Methods and compositions for discovering receptor-ligand specificity through engineered cell entry

Abstract

The present disclosure relates to systems, methods, and compositions for elucidating ligand-receptor interactions, delivering nucleic acids and proteins to target cells, and performing single-cell multiomics. Engineered lentiviruses are disclosed that present ligands that deliver cargo to target cells upon cognate receptor-ligand interaction. Compositions and methods including pMHC or antigen epitope-presenting lentiviruses for identifying pMHC / T cell receptor and antigen / B cell receptor interactions are also disclosed.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 286,507, filed December 6, 2021, and U.S. Provisional Patent Application No. 63 / 382,860, filed November 8, 2022. The disclosures of the foregoing applications, including any drawings, are expressly incorporated by reference in their entireties herein.

[0002] Incorporating sequence tables The accompanying sequence listing material is incorporated by reference into this application. The accompanying sequence listing text file is entitled 078430-538001WO_SequenceListing_ST26.xml, was created on December 1, 2022, and is 17KB in size.

[0003] The present technology relates generally to the field of cell biology. More specifically, the present technology relates to methods and compositions for the discovery and identification of ligand-receptor specificity and gene and protein delivery. The present technology also relates to the elucidation of interactions between T cell receptors and MHC peptides, between antibodies and antigens, or between B cell receptors and B cell antigens (including intracellular / secreted epitopes / cell surface antigen epitopes) and other ligand-receptor interactions. [Background technology]

[0004] Cells communicate with each other through ligand-receptor interactions. Extensive cell-cell communication results in the molecular programming of mammalian cells to direct specific behavior and cell fate decisions. For example, the TCR on the surface of a T cell can recognize and interact with major histocompatibility complex (MHC)-antigen complexes from the surface of an antigen-presenting cell (APC). TCR and antibody genes undergo somatic recombination to produce a large and diverse repertoire (approximately 10 in humans). 16The TCR achieves a set of TCR alpha and beta sequences, which are clonally inherited by daughter cells. The interaction of the T cell receptor with the B cell receptor is highly specific and drives the proliferation and differentiation of antigen-specific T and B cells. Elucidation of TCR-antigen interactions, particularly linking antigen specificity to TCR sequences and T cell state, is essential to understanding how antigen recognition drives T cell fate decisions. To elucidate the antigen specificity of TCRs, diverse approaches have been developed, including: (1) cellular reporter assays to screen T cell-specific MHC antigens using artificial APCs, such as T-scan, SABR, T cell trogocytosis, and cytokine capture assays (Joglekar et al., 2019; Kula et al., 2019; Lee and Meyerson, 2021; Li et al., 2019); (2) yeast display platforms to screen MHC antigens against recombinant TCRs (Birnbaum et al., 2012); (3) T cell-based assays, such as cytokine production upon antigen peptide stimulation (ELISpot) (McCutcheon et al., 1997); and (4) DNA barcoded MHC peptide multimers to capture antigen specificity and TCR sequences by single-cell sequencing (TetTCR-seq; Zhang et al., 2018). Despite the specific advantages of each technique, it remains challenging to rapidly screen immunogenic MHC antigens on primary T cells and simultaneously capture the antigen landscape, paired TCR repertoire, and gene expression of T cell phenotype in a high-throughput manner. Many of the existing methods require re-expression of receptors or ligands on heterologous cells and therefore cannot be directly applied to human clinical samples. Similar challenges apply to the study of B cell receptor-antigen interactions, with the added challenge of addressing known intracellular antigen epitopes recognized by antibodies.

[0005] Despite great advances in characterizing the cellular states of antigen-specific T and B cells, it remains challenging to target these antigen-specific cells and selectively modify their cellular states and behaviors without perturbing other bystander T or B cells. A recent method using pMHC-presenting nanoparticles allows for mRNA delivery in antigen-specific T cells, opening up many possibilities to transiently modulate specific T cells (Su et al., 2022). Another recent study using pMHC pseudotyped viruses allows for genetic modification of antigen-specific T cells (Guo and Elledge, 2022). However, the technology to selectively manipulate antigen-specific B cells besides antigen-specific T cells is still lacking.

[0006] Thus, there is a need for methods and compositions to reliably and rapidly systematically identify ligand-receptor pairings and elucidate receptor specificity. More specifically, there is a need for methods that can scalably (1) present many different types of ligands, (2) match the ligands with receptors on cells, (3) record the information, and (4) engineer cells that express the ligand-matched receptor. There is also a need for methods and compositions to explore ligand-receptor pairings at single-cell resolution and deliver gene or protein payloads in a cell-specific manner. Summary of the Invention

[0007] In one aspect, the present disclosure provides an engineered lentivirus comprising a heterologous ligand displayed on the surface of the lentivirus, a fusogen comprising a modified viral envelope protein (wherein the fusogen is capable of fusing the lentivirus to a host cell, the host cell comprising an endogenous receptor for the ligand), a reporter protein operably linked (e.g., fused) to a lentiviral structural protein, and a barcoded RNA.

[0008] Non-limiting exemplary embodiments of the engineered lentiviruses of the present disclosure can include one or more of the following features: In some embodiments, the fusogen comprises a modified VSV-G viral envelope protein. In some embodiments, the modified VSV-G viral envelope protein comprises one or more amino acid substitutions at any one of positions H8, K47, Y209, and R354 of the VSV-G polypeptide. In some embodiments, the modified VSV-G viral envelope protein comprises a K47Q substitution and a R354A substitution.

[0009] In some embodiments, the ligand is or comprises a protein or epitope.

[0010] In some embodiments, the ligand is or comprises an MHC peptide, an antibody, an antigen, a secreted protein, a cell surface protein, or other form of an antigen that can be expressed by a cell, hi some embodiments, the antigen is or comprises an intracellular antigen.

[0011] In some embodiments, the ligand is operably linked (e.g., fused) to an optimized transmembrane domain, hi some embodiments, the optimized transmembrane domain is a transmembrane domain derived from HLA-DRA, HLA-DRB, HLA-A2, ICAM1, CD43, CD162, CD62L, CD49d, or LFA-1.

[0012] In some embodiments, the ligand is operably linked (eg, fused) to an optimized transmembrane domain and a signal peptide.

[0013] In some embodiments, the engineered lentivirus comprises a defective integrase protein.

[0014] In some embodiments, the reporter protein is GFP or mNeon.

[0015] In some embodiments, the structural protein is a nucleocapsid protein.

[0016] In some embodiments, the structural protein is a Gag protein.

[0017] In some embodiments, the barcoded RNA is encapsulated in a viral particle.

[0018] In some embodiments, the RNA encodes a ligand, eg, a protein ligand.

[0019] In some embodiments, the RNA encodes a gene of interest to be delivered to a target cell, e.g., a host cell.

[0020] In some embodiments, the RNA is read by next generation sequencing techniques.

[0021] In some embodiments, the RNA comprises a capture sequence, e.g., a sequence that can be used to capture or hybridize to an analyte (e.g., DNA, RNA, protein) from or within a sample in a 10x Genomics single cell sequencing workflow.

[0022] In one aspect, the present disclosure further provides a method for identifying a ligand-receptor pair comprising providing at least one engineered lentivirus comprising a heterologous ligand displayed on the surface of the lentivirus, a fusogen comprising a modified viral envelope protein (wherein the fusogen is capable of fusing the lentivirus to a host cell, the host cell comprising an endogenous receptor for the ligand, e.g., an immune receptor), a reporter protein fused to a lentiviral structural protein, and a barcoded RNA; combining the lentivirus with a cell population; and sorting the cell population based on the presence of the reporter gene, thereby identifying the ligand-receptor pair.

[0023] Non-limiting exemplary embodiments of the methods for identifying ligand-receptor pairs of the present disclosure can include one or more of the following features: In some embodiments, the method includes providing a pool of engineered lentiviruses, where the pool displays different ligands.

[0024] In some embodiments, the methods include combining the lentivirus with the cells and incubating the virus / cell mixture at about 4°C.

[0025] In some embodiments, the methods include combining a lentivirus with cells and incubating the virus / cell mixture at room temperature.

[0026] In some embodiments, the methods include combining the lentivirus with the cells and incubating the virus / cell mixture at about 37°C.

[0027] In some embodiments, the method comprises incubating the virus / cell mixture for about 30 minutes, or about 1 hour, or about 2 hours, or any period between about 0.5 hours and about 2.5 hours.

[0028] In some embodiments, the method further comprises performing single-cell sequencing of the viral RNA to identify the ligand sequence.

[0029] In some embodiments, the method further comprises performing single-cell sequencing of the transcriptome of the cell to identify the receptor sequence.

[0030] In one aspect, the present disclosure further provides a method of delivering a molecule of interest, e.g., a nucleic acid or protein of interest, to a user-defined target cell, comprising providing an engineered lentivirus comprising a heterologous ligand displayed on the surface of the lentivirus, a fusogen comprising a modified viral envelope protein (wherein the fusogen is capable of fusing the lentivirus to a host cell, the host cell comprising an endogenous receptor for the ligand, e.g., an immune receptor), a reporter protein fused to a lentiviral structural protein, and a barcoded RNA; and contacting the lentivirus with a cell mixture comprising target cells, such that the nucleic acid or protein is delivered only to the target cells, wherein the target cells express a receptor, e.g., an immune receptor, specific for the ligand on the lentivirus surface.

[0031] Non-limiting exemplary embodiments of the methods for delivering molecules of interest of the present disclosure can include one or more of the following features: In some embodiments, the ligand is modified to deliver the cargo to a user-defined target cell.

[0032] In some embodiments, the nucleic acid of interest is packaged inside an engineered lentiviral particle.

[0033] In some embodiments, the protein of interest is operably linked (eg, fused) to the gag protein of a lentivirus.

[0034] In some embodiments, the protein of interest is replaced with a reporter. In some embodiments, the target cell is in vivo. In some embodiments, the target cell is ex vivo. In some embodiments, the target cell is in vitro. In some embodiments, the target cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the target cell is an immune cell. In some embodiments, the immune cell is a T cell or a B cell. In some embodiments, the immune cell is a T cell and the receptor is a T cell receptor. In some embodiments, the immune cell is a B cell and the receptor is a B cell receptor. In some embodiments, the human cell is a primary human blood cell (PBMC).

[0035] In another aspect, the disclosure further provides a method for identifying an immunogenic antigen comprising providing an engineered lentivirus comprising a heterologous receptor protein displayed on the surface of the lentivirus, a fusogen comprising a modified VSV-G viral envelope protein (wherein the fusogen is capable of fusing the lentivirus to a host cell, the host cell comprising a native antigen for the receptor), a reporter transgene fused to a lentiviral structural protein, and a barcoded RNA (wherein the RNA encodes antigen information), combining the lentivirus with a cell population, and sorting the cell population based on the presence of the reporter.

[0036] Non-limiting exemplary embodiments of the methods for identifying immunogenic antigens of the present disclosure can include one or more of the following features: In some embodiments, the method further includes sequencing the viral RNA to identify the antigen sequence.

[0037] In some embodiments, the method further comprises sequencing the RNA of the cellular receptor.

[0038] In another aspect, the disclosure further provides a method for identifying a T cell receptor and paired MHC peptide comprising providing an engineered lentivirus comprising pMHC displayed on the viral surface, a fusogen comprising a modified VSV-G viral envelope protein (wherein the fusogen is capable of fusing the lentivirus to a host cell, the host cell comprising a T cell receptor for the pMHC), a reporter transgene fused to a lentiviral structural protein, and a barcoded RNA; combining the lentivirus with a cell population; and sorting the cell population based on the presence of the reporter, thereby identifying the T cell receptor.

[0039] Non-limiting exemplary embodiments of the disclosed methods for identifying T cell receptors and paired MHC peptides can include one or more of the following features: In some embodiments, the cell population comprises human primary T cells.

[0040] In some embodiments, the PMHC is encoded by an RNA that includes, in tandem, a signal peptide, a PMHC, a G4S linker, a b2m gene, a G4S linker, and an MH allele.

[0041] In some embodiments, the method further comprises performing single-cell sequencing of the viral RNA to identify the MHC peptide sequence.

