Method and composition for targeted receptor-mediated programmable macromolecule delivery

Abstract

Cells engineered with custom-designed receptors can efficiently transfer membrane-associated macromolecules into recipient cells upon contact, which can be freed from endosomes in recipient cells and optionally functionalized in a trogocytosis-like manner via pH-responsive membrane fusion. A variety of configurations, payload proteins and donor and recipient cells are possible with the methods descried herein.

Description

PATENT Attorney Docket No.110221-1472824-010910WO Client Ref. No. CZB-281S-PC METHOD AND COMPOSITION FOR TARGETED RECEPTOR- MEDIATED PROGRAMMABLE MACROMOLECULE DELIVERY CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] The present application claims benefit of priority to U.S. Provisional Patent Application No.63 / 606,711, filed December 6, 2023, which is incorporated by reference for all purposes. STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with Government support under contracts CA266470, AG077193 and NS137219 awarded by the National Institutes of Health and contract 2046650 awarded by the National Science Foundation. The Government has certain rights in the invention. BACKGROUND OF THE INVENTION

[0003] Genome editing technologies hold great promise for addressing the root causes of various diseases (1, 2). However, realizing their full potential in research and clinical applications calls for major advances in delivery methods. Current approaches, including adeno- associated viruses (AAV) and nanoparticles, face challenges in flexibly programming their cellular specificity. While AAV capsids exhibit a spectrum of tissue tropism, they often differ in relative tropism strength but not absolute specificity (3, 4). Modifying capsids to enhance specificity without compromising virus stability remains non-trivial (5). Nanoparticles tend to accumulate primarily in the liver and spleen, restricting their specificity for other therapeutic sites (6). There is an urgent need for a robust delivery system that allows for easy reprogramming of targeting specificity towards diverse cell types and tissues.

[0004] Engineered cells are promising vehicles for macromolecule delivery, capable of producing biomolecules continuously, responding to external control, and leveraging intercellular communication pathways for cargo delivery to recipient cells. While cell-mediated1 KILPATRICK TOWNSEND 790261771macromolecule secretion, such as cytokines (7) and prodrug-converting enzymes (8), has been demonstrated, specific and programmable macromolecule delivery into recipient cells remains a challenge (9). Natural pathways such as extracellular vesicles and tunneling nanotubes enable intercellular molecule transfer (10–12), and an engineered RNA exporter system showed cell-to- cell mRNA delivery (13), but they lack cell type or tissue specificity.

[0005] Cells have evolved intricate mechanisms of direct material exchange at their interface upon specific ligand-receptor interactions (14–16). One example is trans-synaptic vesicles, including polarized exosomes (17) and synaptic ectosomes (18), which are extracellular vesicles actively formed by T cells at the immunological synapse that can transport molecules across the synaptic cleft (16). Other examples include trans-endocytosis (14, 19–21) or trogocytosis (15), which are recipient cell-mediated processes to acquire molecules from an interacting cell across the synaptic cleft without the need for vesicle generation.

[0006] Among these, trogocytosis uniquely represents the extraction of plasma membrane fragments and associated molecules from one cell onto the surface of the other cell with functional integrity, a desirable trait for biomolecule delivery (22–28). The molecular mechanism underlying trogocytosis remains unclear (29, 30). It has been shown that, chimeric antigen receptors (CAR), a type of synthetic receptor that can reprogram the cancer cell-targeting specificity of T cells (31, 32), can drive the extraction of tumor surface antigen onto the T cell plasma membrane through trogocytosis (22). Trogocytosis has also been shown to be bidirectional in certain cases (33). Based on these observations, we hypothesized that engineered donor cells with a custom-designed receptor can transfer membrane-bound molecules onto recipient cells with a cognate ligand through trogocytosis, to achieve highly specific delivery of molecular cargos. BRIEF SUMMARY OF THE INVENTION

[0007] A variety of aspects are described herein. In some embodiments, a cell is provided comprising: (a) a transmembrane protein comprising (i) an extracellular amino acid sequence that binds to a target protein on a recipient cell and (ii) a transmembrane domain, wherein the transmembrane domain is linked to a payload polypeptide, the transmembrane protein is heterologous to the cell and the transmembrane domain is heterologous to the payload polypeptide; and / or (b) a low pH-dependent fusogenic protein.2 KILPATRICK TOWNSEND 790261771

[0008] In some embodiments, the transmembrane protein further comprises a cleavage sequence between the transmembrane domain and the payload polypeptide, wherein the cleavage sequence is cleaved in a cellular endosome. In some embodiments, the cleavage sequence is cleaved by an endogenous enzyme in the cellular endosome. In some embodiments, the cleavage sequence is cleaved by a heterologous enzyme expressed in or introduced into the cellular endosome.

[0009] In some embodiments, the cleavage sequence is a low pH-dependent cleavage sequence between the transmembrane domain and the payload polypeptide, wherein the low pH-dependent cleavage sequence is self-cleaved at the pH of a cellular endosome but not at the pH of the cytosol of the cell. In some embodiments, the low pH-dependent cleavage sequence is a pH intein.

[0010] In some embodiments, the transmembrane domain is covalently linked to the payload polypeptide. In some embodiments, the transmembrane domain is non-covalently linked to the payload polypeptide.

[0011] In some embodiments, the payload polypeptide is an enzyme or protein-binding polypeptide. In some embodiments, the payload polypeptide comprises a nuclear localization signal sequence or a nuclear export signal sequence.

[0012] In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell.

[0013] In some embodiments, the payload polypeptide is a nucleic acid binding protein. In some embodiments, the nucleic acid binding protein is binding a nucleic acid. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid binding protein is a RNA-guided protein. In some embodiments, the gRNA-guided protein is a CRISPR nuclease or an inactive mutant thereof.

[0014] In some embodiments, the extracellular amino acid sequence comprises an antibody or antigen-binding fragment thereof. In some embodiments, the antibody is a scFv.3 KILPATRICK TOWNSEND 790261771

[0015] In some embodiments, the low pH-dependent fusogenic protein is a type III viral fusion protein or a fusogenic fragment thereof. In some embodiments, the type III viral fusion protein is a Vesicular stomatitis virus G protein or a fusogenic fragment thereof.

[0016] In some embodiments, the cell comprises an expression cassette comprising an inducible promoter operable linked to a polynucleotide encoding the low pH-dependent fusogenic protein.

[0017] Also provided is a method of transferring a payload polypeptide from a first cell to a second cell. In some embodiments, the method comprises, providing the first cell, the first cell comprising: (a) a transmembrane protein comprising (i) an extracellular amino acid sequence that binds to a target protein on a recipient cell and (ii) a transmembrane domain, wherein the transmembrane domain is linked to a payload polypeptide, the transmembrane protein is heterologous to the cell and the transmembrane protein is heterologous to the payload polypeptide; and a low pH-dependent cleavage sequence is between the transmembrane domain and the payload polypeptide, wherein the low pH-dependent cleavage sequence is self-cleaved at the pH of an cellular endosome but not at the pH of the cytosol of the cell; and / or (b) a low pH- dependent fusogenic protein; and contacting the first cell to a second cell under conditions that allow for transfer of a portion of the cell membrane comprising the transmembrane protein and the low pH-dependent fusogenic protein to the second cell and the payload polypeptide is released from an endosome to the cytosol of the second cell following self-cleavage of the low pH-dependent cleavage sequence.

[0018] In some embodiments, the first and second cells are mammalian cells. In some embodiments, the first and second cells are mammalian cells of the same species. In some embodiments, the first and second cells are human cells.

[0019] In some embodiments, the method is performed in vitro, ex vivo or in vivo.

[0020] In some embodiments, the first cell is administered to a mammal and the second cell resides in the mammal and the contacting occurs in the mammal.

[0021] In some embodiments, the second cell is a neuronal cell, immune cell, cancer cell, muscle cell, or stem cell.4 KILPATRICK TOWNSEND 790261771

[0022] In some embodiments, the first cell is a glial cell, immune cell, stem cell, or red blood cell.

[0023] Also provided is a nucleic acid encoding a transmembrane protein comprising (i) an extracellular amino acid sequence that binds to a target protein on a recipient cell and (ii) a transmembrane domain, (iii) a low pH-dependent cleavage sequence, wherein the low pH- dependent cleavage sequence is cleaved in a cellular endosome; and (iv) a payload polypeptide, wherein the transmembrane domain is heterologous to the payload polypeptide. In some embodiments, the cleavage sequence is a low pH-dependent cleavage sequence between the transmembrane domain and the payload polypeptide, wherein the low pH-dependent cleavage sequence is self-cleaved at the pH of a cellular endosome but not at the pH of the cytosol of the cell. In some embodiments, the low pH-dependent cleavage sequence is a pH intein.

[0024] In some embodiments, the transmembrane domain is covalently linked to the payload polypeptide. In some embodiments, the transmembrane domain is non-covalently linked to the payload polypeptide.

[0025] In some embodiments, the payload polypeptide is an enzyme or protein-binding polypeptide.

[0026] In some embodiments, the payload polypeptide comprises a nuclear localization signal sequence or a nuclear export signal sequence.

[0027] In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell.

[0028] In some embodiments, the payload polypeptide is a nucleic acid binding protein. In some embodiments, the nucleic acid binding protein is a RNA-guided protein. In some embodiments, the gRNA-guided protein is a CRISPR nuclease or an inactive mutant thereof.

[0029] In some embodiments, the extracellular amino acid sequence comprises an antibody or antigen-binding fragment thereof. In some embodiments, the antibody is a scFv.

[0030] In some embodiments, the low pH-dependent fusogenic protein is a type III viral fusion protein or a fusogenic fragment thereof. In some embodiments, the type III viral fusion protein is a Vesicular stomatitis virus G protein or a fusogenic fragment thereof.5 KILPATRICK TOWNSEND 790261771

[0031] Also provided is a vector comprising the nucleic acid as described above or elsewhere herein, optionally comprising a promoter operably linked to the nucleic acid. In some embodiments, the vector is a viral vector. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Fig.1: Experimental setup for Jurkat-K562 co-culture. To make the stable CAR- mCherry Jurkat (donor) cell line, construct with EF1a-driven anti-CD19 CAR was packaged into lentivirus and transduced into wild type Jurkat cells. The CAR consists of the FMC63 scFv, signal peptide, hinge and transmembrane domain from CD8, intracellular domains from 4-1BBand CD3 . SP, signal peptide; TM, transmembrane domain; ICD, intracellular domain. The CARwas C-terminally tagged with mCherry via GS linker, and puromycin resistance gene was used as transduction marker. To make the stable CD19-GFP K562 recipient cell line, construct with EF1a-driven CD19-GFP was packaged into lentivirus and transduced into wild type K562 cells. GFP was C-terminally fused to CD19 via GS linker. Puromycin resistance gene and nuclear localized BFP were co-expressed via 2A linkage. Co-culture experiment started at least 3 days after the transduction, during which puromycin treatment lasted for 48h. The engineered donor and recipient cells were co-cultured at 1:1 ratio for 24h in 96-well U-bottom plate and analyzed by flow cytometry afterwards.

[0033] Fig.2A-B: Synthetic receptors enable cell-to-cell transfer with high specificity. (2A) Cartoon depicting potential cell-to-cell molecule transfer between donor cells (Jurkat) with a synthetic receptor (CD19CAR-mCherry) and recipient cells (K562) with a specific ligand (CD19-GFP). Both mCherry and GFP were fused to the C-terminus of the transmembrane proteins, and nuclear-localized BFP was only expressed in recipient cells. (B) Time course of intercellular mCherry transfer upon the above-mentioned co-culture as measured by flow cytometry. n=6 across 2 independent experiments.

[0034] Fig.3A-C: DNA-mediated intercellular molecule transfer. (A-CA) Cartoon depicting the system using DNA to mediate interaction of a pair of donor (Jurkat) and recipient (K562) cells, which displayed mSA2-mCherry (receptor) and FITC-scFv on cell surface, respectively. (B) To fine tune the binding affinity, ssDNA with 3’ FITC or 3’ biotin modifications could base pair and form duplexes in vitro, and bridge a pair of donor and recipient cells through surface binding. X, the length of stem region; Y, the length of overhangs. (C) Cartoon depicting the6 KILPATRICK TOWNSEND 790261771system to fine tune the cellular distance of a pair of donor (Jurkat) and recipient (K562) cells without affecting binding affinity. ssDNA with 3’ FITC or 5’ biotin could base pair and form a branched structure in vitro, and bridge a pair of donor and recipient cells. A pair of unmodified short oligos were added to stabilize the ‘arms’. The length of the ‘arms’ determines the cellular distancing, while the affinity of the ‘stem’ determines the cell-cell binding affinity, respectively.

[0035] Fig.4: Flexible design of transmembrane and cytosolic domain of the receptor. Schematic showing the compositions of receptors with modified transmembrane and cytosolic domains. SP, signal peptide; FMC63, scFv for CD19CAR used; TM, transmembrane domain; ICD, intracellular domain.

[0036] Fig.5: Fusogen-facilitated split GFP reconstitution in cytoplasm. Schematic illustration of constructs in donor HEK293T and recipient K562 cell lines. The synthetic receptor was transduced into HEK293T cells, while the VSV-G was transfected 24h before co-culture. Recipient cell lines with GFP11 localized in cytoplasm were made with lentiviral transduction. LZ2 and LZ2’, a leucine zipper pair. -cyt, no cytosolic domain.

[0037] Fig.6: Fusogen- and pH intein- facilitated split GFP reconstitution in nucleus. Schematic illustration of constructs in donor and recipient cells. pH intein undergoes cleavage in the endosome upon transfer, releasing the GFP1-10 from the membrane-bound portion of the receptor, enabling its nuclear localization. LZ2 and LZ2’, a leucine zipper pair. -cyt, no cytosolic domain.

[0038] Fig.7A-B: ABA-induced functional Cas9 delivery. (7A) Illustration of ABA-inducible membrane recruitment of Cas9 with the ABI-PYL1 protein heterodimerization system. (7B) Percentage of GFP+ cells in all Cas9 reporter recipient cells upon 72h 5:1 co-culture between VSV-G+ FMC63-ABI / PYL1-pH intein-Cas9-mCherry donor (HEK293T) cells and CD19+ Cas9 recipient (HEK293T) cells. The recipient cell contains a frameshifted split GFP that could be restored with genome cutting. FMC63, antiCD19 scFv. ABA was added into co-culture system for indicated period, followed by wash out. n=6 from 2 independent experiments. VSV-G was transfected into the donor cells one day before co-culture.

[0039] Fig.8: Data-driven, programmable macromolecule delivery to various cell types. Specific cell types are first provided by the user, and computational methods are deployed to7 KILPATRICK TOWNSEND 790261771identify unique surface markers for each cell type of interest. A specific binder is defined for each selected marker, which will be expressed on the donor cells for target cell recognition. For experimental validation, the engineered donor cells are co-cultured with the group of input cells in all combinations to determine the cell type specificity.

[0040] Fig.9A-D: TRANSFER enables programmable cell-to-cell transfer to various cell types. (9A) The computed mRNA level of three selected markers in three cell types shown in log2 transformed transcript per million (TPM) values acquired from the CCLE. (9B-D) Percentage of mCherry+ recipient cells upon 24h co-culture with CAR-mCherry donor cells. CD19CAR donor (Jurkat) to recipient ratio was 1:5. HER2CAR donor (Jurkat) to recipient ratio was 5:1. CD3CAR donor (K562) to recipient ratio was 1:5. Gating for mCherry+ population was done separately for each type of recipient cells using mono-cultured recipient cells as a reference. n=6 from 2 independent experiments.

[0041] Fig.10: Conceptual framework for programmable delivery with TRANSFER. Schematic illustrations of the delivery process mediated by TRANSFER. Donor cells are engineered to express the receptor cargo and the fusogen. The cargo consists of 3 domains: Ligand-binding domain, including single-chain variable fragment (scFv); Release domain, a pH- responsive self-cleaving intein that facilitates endosome escape; Effector domain, which can be split GFP, Cre or Cas protein. Upon recognition of the desired recipient cells bearing certain surface ligands, cell-to-cell receptor transfer is triggered, bringing the cargo and associated membrane patches into the endosome. With endosome acidification, the release domain undergoes pH-dependent cleavage and the fusogen undergoes conformational change to facilitate membrane fusion, resulting in the release of effector domain into the cytoplasm. Effector domains including Cre or Cas proteins can subsequently re-localize to the nucleus guided by nuclear localization signal (NLS) and modify the gene of interest (GOI). The membrane-bound portion is re-expressed on recipient cell membrane, like in trogocytosis. DEFINITIONS

[0042] Unless otherwise defined, all terms of art, notations, and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those of ordinary skill in the art. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein8 KILPATRICK TOWNSEND 790261771should not be construed as representing a substantial difference over the definition of the term as generally understood in the art.

[0043] Articles “a” and “an” are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.

[0044] The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements.

[0045] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0046] As used throughout, the terms “nucleic acid,” “nucleic acid sequence,” “oligonucleotide,” “nucleotides,” or other grammatical equivalents as used herein mean at least two nucleotides, either deoxyribonucleotides or ribonucleotides, or analogs thereof, covalently linked together. Polynucleotides are polymers of any length, including, e.g., 20, 50, 100, 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. A polynucleotide described herein generally contains phosphodiester bonds, although in some cases, nucleic acid analogs are included that may have at least one different linkage, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphophoroamidite linkages, and peptide nucleic acid backbones and linkages. Mixtures of naturally occurring polynucleotides and analogs can be made; alternatively, mixtures of different polynucleotide analogs, and mixtures of naturally occurring polynucleotides and analogs may be made. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, cRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any9 KILPATRICK TOWNSEND 790261771sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The term also includes both double- and single-stranded molecules. Unless otherwise specified or required, the term polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues. Polynucleotides as described herein may further comprise detectable labels (such as reporters and affinity purification agents) such as a fluorophore, repeated sequences (polyA sequences, for example) and the like. In embodiments, a polynucleotide as described herein can be at least 80, 85, 90, 95, 98%, or 99% identical to a reference polynucleotide.

[0047] The term "vector" refers to a carrier DNA molecule (i.e., a polynucleotide) into which a DNA sequence can be inserted for introduction into a host cell. In some embodiments, vectors of use according to the present disclosure are those capable of autonomous replication and / or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors". Thus, an "expression vector" is a specialized vector that contains the necessary regulatory regions needed for expression of a gene of interest in a host cell. In some embodiments the gene of interest is operably linked to another sequence in the vector, e.g., a promoter. Vectors include non-viral vectors such as plasmids and viral vectors (for example lentiviral and adenoviral vectors). Vectors10 KILPATRICK TOWNSEND 790261771as described herein additionally may be capable of self-replication and may be able to be passed down from a cell to its daughter progeny.

[0048] A “viral vector” is a viral-derived nucleic acid that is capable of transporting another nucleic acid into a cell. A viral vector is capable of directing expression of a protein or proteins encoded by one or more genes carried by the vector when it is present in the appropriate environment. Examples for viral vectors include lentiviral vectors.

[0049] The term “operably linked” refers to a functional linkage between a first nucleic acid sequence and a second nucleic acid sequence, such that the first and second nucleic acid sequences are transcribed into a single nucleic acid sequence. Operably linked nucleic acid sequences need not be physically adjacent to each other. The term “operably linked” also refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a transcribable nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the transcribable sequence.

[0050] The terms “polypeptide” and “peptide” are used interchangeably herein to refer to a polymer of amino acid residues in a single chain, wherein each amino acid residue is linked to another by a peptide bond unless otherwise specified. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymers. Amino acid polymers may comprise entirely L-amino acids, entirely D-amino acids, or a mixture of L- and D-amino acids. The term “protein” as used herein refers to either a polypeptide or a dimer (i.e., two) or multimer (i.e., three or more) of single chain polypeptides. The single chain polypeptides of a protein may be joined by a covalent bond, e.g., a disulfide bond, or non-covalent interactions. The terms “portion” and “fragment” are used interchangeably herein to refer to parts of a polypeptide, nucleic acid, or other molecular construct. The term “truncated” refers to a portion of a polypeptide that is not a full- length polypeptide when compared to a reference sequence and comprises at least one or more functional elements of that polypeptide. For example, a “truncated” motor polypeptide comprises the localization domain that is necessary to target a localization polypeptide to the proper subcellular location, in addition to any domains necessary for movement along a portion11 KILPATRICK TOWNSEND 790261771of the cytoskeleton to which it is target, but omits other polypeptide sequences comprising elements of the motor protein not required for this functionality. Polypeptides as described herein may further comprise detectable labels (such as reporters and affinity purification agents) such a fluorophore, repeated sequences (His-tag sequences, for example) and the like. In embodiments, a polypeptide as described herein can be at least 80, 85, 90, 95, 98%, or 99% identical to a reference polypeptide.

[0051] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0052] A “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well- known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith & Waterman Add. APL. Math.2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch J. Mol. Biol.48:443 (1970), by the search for similarity method of Pearson & Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerized implementations of these algorithms (e.g., BLAST), or by manual alignment and visual inspection.

[0053] Algorithms that are suitable for determining percent sequence identity and sequence similarity include the BLAST 2.0 algorithm, which is described in Altschul et al. (1990) J. Mol. Biol.215: 403-10.

