

6. The SPA peptide of any one of claims 1 to 5, wherein the multimerization domain is intracellular when expressed by a cell.

7. The SPA peptide of any one of claims 1 to 5, wherein the multimerization domain is extracellular when expressed by a cell.

8. The SPA peptide of any one of claims 1 to 7, wherein the intracellular signaling domain induces phosphorylation of at least one of STAT1, STAT3, and STAT5.

9. 9. The SPA peptide of any one of claims 1 to 8, wherein the intracellular signaling domain comprises a type I cytokine receptor superfamily box 1 (IWPNVDP (SEQ ID NO: 106)) or box 2 (VSVVEIEANDKKP (SEQ ID NO: 107)) peptide motif.

10. 10. The SPA peptide of any one of claims 1 to 9, wherein the intracellular signaling domain comprises a tyrosine phosphorylation motif comprising YXXQ or YXPQ.

11. The SPA peptide of any one of claims 1 to 10, wherein the intracellular signaling domain comprises a polypeptide sequence derived from an interleukin receptor.

12. The SPA peptide of any one of claims 1 to 11, wherein the interleukin receptor comprises a gp130 intracellular signaling domain.

13. 13. The SPA peptide of any one of claims 1 to 12, wherein the intracellular signaling domain comprises a polypeptide sequence comprising amino acids 642 to 918 of gp130 (SEQ ID NO: 59).

14. 14. The SPA peptide of any one of claims 1 to 13, wherein the intracellular signaling domain comprises a polypeptide sequence comprising the sequence set forth as SEQ ID NO:

60.

15. The SPA peptide of any one of claims 1 to 12, wherein the interleukin receptor comprises a truncated gp130 intracellular signaling domain.

16. 16. The SPA peptide of claim 15, wherein the truncated gp130 intracellular signaling domain comprises a deletion of amino acids 771 to 811 of gp130 (SEQ ID NO: 59).

17. 16. The SPA peptide of claim 15, wherein the truncated gp130 intracellular signaling domain comprises a deletion of amino acids 707-755, 771-811, or 818-901 of gp130 (SEQ ID NO: 59).

18. 16. The SPA peptide of claim 15, wherein the truncated gp130 intracellular signaling domain comprises a truncated gp130 intracellular domain of a sequence selected from the group set forth in SEQ ID NOs: 10-16 and 71-77.

19. 19. The SPA peptide of any one of claims 1 to 18, wherein the gp130 intracellular signaling domain further comprises the Y759F mutation of gp130 (SEQ ID NO: 59).

20. 20. The SPA peptide of any one of claims 1 to 19, wherein the intracellular signaling domain further comprises a prenylation motif at the C-terminus.

21. 21. The SPA peptide of any one of claims 1 to 20, wherein the lipid anchor or transmembrane domain comprises a gp130 transmembrane domain, a CD8-alpha transmembrane domain, a prenylation motif, or a myristoylation domain derived from src, fyn, or lck.

22. 22. The SPA peptide of claim 21, wherein the transmembrane domain comprises a gp130 transmembrane domain.

23. 23. The SPA peptide of claim 21 or 22, wherein the transmembrane domain comprises a polypeptide sequence comprising amino acids 620 to 641 of gp130 (SEQ ID NO: 59).

24. 24. The SPA peptide of any one of claims 1 to 23, wherein the transmembrane domain comprises a polypeptide sequence comprising the sequence set forth as SEQ ID NO:

61.

25. 25. The SPA peptide of any one of claims 1 to 24, further comprising a CD8-alpha hinge domain.

26. 26. The SPA peptide of any one of claims 1 to 25, wherein the extracellular domain comprises one or more of a CD34 epitope (e.g., a QBEND10 epitope), a CD34 ectodomain, a BCR ectodomain, a thrombopoietin receptor (TpoR) ectodomain, or an erythropoietin receptor (EpoR) ectodomain.

27. 27. The SPA peptide of claim 26, wherein the thrombopoietin receptor (TpoR) domain or the erythropoietin receptor (EpoR) domain comprises an unpaired cysteine.