[0042] In some embodiments, the method further comprises sequencing the receptor sequences of the cells to identify the MHC peptide and T cell receptor sequences.

[0043] In yet another aspect, the disclosure also provides a method for identifying a B cell receptor or antibody comprising providing an engineered lentivirus comprising an epitope displayed on the lentivirus surface (wherein the epitope is operably linked (e.g., fused) to an ICAM1 transmembrane domain), a fusogen comprising a modified VSV-G viral envelope protein (wherein the fusogen is capable of fusing the lentivirus to a host cell, the host cell comprising a B cell receptor for the intracellular epitope), a reporter transgene fused to a lentiviral structural protein, and a barcoded RNA; combining the lentivirus with a B cell population; and sorting the cell population based on the presence of the reporter, thereby identifying the B cell receptor or antibody.

[0044] Non-limiting exemplary embodiments of the methods for identifying B cell receptors or antibodies of the present disclosure can include one or more of the following features: In some embodiments, the antigen is a cell surface membrane protein, an intracellular protein, a secreted protein, or a glycosylated protein.

[0045] In some embodiments, the method further comprises performing single-cell sequencing of the viral RNA to identify the antigen and matching B cell receptor sequences.

[0046] The present disclosure further provides at least one embodiment of a method for identifying an antigen for a B cell receptor, the method comprising providing an engineered lentivirus comprising a heterologous ligand displayed on the surface of the lentivirus, a fusogen comprising a modified viral envelope protein (wherein the fusogen is capable of fusing the lentivirus to a host cell, the host cell comprising an endogenous receptor for the ligand), a reporter protein fused to a lentiviral structural protein, and a barcoded RNA; combining the lentivirus with a B cell population; and sorting the cell population based on the presence of the reporter, thereby identifying the B cell antigen.

[0047] The present disclosure also provides at least one embodiment of a method for single cell multi-omics comprising providing an engineered lentivirus comprising a heterologous ligand displayed on the surface of the lentivirus, a fusogen comprising a modified VSV-G viral envelope protein (wherein the fusogen is capable of fusing the lentivirus to a host cell, the host cell comprising an endogenous receptor for the ligand), a reporter transgene fused to a lentiviral structural protein, and RNA (wherein the RNA comprises an antigen sequence and a capture tag for single cell sequencing), and simultaneously obtaining transcriptomic and phenotypic information at the single cell level.

[0048] In some embodiments, the single-cell sequencing is a droplet-based platform.

[0049] In some embodiments, the cell phenotype comprises surface markers by CITE-seq.

[0050] In some embodiments, the information includes sequences of the ligand and receptor.

[0051] In some embodiments, single cell multiomics uses whole cells as input.

[0052] In some embodiments, single cell multiomics uses whole cells as input and includes a step of reverse transcription.

[0053] In yet another aspect, the disclosure discloses a method for selectively depleting or enriching a target cell population in a cell mixture, comprising: (a) an engineered lentivirus as described herein; and (b) providing a cell mixture comprising (i) a target cell population that expresses a receptor specific for a ligand displayed on the surface of the engineered lentivirus, and (ii) a non-target cell population that does not express a receptor specific for the ligand displayed on the surface of the engineered lentivirus; contacting the engineered lentivirus with the cell population to deliver a nucleic acid or protein only to the target cells; and adding a reagent that specifically inhibits proliferation of the target cell population or inhibits proliferation of the non-target cell population, thereby selectively depleting or enriching the target cell population. In some embodiments, the receptor is an immune receptor. In some embodiments, the immune receptor is a B cell receptor. In some embodiments, the immune receptor is a T cell receptor.

[0054] Non-limiting exemplary embodiments of the disclosed methods for selectively depleting or enriching a target cell population can include one or more of the following features: In some embodiments, the target cells express a herpes simplex virus thymidine kinase (HSV-TK) transgene and the added reagent comprises or is ganciclovir (GCV). In some embodiments, the target cells express shRNA to reduce expression of the death receptor FAS to prevent cell death in the target population. In some embodiments, the target cell population comprises immune cells. In some embodiments, the immune cells comprise T cells. In some embodiments, the immune cells comprise B cells.

[0055] In some embodiments, the immune cells are autoreactive immune cells. In some embodiments, the immune cells are specific for an antigen associated with a health condition. In some embodiments, the health condition is a proliferative disease, an inflammatory disease, an autoimmune disease, or a microbial infection. In some embodiments, the proliferative disease is cancer. In some embodiments, the microbial infection is a bacterial infection, a viral infection, or a microfungal infection.

[0056] The above summary is illustrative only and is not intended to be limiting in any way. In addition to the exemplary embodiments and features described herein, further aspects, embodiments, objects, and features of the present disclosure will become more fully apparent from the drawings, detailed description, and claims.

[0057] Although various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although for clarity the present disclosure may be described in the context of separate embodiments, the present disclosure may also be provided in a single embodiment. [Brief description of the drawings]

[0058] [Figure 1A] 1A-1G show a viral display platform that displays ligand proteins and fusogens on the viral surface, delivers fluorescent proteins, and records ligand-receptor interactions upon cell entry. FIG. 1A shows a schematic diagram of an exemplary all-in-one platform. Lentiviruses have been engineered in various components, such as (1) user-defined ligand proteins displayed on the viral surface, (2) modified fusogens with intact fusion ability and defective in binding to native receptors, (3) cargo proteins fused with viral structural proteins, and (4) barcoded viral RNA for tracking and gene delivery.

[0059] [Figure 1B] Figure 1B shows a schematic of the experimental setup for GFP expression and flow cytometry analysis 3 days after viral infection. Raji and Jurkat cells were infected with three groups of lentiviruses encoding GFP in the viral RNA: (1) viruses with wild-type VSV-G (left), (2) viruses with receptor-binding mutant VSV-G (center), and (3) viruses with VSV-G mutants and anti-CD19 single-chain antibody variable fragments (scFvs).

[0060] [Figure 1C] Figure 1C shows a schematic diagram of the experimental setup (top). GFP protein is fused to matrix protein (MA-GFP) or nucleocapsid protein (NC-GFP), or viral protein R (VPR-GFP). scFv-CD19-presenting viruses with GFP protein fused to different viral proteins were incubated with Raji (CD19+) cells or Jurkat (CD19-) cells for 2 hours and then subjected to flow cytometry. The bar graph (bottom) shows the percentage of GFP+ cells when viruses with different GFP-fused viral proteins were incubated.

[0061] [Figure 1D] FIG. 1D shows an exemplary flow cytometry plot of GFP signal after transient virus incubation, similar to FIG. 1C.

[0062] [Figure 1E] Figure 1E shows a schematic diagram of the experimental setup and flow cytometry analysis of GFP signals in primary human B cells with or without virus incubation. Naive and activated primary human B cells were incubated with NC-GFP fusion virus and scFv-CD19-presenting virus for 2 hours and then subjected to flow cytometry analysis. B cells were gated on viable CD20+ cells.

[0063] [Figure 1F] FIG. 1F is a histogram analysis of surface CD19 expression of the groups in FIG. 1E.

[0064] [Figure 1G] Figure 1G is a bar graph showing scFv-CD19 virus binding and CD19 surface expression on naive and activated human B cells. P values ​​in Figures 1C and 1F are calculated by unpaired t-test. ****P<0.0001 ***P<0.001.

[0065] [Figure 1H] Figures 1H-1N show how ENTER deciphers the specific ligand-receptor interactions for costimulatory molecules. Figure 1H shows flow cytometry analysis of virus binding and fusion of scFv-CD19-presenting viruses on Raji B cells at different temperatures before and after proteinase K treatment.

[0066] [Figure 1I] FIG. 1I is a bar graph showing the percentage of GFP+ cells in FIG. 1H.

[0067] [Figure 1J] Figure 1J shows a schematic of the experimental setup: CD40-expressing Raji B cells are incubated with GFP viruses displaying either wild-type CD40 ligand (CD40L) or CD40L mutants (K142E, R202E) with reduced binding to its cognate receptor, CD40.

[0068] [Figure 1K] FIG. 1K shows flow cytometry analysis of GFP signals in Raji B cells upon incubation with wild-type CD40E or mutant CD40E-displaying GFP viruses.

[0069] [Figure 1L] Figure 1L is a bar graph showing the percentage of GFP+ cells in Figure 1K. p values ​​were calculated by unpaired t-test. ***P<0.001.

[0070] [Figure 1M] Figure 1M shows a schematic diagram of the immunocapture assay. In this assay, magnetic beads were bound with anti-CD40L, anti-VSV-G, and IgG antibodies and then incubated with CD40L-presenting viruses. The immunocaptured viruses were subjected to viral RNA isolation and qRT-PCR for CD40L.

[0071] [Figure 1N]Figure 1N shows a bar graph showing the qRT-PCR results of ENTER virus enrichment by different antibody-conjugated beads, similar to Figure 1M. All p values ​​were calculated by unpaired t-test. ns not significant p>0.05, *p<0.05, ***p<0.001.

[0072] [Figure 2A] Figures 2A-2F show how ENTER uncovers interactions between MHC peptides (pMHC) and TCRs. Figure 2A shows a schematic of pMHC-presenting viruses and flow cytometry analysis of GFP signals in Jurkat T cells expressing specific TCRs when incubated with pMHC-presenting viruses. GFP-fusion viruses presenting either a 9-mer peptide (NY-ESO-1157-I65) presented by the HLA-A0201 (A2) allele or an 11-mer CMV peptide (pp65363-373) presented by the HLA-A0101 (Al) allele are incubated with T cells expressing a specific TCR (e.g., NY-ESO-1157-165-TCR or CMV-pp65363-373-TCR) that recognizes the cognate antigen. SP: signal peptide; peptide: antigen peptide; B2M: beta-2-mycoglobulin.

[0073] [Figure 2B] Figure 2B shows flow cytometry analysis of GFP signals in Jurkat T cells expressing specific TCRs (e.g., NY-ESO-1157-165-TCR, CMV pp65495-503-TCR, or Flum1(58-66)-TCR) upon incubation with viruses presenting various HLA-A2-presented peptides. HPV16 E782-91 peptide-presenting viruses serve as negative controls.

[0074] [Figure 2C]Figure 2C shows flow cytometry plots of NY-ESO-1 TCR-T cells upon incubation with 2 x 108 ENTER viral particles presenting the ny-eso-1157~166 antigen (left) or with different amounts of ny-eso-1157~166 pMHC tetramer.

[0075] [Figure 2D] Figure 2D shows a comparison of binding efficiency (GFP+%) of viruses presenting antigen variants with different TCR affinities. 1G4wt-TCR T cells were incubated for 2 h with ENTER presenting the wild type of the mutant ny-eso-1157~166 antigen variant. CMV-pp65495~503-TCR T cells were used as a negative control. p24 protein levels were used to normalize viral titers.

[0076] [Figure 2E] Figure 2E shows a schematic diagram of the experimental set-up (top) and flow cytometry analysis (bottom) of the TCR-T cell mixing experiment. Flu-m158~66-TCR T cells were labeled by CellTrace Violet dye and then mixed with CMV-pp65495~503-TCR T cells at different ratios. The mixed T cell populations were incubated with HLA-A2:m1-presenting GFP virus for 2 h and then subjected to flow cytometry. Representative flow cytometry plots showing the GFP signal of the 1:1000 mixture of the two T cell populations and the T cells gated for the Violet+ and Violet- populations.

[0077] [Figure 2F] FIG. 2F is a bar graph showing the signal / noise ratio of ENTER of FIGS. 2E and 2J.

[0078] [Figure 2G] FIG. 2G shows flow cytometry analysis of HLA-A2 and B2M surface expression in wild-type HEK293T, HLA-KO HEK293T, and HLA-A2-rearranged HLA-KO HEK293T cells.

[0079] [Figure 2H] Figure 2H shows flow cytometry plots of CMV-pp65495~503-TCR T cells upon incubation with HLA-A2 peptide-presenting GFP virus for 2 h followed by staining for PE tetramer. M1(58~66) (influenza antigen)-presenting virus and M1(58~66) tetramer are negative controls.

[0080] [Figure 2I] FIG. 2I shows a histogram plot (left) and a bar graph (right) showing tetramer intensity and CD3 surface expression of CMV-pp65495-503-TCR T cells.