[0054] A “fusion protein” refers to a single polypeptide that comprises two heterologous polypeptide sequences that are linked together via a peptide bond and optionally a peptide linker.12 KILPATRICK TOWNSEND 790261771

[0055] The use of “first,” “second,” “third,” etc. in this disclosure is simply for antecedent basis to distinguish other molecules of the same type. For example a “first protein” and a “second protein” means there are two distinguishable proteins. Order is not intended by this usage.

[0056] The phrase “specifically (or selectively) binds” to a peptide tag refers to a binding reaction whereby the affinity agent binds to the peptide tag of interest. In the context of this disclosure, the affinity agent binds to peptide tag in question with a KD that is at least 100-fold greater than its affinity for other peptide tags in the system or other proteins in the cell in question.

[0057] An “affinity agent” refers to a protein sequence or other agent that has specific affinity (specifically binds) to a second affinity agent such that the first and second affinity agent covalently or non-covalently bind. An affinity agent can be any protein known or selected to have specific affinity for a second amino acid sequence. Examples of affinity agents include but are not limited to PYL1 / ABI (in the presence of abscisic acid) or an antibody (which may be a single-chain scfv antibody or a camelid VHH domain) and its target antigen.

[0058] As used herein, the term “heterologous” refers to a molecule (e.g., peptide or protein) that is not normally or naturally produced or expressed by a cell or organism, or that is fused to a second sequence to which it is not fused in a natural cell or organism. DETAILED DESCRIPTION OF THE INVENTION

[0059] It has been discovered that one can transfer the contents of a first cell to a second cell (e.g., mammalian cells) by targeting the first cell using a transmembrane protein anchored to the first cell having an extracellular domain that targets to the mammalian cell. The transmembrane protein is linked (translationally fused or otherwise linked) to an intracellular payload polypeptide. The first cell targets to the second cell and a portion of the first cell’s plasma membrane, including the transmembrane protein and payload polypeptide is transferred to the second cell. In some embodiments, the second cell transports the portion received from the first cell into an endosome. Thus, in some embodiments, a cleavage sequence is included in the transmembrane protein such that the cleavage sequence is not cleaved in a cytosol but is cleaved in an endosome, allowing for release of the payload polypeptide. In some embodiments, the first13 KILPATRICK TOWNSEND 790261771mammalian cell further expresses a fusogenic protein on the cell membrane. The fusogenic protein, which will enter the second cell with the portion comprising the transmembrane protein, will disrupt the endosome, allowing for release of the payload polypeptide into the cytosol of the second cell. The payload protein can be selected for any desired activity in the second cell, as detailed more below. Accordingly, methods and compositions for transfer of payload polypeptides to cells are provided herein.

[0060] One of the components allowing for transfer of a payload from a first cell to a second cells is a transmembrane protein. The transmembrane protein comprises an extracellular targeting domain (typically amino acids but in some embodiments other targeting moieties), a transmembrane sequence to anchor the protein in the cell’s membrane, and an intracellular sequence. In many embodiments, the intracellular sequence comprises the payload polypeptide and optionally the cleavage sequence between the payload protein and the transmembrane sequence. Thus, in some embodiments, the transmembrane polypeptide comprises the extracellular targeting domain, transmembrane sequence, optional cleavage sequence and the payload protein as a single translational fusion protein. In other embodiments, the extracellular targeting domain and transmembrane sequence form a translational fusion that is covalently or non-covalently linked (e.g., via affinity agents) to a second polypeptide comprising the payload protein. In these embodiments, the optional cleavage sequence can be part of either, or both, the intracellular portion or second polypeptide, but allowing for cleavage of the payload protein from the remainder due to cleavage of the cleavage sequence (e.g., in an endosome).

[0061] The extracellular amino acid sequence that binds to a target protein on a recipient cell can be selected from any polypeptide that has affinity for a molecule on the surface of the second (recipient) cell. Exemplary extracellular amino acid sequences can include, but are not limited to, receptor ligands, or antibodies or fragments thereof with binding affinity. In some embodiments the extracellular amino acid sequence specifically binds to a surface protein on the second cell.

[0062] The extracellular amino acid sequence can be selected from any type of binding protein. In some embodiments, the extracellular amino acid sequence is selected from the group consisting of a nanobody (e.g., a single variable domain located on a heavy chain, also known as VHH antibodies), a single chain variable fragment (scFv), an F(ab) fragment, a variable heavy14 KILPATRICK TOWNSEND 790261771chain (VH), a monobody (i.e., a synthetic protein derived from the 10th domain of human fibronectin type III), or other non-antibody scaffold protein.

[0063] In some embodiments, the antibody or antigen binding fragment thereof provided herein can include a heavy (H) chain variable domain sequence (abbreviated herein as VH), and a light (L) chain variable domain sequence (abbreviated herein as VL). In some embodiments, an antibody molecule comprises or consists of a heavy chain and a light chain (referred to as a half antibody). In another example, an antibody molecule includes two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequence, thereby forming twoantigen binding sites, such as Fab, Fab , F(ab )2, Fc, Fd, Fd , Fv, single chain antibodies (scFv,for example), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which may be produced by the modification of whole antibodies or synthesized de novo using recombinant DNA technologies. These functional antibody fragments retain the ability to bind specifically to their respective antigen. Antibodies and antibody fragments can be from any class of antibodies including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass (e.g., IgG1, IgG2, IgG3, and IgG4) of antibodies.

[0064] Antigen binding fragments of an antibody molecule are well known in the art, and include, for example, (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CLand CH1 domains; (ii) a F(ab )2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a diabody (dAb) fragment, which consists of a VH domain; (vi) a camelid or camelized variable domain; (vii) a single chain Fv (scFv) (see e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883); (viii) a single domain antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as are intact antibodies. In some embodiments, the extracellular amino acid sequence comprises an anti-CD19 scFv or other CD19-binding sequence. In some embodiments, the anti-CD19 scFv sequence is at least 80, 85, 90, 95, 98% or 100% identical to SEQ ID NO:6. In some embodiments, the extracellular amino acid sequence comprises an anti-CD3 scFv or other CD3-binding sequence. In some embodiments, the anti-CD3 scFv sequence is at least 80,15 KILPATRICK TOWNSEND 79026177185, 90, 95, 98% or 100% identical to SEQ ID NO:34. In some embodiments, the extracellular amino acid sequence comprises an anti-HER2 scFv or other HER2-binding sequence. In some embodiments, the anti-HER2 scFv sequence is at least 80, 85, 90, 95, 98% or 100% identical to SEQ ID NO:36. In some embodiments, the extracellular amino acid sequence comprises an anti- CD4 scFv or other CD4-binding sequence. In some embodiments, the anti-CD4 scFv sequence is at least 80, 85, 90, 95, 98% or 100% identical to SEQ ID NO:38. In some embodiments, the extracellular amino acid sequence comprises an anti-CD28 scFv or other CD28-binding sequence. In some embodiments, the anti-CD28 scFv sequence is at least 80, 85, 90, 95, 98% or 100% identical to SEQ ID NO:40. In some embodiments, the extracellular amino acid sequence comprises an anti-CD2 scFv or other CD2-binding sequence. In some embodiments, the anti- CD2 scFv sequence is at least 80, 85, 90, 95, 98% or 100% identical to SEQ ID NO:42.

[0065] Antibody molecules can also be single domain antibodies. Single domain antibodies can include antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be any of the art, or any future single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, rat, guinea, pig, human, camel, llama, fish, shark, goat, rabbit, and bovine. Single domain antibodies are described, for example, in International Application Publication No. WO 94 / 04678. For clarity reasons, this variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species (e.g., camel, llama, dromedary, alpaca and guanaco) or other species besides Camelidae.

[0066] In some embodiments, an antigen binding fragment can also be or can also comprise, e.g., a non-antibody, scaffold protein. These proteins are generally obtained through combinatorial chemistry-based adaptation of preexisting antigen-binding proteins. For example, the binding site of human transferrin for human transferrin receptor can be diversified to create a diverse library of transferrin variants, some of which have acquired affinity for different16 KILPATRICK TOWNSEND 790261771antigens. See, e.g., Ali et al. (1999) J. Biol. Chem.274:24066-24073. The portion of human transferrin not involved with binding the receptor remains unchanged and serves as a scaffold, like framework regions of antibodies, to present the variant binding sites. The libraries are then screened, as an antibody library is screened, and in accordance with the methods described herein, against a target antigen of interest to identify those variants having optimal selectivity and affinity for the target antigen. See, e.g., Hey et al. (2005) TRENDS Biotechnol 23(10):514- 522.

[0067] The scaffold portion of the non-antibody scaffold protein can include, e.g., all or part of the Z domain of S. aureus protein A, human transferrin, human tenth fibronectin type III domain, kunitz domain of a human trypsin inhibitor, human CTLA-4, an ankyrin repeat protein, a human lipocalin (e.g., anticalins, such as those described in, e.g., International Application Publication No. WO2015 / 104406), human crystallin, human ubiquitin, or a trypsin inhibitor from E. elaterium.

[0068] In some embodiments, the extracellular amino acid sequence comprises an amino- terminal or carboxy terminal signal sequence allowing for insertion of the transmembrane protein into the cellular membrane. In some embodiments, the signal sequence is at least 80, 85, 90, 95, 98% or 100% identical to SEQ ID NO:4.

[0069] The extracellular amino acid sequence is linked (e.g., fused) to a transmembrane sequence allowing for the protein to be anchored in a cell’s membrane and be presented outside the cell. In some embodiments, a hinge domain that links the extracellular amino acid sequence and the transmembrane domain for positioning the antigen binding domain. Exemplary hinge domains can include the hinge region from the extracellular regions of type 1 membrane proteins such as CD8 alpha, CD4, CD28, PD1 , CD 152, and CD7, which may be wild-type hinge regions from these molecules or may be altered. In some embodiments, the CD8 hinge sequence is at least 80, 85, 90, 95, 98% or 100% identical to SEQ ID NO:8.

[0070] Any transmembrane sequence can be employed. Such transmembrane domains, can include, but are not limited to, all or part of the transmembrane domain of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, the transmembrane sequence is at least 80, 85, 90, 95, 98% or 100% identical to SEQ ID NO:10.17 KILPATRICK TOWNSEND 790261771

[0071] The polypeptide further comprises an intracellular sequence. Optionally the intracellular sequence comprises a linker sequence. In some embodiments, the linker sequence is at least 80, 85, 90, 95, 98% or 100% identical to SEQ ID NO:12 or is another sequence comprising a majority of glycine and serine. In some embodiments the intracellular sequence comprises an affinity sequence allowing for covalent or non-covalent binding to a second polypeptide comprising the payload protein. For example, the extracellular sequence can be translationally fused with (or otherwise covalently or non-covalently associated with) a first affinity sequence, and the payload protein (and optional cleavage sequence) can be translationally fused with (or otherwise covalently or non-covalently associated with) a second affinity sequence such that the first affinity sequence and the second affinity sequence bind or otherwise interact to link the transmembrane polypeptide with the payload protein. .

[0072] In some embodiments, the first and second affinity sequences can interact in a chemically-dependent fashion. Examples of affinity sequence pairs and chemical inducers can include, but are not limited to: the pyrabactin resistance 1-like (PYL):ABI system that utilizes abscisic acid a binding inducer (see, for example, U.S. Patent No.10,221,426; U.S. Patent No. 10,934,559; US Patent No.11,641,857; and International PCT Publication WO 2023 / 141591 A2 for additional information on aspects of the system, including PYL and ABI proteins (and modified variants thereof), as well as ABA and ABA analogs, which are incorporated by reference as if fully set forth herein); a first and a second FKBP12 polypeptide; a first and second FKBP polypeptide that utilizes a chemical inducer such as AP20187; cyclophilin-calcineurin based systems; FKBP-Calcineurin systems; FKBP-CyPFas systems; FKBP-FRB domain of mTOR based systems; Gal-GID1 based systems; SNAPTag-HaloTag based systems; eDHFR- HaloTag based sytems; Bclxl-Fab (AZ1) based systems; GyrB-GyrB based systems; and cyclophilin-based systems. In some embodiments, the ABI protein is at least 80, 85, 90, 95, 98% or 100% identical to SEQ ID NO:14. In some embodiments, the PYL1 protein is at least 80, 85, 90, 95, 98% or 100% identical to SEQ ID NO:18.

[0073] Optically-induced dimerization systems are also contemplated by the present disclosure. Whereas chemically induced dimerization (CID) systems are based on small molecules interacting with two identical proteins (homodimerization) or two different proteins (heterodimerization). Optogenetic dimerization systems employ photosensitive proteins that18 KILPATRICK TOWNSEND 790261771undergo a conformational change upon illumination, and consequently, induce proteininteraction. Chemo optogenetic dimerization systems can use photoactivatable and / or cleavablesmall molecule dimerizers, so that proximity can be induced and / or disrupted by light. Examplesof optically-inducible dimerization systems include UVR8-COP1; UVR8-UVR8 homodimer systems; FKF1-G1; TULIPs; LOVpep-ePDZ; iLID; LOVSsrA-SsrB; Light on; VVD-VVD; and pMag-nMag (VVD variants). As with the CID systems described above, the skilled artisan would readily understand that in any of the aforementioned examples, one component can be the first dimerization handle while the other acts as the second dimerization handle, and vice-versa. Additional information on optically-inducible dimerization systems can be found, for example, in Klewer et al., Chemistry.2019 Sep 25; 25(54): 12452–12463, which is incorporated by reference.

[0074] In some embodiments, the intracellular sequence comprises (as a translational fusion with the extracellular sequence and the transmembrane sequence) the payload protein and optionally a cleavage sequence.

[0075] As noted herein, in some embodiments, a cleavage sequence is present between the transmembrane sequence and the payload protein. The cleavage sequence can be selected such that it remains intact in the first cell but following transfer to the second cell, is cleaved to release the payload protein from the transmembrane sequence. In some embodiments, the transmembrane polypeptide is transferred to the second cell and is introduced into an endosome or other intracellular vesicle having a pH lower than the cytosol of the second cell. Thus, in some embodiments, the cleavage sequence can be selected such that it is selectively cleaved in an endosome. Exemplary cleavage sequences can be recognized by an endosome-specific protease or can be triggered by the reduced pH in the endosome (e.g., pH 4-6) or other vesicle compared to the cytosol of the cells (e.g., pH 7). In some embodiments, the cleavable sequence is a pH intein. Exemplary pH inteins are listed described in Choi et al., Chemical Engineering J. Volume 457, 1 February 2023, 141229. In some embodiments, the intein protein is at least 80, 85, 90, 95, 98% or 100% identical

[0076] Payload proteins can be any protein sequence desired to be delivered to a second cell. In some embodiments, the cleavage sequence is cleaved by an enzyme in the cellular endosome. The enzyme that cleaves the cleavage sequence can be an endogenous enzyme (e.g., a sequence-19 KILPATRICK TOWNSEND 790261771specific protease) in the endosome. Alternatively, the enzyme can be heterologous to the endosome and the cell in which it resides. For example, in some embodiments, the heterologous enzyme is delivered to the endosome or is expressed in the cell from a heterologous expression cassette encoding the enzyme that has been introduced into the cell.

[0077] Payload proteins can be any protein sequence desired to be delivered to a second cell. In some embodiments, the payload protein addresses a deficiency in the second cell or triggers a response in the second cell. For example, if the second cell is an immune cell, the second cell may become more active or less active in response to release of the payload protein in the second cell. Exemplary payload proteins can include but are not limited to enzymes, cytokines, chemokines, other receptor ligands or protein-binding proteins (for example but not limited to antibodies and antigen-binding fragments thereof), or nucleic acid binding proteins. Exemplary nucleic acid binding proteins can include for example, TALENs, zinc finger proteins, CRISPR / Cas proteins, Fanzor, nuclease-dead versions of these proteins, and fusions of these proteins to transcriptional factors, epigenetic factors, transcriptional activation or inhibition domains and base editing modules (e.g., a deaminase that can modify a DNA base). In some embodiments, the base editing module is fused to an inhibitor of uracil DNA glycosylase (UGI) to prevent base excision repair. Yet other possible payload proteins can include, for example, prime editing proteins (e.g., a CRISPR protein o other guide protein fused to a reverse transcriptase (for example but not limited to M-MLV RT), CRISPR-associated transposases (CASTs) (see, e.g., Tou et al., Nature Biotechnology volume 41, 968–979 (2023) or CRISPR- associated integrases (see, e.g., Jakhanwal, et al., Nucleic Acids Res.2021 Apr 6;49(6):3546- 3556).

[0078] The “CRISPR / Cas” system refers to a widespread class of bacterial systems for defense against foreign nucleic acid. CRISPR / Cas systems are found in a wide range of bacterial and archaeal organisms. CRISPR / Cas systems include various types and subtypes based on shared characteristics and evolutionary similarity. These are grouped into two large classes based on the structure of the effector complex that cleaves genomic DNA. The Type II CRISPR / Cas system was the first used for genome engineering, with Type V following. Wild-type type II CRISPR / Cas systems utilize an RNA-mediated nuclease Cas protein or homolog complex with guide RNA to recognize and cleave foreign nucleic acid. The term “Cas nuclease” or “Cas”20 KILPATRICK TOWNSEND 790261771refers to CRISPR associated protein, an RNA-guided nuclease that introduces a double stranded break in nucleic acid. The Cas nuclease can be CRISPR associated protein 9 (“Cas9 nuclease” or “Cas9”) or any other targeted In someembodiments, the programmable nuclease comprises Cas1, Cas1B, Cas2, Cas3,Cas4, Cas5, Cas6, Cas7, Cas8, Csy1, Csy2, Csy3, Cse1, Cse2,Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4,Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3,Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, C2c1, C2c3, Cas12a, Cas12b,Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, or Cas13. In some embodiments, the Cas9 protein is at least 80, 85, 90, 95, 98% or 100% identical to SEQ ID NO:26. Cas9 and some other targeted nuclease proteins also use an activating RNA (also referred to as a transactivating or tracr RNA). Guide RNAs can have activity of either a guide RNA or both a guide RNA and an activating RNA, depending on the type of CRISPR-associated endonuclease used. Dual activity guide RNAs are referred to as a single guide RNA (sgRNA). In this disclosure, the term “sgRNA” is used to refer to an RNA molecule that complexes with a CRISPR-associated endonuclease and localizes the ribonucleoprotein complex to a target DNA sequence. Typically, an sgRNA comprises a “scaffold” sequence for binding the nuclease and a “targeting” sequence that defines the target nucleic acid site (for example, a genomic DNA site). “Activity” in the context of CRISPR / Cas activity, CRISPR-associated endonuclease activity, sgRNA activity, sgRNA:CRISPR-associated endonuclease nuclease activity and the like refers to the ability to bind to a target genetic element. Typically, activity also refers to the ability of the sgRNA:CRISPR-associated endonuclease nuclease complex to make double-strand breaks at a target genomic region. A catalytically inactive variant of Cas endonuclease, such as a catalytically inactive variant of Cas9, which is referred to as “dead Cas9” or “dCas9” in the present disclosure, lacks endonuclease activity. For example, dCas9 is a mutant form of Cas9 whose endonuclease activity is eleminated through point mutations in its endonuclease domains. In the poresence of a guide RNA, such as an sgRNA, the guide RNA and dCas9 generate a DNA recongnition complex that can specifically interfere with transcription of a nucleotide sequence, to which the guide RNA is targeted. CRISPR interference (CRISPRi) methods and systems use dCas9 paired with sgRNA to hinder transcription of a target gene. Similarly CRISPR activation (CRISPRa) can be used to activate transcripton of target genes.

[0079] Optionally a signal sequence can be linked to the payload protein such that the payload protein is targeted to a particular organelle, nucleus or other location in the second cell. For21 KILPATRICK TOWNSEND 790261771example, the signal sequence can be a nuclear localization signal (NLS) sequence or a nuclear export signal (NES) sequence. In some embodiments, the NLS is at least 80, 85, 90, 95, 98% or 100% identical to SEQ ID NO:32. In some embodiments, the NES is at least 80, 85, 90, 95, 98% or 100% identical to SEQ ID NO:16.

[0080] As noted herein, the transmembrane protein can be transferred from a first cell to a second cell. In some embodiments in the transfer, the second cell transports the transmembrane protein to an endosome or other intracellular vesicle or organelle in the second cell. If this is the desired target of the payload protein, then further components may not be included in the first cell. However, if it is desired that the payload protein be delivered to the cytosol of the second cell, it can be beneficial for the first cell to also express a fusogenic protein on the first cell surface such that the fusogenic protein is incorporated into the second cell’s endosome, intracellular vesicle or other organelle with the transmembrane protein and payload protein. A fusogenic protein can merge two opposing membranes. See, e.g., Lozada, et al., Front. Chem., 23 August 2021. Thus, in this case, the fusogenic protein can trigger the fusion of the membrane bounding the endosome, intracellular vesicle or other organelle, allowing the payload protein to be released into the cytosol. This is shown for instance in the examples. In some embodiments, the fusogenic protein is a viral protein (see, e.g., White et al. Crit Rev Biochem Mol Biol.2008; 43(3): 189–219 describing viral fusogenic protein structure), human cell-derived protein (for example but not limited to syncytin, Izumo-Juno, Myomaker and Myomerger) or a de novo engineered fusogenic protein. In some embodiments, the fusogenic protein is a low pH- dependent (for example, fusing at a pH of 5.0 or between 5-6 but not substantially fusing at pH 7 or 7.5) fusogenic protein. In some embodiments, the low pH-dependent fusogenic protein is a type I or III viral fusion protein or a fusogenic fragment thereof. In some embodiments, the type III viral fusion protein is a Vesicular stomatitis virus G protein or a fusogenic fragment thereof. In some embodiments, the type I viral fusogenic protein is an F protein, for example but not limited to from avian metapneumovirus (aMPV) or human metapneumovirus (hMPV). See, e.g., Yun, et al., Scientific Reports volume 5, 15584 (2015). In some embodiments, the fusogenic protein is at least 80, 85, 90, 95 or 98% identical to SEQ ID NO:2 or SEQ ID NO:46.