28. 28. The SPA peptide of any one of claims 1 to 27, wherein the extracellular domain confers constitutive activity to the intracellular signaling domain.

29. 2. The SPA peptide of claim 1, comprising, from the N-terminus to the C-terminus, an extracellular domain comprising a CD34 epitope, a multimerization region comprising unpaired cysteine ​​residues, a gp130 transmembrane domain, and a gp130 intracellular signaling domain.

30. 30. The SPA peptide of any one of claims 1 to 29, comprising a sequence selected from the sequences set forth in SEQ ID NOs: 1 to 58 or 63 to 104.

31. 31. The SPA peptide of claim 30, comprising the sequence set forth in SEQ ID NO:

20.

32. 31. The SPA peptide of claim 30, comprising the sequence set forth in SEQ ID NO:

81.

33. A multimer of the SPA peptide of any one of claims 1 to 32.

34. At least one nucleic acid encoding the SPA peptide of any one of claims 1 to 32.

35. 35. At least one vector comprising at least one nucleic acid according to claim 34.

36. a. a first chimeric polypeptide comprising a priming receptor; b. a second chimeric polypeptide comprising a chimeric antigen receptor (CAR); and c. The SPA peptide of any one of claims 1 to 32. Including, the system.

37. 37. The system of claim 36, wherein binding by the CAR or the priming receptor induces expression of the SPA peptide.

38. 37. The system of claim 36, wherein the SPA peptide is constitutively or inducibly expressed.

39. A cell or population of cells comprising an SPA peptide according to any one of claims 1 to 32, a multimer according to claim 33, a nucleic acid according to claim 34, a vector according to claim 35, or a system according to any one of claims 36 to 38.

40. 40. The cell of claim 39, wherein the cell is an immune cell, optionally wherein the immune cell is a primary human immune cell.

41. 41. A pharmaceutical composition comprising a cell or population of cells according to claim 39 or 40 and a pharmaceutically acceptable excipient.

42. A pharmaceutical composition comprising the nucleic acid of claim 34 or the vector of claim 35 and a pharmaceutically acceptable excipient.

43. 35. A method of editing a cell, comprising inserting the nucleic acid of claim 34 into an insertion site in the genome of the cell.

44. A method for producing a CD11c+ cell, the method comprising inserting the nucleic acid of claim 34 into an insertion site in the genome of the cell.

45. 45. The method of claim 43 or 44, wherein the nucleic acid is introduced into the cell non-virally.

46. 1. A method for editing a cell, comprising: a. providing a nuclease domain and a guide RNA, wherein the nucleic acid comprises the nucleic acid of claim 34, and the 5' and 3' ends of the nucleic acid comprise nucleotide sequences that are homologous to genomic sequences adjacent to an insertion site in the genome of the cell; b. introducing the nuclease domain and the nucleic acid into the cell, wherein the guide RNA specifically hybridizes to a target region of the genome of the cell and the nuclease domain cleaves the target region to create the insertion site in the genome of the cell; c. editing the cell via insertion of the nucleic acid into the insertion site in the genome of the cell; The method comprising:

47. 47. The method of claim 46, wherein the nuclease domain and the nucleic acid are introduced into the cell non-virally.

48. 1. A method for editing immune cells, comprising: a. providing a ribonucleoprotein complex (RNP)-nucleic acid complex, wherein the RNP comprises a nuclease domain and a guide RNA, and the nucleic acid comprises the nucleic acid of claim 34, wherein the 5' and 3' ends of the nucleic acid comprise nucleotide sequences that are homologous to genomic sequences adjacent to 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 in the genome of the primary immune cell and the nuclease domain cleaves the target region to create the insertion site in the genome of the immune cell; c. editing the immune cell via insertion of the nucleic acid of claim 34 into the insertion site in the genome of the immune cell; The method comprising:

49. 49. The method of claim 45, 47, or 48, wherein non-virally introducing comprises electroporation.

50. 50. The method of any one of claims 46 to 49, wherein the nuclease domain comprises a CRISPR-associated endonuclease (Cas), optionally a Cas9 nuclease.