[0081] [Figure 2J] Figure 2J shows a schematic of the experimental design (top) and flow cytometry analysis (bottom) of the T cell mixing experiment. Flu-m158~66-TCR T cells were labeled by CellTrace Violet dye and then mixed with NY-ESO-1157~165-TCR T cells at different ratios. The mixed T cell populations were incubated with HLA-A2:m1-presenting GFP virus for 2 h and then subjected to flow cytometry. Representative flow cytometry plots showing the mixture of the two T cell populations and the GFP signal of T cells gated for Violet+ and Violet- populations.

[0082] [Figure 2K] FIG. 2K shows bar graphs depicting the sensitivity (left) and specificity (right) of ENTER from FIG. 2J.

[0083] [Figure 3A]Figures 3A-3F show the optimization of ENTER to present intracellular antigens on the viral surface and elucidate the interaction between BCR and antigen. Figure 3A shows a schematic of the experimental design. During lentivirus assembly and budding, specific host cell surface proteins can be incorporated into the surface of the virus. The TM domain of a host protein selected from the literature is fused with a B cell epitope derived from the intracellular antigen HPV16 L2. These HPV epitope-presenting GFP viruses are incubated with B cells expressing a BCR targeting this HPV epitope (on-target) or B cells not expressing a BCR (off-target).

[0084] [Figure 3B] Figure 3B shows flow cytometry analysis of GFP signals in B cells incubated with GFP viruses presenting HPV epitopes fused to different TM domains. B cells not expressing a BCR serve as negative controls.

[0085] [Figure 3C] FIG. 3C shows a bar graph indicating the percentage of GFP+ B cells from FIG. 3B.

[0086] [Figure 3D] Figure 3D shows flow cytometry analysis of GFP signal in RBD-BCR+ B cells upon incubation with ENTER viruses presenting SARS-CoV-2 spike RBD antigen or HPV L2 antigen as a negative control (left). Bar graphs show the frequency of GFP+ cells upon incubation with on-target or off-target ENTER viruses (right).

[0087] [Figure 3E]Figure 3E shows a schematic diagram of the experimental setup (top) and flow cytometry analysis (bottom) of the B cell mixing experiment. RBD-BCR+ B cells were labeled with cell trace violet dye and then mixed with HPV-BCR+ B cells at different ratios. The mixed B cell population was incubated with GFP virus presenting RBD antigen fused with ICAM1 TM domain and subjected to flow cytometry. Representative flow cytometry plots show 1:1000 mix of the two B cell populations and GFP signal of B cells gated for violet+ and violet- populations.

[0088] [Figure 3F] FIG. 3F is a bar graph showing the signal / noise ratio of ENTER of FIGS. 3E and 3I.

[0089] [Figure 3G] Figures 3G-3P summarize the results of experiments performed to characterize the antigen specificity of B cells by ENTER and the efficiency and specificity of cargo delivery. Figure 3G shows flow cytometry analysis of GFP+ cells among HER2-BCR+ B cells incubated with HER2- or RBD-presenting viruses.

[0090] [Figure 3H] FIG. 3H is a bar graph showing the percentage of GFP+ cells, similar to FIG. 3G.

[0091] [Figure 3I]Figure 3I shows a schematic diagram of the experimental design (top) and flow cytometry analysis (bottom) of the B cell mixing experiment. RBD-BCR+ B cells were labeled with Cell Trace violet and mixed with HPV-BCR+ B cells at different ratios. The mixed B cell population was incubated with GFP virus presenting RBD antigen fused with ICAM1 TM domain and subjected to flow cytometry. Representative flow cytometry plots show the mixing of the two B cell populations and the GFP signal of B cells gated for Violet+ and Violet- populations.

[0092] [Figure 3J] FIG. 3J is a bar graph showing the sensitivity of ENTER of FIG. 3I.

[0093] [Figure 3K] FIG. 3K is a bar graph showing the sensitivity of ENTER of FIG. 3I.

[0094] [Figure 3L] Figure 3L shows a bar graph showing the delivery efficiency of virus carrying wild-type VSV-G in cell types with different antigen specificities. p values ​​were calculated by unpaired t-test. ns not significant P>0.05.

[0095] [Figure 3M] Figure 3M shows flow cytometry analysis of cargo delivery in a T cell mixing experiment. mScarlet-expressing NY-ESO-1 TCR+ T cells were mixed with CMV-pp65 TCR T cells and then infected with ENTER virus presenting the pp65495-503 antigen and carrying transgenes including HSV-TK and GFP.

[0096] [Figure 3N]Figure 3N shows flow cytometry analysis of cargo delivery in a B cell mixing experiment. mScarlet-expressing RBD BCR+ B cells were mixed with HER2 BCR+ B cells and then infected with ENTER virus presenting HER2 antigen and carrying transgenes including HSV-TK and GFP.

[0097] [Figure 3O] FIG. 3O is a histogram plot showing surface expression of FAS in T cells transduced with various FAS shRNAs or control shRNA.

[0098] [Figure 3P] FIG. 3P is a representative flow plot showing Annexin V and 7-AAD gating of T cells during anti-FAS-induced apoptosis and cell death.

[0099] [Figure 4A] Figures 4A-4M are schematic summaries of the results of experiments performed to show that ENTER allows selective depletion or expansion of antigen-specific T cells or antigen-specific B cells. Figure 4A is a schematic of cargo delivery in antigen-specific T cells (left). CMV-pp65 TCR+ T cells or NY-ESO-1 TCR+ T cells were individually infected with ENTER virus presenting pp65495-503 and carrying the GFP transgene as cargo. Representative histogram plots showing GFP expression between CMV-pp65 TCR+ T cells and NY-ESO-1 TCR+ T cells 2 days after infection.

[0100] [Figure 4B] FIG. 4B is a bar graph showing the percentage of GFP+ cells, similar to FIG. 4A.

[0101] [Figure 4C]Figure 4C is a schematic of cargo delivery in antigen-specific B cells (left). HER2 BCR+ B cells or RBD BCR+ B cells were individually infected with ENTER virus presenting HER2 and carrying a GFP transgene as cargo. Representative histogram plots showing GFP expression between HER2 BCR+ B cells and RBD BCR+ B cells 2 days after infection.

[0102] [Figure 4D] FIG. 4D is a bar graph showing the percentage of GFP+ cells, similar to FIG. 4C.

[0103] [Figure 4E] Figure 4E is a schematic of suicide gene delivery in a pool of different antigen-specific T cells. CMV-pp65 TCR+ T cells and NY-ESO-1 TCR+ T cells expressing mScarlet were mixed together and then infected with ENTER virus presenting pp65495-503 and carrying the herpes simplex virus thymidine kinase (HSV-TK) transgene. Two days after infection, ganciclovir (GCV) drug was added to kill HSV-TK-expressing cells, and cell survival was monitored for 4 days.

[0104] [Figure 4F] FIG. 4F is a bar graph showing the number of viable T cells 4 days after GCV treatment.

[0105] [Figure 4G] Figure 4G shows a schematic summary of the results of kinetic analysis of fold enrichment between the number of CMV-pp65 TCR+ T cells and the number of NY-ESO-1 TCR+ T cells infected with ENTER carrying the GFP gene or ENTER carrying the HSV-TK gene upon GCV drug treatment.

[0106] [Figure 4H]Figure 4H is a schematic of suicide gene delivery in a pool of different antigen-specific B cells. HER2 BCR+ B cells and RBD BCR+ B cells expressing mScarlet were mixed together and then infected with ENTER virus presenting pp65495-503 and carrying the HSV-TK transgene. Two days after infection, GCV drug was added to kill HSV-TK expressing cells, and cell survival was monitored for 4 days.

[0107] [Figure 4I] FIG. 4I is a bar graph showing the number of viable B cells 4 days after GCV treatment.

[0108] [Figure 4J] Figure 4J is a schematic summary of the results of kinetic analysis of fold enrichment between the number of HER2 BCR+ B cells and the number of RBD BCR+ B cells infected with ENTER carrying the GFP gene or ENTER carrying the HSV-TK gene upon GCV drug treatment.

[0109] [Figure 4K] Figure 4K is a schematic of shRNA delivery in pools of different antigen-specific T cells. CMV-pp65 TCR+ T cells and NY-ESO-1 TCR+ T cells expressing mScarlet were mixed together and infected with ENTER virus presenting pp65495-503 and carrying FAS shRNA or control shRNA. Anti-FAS antibody was added to induce apoptosis.

[0110] [Figure 4L] FIG. 4L is a bar graph showing surface expression of FAS in off-target NY-ESO-1 TCR+ T cells (uninfected group) and on-target CMV-pp65 TCR+ T cells transduced with control shRNA or FAS shRNA.

[0111] [Figure 4M]FIG. 4M is a bar graph showing fold enrichment of CMV-pp65 TCR+ T cells / NY-ESO-1 TCR+ T cells normalized by shCtrl group for viable cells (gated by Annexin V and 7-AAD double negative), similar to FIG. 4K. P values ​​are calculated by unpaired t-test. ****P<0.0001; ***P<0.001; **P<0.01; *P<0.05; nsP>0.05.

[0112] [Figure 4N] Figures 4N-4W are a schematic summary of the results of experiments performed to optimize ENTER to detect antigen-specific primary human T cells. Figure 4N is a flow cytometry analysis of GFP signal in T cells expressing TCR (NY-ESO-1 TCR or CMV pp65-TCR) upon incubation with ny-eso-1157-165 antigen peptide-presenting GFP virus (the viral RNA is either intact or has a sequencing capture tag inserted into the viral RNA).

[0113] [Figure 4O] Figure 4O is a bar graph showing the percentage of GFP+ cells in Figure 4N. P values ​​were calculated by unpaired t-test. nS>0.05.

[0114] [Figure 4P] Figure 4P is a schematic of the experimental design. T cells isolated from donors were incubated with pp65495-503-presenting viruses carrying either GFP or mNeon fluorescent protein, then stained with pp65495-503 tetramer and other antibodies, followed by flow cytometry. CD3+CD8+ T cells were first gated to measure pp65-specific T cells with tetramer as a positive control, and then GFP signal in pp65 tetramer+ T cells was monitored.

[0115] [Figure 4Q]FIG. 4Q, like FIG. 4P, is a flow cytometry analysis of tetramer and GFP signals in primary human T cells from a CMV-infected donor.

[0116] [Figure 4R] Figure 4R is a bar graph showing the percentage of GFP+ and GFP- cells among the pp65 tetramer+ T cells in Figure 4Q. P values ​​were calculated by unpaired t-test. *p<0.05.

[0117] [Figure 4S] FIG. 4S is a representative flow cytometry plot showing GFP+ cells among pp65 tetramer+ T cells after incubation with pp65495-503 virus or negative control virus (ny-eso-1157-165).

[0118] [Figure 4T] Figure 4T is a schematic of the experimental design. PBMCs were isolated from CMV seropositive HLA-A2+ donors and incubated with a pool of 12 different CMV antigen peptides (10 μg / mL) for 10 days. Primary T cells before or after peptide-induced expansion were incubated with pp65 antigen-presenting mNeon virus for 2 hours and then stained with antibodies followed by flow cytometry.

[0119] [Figure 4U] Figure 4U is a representative flow cytometry plot showing co-staining of pp65495~503 tetramers with pp65495~503-presenting mNeon virus in pp65495~503 peptide-enriched T cells after 15 days of expansion. These T cells are gated for viable CD8+CD3+ T cells.

[0120] [Figure 4V]FIG. 4V is a representative flow cytometry plot showing the percentage of GFP+ T cells (stained with 12 pooled HLA-A2:CMV antigen mNeon viruses) from four different CMV seropositive donors before or after pooled CMV peptide-induced expansion, similar to FIG. 4T.

[0121] [Figure 4W] Figure 4W is an MA plot showing bulk RNA-seq analysis of CMV-pp65 TCR T cells incubated with pp65495-503 tetramer or pp65495-503-presenting ENTER virus. Genes with log2 fold change and adjusted p-value <0.01 are highlighted in red.