[0081] The first cell (donor) and second (recipient) cell can be selected as desired. In some embodiments, the first and second cells are of the same species. In some embodiments, the cells22 KILPATRICK TOWNSEND 790261771are mammalian cells, e.g., human cells. The cells can be in vitro (e.g., isolated or in culture), ex vivo (isolated from an animal (e.g., a human), optionally primary cells, that will be returned to the same or a different animal (e.g., different human) following transfer described herein) or in vivo (for example, wherein the first cell is introduced into an animal (e.g., human) or wherein one or more nucleic acid encoding the polypeptides described herein are introduced into the animal and expressed in a first cell in the animal that proceeds to transfer the payload to a second cell in the animal).

[0082] Cells as described herein can be a cell of the animal or plant kingdom plant. In embodiments, cells as described herein may be a mammalian cell, for example a human, mouse, or rat cell. Mammalian cells as described herein may be derived from immortalized cell lines or other primary cell lines derived from a subject (for example, a human subject). Mammalian cells, for example, may be derived from any cellular germ layer, for example, mesoderm, endoderm, or ectoderm, or from any organ of the body (for example, liver hepatocytes or kidney cells, such as human embryonic kidney). In embodiments, mammalian cells may be placental or embryonic. Cells may be stem cells (for example, pluripotent cells such as human embryonic cells, or multipotent cells such as hematopoietic stem cells (HSCs) or bone-marrow derived mesenchymal stem cells (BM-MSCs)); bone cells (for example, osteoblasts or osteoclasts); blood cells (for example, white blood cells); muscle cells (also known as myocytes); sperm cells; a female egg; skin cells; endothelial cells; epithelial cells; fat cells; cells of the central or peripheral nervous system (for example, neurons, glia, and pericytes); or cells from an organ in the body, such as kidney or liver.

[0083] Without intending to be limiting, in embodiments, the first or second cell or both can be: a neuron; a glial cell (i.e., an astrocyte, oligodendrocyte, or Schwann cell); immune cell; red blood cell; a pericyte; a fibroblast; an intestinal epithelial cell; a mesenchymal cell; a cancer cell; a stem cell; a chondrocyte; an osteoblast; an osteoclast; an osteocyte; a hematopoietic stem cell; an induced pluripotent stem cells; an embryonic stem cell; a granulocyte; an agranulocyte; a skeletal, cardiac, or smooth muscle myocyte; or an adipocyte (i.e., a white or brown adipocyte). Exemplary neuronal cells can include, for example, motor neurons, ganglion cells, astrocytes, Purkinje cells, and interneurons. Exemplary immune cells include, but are not limited to,23 KILPATRICK TOWNSEND 790261771lymphocytes (e.g., CD4+ or CD8+ T cells, B cells, or NK cells), neutrophils, or monocytes / macrophages.

[0084] Also provided are nucleic acids encoding the transmembrane polypeptides described herein, vectors comprising the nucleic acids and cells comprising the nucleic acids or vectors. Such a vector can be chosen from viral vectors and non-viral vectors, plasmids, cosmids, and artificial chromosomes. By way of example, the vector can be a viral vector, such as a lentiviral vector, adenoviral vector or a retroviral vector. The vector optionally comprises nucleic acid sequences that encode one of or both or a transmembrane polypeptide as described herein and a fusogenic protein. Non-viral vector examples include physical vectors such as electroporation and chemical vectors, such as a lipid nanoparticles. In some embodiments, one or more codon encoding the polypeptides is optimized for expression in a target cell. Various coding sequences for components described herein can be found in odd number sequences numbered (SEQ ID NOs:) 1-31.

[0085] In some embodiments, vectors are delivered to a first cell by any suitable method for introducing DNA into a cell as described herein (for example, lipofection, electroporation, viral transduction, and the like). Transmembrane polypeptides (optionally with payload proteins or as separate polypeptides), fusogenic polypeptides, or nucleic acids encoding the transmembrane polypeptides and / or fusogenic polypeptides can be introduced into a first cell that is in vitro, ex vivo, or in vivo. Examples of ex vivo methods can include obtaining one or more cell from an individual (e.g., a human), optionally sorting or culturing the cells, introducing into the cells the transmembrane polypeptides and / or fusogenic polypeptides or nucleic acids or vectors encoding the polypeptides, and optionally introducing the resulting cells back into the same individual (autologous) or a different individual (allogeneic). In allogeneic methods, matching of MHC / HLA sequences can be performed to reduce chances of transplantation rejection. In vivo methods can involve, for example, introduction of transmembrane polypeptides and / or fusogenic polypeptides or nucleic acids or vectors encoding the polypeptides to cells of interest. This can be achieved for example by direct injection into a cell of interest or for example by including one or more targeting reagent that results in targeting and / or specific expression of the polypeptides in target cells.24 KILPATRICK TOWNSEND 790261771

[0086] The methods described herein allow for transfer of a payload protein from a first (donor) cell to a second (recipient) cell. In some embodiments, the first cell is contacted to the second cell by the user (e.g., by mixing of the cells or other mechanical methods). In other embodiments, the first cell has a natural and / or synthetic targeting mechanism (which can include the extracellular amino acid sequence described here) to target the first cell to the second cell. Consistent with the discussion above, the contacting of a first cell with a second cell can occur in vitro, ex vivo, or in vivo. In some embodiments, the first cell is one or more human primary T cells and the second recipient cell is one or more cancer cells. In some embodiments, the first cell is one or more human primary T-cells and the second recipient cells is one or more muscle stem cells. In some embodiments, the first cell is one or more human primary macrophages and the second recipient cell is one or more motor neurons. In some embodiments, the first cell is one or more human primary mesenchymal stem cells (MSCs) and the second recipient cell is one or more hematopoietic stem cells. In some embodiments, the first cell is one or more human primary MSC and the second recipient cell is one or more T cells / B cells. EXAMPLES

[0087] Cell-specific delivery is crucial for unleashing the therapeutic potential of macromolecules, particularly gene-editing tools. Cells are promising delivery vehicles, capable of producing and transferring biomolecules to interacting cells through intrinsic pathways. A pathway known as trogocytosis represents the cell-to-cell transfer of plasma membrane fragments and associated molecules with function upon cellular contacts. Here, we discovered that cells engineered with custom-designed receptors can efficiently transfer membrane- associated macromolecules into recipient cells upon contact, which can be freed from endosomes and functionalized in a trogocytosis-like manner via pH-responsive membrane fusion. Exploiting these observations, we developed a cell-based delivery system termed TRANSFER, which can deliver gene editing payloads. TRANSFER demonstrates robust, efficient, and specific delivery to various cell types, offering a versatile platform for programmable delivery of macromolecules. Results Synthetic receptors enable cell-to-cell transfer with high specificity

[0088] To characterize the potential molecule transfer from donor cells equipped with a synthetic receptor to recipient cells upon cell-cell interaction, we co-cultured a human Jurkat T lymphocyte cell line stably expressing mCherry-tagged anti-CD19 CAR (CD19CAR) (SEQ ID25 KILPATRICK TOWNSEND 790261771NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:30) with a human K562 lymphoblast cell line stably expressing GFP-tagged CD19 (Fig.1). We chose this system as CD19CAR was shown to facilitate the transfer of CD19 from cancer cells to the T cell surface via trogocytosis, although the process in the opposite direction is not well characterized (22). To be distinguished from donor Jurkat cells, the recipient K562 cells were engineered to stably express a nuclear-localized blue fluorescent protein (BFP).

[0089] After 24 hours of co-culture, we observed a remarkable increase in mCherry level in recipient cells. The level of acquired mCherry was positively correlated with the CD19 expression level in recipient cells. We ruled out the formation of doublets through EDTA treatment, strict single cell gating, and the fact that mCherry in recipient cells showed a distinct distribution from that of donor cells after co-culture. Recipient cells did not express mCherry, indicating that these cells acquired CD19CAR-mCherry from donor cells.

[0090] Live imaging of CD19CAR-mCherry inside recipient cells shortly after interaction with donor cells confirmed that the molecule transfer was not an experimental artifact of separating the cells. More than 40% of recipient cells showed increased mCherry fluorescence within 5 minutes (min) upon co-culture, indicating a rapid and efficient intercellular transfer process (Fig. 2). This time scale is consistent with direct transfer of mCherry protein, instead of mCherry- encoding DNA or mRNA, which would take tens of minutes or longer to translate and mature (34). The mCherry level in recipient cells continued to accumulate until 24 hours (h) of coculture.

[0091] In comparison to donor cells expressing CAR-mCherry, donor cells lacking CAR demonstrated a greatly reduced efficiency in transferring mCherry to recipient cells across various cell ratios, implying that mCherry transfer was CAR-dependent. To test whether mCherry fusion to CAR was necessary for the transfer, we engineered donor cells co-expressing CAR and cytosolic mCherry. In this case, mCherry transfer to recipient cells was almost negligible, indicating that direct fusion was necessary for efficient co-transfer with CAR.

[0092] Furthermore, the observed transfer was specific to recipient cells expressing the cognate ligand and worked across different donor cell types, including Jurkat, HEK293T, and human primary T cells. Co-culture duration with primary T cells in this experiment was limited to 6h to minimize cytotoxicity. In summary, we reveal an efficient process for intercellular26 KILPATRICK TOWNSEND 790261771molecular transfer, mediated by a CAR receptor on donor cells to recognize recipient cells, which offers a potential mechanism for programmable cargo delivery. Extracellular binding is sufficient for cell-to-cell transfer

[0093] Expanding beyond CAR, we asked how the extracellular, transmembrane, and intracellular domains of the synthetic receptor affected transfer efficiency. To flexibly alter the extracellular binding affinity and cell-cell distance, we developed a system using modified single-stranded DNAs (ssDNAs) to mediate interaction between donor (Jurkat) and recipient (K562) cells.

[0094] We switched the extracellular ligand-binding domain of the synthetic receptor to a biotin-binding domain (mSA2), and co-cultured donor cells with recipient cells displaying an anti-FITC single chain variable fragment (scFv) (Fig.3A). To mediate cell-cell interaction, we engineered a pre-annealed double-stranded DNA (dsDNA) scaffold with biotin and FITC modifications at each end. The dsDNA scaffold was designed with a 16 base pair (bp) stem region and 2 bp overhangs to mimic the natural distancing of a T cell immune synapse (35) (Fig. 3B). We observed efficient intercellular transfer of the mCherry-tagged receptor in the presence of the dsDNA scaffold, but not without the scaffold or with a mismatched scaffold. Varying the GC content of dsDNA without changing its length revealed that a higher binding affinity correlated with more efficient transfer.

[0095] The transfer efficiency also showed a dose-dependent increase with dsDNA concentration. In examining the effect of cellular distance, we used a branched DNA scaffold with one ‘stem’ and two ‘arms’, each modified with biotin or FITC and stabilized by a short oligo. Altering the length of the ‘arms’ while keeping the ‘stem’ region constant, we found that the transfer efficiency was insensitive to all cellular spacings tested (Fig.3C).

[0096] To evaluate the importance of the transmembrane domain, we replaced the CD8 transmembrane domain in the CAR molecule with those from mouse Notch, human epidermal growth factor receptor (EGFR), or a glycosylphosphatidylinositol (GPI) membrane anchor (Fig. 4). All transmembrane domains and membrane anchor enabled intercellular transfer, albeit with varying efficiency. The differences in transfer efficiency can be largely attributed to the varying expression levels of synthetic receptors.27 KILPATRICK TOWNSEND 790261771

[0097] As for the cytosolic domain, we either removed or replaced the original cytosolicdomains of choice, 4-1BB and CD3 (SEQ ID NO: 62), of the receptor with 4-1BB alone (SEQID NO: 60), CD28 alone (SEQ ID NO: 58), or CD28-CD3 , tagging their C-terminus withmCherry (Fig. 4). The CD28 alone and CD28-CD3 receptor was constructed with CD28transmembrane domain (SEQ ID NO: 56) as well. When controlled for similar expression levels in donor cells, these alterations in the cytosolic domain did not affect transfer efficiency.

[0098] To further study the role of the cytosolic domain in recipient cells, we removed the cytosolic domain of CD19 while maintaining its expression level. We observed similar transfer efficiency to recipient cells, indicating that the transfer process was independent of CD19 signaling in recipient cells.

[0099] Collectively, our data suggested that the efficiency of molecular transfer mediated by the synthetic receptor is strongly influenced by its binding affinity and avidity to the opposing cell, but not so much by the distance between the cells. The efficient transfer of receptors with various transmembrane and cytosolic domains suggests high flexibility in receptor design, which avoids the use of full-length CARs in donor cells that could confer cytotoxicity towards recipient cells. Additionally, cytosolic domains in the ligand of recipient cells were not necessary for transfer, suggesting flexibility in the selection of ligand to be targeted. Intercellular transferred molecules are endocytosed and degraded

[0100] To functionalize biomolecules delivered via intercellular transfer, we set out to investigate its mechanism using CAR as the receptor. Defining features of trogocytosis include dependence on cell-cell contact, actin polymerization, and the physical transfer of membrane patches onto the recipient cell surface with function (27, 36). We separated CD19CAR-mCherry Jurkat (donor) and CD19+K562 (recipient) with a transwell membrane with 3 m pores, which disrupted direct cellular contacts while allowing cell-derived extracellular vesicles including exosomes (30-100nm in diameter) and ectosomes (50-1000nm in diameter) (37) to freely pass through. In the same well, we also seeded recipient cells into the same chamber as donor cells, both as a positive control and to activate the Jurkat donor cells.

[0101] The transfer of CAR-mCherry was entirely blocked by the transwell membrane, indicating its cell contact dependency. Conditioned media from cultured donor cells showed no28 KILPATRICK TOWNSEND 790261771receptor transfer to the recipient cells, further corroborating that distal extracellular vesicle transport was not responsible for the transfer.

[0102] By pre-treating both donor and recipient cells with a series of inhibitors for cytoskeleton activities, intercellular molecule transfer pathways, and endocytosis pathways without compromising cell viability, we identified several inhibitors that significantly impaired the intercellular receptor transfer efficiency. Most potent inhibitors of transfer include Cytochalasin D (CytD), which disrupts actin polymerization (38); Manumycin-A (Manumycin), a potent inhibitor of Ras farnesylation and activation (39); LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K) activity (40); and Bafilomycin A1 (BafA), an inhibitor of vacuolar ATPase (41). In contrast, inhibitors targeting microtubule (Colchicine) (24), endosomal sorting complexes required for transport (ESCRT)-independent exosome biogenesis (GW4869) (42), Tunneling nanotube formation (L778123) (43), Rho-kinase activity and microvesicle biogenesis (Y27632) (42), clathrin-mediated endocytosis (Pitstop1) and dynamin- mediated endocytosis (Dyngo4A and Dynasore) (44), did not show an effect on intercellular transfer. This revealed that the process is actin-, Ras-, and PI3K-dependent, ruling out the involvement of nanotube-mediated transport and clathrin-mediated endocytosis. BafA has been shown to inhibit both clathrin-mediated and clathrin-independent endocytosis (41), which suggest the role of clathrin-independent endocytosis on the intercellular molecule transfer.

[0103] To differentiate whether donor or recipient cells were responsible for the inhibitory effects, we treated either cell with actin, Ras or PI3K inhibitors before co-culture. This revealed that these activities in donor cells were required for the transfer.

[0104] We also evaluated the presence of synthetic receptor and co-transferred membrane- associated molecules on the recipient cell surface. After 2 h of co-culture with donor (Jurkat) cells, the mCherry level in CD19+recipient (K562) cells increased dramatically (by 14.9 fold). Meanwhile, the surface CAR and CD3 levels increased by only 1.3 fold and 2.1 fold, respectively, and a similar increase was observed with CD19- recipient cells. This indicated that most transferred receptor molecules were internalized instead of displayed on the cell surface. In concordance, we observed that the transferred receptor molecules were co-localized with lysosomes in CD19+K562 or HEK recipient cells via fluorescent microscopy imaging. Using Sephluorin, a pH-sensitive GFP variant quenching at pH=6.5 (45) and C-terminally tagged to29 KILPATRICK TOWNSEND 790261771CD19CAR-mCherry, we found this reporter lost fluorescence relative to the pH-insensitive mCherry upon transfer to recipient cells, despite the continuous receptor transfer during this period. This indicates receptor localization into acidic endosomes or lysosomes in contrast to the pH neutral cytoplasm.

[0105] To investigate whether receptors transferred onto recipient cell plasma membrane remained functional, we established a K562 reporter cell line with genomic-integrated loxP- polyA-loxP-GFP for detecting transferred Cre activity. The reporter also expressed TEV protease (TEVp) fused to CD19. Upon cleavage of the CD19CAR-Cre fusion protein via the TEVp cut site (TCS), Cre would be released from membrane to enter the nucleus for activating GFP. Despite 48h of co-culture with donor cells expressing CD19CAR-TCS-Cre and efficient intercellular transfer observed, there was no GFP turning on in reporter cells compared to controls.

[0106] In summary, we confirmed that the transfer process was dependent on cellular contact, actin polymerization, Ras and PI3K activity. However, unlike trogocytosis, the transferred molecules were not displayed on the recipient cell plasma membrane, but instead endocytosed and targeted for degradation. Fusogen-mediated endosome escape leads to functional biomolecule delivery

[0107] To evaluate potential methods to rescue the function of transferred molecules, we established a sensitive reporter cell expressing one half of the split GFP (GFP11) in either the cytoplasm or the nucleus, enabling the detection of the other half (GFP1-10) in the same compartment. When donor cells stably expressing receptors with GFP1-10 on the C-terminus were co-cultured with the GFP11 recipient cells, various designs incorporating pH-sensitive endosome escape peptide (INF7), endosome protease cut site (NNe) (46), or adding endosome escape-facilitating drug UNC10217938A (47) were unable to generate a reconstituted GFP signal in recipient cells.

[0108] Vesicular stomatitis virus G (VSV-G) protein (SEQ ID NO:2) is a viral-derived fusogen with membrane fusion activity in low pH environment, which facilitates endosome escape of virus and engineered extracellular vesicles (48, 49). By co-expressing VSV-G on the donor cell membrane, we hypothesized that upon cell-to-cell transfer of the membrane patch containing the receptor fusion molecules and endosome acidification, co-transferred VSV-G will30 KILPATRICK TOWNSEND 790261771facilitate the membrane fusion between endosome membrane and the vesicle membrane, thus exposing the encapsulated cargo molecules to the cytoplasm.

[0109] To test this possibility, we first transfected VSV-G into donor HEK293T cells that stably expressed CD19CAR (ligand-binding domain) fused to a C-terminal GFP1-10 (effector domain), and co-cultured them with recipient K562 cells expressing cytoplasm-localized GFP11. As expected, VSV-G expression and co-transfer significantly rescued the transferred GFP1-10 from endosomes, enabling efficient GFP reconstitution (Fig.5), without affecting the molecule transfer efficiency. Compared to wild type VSV-G, expression of a low density lipoprotein receptor (LDLR)-binding deficient variant (VSV-G*, K47Q, R354A) (SEQ IDNO:46) (50) showed similar efficiencies for receptor-cargo transfer and GFP reconstitution. We also observed that donor cells with stably transduced VSV-G were able to functionalize transferred cargo function effectively.

[0110] Moreover, we found that while the transferred mCherry showed up in endosome-like punctae, the reconstituted GFP mainly occurred on the plasma membrane instead of accumulating in punctae, indicating that the transferred membrane receptors were returned to recipient cell plasma membrane, very similar to the natural phenomenon of trogocytosis. Besides, the GFP reconstitution was a rapid process that kicked off at around 6h upon co-culture. Notably, the molecular transfer in the presence of VSV-G was still antigen-specific and contact- dependent.

[0111] Apart from using HEK293T cell as the donor cells, we further confirmed that alternative donor cells (K562, NALM6, Hela and Jurkat), when equipped with anti-CD19 receptor fused with a GFP(1-10) cargo on its C terminus, could transfer the cargo to CD19+recipient HEK cells in a CD19-dependent manner. With VSVG expressed on donor cells, the GFP(1-10) could reconstitute with GFP11 in the recipient cell cytoplasm, giving off GFP signals. The efficacy of delivering functional cargos varies across different donor cell types, with the percentage of GFP+recipient cells ranging from 98% using HEK293T donor cells, 77% using Hela donor cells, 27% using NALM6 donor cells, 30% using K562 donor cells, to 22% using Jurkat donor cells. Notably, adherent cells (HEK293T and Hela) perform better than suspension cells (NALM6, Jurkat, K562) in their ability in transferring functional cargos, which could be31 KILPATRICK TOWNSEND 790261771due to a combination of their higher receptor and VSVG expression levels, and the intrinsic higher stability of cell-cell contact between adherent cells.