51. 51. The method of any one of claims 46 to 50, wherein the target region of the genome of the cell is the genomic safe harbor (GSH) locus or the T-cell receptor alpha constant (TRAC) locus.

52. 52. The method of any one of claims 46 to 51, wherein the target region is the GS94 locus (locus chr11:128340000-128350000).

53. The method of any one of claims 43 to 52, wherein the nucleic acid is a double-stranded nucleic acid or a single-stranded nucleic acid.

54. 54. The method of any one of claims 43 to 53, wherein the nucleic acid is a linear nucleic acid or a circular nucleic acid, optionally wherein the circular nucleic acid is a plasmid.

55. 55. The method of any one of claims 48 to 54, wherein the cells are immune cells, optionally primary human immune cells.

56. The method of any one of claims 48 to 55, wherein the immune cells are autoimmune cells.

57. 56. The method of any one of claims 48 to 55, wherein the immune cells are allogeneic immune cells.

58. 58. The method of any one of claims 48 to 57, 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 precursor cell.

59. The method of any one of claims 48 to 58, wherein the immune cells are primary T cells.

60. 60. The method of any one of claims 48 to 59, wherein the immune cells are primary human T cells.

61. 61. The method of any one of claims 43 to 60, wherein the cells are virus-free.

62. The method of any one of claims 43 to 61, further comprising determining CD11c expression in said cells.

63. 63. The method of any one of claims 43 to 62, further comprising obtaining said cells from a patient and introducing said nucleic acid in vitro.

64. 43. A method of treating a disease in a subject, comprising administering to the subject a cell or population of cells described in claim 39 or 40 or a pharmaceutical composition described in claim 41 or 42.

65. 65. The method of claim 64, wherein the disease is cancer.

66. 66. The method of claim 65, wherein the cancer is a solid cancer or a liquid cancer.

67. 67. The method of claim 65 or 66, wherein the cancer is kidney cancer, clear cell renal cell carcinoma (ccRcc), colon cancer, or lung cancer.

68. 42. A method of inhibiting target cells in a subject, comprising administering to the subject a cell or population of cells described in claim 39 or 40 or a pharmaceutical composition described in claim 41 or 42, wherein the cells inhibit the target cells.

69. 1. A method for modulating the activity of a cell or immune cell, comprising: a. i. an SPA peptide according to any one of claims 1 to 32, ii. The system of any one of claims 36 to 38; iii. A nucleic acid according to claim 34, and / or iv. The vector of claim 35. Obtaining cells or immune cells comprising the b. contacting said cell or said immune cell with a target cell, wherein said synthetic pathway activator modulates the activity of said cell or said immune cell; The method comprising:

70. 1. A method for modulating the activity of a cell or immune cell, comprising: a. i. an SPA peptide according to any one of claims 1 to 32, ii. The system of any one of claims 36 to 38; iii. A nucleic acid according to claim 34, and / or iv. The vector of claim 35. Obtaining cells or immune cells comprising the 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, binding of the chimeric antigen receptor to the CAR antigen on the target cell regulates the activity of the cell or immune cell, and the synthetic pathway activator also regulates the activity of the cell or immune cell; The method comprising:

71. 1. A method of treating a disease in a subject in need thereof, comprising: a. determining or having determined expression of CD11c in a cell comprising a synthetic pathway activator (SPA) peptide of any one of claims 1 to 32 or a nucleic acid of claim 34; b. administering or having administered said cells to said subject; The method comprising:

72. A method for determining SPA expression in a cell, the method comprising expressing one or more SPA peptides according to any one of claims 1 to 32 in said cell and determining CD11c expression in said cell.

73. The method of claim 70 or 72, wherein the expression of CD11c in the cells comprises the mRNA expression level of CD11c or the protein expression level of CD11c.

74. 74. The method of any one of claims 70-73, wherein the cell is an immune cell, a primary human immune cell, a natural killer (NK) cell, a T cell, a CD8+ T cell, a CD4+ T cell, a primary T cell, or a T cell precursor.

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