[0122] [Figure 5A] Figures 5A-5F show schematics of ENTER-seq for massively parallel measurements of antigen peptide sequences, TCR sequences, and transcriptomes. Figure 5A shows a schematic of the ENTER-seq workflow. A library of pooled viruses presenting individual pMHC was incubated with T cells for 2 h. GFP+ T cells are sorted for droplet-based single-cell genomics profiling (e.g., 10x Genomics 5' kit for gene expression and V(D)J immune profiling).

[0123] [Figure 5B] Figure 5B shows the viral RNA engineering strategy for droplet-based single-cell capture. 10x Genomics capture tag is inserted into the linker region between B2M and HLA-A2. 10x Genomics PCR hand is inserted after the CMV promoter. CMV: CMV promoter; SP: signal peptide sequence; Peptide: antigen peptide; B2M: beta 2 myeloglobulin; MHC class I: HLA-A0201 allele; LTR: long terminal repeat; TSO: template switching oligo sequence.

[0124] [Figure 5C]Figure 5C shows a schematic of the T cell mixing experiment for ENTER-seq.

[0125] [Figure 5D] Figure 5D shows the Pp65(495-503)-TCR T cell UMI counts (x-axis) and NY-ESO-1157-165-TCR UMI counts (y-axis) associated with each cell barcode (dot). Colors are assigned as NY-ESO-1157-165-TCR+ T cells (light blue), Pp65(495-503)-TCR+ T cells (red), and doublets (green, both NY-ESO-1157-165-TCR and Pp65(495-503)-TCR).

[0126] [Figure 5E] Figure 5E shows a scatter plot of TCR UMI counts after excluding doublets, colored by the enrichment ratio of pp65(495-503) antigen UMI counts among total UMI counts (left) and the enrichment ratio of nyeso157-165 antigen UMI counts among total UMI counts (right).

[0127] [Figure 5F] Figure 5F shows the pp65(495-503) antigen UMI counts (x-axis) and NY-ESO-1157-165 antigen UMI counts (y-axis) associated with each cell barcode (dot) after excluding doublets, color-assigned as NY-ESO-1157-165-TCR+ T cells (light blue) and Pp65(495-503)-TCR+ T cells (red).

[0128] [Figure 5G] Figures 5G-5N provide a schematic summary of the results of experiments performed to characterize T cell subsets by ENTER-seq. Figure 5G is a UMAP plot showing CMV-specific T cells (yellow) and bystander T cells (blue) by clustering before and after removal of the ENTER-induced gene signature.

[0129] [Figure 5H] FIG. 5H is a UMAP plot showing ten clusters of human CD8 T cell subsets.

[0130] [Figure 5I] FIG. 5I shows a UMAP plot showing the abundance of surface protein CD127 from CITE-seq and expression of genes (CCR7, SELL, and LEF1) in naive T cells.

[0131] [Figure 5J] FIG. 5J shows a UMAP plot showing gene expression of cytolytic molecules.

[0132] [Figure 5K] FIG. 5K shows a UMAP plot showing gene expression of markers of MAIT cells.

[0133] [Figure 5L] FIG. 5L shows a UMAP plot showing the expression of marker genes for each subset / cluster.

[0134] [Figure 5M] FIG. 5M is a UMAP plot showing the origin of the donors.

[0135] [Figure 5N] FIG. 5N shows the percentage of T cell subsets of 10 clusters of CMV antigen-specific T cells (ENTER+) and bystander T cells (ENTER-), separated by donor origin.

[0136] [Figure 6A] Figures 6A-6I summarize the ENTER-seq results of ex vivo expanded CMV-specific primary T cells. Figure 6A shows a schematic of the CMV antigen peptide-induced T cell expansion and ENTER-seq workflow.

[0137] [Figure 6B]FIG. 6B is a UMAP plot showing cells that were bound (ENTER+, colored yellow) or not bound (ENTER-, colored blue) by the CMV antigen-presenting virus.

[0138] [Figure 6C] FIG. 6C shows a UMAP plot showing CITE-seq of surface protein expression of CD45RA (naive marker) and CD45RO (memory marker).

[0139] [Figure 6D] FIG. 6D is a UMAP plot showing ten clusters of human CD8+ T cell subsets labeled with different colors.

[0140] [Figure 6E] FIG. 6E is a bar graph showing the number of CMV antigen-specific T cells that recognize specific CMV antigen epitopes in donor #1 (colored black) and donor #2 (colored gray).

[0141] [Figure 6F] FIG. 6F shows a UMAP plot showing the abundance of CMV antigenic epitopes per cell for the top three CMV antigenic epitopes identified from FIG. 6E.

[0142] [Figure 6G] FIG. 6G is a heatmap showing column-scaled expression of representative genes associated with effector function or Treg signature among different CMV antigen-specific T cells.

[0143] [Figure 6H] Figure 6H is a UMAP plot (left) showing the clonal expansion size of CMV antigen-specific T cells colored by the number of cells in each clonotype. The violin plot (right) shows the distribution of clonal sizes in different CMV antigen-specific T cells colored by antigen epitope.

[0144] [Figure 6I] Figure 6I is a violin plot of cytokine and transcription factor expression in pp65495-503 specific TCR clones color-coded by CDR3 clone. Simplified model (right).

[0145] [Figure 6J] Figures 6J-6P summarize the results of TCR clonotype analysis of ex vivo expanded CMV-specific T cells. Figure 6J shows a UMAP plot showing the clonal expansion size of CMV antigen-specific T cells (ENTER+) and bystander T cells (ENTER-).

[0146] [Figure 6K] Figure 6K: TCR clonotypes of CMV antigen-specific T cells colored by donor. Each circle represents a clonotype with identical CDR3 nucleotide sequences. The size of the circle represents the number of cells of each clonotype.

[0147] [Figure 6L] FIG. 6L shows a scatter plot showing the correlation between clonal expansion size and cytotoxicity gene score.

[0148] [Figure 6M] FIG. 6M shows a scatter plot showing the correlation between clonal expansion size and exhausted or activated gene scores.

[0149] [Figure 6N] FIG. 6N is a summary table of pp65495-503-specific TCR clones showing convergent TCR clonotypes with identical CDR3 amino acid sequences.

[0150] [Figure 6O] FIG. 6O is a summary table of US874-82 and UL100200-208 specific TCR clones.

[0151] [Figure 6P] Figure 6P shows the proportion of T cell subsets in pp65495-503-specific TCR clones separated by CDR3 clones and colored by the 10 clusters. Coefficients and p-values ​​in Figures 6L-6M are calculated by Pearson correlation.

[0152] [Figure 7A] Figures 7A-7K summarize the ENTER-seq results of primary CMV-specific T cells isolated directly (e.g., without in vitro expansion) from the blood of CMV-seropositive patients. Figure 7A shows a schematic of the isolation and ENTER-seq workflow of primary T cells from patient blood.

[0153] [Figure 7B] Figure 7B is a UMAP plot (left) showing cells that bound (ENTER+, colored yellow) or did not bind (ENTER-, colored blue) CMV antigen-presenting virus, and the UMAP plot (right) shows 13 clusters of human CD8+ T cell subsets labeled with different colors.

[0154] [Figure 7C] Figure 7C shows a UMAP plot showing surface protein expression of CD45RA, CD45RO and representative genes from CITE-seq.

[0155] [Figure 7D] FIG. 7D is a heatmap showing the scaled z-scores of expression of genes associated with diverse functions (eg, type I IFN, cytotoxicity, etc.) across different clusters.

[0156] [Figure 7E] FIG. 7E is a UMAP plot showing CMV antigen specificity colored by antigenic epitope.

[0157] [Figure 7F]FIG. 7F is a UMAP plot (left) showing clonal expansion size of CMV antigen-specific T cells colored by the number of cells in each clonotype.

[0158] [Figure 7G] Figure 7G is a violin plot showing the number of pMHC binding per cell in T cells with different clone sizes. P values ​​were calculated by Mann-Whitney test. nsp>0.05; *p<0.05; **p<0.01, ***p<0.001, ****p<0.0001.

[0159] [Figure 7H] FIG. 7H is a CITE-seq density plot showing surface expression of CD45RA and CD45RO on pp65-specific T cells from donor #1 (orange) and donor #2 (blue) before and after peptide-induced expansion.

[0160] [Figure 7I] FIG. 7I shows flow cytometry plots showing CD45RA and CD45RO in pp65-specific T cells from donor #1 and donor #2 before and after peptide-induced expansion.

[0161] [Figure 7J] Figure 7J is a density plot showing the distribution of type I IFN ISG gene scores and cytotoxicity gene scores before and after peptide-induced expansion in the top three TCR clones of pp65-specific T cells (left). Density plots show the expression of IL13 and EOMES before and after peptide-induced expansion in the top three TCR clones of pp65-specific T cells (right).

[0162] [Figure 7K] FIG. 7K is a proposed model of the phenotypic transition of CMV-specific T cells during ex vivo expansion.

[0163] [Figure 7L]Figures 7L-7T summarize the results of intraclonal phenotypic diversity of CMV-specific T cells isolated directly from patient blood. Figure 7L shows UMAP plots showing the expression of key genes associated with diverse functions (type I IFN ISGs, cytotoxicity, chemokines, and transcription factors).

[0164] [Figure 7M] FIG. 7M shows a UMAP plot showing subset clustering of primary T cells isolated directly from patient blood samples before and after depletion of the ENTER-induced gene signature.

[0165] [Figure 7N] FIG. 7N shows a UMAP embedded density plot showing the density of CMV antigen-specific T cells against different CMV antigen epitopes.

[0166] [Figure 7O] FIG. 7O is a bar graph showing the percentage of US874-82-specific T cells with a shared TCR before and after peptide-induced expansion.

[0167] [Figure 7P] Figure 7P shows the number of US874-82 specific T cells isolated from fresh PBMCs before expansion, sorted by donor and colored by TCR CDR3 sequence. Shared TCR sequences before and after expansion are boxed in black.

[0168] [Figure 7Q] FIG. 7Q is a summary table of the top TCR clones of US874-82-specific T cells (identified from E) including indications of cell numbers and abundance ratios before and after expansion.

[0169] [Figure 7R] FIG. 7R shows the number of T cell subsets in pp65495-503-specific TCR clones separated by CDR3 clones and colored by the 13 clusters.

[0170] [Figure 7S] FIG. 7S is a heat map showing the expression of genes associated with diverse functions among different TCR clone-specific T cells.

[0171] [Figure 7T] FIG. 7T shows density plots showing the expression of IL13 and IL4 in different TCR clone-specific T cells before and after peptide-induced expansion. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0172] The present disclosure generally relates to systems, compositions, and methods for identifying and elucidating receptor-ligand pairings, including MHC peptide / T cell receptor and antigen / B cell receptor (antibody) pairs, e.g., for presenting ligand proteins, delivering payloads, and recording receptor specificity. Some embodiments of the present disclosure relate to elucidating interactions between T cell receptors and MHC peptides, between antibodies and antigens, or between B cell receptors and B cell antigens (including intracellular / secreted epitopes / cell surface antigen epitopes), and between other ligand receptors (e.g., CD40 ligand vs. CD40). The present disclosure also relates to compositions and methods for the delivery of proteins or nucleic acids to user-defined target cells. In particular, some embodiments of the present disclosure relate to a modular viral display and delivery platform for elucidating ligand-receptor interactions, delivering cargo to target cells, and linking ligand-receptor interactions to cellular states. In some embodiments, lentiviruses can be engineered at multiple levels, including (1) displaying user-defined ligand proteins on the viral surface, (2) engineering fusogens to achieve receptor-specific cell entry of the cognate ligand-presenting virus, (3) carrying fluorescent proteins to track the engineered virus, (4) delivering cargo upon recognition of paired ligand-receptor, and (5) modifying viral RNA to record ligand information by sequencing. This technology is called "ENTER" ("Lentivirus-Mediated Cell Entry via Engineered Ligand-Receptor Interactions"), and is a novel method for the identification of lentiviruses. e Antiviral-mediated cell nt ry by eENTER, called engineered ligand-receptor interaction (ENTER), can systematically deorphanize interaction pairs including TCR-pMHC, antibody-antigen, costimulatory ligand-receptor, and B cell antigen-BCR. In some embodiments, ENTER allows gene delivery in a receptor-specific manner, allowing selective manipulation of cellular behavior in antigen-specific T and B cells. In some embodiments, ENTER can be combined with droplet-based single-cell genomic profiling (ENTER-seq) to measure antigen specificity, TCR repertoire, gene expression, and surface protein landscape in individual human primary T cells.