[0112] Since macromolecules including gene-editing tools function in the nucleus, we further hypothesized that by incorporating a release domain, the effector domain could more readily dissociate from endosome membrane once it escapes (Fig.6). By inserting an endosome protease cut sites (NNe) between CD19CAR and GFP1-10 and tagging GFP1-10 with a nuclear localization signal (NLS) (SEQ ID NO:32), the transferred GFP1-10 showed effective GFP reconstitution with GFP11 in the nucleus.

[0113] To further improve the efficiency of nuclear localization upon transfer, we incorporated either a pH-sensitive self-cleaving intein (pH intein) (51) (SEQ ID NO:22), a pH intein mutant (N150) (52) (SEQ ID NO: 48), tandem endosome protease cut sites (EndoCut), or a self-cleaving domain (ITIH3) (53) between the C-terminal GFP1-10 and the ligand-binding domain. We observed a significant improvement of GFP reconstitution in the nucleus using pH intein. We further replaced the effector domain with the Cre recombinase (SEQ ID NO: 64) in the pH intein design, and found improved Cre activity in the loxP-polyA-loxP-GFP recipient cells, compared to the group without VSV-G. Cell-to-cell molecular transfer enables Cas9-mediated gene editing in recipient cells

[0114] To efficiently transfer Cas9, we utilized ABI and PYL1, a pair of abscisic acid (ABA)-inducible protein heterodimerization domains. The cytosolic domain (4-1BB-CD3 ) ofCD19CAR was replaced with ABI (SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14), while PYL1 was fused to the N-terminus of Cas9 (SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26). ABA addition would recruit Cas9 to the membrane receptor, allowing its co-transfer into recipient cells (Fig.7A). We engineered stable recipient HEK293T cells expressing a Cas9 gene-editing reporter system and a reporter-targeting single-guide RNA (sgRNA), where Cas9- induced indels can restore the reading frame of a split GFP to turn on the GFP signal. To achieve optimal gene editing, we designed different arrangements of nuclear export signal (NES) and nuclear localization signal (NLS) in the Cas9 construct to balance efficient nuclear export in the donor (HEK293T) cell and efficient nuclear import in the recipient cell after protein cleavage.32 KILPATRICK TOWNSEND 790261771By adding ABA to the co-culture assay, we measured GFP in the recipient cells and chose the design (#5) yielding the highest GFP signal for further experiments.

[0115] We tested whether the system allowed tunable delivery of Cas9. We added ABA into the co-culture system for a certain period, followed by ABA wash out. On day 3 after co-culture, we observed over 50% of recipient cells turning GFP positive with transient or continuous ABA treatment (Fig.7B), while the group without ABA treatment showed significantly lower mCherry-Cas9 delivery and induced GFP signal. The GFP signal without ABA was likely due to the low-level transfer of bystander cytoplasmic proteins. This result presented an example where we can use drug to fine tune the amount of delivered Cas9 to achieve efficient gene editing and control potential side effects.

[0116] Apart from HEK293T as recipient cells, we observed efficient editing with K562 recipient cells in the co-culture assay in the presence of ABA. Due to the similarity of our receptor-mediated cell-to-cell molecule transfer to trogocytosis, and additional engineering to release the transferred molecules for function, we termed our macromolecule delivery technology to be trogocytosis-inspired receptor transfer and functional effector release (TRANSFER).

[0117] To further validate that our cell-to-cell Cas9 transfer system and the gene editing we observed in recipient cells is still dependent on cell-cell contact after the addition of VSVG, we did four additional assays using donor cell expressing VSVG:

[0118] (1) We performed the transwell assay on the Cas9 transfer system, with different arrangements of donor and recipient cells in the transwell (donor and recipient on top, recipient alone at bottom; recipient alone on top, donor and recipient at bottom; donor cell alone on top, recipient alone at bottom). All our results validated that cell contact is essential for cell-to-cell transfer, while no Cas9 transfer happens across the membrane.

[0119] (2) Donor cells and recipient cells seeded on separate coverslips in the same well were allowed to share media, but no molecule transfer was observed, while donor cells and recipient cells co-seeded on the same coverslip yielded significant Cas9 transfer and gene editing.

[0120] (3) Conditioned media produced by donor cells with cell debris removed through either centrifugation or filtering were transferred to recipient cells for 3 consecutive days and no Cas933 KILPATRICK TOWNSEND 790261771molecule transfer or gene editing could be observed, while the co-culture of the same number of donor and recipient cells yielded significant Cas9 transfer and gene editing in recipient cells.

[0121] (4) Donor cells with nuclear mCherry were seeded on the periphery of a well surrounding a removable culture insert in the middle of the well, and on a subsequent day, recipient cells were seeded evenly in the whole well after the removal of culture insert. The reporter GFP signal was found to co-localize with mCherry signal on the periphery of the well, while the middle region without donor cells attached showed low background GFP signals, without significant drop with increasing distance from the donor cells. This confirmed that the cell-to-cell transfer is dominated by cell-cell contact, but not diffusible vesicles. TRANSFER enables programmable cell-to-cell transfer to various cell types

[0122] We expect TRANSFER to be a programmable and specific delivery method for diverse cell types or tissues (Fig.8). The rapid expansion of sequencing data provides a rich cell atlas that defines cell types based on their gene expression signatures (54). We postulate that this dataset can be used as instructions to programmably design cell-specific delivery modalities based on TRANSFER.

[0123] To deliver macromolecules selectively to any cell type of interest among user inputs, we first developed a computational pipeline to identify its unique surface markers, through differential gene expression analysis and filtering of experimentally validated surface proteins. For each marker, a specific binder such as scFv or nanobody could be identified and constructed as the ligand-binding domain of the cargo, so that donor cells can potentially recognize and deliver the cargo molecule to recipient cells in a programmable manner.

[0124] As a proof of concept, we used B cell precursor leukemia cell line NALM6, breast cancer cell line MCF7, and Jurkat cell as the user input. We identified CD19, HER2, and CD3 as unique surface markers of each cell line by analyzing the public gene expression dataset (54, 55) and surface protein dataset (56) (Fig.9A), and used their corresponding scFvs as ligand-binding domains. Upon establishing donor cells expressing the receptor molecules tagged with mCherry, we co-cultured donor cells with recipient cells in all possible combinations. Cell-to-cell molecular transfer was highly specific to the cell type expressing the cognate surface ligand (Fig. 9B-D), demonstrating that TRANSFER is a generalizable and programmable delivery platform (Fig.10).34 KILPATRICK TOWNSEND 790261771

[0125] In addition to ligand-specific transfer of the mCherry cargo to different cell types, we validated that by targeting their endogenous surface ligands (CD3, CD19 and HER2), functional Cas9 and Cre cargos could be transferred into Jurkat, NALM6, and MCF7 recipient cells respectively to edit an integrated reporter cassette in these recipient cells (6.5% NALM6 and 15.2% Jurkat recipient cells showed Cas9 mediated editing, 20% MCF7 recipient cells showed Cre mediated recombination). As expected, the functionalization of Cas9 in Jurkat recipient cells is strictly dependent on the presence of VSVG* in the donor cell.

[0126] Furthermore, we tested whether the specific cell-to-cell transfer applies to primary T cells. Using HEK293T cell as the donor cell and mCherry as the cargo, we demonstrated transfer into primary T cells using receptors targeting CD3 (SEQ ID NO:34), CD28 (SEQ ID NO:40), and CD2 (SEQ ID NO:42), which are endogenous surface markers of T cells. The percentage of recipient cells acquiring the cargo differs across different receptor-ligand pairs, with 99% positive for CD3, 80% for CD28, and 55% for CD2 targeting. In contrast, no transfer was observed when using a receptor targeting CD14, which is not expressed on T cell surface. In addition, donor cells with receptors targeting CD4 (scFv comprising SEQ ID NO:38), which are expressed only on subsets of the T cell population, would selectively transfer cargo into the CD4+T cells in the entire T cell population. Likewise, donor cells with receptors targeting FITC (scFv comprising SEQ ID NO: 44), which can indirectly target CD8 through the binding to FITC-conjugated CD8 antibody, would selectively transfer cargo into the CD8+T cells in the entire T cell population. This further supported the potential of TRANSFER in cell type-specific delivery in complex physiological environment.

[0127] Apart from single surface marker targeting, TRANSFER possesses the unique potential of sensing and processing more than one inputs, and making smart decisions, which is not possible with non-living delivery vehicles. We demonstrated here the potential of TRANSFER to implement AND gate logic, achieving selective delivery of cargo into recipient cells with both two types of ligands present, but not to recipient cells lacking any of the two ligands. Here we used CD19 and CD3 as the two inputs required for implementation of delivery. To achieve that, the donor cell was engineered with an aCD19 synNotch receptor (SEQ ID NO:66) (58) that undergoes intramembrane proteolysis upon recognition of the CD19 ligand on recipient cell surface, followed by nuclear localization of the released C terminal transcription factor. The35 KILPATRICK TOWNSEND 790261771cargo fused to PYL1 is subsequently expressed under the control of the transcription factor, which can associate with a second receptor targeting CD3 on recipient cells through ABA- inducible dimerization. As a result, the cargo will be transferred into recipient cells when both CD19 and CD3 are present in the recipient cells. Indeed, we observed that more than 30% of CD19+CD3+ recipient cells acquired the cargo, while CD19-CD3+, CD19+CD3- and CD19- CD3- recipient cells showed less than 3% mCherry positive rate upon co-culture with the donor cells. Such programmable behavior involving customized sense-response is not achievable with non-living delivery vehicles. TRANSFER is portable across different functional payloads

[0128] Combining all the optimizations, we explored the modularity of the TRANSFER system for different functional payloads and applications. Firstly, we examined the potential of TRANSFER in delivering prodrug converting enzyme for targeted cell ablation. Prodrug converting enzyme Herpes simplex virus thymidine kinase (HSVTK) is able to phosphorylate non-toxic drug Ganciclovir (GCV) into nucleoside monophosphate, which then get phosphorylated into GCV-triphosphate (GCV-3p) that can be incorporated into DNA to cause cell death. By fusing HSVTK (SEQ ID NO:50) to the C terminus of the engineered anti-CD19 receptor in donor cells, HSVTK could be transferred specifically into recipient cells bearing the cognate ligand to mediate cell death (cell count reduced by 79%), while the recipient cells without the cognate ligand were affected to a significantly smaller extent (cell count reduced by 31%).

[0129] Furthermore, we tested the potential of applying TRANSFER to the delivery of a zinc finger nuclease (ZFN) pair (SEQ ID NOs: 52 and 54) by using a HEK293T reporter cell line carrying CD19. The cell line carries reporter loci stably integrated in its genome, so that the ZFN pair-induced gene editing can restore the reading frame of a frameshifted GFP to turn on GFP expression. By engineering a donor cell with aCD19-ABI and PYL1-pH intein-ZFN fusion proteins and co-culturing it with the reporter cells, significant increase in GFP signal was observed when ABA was added. Notably, the donor cell with aCD19-ABI and a nuclear localized ZFN could not transfer ZFN into the reporter cells, and exhibits no response to ABA addition, validating the transfer mechanism of membrane-localized proteins.36 KILPATRICK TOWNSEND 790261771

[0130] To validate the potential of TRANSFER to edit endogenous genetic loci instead of a reporter cassette, we adopted the aCD19-ABI / PYL1-pH intein-Cas9 donor cell line, with VSVG* transfected, and co-cultured it with recipient cells expressing sgRNA targeting endogenous B2M or PDCD1 loci, respectively. We found that upon cell-to-cell transfer facilitated by ABA addition, both B2M and PDCD1 loci could be substantially edited by 70% and 40%. We further validated that recipient cells exhibit 50% B2M knockout upon co-culture at just 1:4 donor-to-recipient ratio.

[0131] Lastly, to demonstrate that the novel cell-to-cell transfer principle we revealed in this work applies to the in vivo context, we used a simple model to transfer Cas9 in donor HEK293T cells to reporter HEK293T cells, using gene editing and activation of the split GFP reporter as readout. Specifically, we implanted CD19+recipient cells with the reporter on one flank of mice, and the 1:1 mixture of the recipient cells with donor cells carrying an antiCD19 receptor and Cas9 into the other flank. After 14 days, we found that only the recipient cells mixed with donor cells showed significant levels of GFP signals (6-12% editing with a donor-to-recipient ratio above 1:5), indicating successful functional protein delivery in vivo, and verified that donor-to- recipient ratio is a critical factor for the percentage of cells acquiring the molecules in vivo. Discussion

[0132] Trogocytosis is a natural cell-cell communication pathway, enabling immune cells to extract functional molecules from their interaction partners (25, 26). Drawing inspiration from natural trogocytosis, we discovered that a ubiquitous cell-to-cell receptor transfer pathway can be engineered to reconstitute a trogocytosis-like process. Based on this discovery, we developed TRANSFER, a novel method that enabled programmable cell-type specific molecular delivery. Our reported trogocytosis-like pathway allowed engineered donor cells to transfer synthetic receptors and linked molecules efficiently into specific recipient cells upon cellular contact. We showed that the transfer was dependent on actin, Ras and PI3K activity, and naturally subjected to rapid degradation. We further identified that pH-responsive fusogen on donor cells facilitated the endosomal release of molecular cargos to the plasma membrane, and pH-responsive self- cleavable protein domain enabled nuclear delivery and gene-editing using Cas9. Different from other delivery approaches including AAV or nanoparticles, TRANSFER achieved programmable and selective delivery of functional molecules into various cell types, and to cellular compartments beyond the plasma membrane. Our data also support that cargo molecule delivery37 KILPATRICK TOWNSEND 790261771is tunable with small molecules using our method, a feature that is often missing from other approaches. Thus, TRANSFER presents a specific, tunable, efficient, and versatile delivery platform for macromolecules including gene-editing tools.

[0133] The mechanism of natural trogocytosis remains unclear. Our research shed light on membrane fusion in endosomes as a potential step in trogocytosis. Supporting the potential role of membrane fusion, a recent paper revealed that the cholesterol metabolism of T cells is changed in tumor microenvironment, increasing the fusion propensity of the membrane and stimulating T cell trogocytosis (57). The existence of other endogenous fusogenic plasma membrane components driving trogocytosis warrants further investigation.

[0134] A more detailed dissection of the molecular mechanism driving the cell-to-cell receptor transfer has been a challenge, as rapid internalization of donor cell-derived membrane-bound materials through direct cell-cell contact is shared by trans-synaptic vesicle uptake, trans- endocytosis, and trogocytosis (16). Nevertheless, our research highlights the importance of donor cell activities in the transfer. It is possible that polarized trans-synaptic vesicles are involved in the synthetic receptor transfer, followed by efficient uptake at the site of cellular interaction by recipient cells. Since trans-synaptic vesicle generation is restricted to immune cells including T cells, there might be differences in the working mechanism when using HEK293T or other cell types as donor cells.

[0135] TRANSFER involves the cell-to-cell transfer of membrane patches. We did not observe significant cell death or cell-cell fusion when expressing VSV-G in HEK293T donor cells. To further mitigate the potential cytotoxicity and cell-cell fusion activities, the fusogen could be chemically induced or conditionally expressed when cells contact each other. Beyond proteins, we also expect the TRANSFER method can be used to deliver mRNA. We believe the programmable nature of TRANSFER should work for a broad spectrum of cell types and macromolecules cargos. Materials and Methods Generation of genetic constructs

[0136] Standard molecular cloning techniques were used to build all plasmids used in the paper and they are included in table S1. Unless otherwise stated, CD19CAR consisted of CD8signal peptide, FMC63 scFv, CD8 hinge domain and transmembrane domain, and 41BB-CD338 KILPATRICK TOWNSEND 790261771signaling domains (from Dr. Crystal Mackal, Stanford University). HER2CAR consisted of 4D5 scFv instead (from Dr. Robbie Majzner, Stanford University). mCherry or GFP was fused to the C terminus of indicated receptors with 8-9 amino acid (aa)-long flexible GS linkers. The mSA2, IgD4 hinge and CD28 sequences were cloned from ordered gBlocks (Integrated DNA technologies). The anti-FITC scFv, Notch and EGFR transmembrane domain, GPI anchor, anti- CD3, Cre, TEVp, TCS, Sephluorin, INF7, NNe, pH intein, endosome cut sites, ITIH3, ABI and PYL1 were cloned from synthesized DNA (Twist Biosciences). loxP-stop-loxP sequence was cloned from pK038.CAG-loxP-stop-loxP-EGFP-ires-tTA-WPRE (Supernova) (Addgene plasmid #85006), a gift from Takuji Iwasato. VSV-G was cloned from the Lentiviral packaging plasmid pMD2G. The Cas9 cutting reporter with frame-shifted GFP was a gift from Dr. Xiaoshu Xu (Stanford University). Plasmids were cloned using InFusion (Takara Bio) into a pHR lentiviral vector and Stellar Competent cells (Takara Bio). Cell lines

[0137] Jurkat Clone E6-1 cells K562 cells and NALM6 cells were obtained from ATCC(TIB-152, CCL-243, CRL-3273). The NALM6 cell line with endogenous CD19 knocked out and expressing truncated CD19 without cytoplasmic tail was a gift from Dr. Robbie Majzer (Stanford University). The Jurkat, K562 and NALM6 were cultured in RPMI 1640 (Thermo Fisher) supplemented with 10% fetal bovine serum (FBS) (Alstem) and 100 U mL-1of penicillin and streptomycin (Gibco) (complete RPMI). Jurkat, K562 and NALM6 cells were passaged every 3 days as cell culture densities approached 1 x 106cells per mL. HEK293T cells (Clontech) were cultured in DMEM + GlutaMAX (Thermo Fisher) supplemented with 10% FBS and 100 U mL-1Pen / Strep (complete DMEM). MCF7 cells were a gift from Dr. Jiangbin Ye (Stanford University), and cultured in DMEM with 1% L-glutamine. HEK293T and MCF7 cells was passaged every 3 days by dissociating in 0.05% trypsin (Life Technologies) for 1-2 min at 37 °C to remove from culture plates. All cells were maintained at 37 °C and 5% CO2and passaged using standard cell culture techniques. Cells were not tested for mycoplasma contamination. Stable cell generation

[0138] Stable Jurkat, K562, NALM6 and HEK293T cell lines were generated using lentiviral transduction. Jurkat, K562, or NALM6 cells were seeded the day of transduction at 1 x 105cells per well in 400 uL complete RPMI in a 48 well plate, while HEK293T cells were seeded 1 day before transduction at 4 x 104cells per well in 400 uL complete DMEM in a 24 well plate.39 KILPATRICK TOWNSEND 790261771

[0139] For lentiviral production, two different protocols were used: (1) 4.5 x 105HEK293T cells were seeded per well in a 6-well plate in 2 mL complete DMEM 1 day before transfection. On the day of transfection, HEK293T cells were transfected with 1.51 g of pHR vector, 1.32 g dR8.91 and 165 ng of pMD2g with 7.5 uL of Mirus TransIT-LT1 in 250 L OptiMEM (Thermo Fisher). Three days post transfection, lentivirus was harvested and filtered through a 0.45 m polyvinylidene fluoride filter (Millipore). After filtration, lentivirus was mixed 4:1 with Lentivirus Precipitation Solution (Alstem) and refrigerated overnight. Lentivirus was pelleted at 1,500 x g for 30 min at 4 C and resuspended in complete RPMI. (2) 1.5 x 106HEK293T cells per well were seeded in a 6-well plate in 2mL complete DMEM 1 day before transfection. On the day of transfection, 45% of media was removed from each well, and cells were transfected with1.76 g of pHR vector, 1.26 g of psPAX2 vector, 0.55 g of pMD2G plasmid with 10.7 uL ofMirus TransIT-LT1 in 420 uL OptiMEM (Thermo Fisher). After 6h of incubation, media was removed and changed into 2ml complete DMEM with 1xViralBoost reagent (Alstembio). After 18h of incubation at 37C, lentiviral supernatant was collected, filtered, and precipitated as mentioned above.