[0173] I. General Techniques The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are described in Sambrook, J., & Russell, DW (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russell, DW (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). “Sambrook”);Ausubel,FM(1987).Current Protocols in Molecular Biology.New York,NY:Wiley(including supplements through 2014);Bollag,DMet al.(1996).Protein Methods.New York,NY:Wiley-Liss;Huang,L.et al.(2005).Nonviral Vectors for Gene Therapy.San Diego:Academic Press;Kaplitt,MGet al.(1995).Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, KB, Ferre, F.& Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, EA (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, SLet al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements through 2014); and Makrides, SC (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences BV, the disclosures of which are incorporated herein by reference.

[0174] II. Definition Unless otherwise defined, all technical terms, notations, and other scientific terms used herein are intended to have the meaning commonly understood by those skilled in the art to which this disclosure pertains. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly used by those skilled in the art using conventional methodology.

[0175] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes one or more cells (including mixtures thereof). As used herein, "A and / or B" is used to include all of the following options: "A," "B," "A or B," and "A and B."

[0176] " Lentivirus " as used herein refers to a genus of the Retroviridae family. Lentivirus is unique among retroviruses in that it can infect non-dividing cells, and can deliver large amounts of genetic information to the DNA of host cells, making it one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentivirus. Lentivirus-derived vectors provide a means to achieve significant gene transfer levels in vivo.

[0177] The terms "cell", "cell culture", and "cell line" refer not only to a particular subject cell, cell culture, or cell line, but also to the progeny or potential progeny of such a cell, cell culture, or cell line, regardless of the number of transplants or passages in culture. It should be understood that not all progeny are completely identical to the parent cell. This is because certain modifications may occur in subsequent generations due to mutations (such as deliberate or accidental mutations) or environmental influences (such as methylation or other epigenetic modifications), so that the progeny may not actually be identical to the parent cell, but still be within the scope of the terms used herein, so long as the progeny retain the same function as the original cell, cell culture, or cell line.

[0178] The term "exogenous" refers to any substance that is introduced into or produced outside an organism, cell, tissue, or system.

[0179] As used herein, the term "linker", also referred to as "spacer" or "spacer domain", refers to an amino acid or amino acid sequence that is optionally placed between two amino acid sequences in the fusion protein of the present invention.

[0180] The term "biological sample" or "sample" refers to any solid or liquid sample isolated from an individual or subject. For example, it can refer to any solid (e.g., tissue sample) or liquid sample (e.g., blood) isolated from an animal (e.g., human), including, but not limited to, a biopsy (e.g., solid tissue sample) or blood (e.g., whole blood). Such samples may be, for example, fresh, fixed (e.g., fixed in formalin, alcohol, or acetone), paraffin-embedded, or frozen prior to analysis. In some embodiments, the biological sample is obtained from a tumor (e.g., pancreatic cancer). A "test biological sample" is a biological sample that has been the subject of analysis, monitoring, or observation. A "reference biological sample," including the same type of biological sample (e.g., the same type of tissue or cell), is a control for the test biological sample.

[0181] The term "operably linked" as used herein refers to a physical or functional linkage between two or more elements, e.g., polypeptide or polynucleotide sequences, such that they can operate in an intended manner. For example, the term "operably linked" when used in the context of coding and promoter sequences in a nucleic acid construct (e.g., lentiviral vector) or nucleic acid molecule described herein means that the coding and promoter sequences are in frame and appropriately separated in space and distance to allow binding of each by a transcription factor or RNA polymerase to affect transcription. It should be understood that operably linked elements may be contiguous or non-contiguous (e.g., linked to each other via a linker). In the context of a polypeptide construct, "operably linked" refers to a physical linkage (e.g., directly or indirectly linked) between amino acid sequences (e.g., different segments, portions, regions, or domains) to provide a described activity of the construct. The operably linked segments, portions, regions, and domains of the polypeptide or nucleic acid molecules disclosed herein may be contiguous or non-contiguous (e.g., linked to each other via a linker). In some embodiments, operably linked segments, portions, regions, and domains of the polypeptides described herein are fused in-frame to each other.

[0182] All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species to which the compositions and methods disclosed herein may be applied. Thus, these terms include, but are not limited to, genes and gene products from human and mouse. Where genes or gene products from a particular species are disclosed, it is understood that this disclosure is intended to be illustrative only and should not be construed as limiting unless the context in which it appears clearly indicates so. Thus, for example, in some embodiments, genes or gene products disclosed herein with respect to mammalian nucleic acid and amino acid sequences are intended to encompass homologous and / or orthologous genes and gene products from other animals, including, but not limited to, other mammals, fish, amphibians, reptiles, and birds. In some embodiments, the genes, nucleic acid sequences, amino acid sequences, peptides, polypeptides, and proteins are human. The term "gene" is intended to include variants thereof.

[0183] The cell populations described herein may be any mammalian cell population. In some embodiments, the cell population is a human, mouse, rat, or non-human primate cell population. In some embodiments, the cell population is a somatic cell population or a germ cell population. In some embodiments, the cell population comprises antigen-specific cells (e.g., cells that bind to a particular antigen). In some embodiments, the population of antigen-specific cells comprises immune cells. In some embodiments, the population of antigen-specific cells comprises B cells and / or T cells. In some embodiments, the cell population comprises a homogenous cell population. In some embodiments, the cell population comprises a heterogeneous cell population. In some embodiments, the cell population is a cell population isolated from a subject. The subject may be a human subject (e.g., a human subject suffering from a disease), a mouse subject, a rat subject, or a non-human primate subject. In some embodiments, the cell population is isolated from the subject's blood or tumor.

[0184] Where a range of values ​​is provided, unless the context clearly indicates otherwise, it is understood that each intervening value between the upper and lower limit of that range (to the nearest tenth of the lower limit) and any other stated or intervening value in the stated range is included in the disclosure. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are included within the disclosure subject to any limit specifically excluded in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0185] As will be understood by one of ordinary skill in the art, for all purposes, including as to providing descriptive requirements, all ranges disclosed herein include any and all possible subranges and combinations of those subranges. Any recited range is fully described and can be readily recognized as being capable of dividing the same range into at least one half, one third, one quarter, one fifth, one tenth, etc., in equal parts. As a non-limiting example, each range discussed herein can be readily divided into a lower third, a middle third, and an upper third, etc. As will be understood by one of ordinary skill in the art, all terms such as "up to," "at least," "greater than," "less than," etc. refer to ranges that are inclusive of the recited numbers and can subsequently be divided into subranges as described above. Additionally, as will be understood by one of ordinary skill in the art, ranges include individual members. Thus, for example, a group having 1-3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1-5 items refers to a group having 1, 2, 3, 4, or 5 items, etc.

[0186] In this specification, a particular range is provided using numerical values ​​following the term "about". In this specification, the term "about" is used to provide literal support for the exact numerical value following the term and a numerical value close to or approximately the numerical value following the term. When determining whether a number is close to or close to a specifically stated number, the unstated number that is close or close may be a number that provides a substantial equivalent to the specifically stated number in the context in which it is presented. If the degree of approximation is not otherwise clear from the context, "about" means either within plus or minus 10% of the provided value or a value rounded to the nearest significant figure, and includes the provided value in all cases. In some embodiments, the term "about" refers to the specified value ± up to 10%, up to ± 5%, or up to ± 1%.

[0187] It is understood that the aspects and embodiments of the disclosure described herein include those that "comprise," "consist," and "consist essentially of." As used herein, "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional unrecited elements or method steps. As used herein, "consisting" excludes any element, step, or ingredient not specified in the claimed composition or method. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any recitation herein of the term "comprising," particularly in a description of a component of a composition or a description of a step of a method, is understood to encompass compositions and methods that consist essentially of the recited components or steps.

[0188] Headings, e.g., (a), (b), (i), etc., are provided solely to facilitate the reading of the specification and claims. The use of headings in the specification or claims does not require that the steps or elements be performed in alphabetical or numerical order or in the order in which they are presented.

[0189] III. Compositions of the Present Disclosure Engineered lentiviruses Provided herein are compositions comprising lentiviruses engineered to a) present a specific ligand on the cell surface, b) have mutated fusogens that enable the virus to fuse with and enter only host cells that have a receptor that naturally pairs with the ligand, c) deliver a reporter to the host cell, and d) tag the viral RNA so that single-cell sequencing can be performed.

[0190] In some embodiments, lentiviruses can be further engineered to contain a defective integrase such that the viral RNA does not integrate into the host cell's genome, thereby avoiding integration-induced mutations of the host genome.

[0191] Ligand In some embodiments, the engineered lentivirus of the present disclosure comprises one or more user-defined ligands displayed on the viral cell surface. In some embodiments, the ligand is heterologous to the lentivirus that displays the ligand on its surface, e.g., the ligand is derived from a heterologous source, such as another cell or another virus. Non-limiting examples of suitable ligand species include cell surface receptors, adhesion proteins, glycoproteins, carbohydrates, lipids, glycolipids, lipoproteins, and surface-bound lipopolysaccharides, integrins, mucins, and lectins. In some embodiments, the ligand is or can include a protein. In some embodiments, the ligand is or can include an epitope. Those skilled in the art will understand that the term "epitope" refers to a specific antigen-binding site of an antigen-binding polypeptide, e.g., an antigenic determinant that interacts with a variable region of an antibody molecule known as a paratope. A single antigen can have more than one epitope. Thus, different antibodies can bind to different regions on an antigen and result in different biological effects. The term "epitope" also refers to a site on an antigen to which B cells respond. It also refers to the region of the antigen to which the antibody binds. Epitopes can be defined as structural or functional epitopes. Functional epitopes are generally a subset of structural epitopes and have residues that directly contribute to the affinity of the interaction. Epitopes can be linear or conformational, i.e., composed of non-linear amino acids. In certain embodiments, epitopes include determinants that are chemically active surface molecular groups of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments can have specific three-dimensional structural characteristics and / or specific charge characteristics. In some embodiments, the ligand can be or include a cell surface protein or intracellular protein or part thereof. In some embodiments, the ligand can be or include an MHC peptide, an antibody, an intracellular antigen, a secreted protein, or other form of protein or peptide.

[0192] In some embodiments, if the ligand does not contain a native transmembrane domain, a signal peptide and a transmembrane domain are added and fused to the ligand. Thus, in some embodiments, the transmembrane domain is operably linked to the transmembrane domain. In some embodiments, the TM domain can promote viral carryover efficiency. In some embodiments, the transmembrane domain is a heterologous transmembrane domain. In some embodiments, the transmembrane domain is a heterologous transmembrane domain derived from HLA-DRA, HLA-DRB, HLA-A2, ICAM1, CD43, CD162, CD62L, CD49d, or LFA-1. In some embodiments, the TM is replaced with an optimized TM (e.g., TM derived from ICAM1, PDGFR, etc.).

[0193] In some embodiments, the ligand can be engineered to be initially displayed on the surface of a cell line suitable for lentivirus production (e.g., HEK293T, a common cell type for producing lentiviruses). The ligand can be carried over onto the viral surface during viral budding to generate viral particles.

[0194] In some embodiments, an engineered lentivirus of the present disclosure comprises a defective integrase protein.

[0195] Reporter genes In some embodiments, the lentiviruses described herein may include a reporter (e.g., a reporter protein). In some embodiments, the lentivirus includes a nucleic acid encoding a reporter (e.g., a reporter protein). As used herein, a reporter is generally a protein or gene that can be detected when expressed in a retrovirus and / or a target cell. In some embodiments, the presence or absence of the reporter in a target cell or a subset of target cells in a cell population allows the cells to be sorted (e.g., using flow cytometry and / or fluorescence activated cell sorting).

[0196] In some embodiments, the reporter is a fluorescent protein. The fluorescent protein may be green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP). Exemplary fluorescent proteins may be those described in U.S. Pat. No. 7,060,869. Engineered lentiviral particles that display specific ligands deliver fluorescent proteins to target cells upon cognate receptor-ligand interaction.

[0197] In some embodiments, the reporter is mNeon, a monomeric green fluorescent protein that is significantly brighter than GFP.

[0198] In some embodiments, the reporter can be operably linked (e.g., fused) to a viral structural gene. In some embodiments, the structural gene can be a nucleocapsid protein (NC) or a Gag protein. Those skilled in the art will recognize that other structural genes may be used without departing from the teachings of the present disclosure.

[0199] In some embodiments, the barcoded RNA is encapsulated in a viral particle, e.g., a viral particle produced by an engineered lentivirus. In some embodiments, the RNA encodes a ligand. In some embodiments, the RNA encodes a gene of interest to be delivered to a target cell, e.g., a host cell. In some embodiments, the RNA is read by next-generation sequencing technology. In some embodiments, the RNA comprises a capture sequence.

[0200] Fusogen The term "fusogen" or "fusion molecule" as used herein generally refers to any molecule that can promote, catalyze, or induce membrane fusion when present on the surface of a virus. Non-limiting examples of fusogens suitable for the compositions and methods of the present disclosure include fusogens derived from viruses or fusogens endogenously expressed in host cells, e.g., mammalian cells (e.g., human cells). In some embodiments, the fusogens of the present disclosure are glycoproteins. In some embodiments, the fusogens of the present disclosure are viral glycoproteins. Exemplary viral fusogens suitable for the compositions and methods disclosed herein include those belonging to class I, II, and III viral fusion proteins that are produced by enveloped viruses to promote viral-host membrane fusion. Additional information on this can be found, for example, in Vance TDR and Lee JE (Curr.Biol.Jul 6,30(13),R750-R754,2020), which is incorporated herein by reference. In some embodiments, the fusogens of the present disclosure belong to class I viral fusion proteins, i.e., those capable of forming coiled-coil trimers, including but not limited to those derived from influenza virus, coronavirus, HIV, and Ebola virus. In some embodiments, the fusogens of the present disclosure belong to class II viral fusion proteins, i.e., those capable of generating an elongated extracellular domain composed primarily of β-sheets that transition from dimers to trimers during fusion and become hairpin trimers after fusion. Suitable class II viral fusion proteins include but are not limited to those derived from Dengue virus, West Nile virus, Zika virus, and tick-borne encephalitis virus. In some embodiments, the fusogens of the present disclosure belong to class III viral fusion proteins, i.e., those capable of combining elements from the two aforementioned classes and adopting a post-fusion conformation that includes both a coiled-coil trimerization region similar to class I and an elongated hairpin trimer like class II.Suitable class III viral fusion proteins include, but are not limited to, those derived from vesicular stomatitis virus (VSV), herpes simplex virus 1 (HSV1), and rabies virus. In some embodiments, the fusogen of the compositions and methods disclosed herein is or comprises a vesicular stomatitis virus G (VSV-G) protein. In some embodiments, the fusogen of the compositions and methods disclosed herein is or comprises a viral fusion protein derived from a human endogenous retrovirus (HERV), such as measles virus, Sindbis virus, baboon endogenous retrovirus (BaEV), murine leukemia virus, rabies virus, Nipah virus, RD114 retrovirus, gibbon ape leukemia virus (GALV), Tupaia paramyxovirus (TPMV), or ERVW-1 (e.g., Syncytin-1).

[0201] In some embodiments, the engineered lentivirus can include modified fusogens, for example, to promote fusion of the virus with a cell membrane. The fusogens can be modified to promote fusion of the virus with a cell membrane without the need for a viral surface glycoprotein. In some embodiments, the fusogens include modified vesicular stomatitis virus G (VSV-G) viral envelope protein. In some embodiments, the VSV-G polypeptide is or includes the sequence of SEQ ID NO: 56. In some embodiments, the modified VSV-G viral envelope protein includes one or more substitutions, for example, substitutions that abolish binding of VSV-G to a cellular receptor. In some embodiments, the VSV-G envelope protein can include one or more amino acid substitutions at positions corresponding to any one of positions H8, K47, Y209, and R354 of the VSV-G polypeptide. It is within the knowledge of one of skill in the art to know how to align amino acid sequences, for example, sequences of multiple VSV-G polypeptides, to determine which amino acids at specific positions referred to herein "correspond" to amino acids of another VSV-G amino acid sequence not described herein. Thus, the term "corresponding position" as used herein is well known within the art.

[0202] The modified VSV-G viral envelope proteins disclosed herein can also include conservative modifications and substitutions at other positions of VSV-G (e.g., those that abolish binding of VSV-G to cellular receptors). Such conservative substitutions include those described in Dayhoff 1978 (supra) and Argos 1989 (supra). For example, amino acids belonging to one of the following groups represent conservative changes: Group I: Ala, Pro, Gly, Gln, Asn, Ser, Thr; Group II: Cys, Ser, Tyr, Thr; Group III: Val, Ile, Leu, Met, Ala, Phe; Group IV: Lys, Arg, His; Group V: Phe, Tyr, Trp, His; and Group VI: Asp, Glu. In some embodiments, the amino acid substitutions in the amino acid sequence of the modified VSV-G viral envelope protein disclosed herein are independently selected from the group consisting of alanine (A) substitutions, arginine (R) substitutions, asparagine (N) substitutions, aspartic acid (D) substitutions, leucine (L) substitutions, lysine (K) substitutions, phenylalanine (F) substitutions, lysine substitutions, glutamine (Q) substitutions, glutamic acid (E) substitutions, serine (S) substitutions, and threonine (T) substitutions, and any combination thereof. In some embodiments, the amino acid substitutions in the amino acid sequence of the modified VSV-G viral envelope protein disclosed herein include alanine substitutions.

[0203] In some embodiments, the modified VSV-G viral envelope protein comprises an amino acid substitution corresponding to the K47Q or R354A substitution of the sequence of SEQ ID NO: 56. In some embodiments, the modified VSV-G viral envelope protein comprises a K47Q and R354A substitution. As described above, other viral fusogens capable of fusing viral particles with cell membranes may also be suitable for use in ENTER.

[0204] Single Cell Sequencing ENTER can be applied to combine with any single-cell method that uses whole cells as input and includes a step of reverse transcription. For example, it is compatible with any published or commercially available single-cell sequencing technology, such as any single-cell RNA-seq (such as 10x Genomics) including 5' or 3' approaches, scTCR / BCR-seq (10x Genomics) to identify immune VDJ recombination in B and T cells, CITE-seq / ECCITE to identify surface markers using barcoded antibodies, and single-cell CRISPR perturb-seq to perturb genes using CRISPR combined with single-cell transcriptome.

[0205] IV. Methods of the Disclosure Methods for identifying ligand-receptor pairs Provided herein are methods for identifying ligand-receptor pairs by (i) providing at least one engineered lentivirus disclosed herein, (ii) combining the lentivirus with a cell population, and (iii) sorting the cell population based on the presence of a reporter gene, thereby identifying the ligand-receptor pair. In some embodiments, the engineered lentivirus comprises a ligand displayed on the surface of the lentivirus (wherein the ligand is heterologous to the lentivirus), a fusogen comprising a modified viral envelope protein (wherein the fusogen is capable of fusing the lentivirus to a host cell (e.g., capable of promoting, catalyzing, or inducing fusion of the lentivirus with a host cell), and the host cell comprises an endogenous receptor for the ligand, a reporter protein operably linked (e.g., fused) to a lentiviral structural protein, and a barcoded RNA.

[0206] The lentivirus can be combined with the cell population for a defined period of time. In some embodiments, the period may be measured in seconds, minutes, hours, or days. In some embodiments, the period is 0-30 seconds, 15-45 seconds, 30-60 seconds, 45-90 seconds, 60-90 seconds, or 60-120 seconds. In some embodiments, the virus is combined with the cell population and contacted for 0-30 seconds, 15-45 seconds, 30-60 seconds, 45-90 seconds, 60-90 seconds, or 60-120 seconds. In some embodiments, the period is 1-2 minutes, 1-5 minutes, 1-10 minutes, 2-10 minutes, 5-10 minutes, 5-20 minutes, 10-20 minutes, 25-30 minutes, 25-60 minutes, 30-45 minutes, 30-40 minutes, 40-60 minutes, 50-70 minutes, or 60-120 minutes. In some embodiments, the retrovirus is combined with the cell population and contacted for 1-2 minutes, 1-5 minutes, 1-10 minutes, 2-10 minutes, 5-10 minutes, 5-20 minutes, 10-20 minutes, 25-30 minutes, 25-60 minutes, 30-45 minutes, 30-40 minutes, 40-60 minutes, 50-70 minutes, or 60-120 minutes. In some embodiments, the period is 1-2 hours, 1-5 hours, 1-3 hours, 2-5 hours, 3-6 hours, 3-12 hours, 6-12 hours, 12-18 hours, 12-24 hours, 15-30 hours, 18-24 hours, 24-48 hours, 24-36 hours, or 36-50 hours. In some embodiments, the virus is combined with the cell population and contacted for 1-2 hours, 1-5 hours, 1-3 hours, 2-5 hours, 3-6 hours, 3-12 hours, 6-12 hours, 12-18 hours, 12-24 hours, 15-30 hours, 18-24 hours, 24-48 hours, 24-36 hours, or 36-50 hours. In some embodiments, the period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 5-15 days. In some embodiments, the virus is combined with the cell population and contacted for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 5-15 days. In one aspect, the method includes combining the lentivirus with the cells for 2 hours to identify ligand-receptor pairs.

[0207] In some embodiments, the lentivirus may be combined with the cell population at a temperature ranging from 4°C to 42°C, 4°C to 8°C, 4°C to 10°C, 8°C to 15°C, 10°C to 20°C, 18°C ​​to 23°C, 20°C to 30°C, 25°C to 35°C, 30°C to 40°C, or 37°C to 42°C.

[0208] In some embodiments, to identify ligand-receptor pairs, the cell population and virus can be incubated for about 2 hours at 37° C. However, modifications are within the scope of this disclosure.

[0209] Methods for identifying T cell receptors and paired pMHC Further provided herein is a method for identifying a T cell receptor and paired pMHC, comprising: (i) providing an engineered lentivirus as disclosed herein; (ii) combining the lentivirus with a cell population; and (iii) sorting the cell population based on the presence of a reporter, thereby identifying the T cell receptor. In some embodiments, the engineered lentivirus comprises a pMHC displayed on the viral surface, a fusogen comprising a modified VSV-G viral envelope protein (wherein the fusogen is capable of fusing the lentivirus to a host cell, the host cell comprising a T cell receptor for the pMHC), a reporter transgene operably linked (e.g., fused) to the lentiviral structural protein, and a barcoded RNA. In some embodiments, the method provides a mixture of viruses presenting different MHC peptides (e.g., a pool of MHC peptides). In some embodiments, the T cells can be a population of cell lines. In another embodiment, the cells are human primary T cells.

[0210] In some embodiments, the presented pMHC can be engineered to use a single-chain format (Figure 2A), i.e., the RNA can tandemly encode the signal peptide, the antigenic peptide, a G4S linker, a b2m gene, a G4S linker, and the MH allele.

[0211] In some embodiments, the method may further comprise performing single cell sequencing of viral RNA and cellular receptor sequences to identify MHC peptide sequences and TCR receptor information.

[0212] In addition to identifying novel TCR receptors using known pMHC pools, the methods provided herein can be used to identify matching MHC peptides against known T cell receptors. For example, as shown in the Examples below, if the alpha and beta chains of a known T cell receptor are expressed in a TCR negative cell line such as Jurkat-76, a pool of viruses bearing pMHC candidates can be mixed with the cells.