[0140] Cells were treated with 50 - 100 uL of 10x concentrated lentivirus for 18 - 24 h. After 24 h, fresh media was exchanged and transduced cells were cultured for at least 3 days beforeassay. Cell lines expressing puromycin resistance gene were selected in 2 g / ml puromycin for 2days, and then changed into fresh media. Different viral titers were tested for each construct to ensure the transduction level indicated by fluorescence marker intensity were comparable between cell lines used in the same experiment. Transfection of VSV-G expressing plasmid

[0141] For transfection of non-viral packaging purposes, HEK293T cells were seeded into 6 well plate at 500k / well one day prior to transfection. On the day of transfection, 250uL of Opti-Mem was mixed with 2.5 g DNA and 7.5 L of Mirus TransIT-LT1 reagent, and then incubatedat room temperature for 15–30 min, before being added dropwise to the seeded cells.Experiments were performed one day post-transfection. Generation of CAR expressing primary T cells

[0142] Mixed CD4+and CD8+primary T cells were isolated with the Dynabeads™ Human T- Activator CD3 / CD28 for T Cell Expansion and Activation (Thermo Fisher) from healthy donor whole blood obtained from the Stanford Blood Center (Stanford, CA) and stored in LN2.40 KILPATRICK TOWNSEND 790261771Thawed T-cells were cultured in complete RPMI 1640 with 200 U / mL rIL-2 (Thermo Fisher). T cells stimulated with CD3 / CD28 Dynabeads (Thermo Fisher Scientific) at a 1:1 cell:bead ratio for 1 d were seeded at 5 x 105per well in a 48 well plate and transduced with 20uL of 100x concentrated lentivirus. Dyna beads were removed after 2 d of culture and cell density was maintained at 1 x 106- 2 x 106cells per mL. All T cell co-culture assays were performed on day 7 after bead activation. Synthesis of small molecule-conjugated DNA origami

[0143] DNA structures were designed and predicted with the online tool NUPACK with salt concentration [Na+]=0.1437M, [Mg2+]=0.83mM (calculated for complete RPMI). The yields for designed structures were all >95%. Modified ssDNA strands were synthesized by Stanford Protein and Nucleic Acid facility (PAN). For the double-strand DNA structures, DNA origami structures were prepared immediately before use by annealing complementary ssDNA strands at 100 M in duplex buffer (IDT) using the following program: 95 °C for 5 min, 65 °C for 5 min, 60 °C for 5 min followed by slowly lowering temperature to 4 °C. For the branched structure,ssDNA strands were annealed at 20 M in duplex buffer using the same program. DNA origamiwas stored at 4 °C until used. The designed DNA oligo sequences were listed in table S3. Co-culture assay

[0144] Unless otherwise stated, donor and recipient cells were co-cultured at 1:1 ratio for 24h. Jurkat cells and K562 cells expressing the indicated constructs were mixed, with total cell number at 105per well in a 96-well U-bottom plate in 100uL fresh complete RPMI. For co- cultures involving adherent cells, the co-culture was conducted in 96-well U-bottom plate in 100uL fresh complete DMEM.96-well flat-bottom plate was used for adherent cells instead if co-culture exceeded 48h. Cells were mixed by pipetting before incubation at 37°C for indicated durations. For co-culture below 2h, the plate was spinned at 700rpm for 30s to settle the cells.For co-culture in transwell (Corning), the wells were with 3 m pore size, and were equilibratedin complete RPMI for 1h at 37°C before experiment. For the experiments where donor cells were activated by recipient cells, 5x104donor cells and 5x104recipient cells in 75uL media were added into the upper chamber, while 5x104recipient cells in 235uL media were added into the lower chamber. For the experiment where donor cells were not active by recipient cells, 5x104cells in 75uL or 235uL media were added into the upper and lower chamber, respectively. For co-culture of primary T cell and recipient cell, co-culture period was shortened to 2h or 6h to41 KILPATRICK TOWNSEND 790261771reduce the influence from cytotoxicity, and live / dead staining was done to gate on the live cells for analysis. Conditioned media collection

[0145] Conditioned media was collected from donor Jurkat cells cultured in 12 well plate until confluency. Cell culture was spinned at 1000rpm for 5min, and supernatant was collected. Recipient cells were subsequently cultured in the conditioned media for 24h. Drug treatment

[0146] Cells were incubated with the inhibitors at 37°C for indicated period of time in mono- culture and seeded for co-culture as mentioned above for 2h at 37°C, with the inhibitors remaining in the co-culture. Specific details of inhibitor usage are listed in table S4. Inhibitor titration and live / dead staining was done to ensure the concentration used in co-culture did not compromise cell viability for the duration of experiment. For inhibitor treatment of one specific type of cell, the treated cells were cultured alone with the inhibitor for 6h, and then co-cultured with same numbers of the other cell type (untreated) for 1h. During the 1h co-culture, the inhibitor remained in the co-culture system.

[0147] For ABA treatment experiments, ABA was added to the co-cultured cells at 250 M.For ABA washout, co-cultured cells were spinned at 1000rpm for 5min in 96 well U bottom plate, with media removed and resuspension in 150uL PBS, followed by another round of spinning and resuspension in ABA-free media. Live / dead staining

[0148] The viability after drug treatment was measured using viability dyes staining (ThermoFisher). Cells are spinned at 1000rpm for 5min, washed with 150uL per well of ice-cold PBS buffer. The stain was diluted 1:1000 into 100uL of PBS-resuspended single cell solution and incubated on ice for 30min in dark before the rest of flow cytometry measurements. Flow cytometry

[0149] For flow measurement of co-cultured suspension cells without antibody staining, co- cultured cells were spinned at 1000rpm for 5min, washed with 150uL per well of ice-cold FACS buffer (PBS + 2% FBS) containing 5mM EDTA (FACS-EDTA), and eventually resuspended in 100uL per well of FACS-EDTA for flow cytometry measurement (Beckman-Coulter Cytoflex S). For co-culture involving adherent cells, co-cultured cells were first spinned at 1000rpm for 5min and digested with 70uL 0.05% Trypsin for 5min. Afterwards, cells were spinned at42 KILPATRICK TOWNSEND 7902617711000rpm for 5min, washed with 150uL per well of ice-cold FACS-EDTA, and eventually resuspended in 100uL per well of FACS-EDTA.

[0150] For measurement with antibody staining, the following dye-conjugated antibodies wereused: FITC AffiniPure F(ab') Fragment Goat Anti-Mouse IgG, F(ab') fragment specific(polyclonal, Jackson Immunoresearch #115-096-006), FITC anti-human CD3 (clone UCHT1, Biolegend #300405), FITC Mouse IgG1, Isotype Ctrl Antibody (clone MOPC-21, Biolegend #400107). After the initial washing step with 150uL FACS-EDTA, cells were incubated with antibodies diluted 1:500 in 100uL FACS-EDTA for 30min on ice in dark, followed by another wash with 150uL FACS-EDTA and resuspension in 100uL FACS-EDTA before flow cytometry measurement.

[0151] We collected 10,000 single cells for analysis. Data were analyzed using FlowJo v.10.8.1 (BD Biosciences). Unless otherwise stated, FACS analysis were performed on gated single cells. Mono-cultured recipient cells were used as control for gating mCherry+and GFP+cells, and were used as basal level for normalizing MFI mCherry or MFI GFP. A threshold for positive and negative population was drawn so that no more than 0.5% of the same population in control group was positive. Fluorescence microscopy

[0152] All Fluorescence microscopy was performed on Leica DFC9000 GT equipped with a 4.2 MP sCMOS monochrome fluorescence camera, using 405nm, 488nm, and 561nm lasers, and 63x or 100x oil objective lens. To visualize the localization of the transferred cargos, glass- bottom 24-well plate was coated with poly-lysine for 30min at 37 °C and washed with 1ml water for three times. Cells co-cultured in 96-well U-bottom plate as mentioned above were mixed with 1000x LysoView dye (biotium) and plated onto the glass-bottom plate. Adherant cells were digested with trypsin first before reseeding into the imaging plate. Cells were settled for 30min before imaging.

[0153] Live microscopy was taken in poly-lysine coated 96-well glass-bottom format in a humidified chamber at 37°C with 5% CO2, with 5x104K562 and 5x104Jurkat cells added per well, followed by 700rpm spin for 1min without pre-co-culture. Movies were taken with the 63xoil objective (NA=1.4), with Z stacks at 0.5 m steps, 1 image per field per minute. Images weretaken using Leica LAS X software. The camera pixel size is 0.240 m / pixel. Only one Z slice is43 KILPATRICK TOWNSEND 790261771used for all images shown. Images were analyzed by Fiji, where the brightness and contrast for each channel were adjusted to be the same for all samples in the same experiment. Images of mCherry channel were overexposed to reveal the relatively weak signal in the recipient cells. Incucyte measurement

[0154] For monitoring co-culture, cells were seeded into 96-well flat-bottom plate. Wells were mixed well and cells were allowed to settle for 1 - 2 h. Four images per well were acquired at 10x magnification in the green channel (300 ms exposure) and the red channel (400 ms exposure) every 30min using an Incucyte (Sartorius). The integrated intensity of fluorescence per well was calculated for each time point. Cell sorting

[0155] Cells were sorted with Sony SH800S cell sorter using 100 m chip where indicated. Computational analysis of surface markers

[0156] The gene expression TPM values for cancer cell lines were downloaded from CCLE (Expression Public 23Q2). Values were inferred from RNA-seq data using the RSEM tool and are reported after log2 transformation, using a pseudo-count of 1; log2(TPM+1). Upon input of a list of cell types, gene enrichment score in cell type A over the rest of the cell types (B, C, D…) were computed using the formula m ,a ,x …{log2(TPM+1)_A- log2(TPM+1)_i}. Genes were sorted in decreasing order of the enrichment score and filtered for cell surface proteins by intersecting with a list of protein from mass spectrometric-derived cell surface protein atlas. Cell culture on coverslips

[0157] At the cell seeding stage, 600k donor or 600k recipient HEK293T cells were seeded evenly in a 6-well plate well with a tissue-culture treated Microscope Cover Glass (Universal Medical, cut into identical size and shape with glass cutter) at the bottom. Unnecessary motions were avoided during the cell sedimentation period. One day later, the coverslip covered by cells on its top surface were transferred carefully with a forcep into a new 6-well plate well to initiate the co-culture as specified, with 10uM ABA added. To have donor cells and recipient cell co- seeded on the same coverslip, a coverslip covered with donor cells was transferred into an empty well with another empty coverslip, and 600k recipient cells were seeded evenly on top.3 days later, each coverslip was transferred into a blank well, and cells attached to the coverslip was lifted with 0.05% trypsin and measured by flow cytometry.44 KILPATRICK TOWNSEND 790261771Cell culture using culture inserts

[0158] On the first day of experiment, a culture-insert (ibidi, Cat# 80369) was installed in the middle of a 6-well plate well, with 500k donor HEK293T cells evenly seeded surrounding the insert on the periphery of the well. One day later, the culture insert was carefully removed with a forcep, and 500k recipient HEK293T cells were evenly seeded in the whole well, with 10uM ABA added. mCherry and GFP signals from the whole well were monitored with Incucyte every 24h for 3 days. For image analysis, integrated mCherry signals and GFP+ cell count were quantified in selected regions of the same size and shape in the image. Media transfer experiment

[0159] At the cell seeding stage, 100k donor HEK293T cells and 100k recipient HEK293T cells were seeded into separate 24-well plate wells. For comparison, the same number of donor and recipient cells were co-cultured in the same 24-well plate well. On the next day, supernatant from the donor cell well were collected, either spinned at 1000rpm for 5min, or filtered through 0.45um sterilized Millex-HV Syringe Filter to get rid of cell debris. The old media in the recipient cell well was replaced by the supernatant from donor cells, with 10uM ABA added. On the second day and third day, the procedure was repeated, without removal of old media in the recipient cell well. On the fourth day, the cells from the recipient cell well were measured by flow cytometry, using the recipient cells co-cultured with the same number of donor cells for 3 days as control. Recipient cell number quantification with counting beads

[0160] After trypsinization of the co-culture cells, 2000 counting beads were added per sample and evenly mixed. For each sample, at least 1000 bead events were collected, and the recipient cell count was determined based on the ratio of BFP+ events to bead events. Animal models

[0161] Immunocompromised NSG mice (NOD.Cg-PrkdcscidJAX #005557) mice were purchased from the Jackson Laboratory and housed compliant with Stanford University Laboratory Animal Care (APLAC) protocols. Mice were housed at Stanford University with a 12-hour light / dark cycle and monitored daily. Healthy male mice were used for in vivo experiments. Mice were 4 weeks old at tumor engraftment and had not been involved in45 KILPATRICK TOWNSEND 790261771previous procedures, drug treatments, or experiments. For tumor implantation, 10 million of recipient cells, or 10 millions of donor cells mixed with 10 millions of recipient cells were injected subcutaneously in a 1:1 mixture with Matrigel (Fisher Scientific Cat# CB-40234). For ABA administration, 400uL of 4g / L ABA dissolved in 0.9% saline was injected intraperitoneally for twice per day for 14 days from the day of tumor implantation. Genomic DNA extraction, targeted amplification and Sanger sequencing

[0162] Genomic DNA (gDNA) was extracted from sorted recipient cells using the DNeasy Blood and Tissue kit (QIAGEN). Cells were lysed using Buffer AL and the cell lysate supernatant was transferred into a clean tube before proceeding with the manufacturer’s protocol for gDNA isolation. Primers flanking the gRNA binding region on the genome were used for targeted amplification of the gene editing loci, and reactions were set up with NEB Phusion® High-Fidelity DNA Polymerase. The PCR product was sent for Sanger sequencing, and Cas- mediated indel rate was quantified using the TIDE software. Statistics

[0163] Statistical analysis and data visualizations were performed in GraphPad Prism softwareversion 9. Data are reported as individual values and mean ± standard deviation. n is the numberof replicates per experiment. MFI indicates median fluorescence intensity. Equal variance between populations was not assumed. To account for unequal variance among conditions, Welch’s two-sided t test was performed when comparing two conditions. For incucyte data, statistical analysis was performed with repeated measures one-way ANOVA. All statistical dataanalyses are compiled in table S2. p<0.0001, ****; 0.0001 p<0.001, ***; 0.001 p<0.01, **;0.01 p<0.05, *; p 0.05, non-significant (ns).Supplementary Tables Table S1. Constructs used in the study.46 KILPATRICK TOWNSEND 79026177147 KILPATRICK TOWNSEND 79026177148 KILPATRICK TOWNSEND 790261771Table S2. Significance level for statistical tests.49 KILPATRICK TOWNSEND 790261771Table S3. DNA oligos used in the study.50 KILPATRICK TOWNSEND 79026177151 KILPATRICK TOWNSEND 790261771Table S4. Inhibitors and drugs used in the study.52 KILPATRICK TOWNSEND 790261771Cited references 1. J. A. Doudna, The promise and challenge of therapeutic genome editing. Nature Research [Preprint] (2020). https: / / doi.org / 10.1038 / s41586-020-1978-5. 2. M. Chavez, X. Chen, P. B. Finn, L. S. Qi, Advances in CRISPR therapeutics. Nature Research [Preprint] (2023). https: / / doi.org / 10.1038 / s41581-022-00636-2. 3. D. Wang, F. Zhang, G. Gao, CRISPR-Based Therapeutic Genome Editing: Strategies and In Vivo Delivery by AAV Vectors. Cell Press [Preprint] (2020). https: / / doi.org / 10.1016 / j.cell.2020.03.023. 4. C. Li, R. J. Samulski, Engineering adeno-associated virus vectors for gene therapy. Nature Research [Preprint] (2020). https: / / doi.org / 10.1038 / s41576-019-0205-4. 5. S. Zolotukhin, L. H. Vandenberghe, AAV capsid design: A Goldilocks challenge. Elsevier Ltd [Preprint] (2022). https: / / doi.org / 10.1016 / j.molmed.2022.01.003. 6. M. J. Mitchell, M. M. Billingsley, R. M. Haley, M. E. Wechsler, N. A. Peppas, R. Langer, Engineering precision nanoparticles for drug delivery. Nature Research [Preprint] (2021). https: / / doi.org / 10.1038 / s41573-020-0090-8. 7. Y. Liu, K. Adu-Berchie, J. M. Brockman, M. Pezone, D. K. Y. Zhang, J. Zhou, J. W. Pyrdol, H. Wang, K. W. Wucherpfennig, D. J. Mooney, Cytokine conjugation to enhance T cell therapy. Proc Natl Acad Sci U S A 120 (2023). 8. T. J. Gardner, J. P. Lee, C. M. Bourne, D. Wijewarnasuriya, N. Kinarivala, K. G. Kurtz, B. C. Corless, M. M. Dacek, A. Y. Chang, G. Mo, K. M. Nguyen, R. J. Brentjens, D. S. Tan, D. A. Scheinberg, Engineering CAR-T cells to activate small-molecule drugs in situ. Nat Chem Biol 18, 216–225 (2022). 9. L. M. Bush, C. P. Healy, S. B. Javdan, J. C. Emmons, T. L. Deans, Biological Cells as Therapeutic Delivery Vehicles. Elsevier Ltd [Preprint] (2021). https: / / doi.org / 10.1016 / j.tips.2020.11.008. 10. R. Kojima, D. Bojar, G. Rizzi, G. C. El Hamri, M. D. El-Baba, P. Saxena, S. Ausländer, K. R. Tan, M. Fussenegger, Designer exosomes produced by implanted cells53 KILPATRICK TOWNSEND 790261771intracerebrally deliver therapeutic cargo for Parkinson’s disease treatment. Nat Commun 9 (2018). 11. L. R. Johnson, D. Y. Lee, J. S. Eacret, D. Ye, C. H. June, A. J. Minn, The immunostimulatory RNA RN7SL1 enables CAR-T cells to enhance autonomous and endogenous immune function. Cell 184, 4981-4995.e14 (2021). 12. C. Zhang, R. Schekman, Syncytin-mediated open-ended membrane tubular connections facilitate the intercellular transfer of cargos including Cas9 protein. Elife 12 (2023). 13. F. Horns, J. A. Martinez, C. Fan, M. Haque, J. M. Linton, V. Tobin, L. Santat, A. O. Maggiolo, P. J. Bjorkman, C. Lois, M. B. Elowitz, Engineering RNA export for measurement and manipulation of living cells. Cell, doi: 10.1016 / j.cell.2023.06.013 (2023). 14. O. S. Qureshi, Y. Zheng, K. Nakamura, K. Attridge, C. Manzotti, E. M. Schmidt, J. Baker, L. E. Jeffery, S. Kaur, Z. Briggs, T. Z. Hou, C. E. Futter, G. Anderson, L. S. K. Walker, D. M. Sansom, “Trans-Endocytosis of CD80 and CD86: A Molecular Basis for the Cell- Extrinsic Function of CTLA-4;” https: / / www.science.org. 15. What is trogocytosis and what is its purpose? 16. P. F. Céspedes, A. Jainarayanan, L. Fernández-Messina, S. Valvo, D. G. Saliba, E. Kurz, A. Kvalvaag, L. Chen, C. Ganskow, H. Colin-York, M. Fritzsche, Y. Peng, T. Dong, E. Johnson, J. A. Siller-Farfán, O. Dushek, E. Sezgin, B. Peacock, A. Law, D. Aubert, S. Engledow, M. Attar, S. Hester, R. Fischer, F. Sánchez-Madrid, M. L. Dustin, T-cell trans-synaptic vesicles are distinct and carry greater effector content than constitutive extracellular vesicles. Nat Commun 13 (2022). 17. M. Mittelbrunn, C. Gutiérrez-Vázquez, C. Villarroya-Beltri, S. González, F. Sánchez- Cabo, M. Á. González, A. Bernad, F. Sánchez-Madrid, Unidirectional transfer of microRNA- loaded exosomes from T cells to antigen-presenting cells. Nat Commun 2 (2011). 18. K. Choudhuri, J. Llodrá, E. W. Roth, J. Tsai, S. Gordo, K. W. Wucherpfennig, L. C. Kam, D. L. Stokes, M. L. Dustin, Polarized release of T-cell-receptor-enriched microvesicles at the immunological synapse. Nature 507, 118–123 (2014).54 KILPATRICK TOWNSEND 79026177119. B. W. Heck, D. Devenport, Trans-endocytosis of Planar Cell Polarity Complexes during Cell Division. Current Biology 27, 3725-3733.e4 (2017). 20. J. Spacek, K. M. Harris, Trans-Endocytosis via Spinules in Adult Rat Hippocampus. Journal of Neuroscience 24, 4233–4241 (2004). 21. S. Kusakari, H. Ohnishi, F. J. Jin, Y. Kaneko, T. Murata, Y. Murata, H. Okazawa, T. Matozaki, Trans-endocytosis of CD47 and SHPS-1 and its role in regulation of the CD47-SHPS- 1 system. J Cell Sci 121, 1213–1223 (2008). 22. M. Hamieh, A. Dobrin, A. Cabriolu, S. J. C. van der Stegen, T. Giavridis, J. Mansilla- Soto, J. Eyquem, Z. Zhao, B. M. Whitlock, M. M. Miele, Z. Li, K. M. Cunanan, M. Huse, R. C. Hendrickson, X. Wang, I. Rivière, M. Sadelain, CAR T cell trogocytosis and cooperative killing regulate tumour antigen escape. Nature 568, 112–116 (2019). 23. V. D. Gonzalez, Y. W. Huang, A. Delgado-Gonzalez, S. Y. Chen, K. Donoso, K. Sachs, A. J. Gentles, G. M. Allard, K. S. Kolahi, B. E. Howitt, E. Porpiglia, W. J. Fantl, High- grade serous ovarian tumor cells modulate NK cell function to create an immune-tolerant microenvironment. Cell Rep 36 (2021). 24. B. Vanherberghen, K. Andersson, L. M. Carlin, E. N. M. Nolte-’t Hoen, G. S. Williams, P. Hö Glund † §, D. M. Davis, “Human and murine inhibitory natural killer cell receptors transfer from natural killer cells to target cells” (2004); www.pnas.orgcgidoi10.1073pnas.0406240101. 25. S. S. Somanchi, A. Somanchi, L. J. N. Cooper, D. A. Lee, Engineering lymph node homing of ex vivo-expanded human natural killer cells via trogocytosis of the chemokine receptor CCR7. Blood 119, 5164–5172 (2012). 26. F. N. Cho, T. H. Chang, C. W. Shu, M. C. Ko, S. K. Liao, K. H. Wu, M. S. Yu, S. J. Lin, Y. C. Hong, C. H. Chen, C. H. Hung, Y. H. Chang, Enhanced cytotoxicity of natural killer cells following the acquisition of chimeric antigen receptors through trogocytosis. PLoS One 9 (2014). 27. M. S. Hasim, M. Marotel, J. J. Hodgins, E. Vulpis, O. J. Makinson, S. Asif, H. Y. Shih, A. K. Scheer, O. MacMillan, F. G. Alonso, K. P. Burke, D. P. Cook, R. Li, M. T. Petrucci, A.55 KILPATRICK TOWNSEND 790261771Santoni, P. G. Fallon, A. H. Sharpe, G. Sciumè, A. Veillette, A. Zingoni, D. A. Gray, A. McCurdy, M. Ardolino, When killers become thieves: Trogocytosed PD-1 inhibits NK cells in cancer. Sci Adv 8 (2022). 28. E. A. Rossi, D. M. Goldenberg, R. Michel, D. L. Rossi, D. J. Wallace, C.-H. Chang, Trogocytosis of multiple B-cell surface markers by CD22 targeting with epratuzumab. doi: 10.1182 / blood-2012-12 (2013). 29. S. Zhao, L. Zhang, S. Xiang, Y. Hu, Z. Wu, J. Shen, Gnawing Between Cells and Cells in the Immune System: Friend or Foe? A Review of Trogocytosis. Frontiers Media S.A. [Preprint] (2022). https: / / doi.org / 10.3389 / fimmu.2022.791006. 30. A. Bettadapur, H. W. Miller, K. S. Ralston, “Biting Off What Can Be Chewed: Trogocytosis in Health, Infection, and Disease” (2020); https: / / doi.org / 10. 31. L. Labanieh, R. G. Majzner, C. L. Mackall, Programming CAR-T cells to kill cancer. Nature Publishing Group [Preprint] (2018). https: / / doi.org / 10.1038 / s41551-018-0235-9. 32. R. G. Majzner, C. L. Mackall, Clinical lessons learned from the first leg of the CAR T cell journey. Nature Publishing Group [Preprint] (2019). https: / / doi.org / 10.1038 / s41591-019- 0564-6. 33. G. Li, M. T. Bethune, S. Wong, A. V. Joglekar, M. T. Leonard, J. K. Wang, J. T. Kim, D. Cheng, S. Peng, J. M. Zaretsky, Y. Su, Y. Luo, J. R. Heath, A. Ribas, O. N. Witte, D. Baltimore, T cell antigen discovery via trogocytosis. Nat Methods 16, 183–190 (2019). 34. E. Balleza, J. M. Kim, P. Cluzel, Systematic characterization of maturation time of fluorescent proteins in living cells. Nat Methods 15, 47–51 (2018). 35. M. J. Taylor, K. Husain, Z. J. Gartner, S. Mayor, R. D. Vale, A DNA-Based T Cell Receptor Reveals a Role for Receptor Clustering in Ligand Discrimination. Cell 169, 108- 119.e20 (2017). 36. D. Hudrisier, A. Aucher, A.-L. Puaux, C. Bordier, E. Joly, Capture of Target Cell Membrane Components via Trogocytosis Is Triggered by a Selected Set of Surface Molecules on T or B Cells. The Journal of Immunology 178, 3637–3647 (2007).56 KILPATRICK TOWNSEND 79026177137. G. Van Niel, G. D’Angelo, G. Raposo, Shedding light on the cell biology of extracellular vesicles. Nature Publishing Group [Preprint] (2018). https: / / doi.org / 10.1038 / nrm.2017.125. 38. N. Martínez-Martín, E. Fernández-Arenas, S. Cemerski, P. Delgado, M. Turner, J. Heuser, D. J. Irvine, B. Huang, X. R. Bustelo, A. Shaw, B. Alarcón, T Cell Receptor Internalization from the Immunological Synapse Is Mediated by TC21 and RhoG GTPase- Dependent Phagocytosis. Immunity 35, 208–222 (2011). 39. A. Datta, H. Kim, M. Lal, L. McGee, A. Johnson, A. A. Moustafa, J. C. Jones, D. Mondal, M. Ferrer, A. B. Abdel-Mageed, Manumycin A suppresses exosome biogenesis and secretion via targeted inhibition of Ras / Raf / ERK1 / 2 signaling and hnRNP H1 in castration- resistant prostate cancer cells. Cancer Lett 408, 73–81 (2017). 40. A. Delgado-Gonzalez, Y. W. Huang, E. Porpiglia, K. Donoso, V. D. Gonzalez, W. J. Fantl, Measuring trogocytosis between ovarian tumor and natural killer cells. STAR Protoc 3 (2022). 41. J. Xu, H. T. Feng, C. Wang, K. H. M. Yip, N. Pavlos, J. M. Papadimitriou, D. Wood, M. H. Zheng, Effects of bafilomycin A1: An inhibitor of vacuolar H (+)-ATPases on endocytosis and apoptosis in RAW cells and RAW cell-derived osteoclasts. J Cell Biochem 88, 1256–1264 (2003). 42. M. Catalano, L. O’Driscoll, Inhibiting extracellular vesicles formation and release: a review of EV inhibitors. Taylor and Francis Ltd. [Preprint] (2020). https: / / doi.org / 10.1080 / 20013078.2019.1703244. 43. T. Saha, C. Dash, R. Jayabalan, S. Khiste, A. Kulkarni, K. Kurmi, J. Mondal, P. K. Majumder, A. Bardia, H. L. Jang, S. Sengupta, Intercellular nanotubes mediate mitochondrial trafficking between cancer and immune cells. Nat Nanotechnol 17, 98–106 (2022). 44. O. Rechavi, I. Goldstein, H. Vernitsky, B. Rotblat, Y. Kloog, Intercellular transfer of oncogenic H-Ras at the immunological synapse. PLoS One 2 (2007).57 KILPATRICK TOWNSEND 79026177145. M. Koivusalo, C. Welch, H. Hayashi, C. C. Scott, M. Kim, T. Alexander, N. Touret, K. M. Hahn, S. Grinstein, Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling. Journal of Cell Biology 188, 547–563 (2010). 46. S. Yu, H. Yang, T. Li, H. Pan, S. Ren, G. Luo, J. Jiang, L. Yu, B. Chen, Y. Zhang, S. Wang, R. Tian, T. Zhang, S. Zhang, Y. Chen, Q. Yuan, S. Ge, J. Zhang, N. Xia, Efficient intracellular delivery of proteins by a multifunctional chimaeric peptide in vitro and in vivo. Nat Commun 12 (2021). 47. N. Heath, X. Osteikoetxea, T. M. De Oliveria, E. Lázaro-Ibáñez, O. Shatnyeva, C. Schindler, N. Tigue, L. M. Mayr, N. Dekker, R. Overman, R. Davies, Endosomal escape enhancing compounds facilitate functional delivery of extracellular vesicle cargo. Nanomedicine 14, 2799–2814 (2019). 48. P. E. Mangeot, S. Dollet, M. Girard, C. Ciancia, S. Joly, M. Peschanski, V. Lotteau, Protein transfer into human cells by vsv-g-induced nanovesicles. Molecular Therapy 19, 1656– 1666 (2011). 49. L. A. Campbell, L. M. Coke, C. T. Richie, L. V. Fortuno, A. Y. Park, B. K. Harvey, Gesicle-Mediated Delivery of CRISPR / Cas9 Ribonucleoprotein Complex for Inactivating the HIV Provirus. Molecular Therapy 27, 151–163 (2019). 50. J. Nikolic, L. Belot, H. Raux, P. Legrand, Y. Gaudin, A. A. Albertini, Structural basis for the recognition of LDL-receptor family members by VSV glycoprotein. Nat Commun 9 (2018). 51. D. W. Wood, W. Wu, G. Belfort, V. Derbyshire, M. Belfort, A genetic system yields self-cleaving inteins for bioseparations. Nat Biotechnol 17, 889–892 (1999) 52. Choi, S., Lee, Y., Hwang, J., Chun, D., Koo, H., & Lee, Y. (2023). Self-cleaving protein linkers with modulated pH-responsiveness: A new platform for selective control of protein drug function. Chemical Engineering Journal, 457, 141229.53. L. Zhuo, K. Kimata, Structure and function of inter- -trypsin inhibitor heavy chains.[Preprint] (2008). https: / / doi.org / 10.1080 / 03008200802325458.58 KILPATRICK TOWNSEND 79026177154. P. J. Thul, C. Lindskog, The human protein atlas: A spatial map of the human proteome. Protein Science 27, 233–244 (2018). 55. M. Ghandi, F. W. Huang, J. Jané-Valbuena, G. V. Kryukov, C. C. Lo, E. R. McDonald, J. Barretina, E. T. Gelfand, C. M. Bielski, H. Li, K. Hu, A. Y. Andreev-Drakhlin, J. Kim, J. M. Hess, B. J. Haas, F. Aguet, B. A. Weir, M. V. Rothberg, B. R. Paolella, M. S. Lawrence, R. Akbani, Y. Lu, H. L. Tiv, P. C. Gokhale, A. de Weck, A. A. Mansour, C. Oh, J. Shih, K. Hadi, Y. Rosen, J. Bistline, K. Venkatesan, A. Reddy, D. Sonkin, M. Liu, J. Lehar, J. M. Korn, D. A. Porter, M. D. Jones, J. Golji, G. Caponigro, J. E. Taylor, C. M. Dunning, A. L. Creech, A. C. Warren, J. M. McFarland, M. Zamanighomi, A. Kauffmann, N. Stransky, M. Imielinski, Y. E. Maruvka, A. D. Cherniack, A. Tsherniak, F. Vazquez, J. D. Jaffe, A. A. Lane, D. M. Weinstock, C. M. Johannessen, M. P. Morrissey, F. Stegmeier, R. Schlegel, W. C. Hahn, G. Getz, G. B. Mills, J. S. Boehm, T. R. Golub, L. A. Garraway, W. R. Sellers, Next-generation characterization of the Cancer Cell Line Encyclopedia. Nature 569, 503–508 (2019). 56. D. Bausch-Fluck, A. Hofmann, T. Bock, A. P. Frei, F. Cerciello, A. Jacobs, H. Moest, U. Omasits, R. L. Gundry, C. Yoon, R. Schiess, A. Schmidt, P. Mirkowska, A. Härtlová, J. E. Van Eyk, J. P. Bourquin, R. Aebersold, K. R. Boheler, P. Zandstra, B. Wollscheid, A mass spectrometric-derived cell surface protein atlas. PLoS One 10 (2015). 57. Z. Lu, N. McBrearty, J. Chen, V. S. Tomar, H. Zhang, G. De Rosa, A. Tan, A. M. Weljie, D. P. Beiting, Z. Miao, S. S. George, A. Berger, G. Saggu, J. A. Diehl, C. Koumenis, S. Y. Fuchs, ATF3 and CH25H regulate effector trogocytosis and anti-tumor activities of endogenous and immunotherapeutic cytotoxic T lymphocytes. Cell Metab 34, 1342-1358.e7 (2022). 58. Hernandez-Lopez, R. A., Yu, W., Cabral, K. A., Creasey, O. A., Lopez Pazmino, M. D. P., Tonai, Y., ... & Lim, W. A. T cell circuits that sense antigen density with an ultrasensitive threshold. Science, 371(6534), 1166-1171 (2021).