[0213] Methods for identifying B cell receptors and paired antigens - Patents.com Also provided herein is a method for identifying a B cell receptor or antibody, the method comprising: (ii) providing an engineered lentivirus as disclosed herein; (iii) combining the lentivirus with a B cell population; and (iv) sorting the cell population based on the presence of a reporter, thereby identifying the B cell antigen. In some embodiments, the engineered lentivirus comprises an epitope displayed on the lentivirus surface (wherein the epitope is operably linked (e.g., fused) to an ICAM1 transmembrane domain), a fusogen comprising a modified VSV-G viral envelope protein (wherein the fusogen is capable of fusing the lentivirus to a host cell (e.g., capable of promoting, catalyzing, or inducing fusion of the lentivirus with a host cell), the host cell comprising a B cell receptor for the intracellular epitope), a reporter transgene operably linked (e.g., fused) to a lentiviral structural protein, and a barcoded RNA. In some embodiments, the antigen is a cell surface membrane protein, an intracellular protein, a secreted protein, or in other forms capable of being expressed on the cell surface (eg, a glycosylated molecule).

[0214] In some embodiments, the method may further comprise a step of single-cell sequencing of viral RNA and cell receptor sequences to identify antigens and matching B-cell receptor (BCR, antibody) information. In a further aspect, the method may identify novel antibody / BCR and antigen pairs.

[0215] Methods for intracellular delivery of molecules of interest - Patents.com In some embodiments, the disclosure further provides a method for delivering a molecule of interest, e.g., a nucleic acid or protein of interest, to a user-defined target cell, comprising providing an engineered lentivirus comprising a heterologous ligand displayed on the surface of the lentivirus, a fusogen comprising a modified viral envelope protein (wherein the fusogen is capable of fusing the lentivirus to a host cell (e.g., capable of promoting, catalyzing, or inducing fusion of the lentivirus with a host cell), the host cell comprising an endogenous receptor for the ligand), a reporter protein operably linked (e.g., fused) to a lentiviral structural protein, and a barcoded RNA; and contacting the lentivirus with a cell mixture comprising target cells to deliver the nucleic acid or protein only to the target cells, the target cells expressing a receptor specific for the ligand on the lentiviral surface. In some embodiments, the ligand is modified to deliver the cargo to the user-defined target cell. In some embodiments, the nucleic acid of interest is packaged within the engineered lentiviral particle. In some embodiments, the protein of interest is operably linked (e.g., fused) to a gag protein of the lentivirus. In some embodiments, the protein of interest is replaced with a reporter. In some embodiments, the target cell is in vivo. In some embodiments, the target cell is ex vivo. In some embodiments, the target cell is in vitro. In some embodiments, the target cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the target cell is an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a T cell and the receptor is a T cell receptor. In some embodiments, the immune cell is a B cell. In some embodiments, the immune cell is a B cell and the receptor is a B cell receptor. In some embodiments, the human cell is a primary human blood cell (PBMC).

[0216] Methods for delivering molecules of interest to cells As outlined above, one aspect of the disclosure is a method for selectively depleting or enriching a target cell population in a cell mixture, the method comprising: (a) providing an engineered lentivirus according to any one of claims 1 to 17; and (b) providing a cell mixture comprising (i) a target cell population expressing a receptor specific for a ligand displayed on the surface of the engineered lentivirus, and (ii) a non-target cell population that does not express the receptor; contacting the engineered lentivirus with the cell population to deliver nucleic acid or protein only to the target cells; and adding a reagent that specifically inhibits proliferation of the target cell population or inhibits proliferation of the non-target cell population, thereby selectively depleting or enriching the target cell population. In some embodiments, the receptor is an immune receptor. In some embodiments, the immune receptor is a B cell receptor. In some embodiments, the immune receptor is a T cell receptor.

[0217] Non-limiting exemplary embodiments of the disclosed methods for selectively depleting or enriching a target cell population can include one or more of the following features: In some embodiments, the target cells express a herpes simplex virus thymidine kinase (HSV-TK) transgene and the added reagent comprises or is ganciclovir (GCV). In some embodiments, the target cell population comprises immune cells. In some embodiments, the target cells express shRNA to reduce expression of the death receptor FAS to prevent cell death in the target population. In some embodiments, the immune cells comprise T cells. In some embodiments, the immune cells comprise B cells.

[0218] In some embodiments, the immune cells are autoreactive immune cells. In some embodiments, the immune cells are specific for an antigen associated with a health condition. In some embodiments, the health condition is a proliferative disorder, an inflammatory disorder, an autoimmune disorder, or a microbial infection. In some embodiments, the proliferative disorder is cancer. In some embodiments, the microbial infection is a bacterial infection, a viral infection, or a microfungal infection.

[0219] V. System Also provided herein is a system termed ENTER (lentivirus-mediated cell entry by engineered receptor-ligand interactions), which has been shown to be a versatile platform capable of achieving a variety of applications, such as for presenting ligands, delivering cargo, and recording interactions. Exemplary applications of ENTER include elucidating ligand-receptor interactions, correlating receptor interactions with cellular states at the single-cell level, and delivering cargo in a receptor-specific manner. As described in more detail herein, the ability to centralize multiple functions into one platform is a major advantage. ENTER provides users with one platform that solves many important problems, rather than assembling and juggling multiple separate single-purpose technologies. First, in some embodiments of the present disclosure, lentiviruses have been engineered to enable the presentation of heterologous cell surface proteins, intracellular and extracellular epitopes, including pMHC complexes, antibodies, costimulatory molecules, and B-cell antigens. ENTER has several advantages over yeast or phage display platforms (see, e.g., Table 1). Glycosylation patterns in yeast / phage display platforms are different from mammalian systems, which may prevent correct MHC presentation and recognition of paired TCRs. Moreover, yeast or phage display require significant optimization to achieve correct folding, stability, and MHC presentation. Moreover, ENTER is performed in human cells, allowing for human glycosylation and protein folding patterns, so that the applicants can demonstrate that multiple HLA peptide combinations can be presented. Furthermore, screening in yeast and phage display platforms requires soluble recombinant TCRs, making it difficult to test multiple TCRs in parallel. In contrast, ENTER allows researchers to screen primary T cell samples, which allows for the broad diversity of the human TCR repertoire to be examined (see, for example, Table 1).ENTER is also superior to cytolytic T cell reporter assays such as T-scan for elucidating pMHC-TCR interactions, because the latter cannot record pMHC–TCR pairing at the single-cell level.

[0220] Second, in some embodiments, ENTER can be engineered to deliver cargo in a receptor-specific manner. Lentiviruses were engineered to drive viral fusion and infection by receptor-ligand interactions. The system was further engineered to allow researchers to flexibly use defective integration mechanisms to choose to deliver cargo transiently or stably. In some embodiments, ENTER can achieve superior cell type specificity compared to existing modalities such as AAV, and thus may have applications in gene therapy or RNA medicine. In some embodiments, ENTER can selectively deplete or expand antigen-specific T cells based on the specific delivery of genetic cargo that induces or protects against cell death (see, for example, FIG. 4). ENTER allows for the depletion of antigen-specific B cells, which can eradicate pathogenic autoantigen-specific B cells to potentially treat autoimmune disorders.

[0221] [Table 1]

[0222] In some embodiments, ENTER as described herein can be used to correlate ligand-receptor interactions with molecular blueprints at the single-cell level. For example, ENTER-seq combines the ability to elucidate ligand-receptor interactions with the power of single-cell genomics to elucidate cell-cell communication and cell states in a massively parallel fashion. ENTER-seq for pMHC is conceptually similar to DNA barcoded libraries of pMHC tetramer molecules, but has several potential advantages. In addition, ENTER can be less costly compared to commercially available DNA barcoded pMHC tetramers. ENTER can be easily performed by any laboratory compared to in-house generation of pMHC tetramers with DNA barcodes (see Table 2). While DNA binding to pMHC tetramers can suffer from uneven loading of barcode oligonucleotides during the binding reaction, ENTER-seq libraries leverage lentiviral biology, which ensures two copies of barcoded viral RNA for each viral particle. The uniform distribution of DNA barcodes per virus-like particle allows quantification of pMHC binding strength by ENTER-seq, which has not been examined in studies using DNA-barcoded pMHC tetramers. The experimental data described herein revealed that highly expanded TCR clones were associated with higher pMHC binding (see, e.g., FIG. 7G), likely due to higher TCR affinity for pMHC or higher TCR surface density in the expanded clones.

[0223] [Table 2-1] [Table 2-2]

[0224] Finally, as described in more detail herein, ENTER is more sensitive than pMHC tetramers on a moles per reagent basis (see FIG. 2C). In some embodiments, the superior sensitivity may be due to the higher number of pMHCs presented in ENTER. HIV-based lentiviral particles present 14-100 envelope protein molecules per viral particle, whereas pMHC tetramers are by definition four linked molecules. Taken together, ENTER-seq allows researchers to document ligand-receptor specificity and read out the biological outcome of this interaction, such as the fate of antigen-dependent T cells, including activation of naive cells, proliferation of effector cells, formation of memory cells, or exhaustion of T cells. Similarly, ENTER-seq may be used to understand the molecular programs of antigen-specific B cells in the context of infectious disease and autoimmunity.

[0225] ENTER-seq analysis of primary CMV-specific T cells demonstrates the power of the platform to link antigen epitopes, TCR repertoires, gene expression programs, and surface protein phenotypic landscapes across tens of thousands of primary T cells in a single experiment. Massively parallel profiling of such diverse modalities revealed donor-specific antigen specificity and immunogenicity of viral epitopes. In some embodiments, ENTER-seq of T cells before and after peptide stimulation revealed transcriptional changes during proliferation and phenotypic diversity among clones responding to the same antigen. Such transcriptional changes and clonal diversity in Th2 cytokine expression may be influenced by different TCR affinity / avidity / density for the same pMHC antigen or different priming environments from antigen-presenting cells. A recent study of single-cell profiling of CD19-CART cells in patients with acute lymphoblastic leukemia showed that induction of Th2 expression positively correlated with clinical benefit in sustained responders compared to relapsed patients (Bai et al., 2022). However, it is unclear how Th2 cytokines can enhance CD8 T cell effector function to achieve long-term remission and whether such benefits can be generalized to infectious diseases. The experimental data described herein showing TCR clone-specific induction of Th2 cytokine expression may inform the selection of TCRs for engineering TCR-T cells for adoptive T cell therapy. Furthermore, ENTER-seq described herein provides insights for a comprehensive understanding of how T cell clonality and specificity affect the molecular phenotype and physiological function of antigen-specific T cells.

[0226] In some embodiments, the ENTER described herein may be used to isolate and enrich tumor antigen-reactive T cells for reinfusion into patients. In some embodiments, the ENTER described herein may be further used in a discovery context to screen immunogenic antigens or superior TCRs for vaccine development or rational design of cancer immunotherapy. In some embodiments, the ENTER described herein may also be applied to screen BCRs targeting viral antigens, facilitating the development of therapeutic antibodies to prevent viral infection. In some embodiments, the ENTER described herein allows for antigen-specific delivery of cargoes such as genes and shRNAs, which allow for the perturbation and manipulation of antigen-specific T and B cells. This targeted delivery strategy may be applied to reactivate exhausted anti-tumor T cells without causing immune-related adverse events, or to deplete autoreactive T or B cells to treat autoimmunity. In yet another embodiment, the ENTER may be extended to additional receptor-ligand pairs such as G protein-coupled receptors, adhesion molecules, or protocadherins. Thus, the ENTER may be used to address cell-cell associations beyond the immune system.

[0227] ENTER-seq can combine the power of single-cell genomics with the ability to elucidate ligand-receptor interactions to elucidate cell types and cell states in a massively parallel manner. ENTER-seq for pMHC is conceptually similar to DNA barcoded libraries of MHC tetramer molecules, but has several potential advantages. MHC tetramer libraries require individual peptides to be synthesized and then loaded onto MHC tetramers, resulting in higher costs, longer lead times, and lower throughput compared to ENTER-seq libraries, which can be prepared by massively parallel DNA synthesis. While DNA binding to MHC tetramers can be subject to uneven loading of barcode oligonucleotides during the binding reaction, ENTER-seq libraries leverage lentiviral biology, which ensures two copies of barcoded viral RNA for each viral particle. Finally, ENTER-seq can be more sensitive compared to MHC tetramers. HIV-based lentiviral particles display 14-100 Env protein molecules per viral particle, whereas MHC tetramers are by definition four linked molecules.

[0228] ENTER-seq allows researchers to document ligand-receptor specificity and read out the biological outcome of this interaction, e.g., antigen-dependent T cell fate, such as activation of naive cells, proliferation of effector cells, formation of memory cells, or T cell exhaustion. Similarly, ENTER-seq may be used to understand the molecular program of autoantibody-producing B cells in autoimmunity.