[0164] All publications, patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of59 KILPATRICK TOWNSEND 790261771understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.60 KILPATRICK TOWNSEND 790261771INFORMAL SEQUENCE LISTING SEQ ID NO:1 Fusogenic protein coding sequence atgaagtgccttttgtacttagcctttttattcattggggtgaattgcaagttcaccatagtttttccacacaaccaaaaaggaaactggaaaaatg ttccttctaattaccattattgcccgtcaagctcagatttaaattggcataatgacttaataggcacagccttacaagtcaaaatgcccaagagtc acaaggctattcaagcagacggttggatgtgtcatgcttccaaatgggtcactacttgtgatttccgctggtatggaccgaagtatataacacat tccatccgatccttcactccatctgtagaacaatgcaaggaaagcattgaacaaacgaaacaaggaacttggctgaatccaggcttccctcct caaagttgtggatatgcaactgtgacggatgccgaagcagtgattgtccaggtgactcctcaccatgtgctggttgatgaatacacaggaga atgggttgattcacagttcatcaacggaaaatgcagcaattacatatgccccactgtccataactctacaacctggcattctgactataaggtca aagggctatgtgattctaacctcatttccatggacatcaccttcttctcagaggacggagagctatcatccctgggaaaggagggcacaggg ttcagaagtaactactttgcttatgaaactggaggcaaggcctgcaaaatgcaatactgcaagcattggggagtcagactcccatcaggtgtc tggttcgagatggctgataaggatctctttgctgcagccagattccctgaatgcccagaagggtcaagtatctctgctccatctcagacctcag tggatgtaagtctaattcaggacgttgagaggatcttggattattccctctgccaagaaacctggagcaaaatcagagcgggtcttccaatctc tccagtggatctcagctatcttgctcctaaaaacccaggaaccggtcctgctttcaccataatcaatggtaccctaaaatactttgagaccagat acatcagagtcgatattgctgctccaatcctctcaagaatggtcggaatgatcagtggaactaccacagaaagggaactgtgggatgactgg gcaccatatgaagacgtggaaattggacccaatggagttctgaggaccagttcaggatataagtttcctttatacatgattggacatggtatgtt ggactccgatcttcatcttagctcaaaggctcaggtgttcgaacatcctcacattcaagacgctgcttcgcaacttcctgatgatgagagtttatt ttttggtgatactgggctatccaaaaatccaatcgagcttgtagaaggttggttcagtagttggaaaagctctattgcctcttttttctttatcatag ggttaatcattggactattcttggttctccgagttggtatccatctttgcattaaattaaagcacaccaagaaaagacagatttatacagacatag agatgaaccgacttggaaagtaa SEQ ID NO:2 Fusogenic protein sequence MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQ VKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTK QGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICP TVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKA CKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVE RILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPI LSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLH LSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLII GLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK* The following parts of sequence constitute the Cargo (receptor portion) in order: signal peptide- Ligand binding domain -CD8 hinge-CD8 transmembrane-GS linker-ABI SEQ ID NO:3 Signal peptide DNA sequence: atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccg SEQ ID NO:4 Signal peptide amino acid sequence: MALPVTALLLPLALLLHAARP61 KILPATRICK TOWNSEND 790261771SEQ ID NO:5 Ligand-binding domain (anti-CD19 ScFv) coding sequence gacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaa atatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttca gtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgct tccgtacacgttcggaggggggaccaagctggagatcacaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgag gtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgact atggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagct ctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttact actgtgccaaacattattactacggtggtagctatgctatggactactggggccaaggaacctcagtcaccgtctcctca SEQ ID NO:6 Ligand-binding domain (anti-CD19 ScFv) amino acid sequence: DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPS RFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGG GSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETT YYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTS VTVSS SEQ ID NO:7 CD8 hinge coding sequence ttcgtgccggtcttcctgccagcgaagcccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccc tgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgat SEQ ID NO:8 CD8 hinge amino acid sequence: FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD SEQ ID NO:9 CD8 transmembrane coding sequence Atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc SEQ ID NO:10 CD8 transmembrane amino acid sequence IYIWAPLAGTCGVLLLSLVITLYC SEQ ID NO:11 GS linker coding sequence aaaGGAGGTTCCGGAGGTTCCGGCAGCAGCGGATCAGGAGGTTCCGGCAGCAGCGGA TCC62 KILPATRICK TOWNSEND 790261771SEQ ID NO:12 GS linker amino acid sequence: KGGSGGSGSSGSGGSGSSGS SEQ ID NO:13 ABI coding sequence: ACGCGTGTGCCTTTGTATGGTTTTACTTCGATTTGTGGAAGAAGACCTGAGATGGAA GcTGCTGTTTCGACTATACCAAGATTCCTTCAATCTTCCTCTGGTTCGATGTTAGATG GTCGGTTTGATCCTCAATCCGCCGCTCATTTCTTCGGTGTTTACGACGGCCATGGCGG TTCTCAGGTAGCGAACTATTGTAGAGAGAGGATGCATTTGGCTTTGGCGGAGGAGAT AGCTAAGGAGAAACCGATGCTCTGCGATGGTGATACGTGGCTGGAGAAGTGGAAGA AAGCTCTTTTCAACTCGTTCCTGAGAGTTGACTCGGAGATTGAGTCAGTTGCGCCGG AGACGGTTGGGTCAACGTCGGTGGTTGCCGTTGTTTTCCCGTCTCACATCTTCGTCGC TAACTGCGGTGACTCTAGAGCCGTTCTTTGCCGCGGCAAAACTGCACTTCCATTATC CGTTGACCATAAACCGGATAGAGAAGATGAAGCTGCGAGGATTGAAGCCGCAGGAG GGAAAGTGATTCAGTGGAATGGAGCTCGTGTTTTCGGTGTTCTCGCCATGTCGAGAT CCATTGGCGATAGATACTTGAAACCATCCATCATTCCTGATCCGGAAGTGACGGCTG TGAAGAGAGTAAAAGAAGATGATTGTCTGATTTTGGCGAGTGACGGGGTTTGGGAT GTAATGACGGATGAAGAAGCGTGTGAGATGGCAAGGAAGCGGATTCTCTTGTGGCA CAAGAAAAACGCGGTGGCTGGGGATGCATCGTTGCTCGCGGATGAGCGGAGAAAGG AAGGGAAAGATCCTGCGGCGATGTCCGCGGCTGAGTATTTGTCAAAGCTGGCGATA CAGAGAGGAAGCAAAGACAACATAAGTGTGGTGGTGGTTGATTTGAAG SEQ ID NO:14 ABI amino acid sequence: TRVPLYGFTSICGRRPEMEAAVSTIPRFLQSSSGSMLDGRFDPQSAAHFFGVYDGHGGSQ VANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETVG STSVVAVVFPSHIFVANCGDSRAVLCRGKTALPLSVDHKPDREDEAARIEAAGGKVIQW NGARVFGVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEAC EMARKRILLWHKKNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISV VVVDLK The following parts of sequence constitute an exemplary Cargo (payload portion): NES-PYL1- linker-NES-linker-cleavage domain-linker-NLS-Cas9-linker-mCherry-NLS SEQ ID NO:15. NES coding sequence: atgCTGCCTCCGCTTGAAAGACTTACACTCGGCGGAGGGAGCCTTCCTCCGTTGGAGC GGTTGACGCTCTCCGGCGGTAGT SEQ ID NO:16. NES amino acid sequence:63 KILPATRICK TOWNSEND 790261771MLPPLERLTLGGGSLPPLERLTLSGGS SEQ ID NO:17 PYL1 coding sequence: ggtgggggcgcgccaactcaagacgaattcacccaactctcccaatcaatcgccgagttccacacgtaccaactcggtaacggccgttgct catctctcctagctcagcgaatccacgcgccgccggaaacagtatggtccgtggtgagacgtttcgataggccacagatttacaaacacttc atcaaaagctgtaacgtgagtgaagatttcgagatgcgagtgggatgcacgcgcgacgtgaacgtgataagtggattaccggcgaatacgt ctcgagagagattagatctgttggacgatgatcggagagtgactgggtttagtataaccggtggtgaacataggctgaggaattataaatcg gttacgacggttcatagatttgagaaagaagaagaagaagaaaggatctggaccgttgttttggaatcttatgttgttgatgtaccggaaggta attcggaggaagatacgagattgtttgctgatacggttattagattgaatcttcagaaacttgcttcgatcactgaagctatgaac SEQ ID NO:18 PYL1 amino acid sequence: GGGAPTQDEFTQLSQSIAEFHTYQLGNGRCSSLLAQRIHAPPETVWSVVRRFDRPQIYKH FIKSCNVSEDFEMRVGCTRDVNVISGLPANTSRERLDLLDDDRRVTGFSITGGEHRLRNY KSVTTVHRFEKEEEEERIWTVVLESYVVDVPEGNSEEDTRLFADTVIRLNLQKLASITEA MN SEQ ID NO:19 Linker-NES-linker coding sequence GGAGGTTCCGGCTCAaaaGCCCGGGCCTTGGCTCTCAAACTCGCGGGGCTCGACATTG GAGGTTCCGGCAGCAGCGGTTTAAACGGC SEQ ID NO:20 Linker-NES-linker amino acid sequence: GGSGSKARALALKLAGLDIGGSGSSGLNG SEQ ID NO:21 Cleavage domain (pH intein) coding sequence GCGTTGGCAGAGGGAACGCGGATTTTTGACCCAGTAACGGGGACTACTCATCGAAT AGAGGATGTTGTGGGAGGAAGAAAGCCGATTCATGTAGTAGCTGCAGCAAAGGACG GGACACTCCATGCGAGGCCAGTCGTCTCTTGGTTTGATCAGGGAACGCGAGATGTCA TCGGACTTAGGATTGCAGGTGGTGCAATTCTCTGGGCCACACCTGACCACAAAGTCC TGACCGAATATGGTTGGAGAGCTGCGGGGGAGCTGCGGAAAGGCGACCGAGTTGCC CAACCCAGACGGTTTGATGGCTTCGGAGACTCCGCTCCCATCCCTGCGAGAGTTCAA GCGTTGGCTGACGCGCTCGACGATAAATTCCTTCACGATATGCTCGCGGAGGAGCTT CGCTACAGTGTGATACGAGAAGTGCTCCCGACCAGAAGAGCTAGGACGTTCGGACT TGAGGTGGAAGAGCTCCACACACTGGTAGCGGAAGGAGTGGTTGTCCACAAT SEQ ID NO:22 Cleavage domain (pH intein) amino acid sequence:64 KILPATRICK TOWNSEND 790261771ALAEGTRIFDPVTGTTHRIEDVVGGRKPIHVVAAAKDGTLHARPVVSWFDQGTRDVIGL RIAGGAILWATPDHKVLTEYGWRAAGELRKGDRVAQPRRFDGFGDSAPIPARVQALAD ALDDKFLHDMLAEELRYSVIREVLPTRRARTFGLEVEELHTLVAEGVVVHN SEQ ID NO:23 Linker-NLS coding sequence: GGCAGTGGCTCTGGTGGTGGCAGTCCAAAGAAGAAGCGGAAGGTCGGTAGCGGTTC A SEQ ID NO:24 Linker-NLS amino acid sequence: GSGSGGGSPKKKRKVGSGS SEQ ID NO: 25 Cas9 coding sequence GACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGT GATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCG ACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAA ACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGA AGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGAC GACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCA CGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGT ACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGAC CTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTG ATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCT GGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGG ACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTG ATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCT GAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCA AACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAG ATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATC CTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGC CTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTC TCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAG AACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTT CATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGA ACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCAC CAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCC ATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCT ACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAG AGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTC CGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGA AGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGA CCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAG65 KILPATRICK TOWNSEND 790261771CAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAA GCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCT CCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAA ATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGA TATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGA AAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGA TACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCA GTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTT CATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCC AGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGC CCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAA AGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAAC CAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAG AGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACAC CCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGT ACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATC GTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAG CGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAG ATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTT CGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCT TCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATC CTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGT GAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTT TTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACG CCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTG TACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGA AATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAA GACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAA ACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGG AAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGG CGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCA GAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCC TATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAG TGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATC CCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATC AAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGC CTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGA ACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAAT GAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGA GCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAG TGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAAT ATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTG ACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACC CTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTG GGAGGCGAC66 KILPATRICK TOWNSEND 790261771SEQ ID NO: 26 Cas9 amino acid sequence: DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGN IVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDV DKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLI ALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAG YIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAI LRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS GEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRE RMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDV DHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVI TLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYK VYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWD KGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGG FDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKK DLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHL FTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD SEQ ID NO: 27 Linker coding sequence GGTAGCGGTAGTGGCAGTGGAGGATCCTCCGGTTTAAAC SEQ ID NO: 28 Linker amino acid sequence: GSGSGSGGSSGLN SEQ ID NO: 29 mCherry coding sequence: Atggtgagcaagggcgaggaggataacatggccatcatcaaggagttcatgcgcttcaaggtgcacatggagggctccgtgaacggcc acgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggtggcccc ctgcccttcgcctgggacatcctgtcccctcagttcatgtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaa gctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtggtgaccgtgacccaggactcctccctgca ggacggcgagttcatctacaaggtgaagctgcgcggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctggg aggcctcctccgagcggatgtaccccgaggacggcgccctgaagggcgagatcaagcagaggctgaagctgaaggacggcggccact acgacgctgaggtcaagaccacctacaaggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttggacatcacc tcccacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggcatggacgagctgtacaag67 KILPATRICK TOWNSEND 790261771SEQ ID NO: 30 mCherry amino acid sequence: MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGP LPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDS SLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKD GGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMD ELYK SEQ ID NO: 31 NLS coding sequence GGGGGCGGTTCACCTGCGGCTAAACGAGTCAAACTGGAC SEQ ID NO: 32 NLS amino acid sequence: GGGSPAAKRVKLD SEQ ID NO: 33 Ligand-binding domain (anti-CD3 ScFv) coding sequence gatatccagatgacccagtccccgagctccctgtccgcctctgtgggcgatagggtcaccatcacctgtcgtgccagtcaggacatccgtaa ttatctcaactggtatcaacagaaaccaggaaaagctccgaaactactgatttactatacctcccgcctggagtctggagtcccttctcgcttct ctggttctggttctgggacggattacactctgaccatcagcagtctgcaaccggaggacttcgcaacttattactgtcagcaaggtaatactct gccgtggacgttcggacagggcaccaaggtggagatcaaaggtggaggcggttcaggcggaggtggctctggcggtggcggatcgga ggttcagctggtggagtctggcggtggcctggtgcagccagggggctcactccgtttgtcctgtgcagcttctggctactcctttaccggcta cactatgaactgggtgcgtcaggccccaggtaagggcctggaatgggttgcactgattaatccttataaaggtgtttccacctataaccagaa attcaaggatcgtttcacgatatccgtagataaatccaaaaacacagcctacctgcaaatgaacagcctgcgtgctgaggacactgccgtcta ttattgtgctagaagcggatactacggcgatagcgactggtattttgacgtctggggtcaaggaaccctggtcaccgtctcctcg SEQ ID NO: 34 Ligand-binding domain (anti-CD3 ScFv) amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPS RFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGG GGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPY KGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDV WGQGTLVTVSS SEQ ID NO: 35 Ligand-binding domain (anti-HER2 ScFv) coding sequence GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTC ACCATCACCTGCCGTGCCAGTCAGGATGTGAATACTGCTGTAGCCTGGTATCAACAG AAACCAGGAAAAGCTCCGAAACTACTGATTTACTCGGCATCCTTCCTTGAGTCTGGA GTCCCTTCTCGCTTCTCTGGATCTAGATCTGGGACGGATTTCACTCTGACCATCAGCA GTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAACATTATACTACTCCTC CCACGTTCGGACAGGGTACCAAGGTGGAGATCAAAGGGTCTACATCTGGATCTGGG AAGCCGGGTTCTGGTGAGGGTTCTGGTGAGGTTCAGCTGGTGGAGTCTGGCGGTGGC CTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACATT AAAGACACCTATATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGT68 KILPATRICK TOWNSEND 790261771TGCAAGGATTTATCCTACGAATGGTTATACTAGATATGCCGATAGCGTCAAGGGCCG TTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTGCAGATGAACAGCC TGCGTGCTGAGGACACTGCCGTCTATTATTGTTCTAGATGGGGAGGGGACGGCTTCT ATGCTATGGACGTGTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 36 Ligand-binding domain (anti-HER2 ScFv) amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLESGVPS RFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGSTSGSGKPGSGEG SGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNG YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQ GTLVTVSS SEQ ID NO: 37 Ligand-binding domain (anti-CD4 ScFv) coding sequence gacatccagctgacccagtctccatcctccctgtctgcatctgtaggtgaccgggtcaccatcacttgtcgggccagccaaagtgtcagtatc tctagccatgatctcatgcagtggtatcagcagaaaccagggaaagcccctaagctcctgatctatgatgcattcaacctggcatctggggtc ccatcacggttcagcggcagtggttctgggacagatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgccagc agagtaaggatgatccgtacacgttcggccaggggaccaagctggagatcaaaggtggcggtggctcgggcggtggtgggtcgggtgg cggcggatctgaggtgcagctggtggagtctgggggcggcttggtacagcctgggcggtccctgcggctctcctgtgcagcctctgggtt cacctttagtaactatggcatggcctgggtccgccaggctccagggaaggggctggagtgggtcgcaaccattagttatgatggcagtatc acttattatcgagactccgtgaagggccggttcaccatctcccgggacaattccaagaacacgctgtacctgcaaatgaacagcctgcgag ccgaggacacggccgtatattactgtgcgcgggaggaacaatatagcagctggtactttgacttctggggccaggggaccctggtcaccgt ctcctca SEQ ID NO: 38 Ligand-binding domain (anti-CD4 ScFv) amino acid sequence DIQLTQSPSSLSASVGDRVTITCRASQSVSISSHDLMQWYQQKPGKAPKLLIYDAFNLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSKDDPYTFGQGTKLEIKGGGGSGGGG SGGGGSEVQLVESGGGLVQPGRSLRLSCAASGFTFSNYGMAWVRQAPGKGLEWVATIS YDGSITYYRDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREEQYSSWYFDFW GQGTLVTVSS SEQ ID NO: 39 Ligand-binding domain (anti-CD28 ScFv) coding sequence GACATACAGATGAATCAGAGCCCTTCCTCCCTCTCAGCGAGCCTCGGCGATACCATA ACCATAACATGTCACGCATCCCAGAATATCTACGTTTGGCTTAATTGGTACCAGCAA AAACCCGGTAATATTCCGAAACTCCTTATATATAAGGCATCTAATTTGCATACCGGT GTGCCATCAAGATTCTCCGGCTCTGGATCAGGAACAGGCTTTACGCTGACTATCAGC AGTCTTCAGCCTGAGGACATTGCAACTTACTACTGCCAGCAAGGACAGACCTACCCG TATACATTCGGAGGGGGGACTAAGCTCGAAATTAAGAGAGCTGACGCCGCCCCAAC AGTCAGTATATTTCCACCTAGCTCTGAGCAACTTACATCAGGTGGGGCAAGTGTTGT CTGTTTTTTGAATAATTTTTATCCGAAGGATATTAATGTCAAATGGAAGATCGACGG GTCAGAGCGCCAGAACGGGGTTCTCAATTCTTGGACGGATCAGGATAGCAAAGATA GCACGTATTCTATGAGTTCAACTCTCACTTTGACGAAAGATGAGTACGAAAGGCATA ATTCCTACACATGCGAGGCTACTCATAAGACCTCAACTAGCCCTATAGTGAAATCCT TTAATAGGAATGGCGGCGGTGGCAGTGGTGGAGGCGGGTCCGGCGGTGGGGGTTCT CAGGTCCAACTGCAACAAAGCGGTCCCGAACTCGTAAAGCCAGGGACAAGCGTCCG GATCAGCTGTGAGGCAAGTGGATATACTTTCACATCCTATTATATTCACTGGGTGAA ACAGCGACCCGGACAAGGACTCGAATGGATTGGCTGTATATATCCGGGTAACGTAA69 KILPATRICK TOWNSEND 790261771ACACAAACTACAATGAAAAGTTTAAGGATAAAGCAACACTTATTGTTGATACTAGT AGTAATACTGCCTATATGCAGTTGTCTCGGATGACGAGCGAGGACTCCGCAGTGTAT TTCTGCACGAGAAGTCATTACGGACTGGACTGGAACTTTGACGTATGGGGCGCAGG CACAACCGTTACCGTAAGCAGTGCAAAAACAACCCCTCCATCCGTGTACCCACTTGC ACCGGGAAGTGCGGCGCAGACGAACAGCATGGTGACTCTGGGGTGTCTTGTAAAGG GCTACTTCCCGGAGCCCGTGACCGTTACCTGGAATAGTGGATCACTCAGTTCAGGAG TGCATACGTTCCCCGCGGTGTTGCAAAGTGATCTGTATACCCTGTCCTCATCAGTAA CCGTACCGAGTTCAACGTGGCCATCAGAAACGGTTACGTGTAACGTAGCACACCCC GCCAGCAGTACAAAGGTGGATAAGAAGATTGTTCCGAGAGATTGT SEQ ID NO: 40 Ligand-binding domain (anti-CD28 ScFv) amino acid sequence DIQMNQSPSSLSASLGDTITITCHASQNIYVWLNWYQQKPGNIPKLLIYKASNLHTGVPS RFSGSGSGTGFTLTISSLQPEDIATYYCQQGQTYPYTFGGGTKLEIKRADAAPTVSIFPPSS EQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLT LTKDEYERHNSYTCEATHKTSTSPIVKSFNRNGGGGSGGGGSGGGGSQVQLQQSGPELV KPGTSVRISCEASGYTFTSYYIHWVKQRPGQGLEWIGCIYPGNVNTNYNEKFKDKATLIV DTSSNTAYMQLSRMTSEDSAVYFCTRSHYGLDWNFDVWGAGTTVTVSSAKTTPPSVYP LAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVT VPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC SEQ ID NO: 41 Ligand-binding domain (anti-CD2 ScFv) coding sequence AACATTATGATGACACAGTCGCCATCATCTCTGGCTGTGTCTGCAGGAGAAAAGGTC ACTATGACCTGTAAGTCCAGTCAAAGTGTTTTATACAGTTCAAATCAGAAGAACTAC TTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTACTGATCTACTGGGCA TCCACTAGGGAATCTGGTGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGAT TTTACTCTTACCATCAGCAGTGTGCAACCTGAAGACCTGGCAGTTTATTACTGTCATC AATACCTCTCCTCGCACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGGT GGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTCAACTGCAGCAGCC TGGGGCTGAGCTGGTGAGGCCTGGGTCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGG CTACACCTTCACCAGGTACTGGATACATTGGGTGAAGCAGAGGCCTATACAAGGCCT TGAATGGATTGGTAACATTGATCCTTCTGATAGTGAAACTCACTACAATCAAAAGTT CAAGGACAAGGCCACATTGACTGTAGACAAATCCTCCGGCACAGCCTACATGCAGC TCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAACAGAGGATCTTT ACTATGCTATGGAGTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCT SEQ ID NO: 42 Ligand-binding domain (anti-CD2 ScFv) amino acid sequence NIMMTQSPSSLAVSAGEKVTMTCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWAS TRESGVPDRFTGSGSGTDFTLTISSVQPEDLAVYYCHQYLSSHTFGGGTKLEIKRGGGGS GGGGSGGGGSQLQQPGAELVRPGSSVKLSCKASGYTFTRYWIHWVKQRPIQGLEWIGNI DPSDSETHYNQKFKDKATLTVDKSSGTAYMQLSSLTSEDSAVYYCATEDLYYAMEYW GQGTSVTVSS SEQ ID NO: 43 Ligand-binding domain (anti-FITC ScFv) coding sequence CAAGTTCAGCTCGTGGAGAGTGGGGGTAATCTGGTCCAGCCGGGCGGTTCACTCCGC CTGTCATGTGCAGCATCTGGCTTTACATTCGGCTCTTTCTCTATGTCTTGGGTACGAC AAGCGCCCGGCGGGGGGCTCGAATGGGTCGCTGGATTGTCAGCCCGGTCCAGTCTG70 KILPATRICK TOWNSEND 790261771ACTCATTACGCTGACTCCGTTAAGGGGAGATTTACAATTAGCCGAGACAATGCCAAA AACAGTGTGTATTTGCAGATGAATTCTCTTAGAGTAGAGGATACAGCTGTGTATTAC TGTGCGCGAAGGTCTTATGATAGCTCAGGATATTGGGGACACTTTTATTCCTACATG GACGTATGGGGCCAGGGGACTCTGGTTACTGTGAGTGGTGGGGGAGGGAGTGGCGG AGGGGGTAGCGGAGGAGGGGGTTCTTCAGTTCTTACTCAGCCCAGTTCTGTTTCCGC AGCGCCGGGTCAAAAGGTCACTATTTCATGTAGTGGTTCTACGAGTAATATTGGAAA TAATTATGTGAGTTGGTATCAACAGCACCCGGGTAAGGCGCCTAAGCTCATGATATA TGACGTAAGTAAGAGACCGAGTGGGGTCCCGGACCGGTTCAGCGGCTCCAAGTCAG GTAACTCCGCTTCCTTGGATATATCTGGCTTGCAGTCTGAGGATGAGGCCGATTACT ACTGTGCCGCGTGGGACGACTCACTCTCTGAGTTTCTGTTCGGAACGGGGACTAAAT TGACAGTTCTCGGA SEQ ID NO: 44 Ligand-binding domain (anti-FITC ScFv) amino acid sequence QVQLVESGGNLVQPGGSLRLSCAASGFTFGSFSMSWVRQAPGGGLEWVAGLSARSSLT HYADSVKGRFTISRDNAKNSVYLQMNSLRVEDTAVYYCARRSYDSSGYWGHFYSYMD VWGQGTLVTVSGGGGSGGGGSGGGGSSVLTQPSSVSAAPGQKVTISCSGSTSNIGNNYV SWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNSASLDISGLQSEDEADYYCAAW DDSLSEFLFGTGTKLTVLG SEQ ID NO: 45 Mutated VSVG (K47Q, R354A) coding sequence atgaagtgccttttgtacttagcctttttattcattggggtgaattgcaagttcaccatagtttttccacacaaccaaaaaggaaactggaaaaatg ttccttctaattaccattattgcccgtcaagctcagatttaaattggcataatgacttaataggcacagccttacaagtcaaaatgccccagagtc acaaggctattcaagcagacggttggatgtgtcatgcttccaaatgggtcactacttgtgatttccgctggtatggaccgaagtatataacacat tccatccgatccttcactccatctgtagaacaatgcaaggaaagcattgaacaaacgaaacaaggaacttggctgaatccaggcttccctcct caaagttgtggatatgcaactgtgacggatgccgaagcagtgattgtccaggtgactcctcaccatgtgctggttgatgaatacacaggaga atgggttgattcacagttcatcaacggaaaatgcagcaattacatatgccccactgtccataactctacaacctggcattctgactataaggtca aagggctatgtgattctaacctcatttccatggacatcaccttcttctcagaggacggagagctatcatccctgggaaaggagggcacaggg ttcagaagtaactactttgcttatgaaactggaggcaaggcctgcaaaatgcaatactgcaagcattggggagtcagactcccatcaggtgtc tggttcgagatggctgataaggatctctttgctgcagccagattccctgaatgcccagaagggtcaagtatctctgctccatctcagacctcag tggatgtaagtctaattcaggacgttgagaggatcttggattattccctctgccaagaaacctggagcaaaatcagagcgggtcttccaatctc tccagtggatctcagctatcttgctcctaaaaacccaggaaccggtcctgctttcaccataatcaatggtaccctaaaatactttgagaccagat acatcagagtcgatattgctgctccaatcctctcaagaatggtcggaatgatcagtggaactaccacagaagccgaactgtgggatgactgg gcaccatatgaagacgtggaaattggacccaatggagttctgaggaccagttcaggatataagtttcctttatacatgattggacatggtatgtt ggactccgatcttcatcttagctcaaaggctcaggtgttcgaacatcctcacattcaagacgctgcttcgcaacttcctgatgatgagagtttatt ttttggtgatactgggctatccaaaaatccaatcgagcttgtagaaggttggttcagtagttggaaaagctctattgcctcttttttctttatcatag ggttaatcattggactattcttggttctccgagttggtatccatctttgcattaaattaaagcacaccaagaaaagacagatttatacagacatag agatgaaccgacttggaaagtaa SEQ ID NO: 46 Mutated VSVG (K47Q, R354A) amino acid sequence MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQ VKMPQSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTK QGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICP TVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKA CKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVE RILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPI LSRMVGMISGTTTEAELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLH71 KILPATRICK TOWNSEND 790261771LSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLII GLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK* SEQ ID NO: 47 Mutated pH intein (N150) coding sequence GCGTTGGCAGAGGGAACGCGGATTTTTGACCCAGTAACGGGGACTACTCATCGAAT AGAGGATGTTGTGGGAGGAAGAAAGCCGATTCATGTAGTAGCTGCAGCAAAGGACG GGACACTCCATGCGAGGCCAGTCGTCTCTTGGTTTGATCAGGGAACGCGAGATGTCA TCGGACTTAGGATTGCAGGTGGTGCAATTCTCTGGGCCACACCTGACCACAAAGTCC TGACCGAATATGGTTGGAGAGCTGCGGGGGAGCTGCGGAAAGGCGACCGAGTTGCC CAACCCAGACGGTTTGATGGCTTCGGAGACTCCGCTCCCATCCCTGCGAGAGTTCAA GCGTTGGCTGACGCGCTCGACGATAAATTCCTTCACGATATGCTCGCGGAGGAGCTT CGCTACAGTGTGATACGAGAAGTGCTCCCGACCAGAAGAGCTAGGACGTTCAATCT TGAGGTGGAAGAGCTCCACACACTGGTAGCGGAAGGAGTGGTTGTCCACAAT SEQ ID NO: 48 Mutated pH intein (N150) amino acid sequence ALAEGTRIFDPVTGTTHRIEDVVGGRKPIHVVAAAKDGTLHARPVVSWFDQGTRDVIGL RIAGGAILWATPDHKVLTEYGWRAAGELRKGDRVAQPRRFDGFGDSAPIPARVQALAD ALDDKFLHDMLAEELRYSVIREVLPTRRARTFNLEVEELHTLVAEGVVVHN SEQ ID NO: 49 HSVTK coding sequence Atggcttcgtacccctgccatcaacacgcgtctgcgttcgaccaggctgcgcgttctcgcggccatagcaaccgacgtacggcgttgcgc cctcgccggcagcaagaagccacggaagtccgcctggagcagaaaatgcccacgctactgcgggtttatatagacggtcctcacgggat ggggaaaaccaccaccacgcaactgctggtggccctgggttcgcgcgacgatatcgtctacgtacccgagccgatgacttactggcaggt gctgggggcttccgagacaatcgcgaacatctacaccacacaacaccgcctcgaccagggtgagatatcggccggggacgcggcggtg gtaatgacaagcgcccagataacaatgggcatgccttatgccgtgaccgacgccgttctggctcctcatatcgggggggaggctgggagc tcacatgccccgcccccggccctcaccctcatcttcgaccgccatcccatcgccgccctcctgtgctacccggccgcgcgataccttatgg gcagcatgaccccccaggccgtgctggcgttcgtggccctcatcccgccgaccttgcccggcacaaacatcgtgttgggggcccttccgg aggacagacacatcgaccgcctggccaaacgccagcgccccggcgagcggcttgacctggctatgctggccgcgattcgccgcgtttac gggctgcttgccaatacggtgcggtatctgcagggcggcgggtcgtggcgggaggattggggacagctttcggggacggccgtgccgc cccagggtgccgagccccagagcaacgcgggcccacgaccccatatcggggacacgttatttaccctgtttcgggcccccgagttgctgg cccccaacggcgacctgtacaacgtgtttgcctgggccttggacgtcttggccaaacgcctccgtcccatgcacgtctttatcctggattacg accaatcgcccgccggctgccgggacgccctgctgcaacttacctccgggatggtccagacccacgtcaccacccccggctccataccg acgatctgcgacctggcgcgcacgtttgcccgggagatgggggaggctaactga SEQ ID NO: 50 HSVTK amino acid sequence MASYPCHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRLEQKMPTLLRVYIDGPH GMGKTTTTQLLVALGSRDDIVYVPEPMTYWQVLGASETIANIYTTQHRLDQGEISAGDA AVVMTSAQITMGMPYAVTDAVLAPHIGGEAGSSHAPPPALTLIFDRHPIAALLCYPAAR YLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQRPGERLDLAMLAAIR RVYGLLANTVRYLQGGGSWREDWGQLSGTAVPPQGAEPQSNAGPRPHIGDTLFTLFRA PELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPAGCRDALLQLTSGMVQTH VTTPGSIPTICDLARTFAREMGEAN* SEQ ID NO: 51 CCR5 ZFN1 coding sequence CATATGCCAAAGAAAAAAAGGAAAGTAGCCCAGGCCGCGATGGCAGAACGGCCGT TCCAGTGTAGAATCTGCATGAGAAACTTTTCTGACAGGAGCAATTTGAGCAGACACA72 KILPATRICK TOWNSEND 790261771TCCGCACACACACGGGTGAGAAACCTTTTGCTTGCGACATATGCGGGCGGAAATTTG CTATTAGTTCTAACTTGAACTCTCATACGAAAATTCATACTGGGTCCCAGAAACCGT TTCAGTGTCGGATCTGCATGCGCAACTTCAGTCGGAGCGATAACTTGGCGAGGCACA TCCGCACGCACACAGGTGAAAAACCATTTGCATGTGACATATGTGGGCGCAAATTTG CAACATCCGGTAATCTGACCCGGCATACAAAGATACATCTGCGAGGAAGCCAGTTG GTTAAATCCGAACTCGAGGAAAAAAAATCTGAGCTCCGGCACAAACTCAAATACGT ACCCCACGAGTACATCGAGCTGATAGAAATTGCTCGCAACCCTACCCAAGACCGGA TACTTGAAATGAAGGTCATGGAGTTCTTTATGAAAGTTTATGGATACAGAGGAGAGC ACCTGGGAGGCTCCAGAAAGCCCGATGGTGCGATTTACACTGTGGGAAGCCCCATC GACTATGGAGTGATCGTGGACACTAAAGCATACTCCGGAGGTTACAATCTCCCTATA GGACAAGCAGACGAGATGCAGCGGTACGTCGAAGAAAACCAGACGAGAAATAAAC ATATTAATCCTAACGAATGGTGGAAAGTTTATCCGAGTTCTGTAACAGAATTCAAGT TCCTCTTTGTATCAGGGCACTTCAAGGGCAACTACAAAGCGCAACTCACGCGACTGA ACCACATAACAAATTGTAATGGGGCTGTGTTGAGTGTGGAAGAACTGTTGATCGGG GGAGAAATGATCAAGGCTGGAACCCTGACCCTTGAGGAGGTCCGCAGAAAGTTTAA TAATGGAGAAATCAATTTC SEQ ID NO: 52 CCR5 ZFN1 amino acid sequence HMPKKKRKVAQAAMAERPFQCRICMRNFSDRSNLSRHIRTHTGEKPFACDICGRKFAIS SNLNSHTKIHTGSQKPFQCRICMRNFSRSDNLARHIRTHTGEKPFACDICGRKFATSGNL TRHTKIHLRGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNPTQDRILEMKVMEFF MKVYGYRGEHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVE ENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSV EELLIGGEMIKAGTLTLEEVRRKFNNGEINF SEQ ID NO: 53 CCR5 ZFN2 coding sequence CACATGCCAAAAAAGAAACGGAAGGTGGCTCAGGCGGCGATGGCAGAGCGACCAT TCCAGTGCCGAATCTGTATGAGGAACTTTAGCAGGTCTGATAACCTTAGTGTTCACA TACGGACGCACACGGGAGAGAAGCCCTTCGCCTGTGATATTTGTGGACGCAAGTTC GCTCAGAAGATAAACCTGCAAGTACATACAAAAATACACACGGGTGAAAAGCCATT TCAGTGTAGAATTTGCATGAGGAATTTTTCACGCTCCGACGTTTTGTCCGAACACATT CGGACCCACACTGGAGAAAAGCCCTTTGCGTGTGATATTTGTGGTAGAAAATTCGCG CAGCGCAACCACAGGACCACCCATACCAAGATTCATCTGAGGGGGAGCCAACTGGT AAAATCTGAACTTGAGGAAAAGAAATCCGAATTGCGGCATAAACTGAAGTATGTCC CGCATGAATACATAGAGCTTATCGAGATTGCTCGAAATCCGACCCAGGATAGGATTT TGGAAATGAAGGTGATGGAGTTCTTTATGAAAGTATACGGGTATAGAGGCGAGCAC TTGGGGGGCTCCCGAAAACCAGACGGTGCAATCTATACAGTAGGGTCCCCGATTGA TTACGGGGTTATAGTAGACACAAAAGCCTACAGTGGTGGTTATAACTTGCCGATTGG ACAAGCAGACGAGATGCAACGCTACGTCGAAGAAAACCAAACTCGGAATAAACAC ATAAACCCGAACGAATGGTGGAAGGTATACCCCAGTAGCGTTACTGAATTTAAGTTC CTCTTCGTGTCCGGACATTTTAAAGGGAATTACAAAGCGCAATTGACCAGGCTCAAC CATATAACGAACTGCAACGGTGCTGTACTTAGTGTCGAAGAGCTTCTCATAGGGGGC GAAATGATCAAAGCAGGCACATTGACACTCGAGGAGGTCAGACGCAAGTTCAACAA TGGCGAAATCAATTTT SEQ ID NO: 54 CCR5 ZFN2 amino acid sequence73 KILPATRICK TOWNSEND 790261771HMPKKKRKVAQAAMAERPFQCRICMRNFSRSDNLSVHIRTHTGEKPFACDICGRKFAQ KINLQVHTKIHTGEKPFQCRICMRNFSRSDVLSEHIRTHTGEKPFACDICGRKFAQRNHR TTHTKIHLRGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNPTQDRILEMKVMEFFM KVYGYRGEHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEE NQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVE ELLIGGEMIKAGTLTLEEVRRKFNNGEINF SEQ ID NO: 55 CD28 transmembrane domain coding sequence ttctgggtgctggtggtggtcggaggcgtgctggcctgctacagcctgctggtcaccgtggccttcatcatcttttgggtg SEQ ID NO: 56 CD28 transmembrane domain amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWV SEQ ID NO: 57 CD28 cytosolic domain coding sequence Aggagtaagaggagcaggggtggacacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccc tatgccccaccacgcgacttcgcagcctatcgctcc SEQ ID NO: 58 CD28 cytosolic domain amino acid sequence RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO: 5941BB cytosolic domain coding sequence cggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgattt ccagaagaagaagaaggaggatgtgaa SEQ ID NO: 6041BB cytosolic domain amino acid sequence RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE SEQ ID NO: 61 CD3z cytosolic domain coding sequence ctgcgggtgaagttcagcagaagcgccgacgcccctgcctaccagcagggccagaatcagctgtacaacgagctgaacctgggcagaa gggaagagtacgacgtcctggataagcggagaggccgggaccctgagatgggcggcaagcctcggcggaagaacccccaggaaggc ctgtataacgaactgcagaaagacaagatggccgaggcctacagcgagatcggcatgaagggcgagcggaggcggggcaagggcca cgacggcctgtatcagggcctgtccaccgccaccaaggatacctacgacgccctgcacatgcaggccctgcccccacgC SEQ ID NO: 62 CD3z cytosolic domain amino acid sequence LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 63 Cre coding sequence Atgggcccaaagaagaagagaaaggtttcgaatttactgaccgtacaccaaaatttgcctgcattaccggtcgatgcaacgagtgatgagg ttcgcaagaacctgatggacatgttcagggatcgccaggcgttttctgagcatacctggaaaatgcttctgtccgtttgccggtcgtgggcgg catggtgcaagttgaataaccggaaatggtttcccgcagaacctgaagatgttcgcgattatcttctatatcttcaggcgcgcggtctggcagt aaaaactatccagcaacatttgggccagctaaacatgcttcatcgtcggtccgggctgccacgaccaagtgacagcaatgctgtttcactggt tatgcggcggatccgaaaagaaaacgttgatgccggtgaacgtgcaaaacaggctctagcgttcgaacgcactgatttcgaccaggttcgtt cactcatggaaaatagcgatcgctgccaggatatacgtaatctggcatttctggggattgcttataacaccctgttacgtatagccgaaattgc caggatcagggttaaagatatctcacgtactgacggtgggagaatgttaatccatattggcagaacgaaaacgctggttagcaccgcaggt gtagagaaggcacttagcctgggggtaactaaactggtcgagcgatggatttccgtctctggtgtagctgatgatccgaataactacctgtttt74 KILPATRICK TOWNSEND 790261771gccgggtcagaaaaaatggtgttgccgcgccatctgccaccagccagctatcaactcgcgccctggaagggatttttgaagcaactcatcg attgatttacggcgctaaggatgactctggtcagagatacctggcctggtctggacacagtgcccgtgtcggagccgcgcgagatatggcc cgcgctggagtttcaataccggagatcatgcaagctggtggctggaccaatgtaaatattgtcatgaactatatccgtaacctggatagtgaa acaggggcaatggtgcgcctgctggaagatggcgat SEQ ID NO: 64 Cre amino acid sequence MGPKKKRKVSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVC RSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPR PSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWI SVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYL AWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED GD SEQ ID NO: 65 antiCD19 synNotch coding sequence ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCC AGGCCGGAGCAGAAGCTGATCAGCGAGGAGGACCTGGACATCCAGATGACACAGA CTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAA GTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTA AACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTG GCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGAT ATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGG ACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGG CGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGA GCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCT GGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGT GAAACCACATACTATAATTCAGCTCTCAAATCCAGActgACCATCATCAAGGACAACT CCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTT ACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCC AAGGAACCTCAGTCACCGTCTCCTCAatcctggactacagcttcacaggtggcgctgggcgcgacattcccccac cgcagattgaggaggcctgtgagctgcctgagtgccaggtggatgcaggcaataaggtctgcaacctgcagtgtaataatcacgcatgtgg ctgggatggtggcgactgctccctcaacttcaatgacccctggaagaactgcacgcagtctctacagtgctggaagtattttagcgacggcc actgtgacagccagtgcaactcggccggctgcctctttgatggcttcgactgccagctcaccgagggacagtgcaaccccctgtatgacca gtactgcaaggaccacttcagtgatggccactgcgaccagggctgtaacagtgccgaatgtgagtgggatggcctagactgtgctgagcat gtacccgagcggctggcagccggcaccctggtgctggtggtgctgcttccacccgaccagctacggaacaactccttccactttctgcggg agctcagccacgtgctgcacaccaacgtggtcttcaagcgtgatgcgcaaggccagcagatgatcttcccgtactatggccacgaggaag agctgcgcaagcacccaatcaagcgctctacagtgggttgggccacctcttcactgcttcctggtaccagtggtgggcgccagcgcaggg agctggaccccatggacatccgtggctccattgtctacctggagatcgacaaccggcaatgtgtgcagtcatcctcgcagtgcttccagagt gccaccgatgtggctgccttcctaggtgctcttgcgtcacttggcagcctcaatattccttacaagattgaggccgtgaagagtgagccggtg gagcctccgctgccctcgcagctgcacctcatgtacgtggcagcggccgccttcgtgctcctgttctttgtgggctgtggggtgctgctgtcc cgcaagcgccggcggatgaagctgctgagcagcatcgagcaggcctgtgacatctgccggctgaagaaactgaagtgcagcaaagaaa agcccaagtgcgccaagtgcctgaagaacaactgggagtgccggtacagccccaagaccaagagaagccccctgaccagagcccacc tgaccgaggtggaaagccggctggaaagactggaacagctgtttctgctgatcttcccacgcgaggacctggacatgatcctgaagatgg acagcctgcaggacatcaaggccctgctgaccggcctgttcgtgcaggacaacgtgaacaaggacgccgtgaccgacagactggccag cgtggaaaccgacatgcccctgaccctgcggcagcacagaatcagcgccaccagcagcagcgaggaaagcagcaacaagggccagc ggcagctgacagtgtctgctgctgcaggcggaagcggaggctctggcggatctgatgccctggacgacttcgacctggatatgctgggca75 KILPATRICK TOWNSEND 790261771gcgacgccctggatgattttgatctggacatgctgggatctgacgctctggacgatttcgatctcgacatgttgggatcagatgcactggatg actttgacctggacatgctcggatcatga SEQ ID NO: 66 antiCD19 synNotch amino acid sequence MALPVTALLLPLALLLHAARPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDIS KYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQ GNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGV SLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQ TDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSILDYSFTGGAGRDIPPPQIEEACEL PECQVDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDS QCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEH VPERLAAGTLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGHE EELRKHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDIRGSIVYLEIDNRQCVQSSSQCF QSATDVAAFLGALASLGSLNIPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCG VLLSRKRRRMKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLT RAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDR LASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVSAAAGGSGGSGGSDALDDFDLDM LGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGS*76 KILPATRICK TOWNSEND 790261771

Claims

1. A cell comprising: (a) a transmembrane protein comprising (i) an extracellular amino acid sequence that binds to a target protein on a recipient cell and (ii) a transmembrane domain, wherein the transmembrane domain is linked to a payload polypeptide, the transmembrane protein is heterologous to the cell and the transmembrane domain is heterologous to the payload polypeptide; and (b) a low pH-dependent fusogenic protein.

2. The cell of claim 1, wherein the transmembrane protein further comprises a cleavage sequence between the transmembrane domain and the payload polypeptide, wherein the cleavage sequence is cleaved in a cellular endosome.

3. The cell of claim 2, wherein the cleavage sequence is cleaved by an endogenous enzyme in the cellular endosome.

4. The cell of claim 2, wherein the cleavage sequence is cleaved by a heterologous enzyme expressed in or introduced into the cellular endosome.

5. The cell of claim 2, wherein the cleavage sequence is a low pH-dependent cleavage sequence between the transmembrane domain and the payload polypeptide, wherein the low pH-dependent cleavage sequence is self-cleaved at the pH of a cellular endosome but not at the pH of the cytosol of the cell.

6. The cell of claim 5, wherein the low pH-dependent cleavage sequence is a pH intein.

7. The cell of claim 1 or 2, wherein the transmembrane domain is covalently linked to the payload polypeptide.

8. The cell of claim 1 or 2, wherein the transmembrane domain is non- covalently linked to the payload polypeptide.77 KILPATRICK TOWNSEND 7902617719. The cell of any one of claims 1-8, wherein the payload polypeptide is an enzyme or protein-binding polypeptide.

10. The cell of any one of claims 1-8, wherein the payload polypeptide comprises a nuclear localization signal sequence or a nuclear export signal sequence.

11. The cell of any one of claims 1-10, wherein the cell is a mammalian cell.

12. The cell of claim 11, wherein the mammalian cell is a human cell.

13. The cell of any one of claims 1-12, wherein the payload polypeptide is a nucleic acid binding protein.

14. The cell of claim 13, wherein the nucleic acid binding protein is binding a nucleic acid.

15. The cell of claim 14, wherein the nucleic acid is RNA.

16. The cell of claim 14, wherein the nucleic acid is DNA.

17. The cell of claim 14 or 15, wherein the nucleic acid binding protein is a RNA-guided protein.

18. The cell of claim 17, wherein the gRNA-guided protein is a CRISPR nuclease or an inactive mutant thereof.

19. The cell of any one of claims 1-18, wherein the extracellular amino acid sequence comprises an antibody or antigen-binding fragment thereof.

20. The cell of claim 19, wherein the antibody is a scFv.

21. The cell of any one of claims 1-20, wherein the low pH-dependent fusogenic protein is a type III viral fusion protein or a fusogenic fragment thereof.

22. The cell of claim 21, wherein the type III viral fusion protein is a Vesicular stomatitis virus G protein or a fusogenic fragment thereof.78 KILPATRICK TOWNSEND 79026177123. The cell of any one of claims 1-22, wherein the cell comprises an expression cassette comprising an inducible promoter operable linked to a polynucleotide encoding the low pH-dependent fusogenic protein.

24. A method of transferring a payload polypeptide from a first cell to a second cell, the method comprising, providing the first cell, the first cell comprising: (a) a transmembrane protein comprising (i) an extracellular amino acid sequence that binds to a target protein on a recipient cell and (ii) a transmembrane domain, wherein the transmembrane domain is linked to a payload polypeptide, the transmembrane protein is heterologous to the cell and the transmembrane protein is heterologous to the payload polypeptide; and a low pH-dependent cleavage sequence is between the transmembrane domain and the payload polypeptide, wherein the low pH-dependent cleavage sequence is self-cleaved at the pH of an cellular endosome but not at the pH of the cytosol of the cell; (b) a low pH-dependent fusogenic protein; and contacting the first cell to a second cell under conditions that allow for transfer of a portion of the cell membrane comprising the transmembrane protein and the low pH-dependent fusogenic protein to the second cell and the payload polypeptide is released from an endosome to the cytosol of the second cell following self-cleavage of the low pH-dependent cleavage sequence.

25. The method of claim 24, wherein the first and second cells are mammalian cells 26. The method of claim 24, wherein the first and second cells are mammalian cells of the same species.79 KILPATRICK TOWNSEND 79026177127. The method of claim 24, wherein the first and second cells are human cells.

28. The method of any one of claims 24-27, wherein the method is performed in vitro.

29. The method of any one of claims 24-27, wherein the first cell is administered to a mammal and the second cell resides in the mammal and the contacting occurs in the mammal.

30. The method of any one of claims 24-29, wherein the second cell is a neuronal cell, immune cell, cancer cell, muscle cell, or stem cell 31. The method of any one of claims 24-29, wherein the first cell is a glial cell, immune cell, stem cell, or red blood cell.

32. A nucleic acid encoding a transmembrane protein comprising (i) an extracellular amino acid sequence that binds to a target protein on a recipient cell and (ii) a transmembrane domain, (iii) a low pH-dependent cleavage sequence, wherein the low pH- dependent cleavage sequence is cleaved in a cellular endosome; and (iv) a payload polypeptide, wherein the transmembrane domain is heterologous to the payload polypeptide.

33. The nucleic acid of claim 32, wherein the cleavage sequence is a low pH- dependent cleavage sequence between the transmembrane domain and the payload polypeptide, wherein the low pH-dependent cleavage sequence is self-cleaved at the pH of a cellular endosome but not at the pH of the cytosol of the cell.

34. The nucleic acid of claim 33, wherein the low pH-dependent cleavage sequence is a pH intein.

35. The nucleic acid of claim 32 or 33, wherein the transmembrane domain is covalently linked to the payload polypeptide.80 KILPATRICK TOWNSEND 79026177136. The nucleic acid of claim 32 or 33, wherein the transmembrane domain is non-covalently linked to the payload polypeptide.

37. The nucleic acid of any one of claims 32-36, wherein the payload polypeptide is an enzyme or protein-binding polypeptide.

38. The nucleic acid of any one of claims 32-37, wherein the payload polypeptide comprises a nuclear localization signal sequence or a nuclear export signal sequence.

39. The nucleic acid of any one of claims 32-38, wherein the cell is a mammalian cell.

40. The nucleic acid of claim 39, wherein the mammalian cell is a human cell.

41. The nucleic acid of any one of claims 32-40, wherein the payload polypeptide is a nucleic acid binding protein.

42. The nucleic acid of claim 41, wherein the nucleic acid binding protein is a RNA-guided protein.

43. The nucleic acid of claim 42, wherein the gRNA-guided protein is a CRISPR nuclease or an inactive mutant thereof.

44. The nucleic acid of any one of claims 32-43, wherein the extracellular amino acid sequence comprises an antibody or antigen-binding fragment thereof.

45. The nucleic acid of claim 44, wherein the antibody is a scFv.

46. The nucleic acid of any one of claims 32-45, wherein the low pH- dependent fusogenic protein is a type III viral fusion protein or a fusogenic fragment thereof.

47. The nucleic acid of claim 46, wherein the type III viral fusion protein is a Vesicular stomatitis virus G protein or a fusogenic fragment thereof.

48. A vector comprising the nucleic acid of any one of claims 32-47, optionally comprising a promoter operably linked to the nucleic acid.81 KILPATRICK TOWNSEND 79026177149. The vector of claim 48, wherein the vector is a viral vector.82 KILPATRICK TOWNSEND 790261771

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