[0229] ENTER links ligand-receptor interactions with molecular blueprints at single-cell resolution and has the advantage over cytolytic T cell reporter assays such as T-scan, because the latter cannot record peptide-MHC and TCR pairing at the single-cell level, which precludes pooled analysis.

[0230] ENTER may have translational applications in immunology and beyond. ENTER may be used to isolate and enrich tumor antigen-reactive T cells for reinfusion into patients. The non-integrating properties of ENTER facilitate adoptive T cell therapy. ENTER may further be used in the context of discovery to screen immunogenic antigens or superior TCRs for vaccine development and rational design of cancer immunotherapy.

[0231] For clarity, it is understood that certain features of the present disclosure that are described in the context of separate embodiments can also be provided in combination in a single embodiment. Conversely, various features of the disclosure that are, for brevity, described in the context of a single embodiment can also be provided separately or in any suitable subcombination. All combinations of the embodiments related to the present disclosure are specifically embraced by the present disclosure and are disclosed herein as if each and every combination were individually and expressly disclosed herein. Moreover, all subcombinations of the various embodiments and elements thereof are specifically embraced by the present disclosure and are disclosed herein as if each such subcombination were individually and expressly disclosed herein.

[0232] The general method discussion provided herein is intended for illustrative purposes only: other alternative methods and substitutes will be apparent to those of skill in the art upon review of this disclosure, and are intended to be within the spirit and scope of this application.

[0233] Although the present disclosure has been described in some detail by way of illustration and example, for purposes of clarity and understanding, it will be apparent that certain changes and modifications can be practiced within the scope of the appended claims.

[0234] Throughout this specification, various patents, patent applications, and other types of publications are referenced, such as original articles, electronic database entries, etc. The disclosures of all patents, patent applications, and other publications cited herein are incorporated by reference in their entirety for all purposes. EXAMPLES

[0235] Example 1 This example describes the results of experiments performed to demonstrate an exemplary ENTER, in accordance with several embodiments of the present disclosure, engineered as a modular viral display and delivery platform to capture and elucidate ligand-receptor interactions, deliver cargo to target cells, and correlate ligand-receptor interactions with cellular states.

[0236] Lentiviruses were engineered at multiple levels, including (i) ligand proteins displayed on the viral surface, (ii) host receptor-targeted viral entry via displayed ligands and modified fusogens, (iii) fluorescent protein delivery via fusion of the viral capsid, and (iv) tagged viral RNA for single-cell sequencing (see, e.g., Figure 1A).

[0237] To achieve specific ligand-receptor interactions between lentiviruses and host cells, for example by using viruses that display user-defined ligand proteins, viral envelope proteins (termed fusogens) in which the binding of the natural receptor is broken while maintaining intact fusion ability are designed to cooperate with the user-defined ligand protein displayed on the viral surface. The cooperation of two separate modules (ligand protein + fusogen) allows interaction of the virus-displayed ligand with the host receptor, further promoting fusion of the virus to the host cell by the fusogen (see Figure 1A). The viral envelope protein, vesicular stomatitis virus G protein (VSV-G), is used to pseudotype lentiviruses. VSV-G pseudotyped viruses have a broad tropism because VSV-G can recognize and interact with the low-density lipoprotein receptor (LDLR) expressed in many cell types.

[0238] In these experiments, Jurkat T cells and Raji B cells were infected with VSV-G pseudotyped lentivirus carrying a GFP transgene. Although strong GFP expression was observed in these Jurkat T cells and Raji B cells, the transduction efficiency was different, which may be due to the variable expression of LDLR in various cell types (see FIG. 1B).

[0239] A VSV-G mutant with two point mutations (K47Q, R354A) was designed to prevent recognition and interaction with LDLR on host cells (Nikolic et al., 2018). Minimal GFP expression was observed in Raji (0.1%) and Jurkat (0.6%) cells using mutant VSV-G pseudotyped viruses (see Figure 1B), suggesting that viral recognition of specific receptors on host cells is the first critical step for viral entry and integration. To test whether VSV-G mutants are good fusogen candidates that cooperate with user-defined ligands on the viral surface for viral infection in host cells expressing the paired receptor, we co-expressed a well-established CD19-CAR (chimeric antigen receptor) containing an anti-CD19 single-chain antibody variable fragment (sc-Fv) in HEK293T cells with the VSV-G mutants, a GFP transgene in a viral transfer vector, and lentivirus packaging components, and collected the virus from the supernatant. As expected, such viruses specifically infected Raji B cells, which express high levels of CD19, but did not infect CD19-negative Jurkat T cells (see FIG. 1B). Thus, applicants have developed a viral display platform that can reprogram viral fusion from cell entry dependent on the natural VSV-G / LDLR interaction to one dependent on the interaction between a user-defined ligand and a host cell receptor paired with the virus.

[0240] To capture the ligand-receptor interaction while avoiding viral integration and integration-induced mutations in the host genome (Ranzani et al., 2013), we engineered a viral integrase mutant (D64V) that cannot integrate into the host genome (Certo et al., 2011), fused GFP protein with viral structural proteins, and tracked the ligand-presenting virus. Instead of viral integration to express GFP, we used a ligand-presenting virus with GFP protein to measure transient viral entry into host cells expressing the paired receptor. To identify viral proteins that function as optimal fusion partners for GFP, we tested three viral proteins, including matrix protein (MA), nucleocapsid protein (NC), and an HIV accessory protein called viral protein R (VPR) (see Figure 1C). During virus assembly and synthesis, MA and NC are processed from Gag precursor proteins, which can be assembled in cis as 3000 copies of MA or NC per virus particle (De Guzman et al., 1998; Kutluay et al., 2014). VPR is incorporated in trans into virus particles by interacting with Gag proteins as 500 copies of VPR per virus particle (Wuet et al., 1995). We observed that 80% of Raji cells were bound by CD19-CAR-presenting virus when GFP was fused with NC, which performed significantly better than MA-GFP and VPR-GFP (see Figure 1C).

[0241] To examine whether CD19-CAR-presenting NC-GFP viruses can recognize and bind to primary CD19+ B cells derived from human blood, these viruses were incubated with naive or activated human primary B cells for 2 h and GFP signals on these B cells were detected by flow cytometry. Similar to the Raji B cell line, 80% of activated human primary B cells were bound by NC-GFP-labeled CD19-CAR-presenting viruses, whereas 60% of naive B cells are GFP+ (see, e.g., Figure 1D). The difference in CD19 expression between naive and activated B cells may be the cause of the discrepancy in binding of CD19-CAR viruses. Indeed, the flow cytometry results showed that the surface expression of CD19 on activated B cells was significantly higher than that on naive B cells (see, e.g., Figure 1E-1F), consistent with the higher binding of the virus on activated B cells. This result indicates that the binding of ligand-presenting viruses to receptor-expressing cells is quantitatively correlated with the expression level of the receptor paired with the ligand. Furthermore, after incubation of CD19-CAR virus, CD19 surface expression was dramatically reduced, indicating that CD19-CAR virus specifically binds to CD19 and prevents subsequent binding of flow cytometry antibodies targeting CD19, either by masking the CD19 antigen or by inducing internalization of surface CD19 (Figures 1E-1F). To determine whether the loss of surface CD19 was due to viral fusion-induced internalization of surface CD19 or specific viral binding to CD19 that prevented subsequent CD19 antibody staining, viral binding and fusion assays were performed (see, e.g., Figure 1H). Results showed that there were only 5% GFP+ cells after proteinase K treatment, indicating that only a few cells had undergone viral fusion to prevent proteinase K-mediated degradation of surface-bound GFP-labeled virus (see Figure 1I). Thus, the reduction in surface CD19 results primarily from specific CD19 binding / masking from ENTER viruses rather than internalization, further emphasizing that ligand-presenting viruses are highly specific for their targeted receptors.

[0242] To further determine whether the ligand-presenting GFP fusion virus could specifically bind target cells through other ligand-receptor interactions, the virus was engineered to present either wild-type CD40 ligand (CD40L) or mutant CD40L. The CD40L mutant contains two point mutations (K142E, R202E) that result in reduced binding affinity to CD40 (Pasqual et al., 2018). Flow cytometry results showed that GFP+Raji cells were significantly reduced when incubated with viruses presenting the CD40L mutant compared to wild-type CD40L (Figures 1J-1L).

[0243] To confirm that the ligand protein and fusogen were incorporated onto the viral particle surface, an immunocapture assay was performed to pull down the virus presenting the desired protein by antibody-coated magnetic beads (see, for example, FIG. 1M). Specifically, viruses presenting CD40L and fusogen (VSV-G mutant) were generated and then incubated with anti-CD40L beads, anti-VSV-G beads, and IgG beads, respectively. After incubation and extensive washing, viral RNA was extracted for subsequent qRT-PCR analysis. Compared with the IgG negative control, a significant enrichment of viral RNA was found in the anti-CD40L and anti-VSV-G groups, confirming that the ligand protein and fusogen were incorporated onto the virion surface (see FIG. 1N).

[0244] Collectively, these results suggest that Applicant's viral display platform, e.g., ENTER, can capture highly specific ligand-receptor interactions in transient viral binding assays and can be applied to multiple categories of receptor-ligand interactions.

[0245] Thus, engineered lentiviral particles displaying specific ligands deliver fluorescent proteins to target cells upon cognate receptor-ligand interaction, without genomic integration and transcription of the transgene.

[0246] Example 2 In this example, we describe the results of experiments performed to demonstrate how ENTER using viruses presenting MHC peptides (pMHC) maps TCR specificity, and in particular how this viral display platform captures the interaction between pMHC and the TCR.

[0247] Applicants engineered the virus to present a single chain of MHC and covalently linked peptide fused with beta 2 myeloglobulin (B2M) (see, e.g., FIG. 2A). To prevent interference of endogenous human leukocyte antigen (HLA, human MHC locus) from HEK293T cells when producing HLA peptide-presenting viruses, Applicants generated a stable HLA knockout (KO) HEK293T cell line by knocking out all potential HLA class I alleles (HLA-A / B / C) by CRISPR-Cas9. Surface expression of B2M was absent in HLA KO cells, suggesting that all endogenous HLA alleles were successfully deleted (see FIG. 2G). HLA-A fused with B2M and peptide * Single chains of 0201 (HLA-A2) were overexpressed in HLA KO cells, and high levels of surface expression of HLA-A2 and B2M were observed (see, e.g., FIG. 2G).

[0248] Applicants engineered a GFP-fusion reporter virus to present pMHC on the surface by co-expressing viral Gag proteins containing a single-chain trimer of pMHC, mutant VSV-G, and NC-GFP in HLA KO HEK293T cells. Viruses were harvested and incubated with Jurkat T cells expressing a TCR targeting the cognate pMHC antigen. Using these modular components, we successfully produced a virus presenting NY-ESO-1, a well-established cancer-testis antigen, as a 9-mer peptide (SLLMWITQC) on HLA-A2, the most prevalent HLA allele in humans (Jager et al., 1998) (see Figure 2A). 88.2% of cognate NY-ESO-1 TCR-expressing T cells were labeled by the GFP virus bearing the NY-ESO-1 antigen, compared to 1.26% of T cells specific for known cytomegalovirus (CMV) epitopes (Lee and Meyerson, 2021). Similarly, different HLA alleles HLA-A * Viruses presenting CMV antigens as 11-mer (YSEHPTFTSQY) peptides on 01:01 specifically entered CMV TCR-T cells but not NY-ESO-1 TCR-T cells (see FIG. 2A). Applicants further demonstrated that the cancer-testis antigen, CMV pp65 antigen (ny-eso-1), all presented on HLA-A2 alleles, specifically entered CMV TCR-T cells but not NY-ESO-1 TCR-T cells (see FIG. 2A). 157~165 ), CMV pp65 antigen (pp65 495~503 ), and influenza matrix protein antigen (m1 58~66We engineered viruses presenting diverse 9-mer antigen epitopes derived from the ENTER platform (Gotch et al., 1987; Wills et al., 1996). Results showed that after 2 h of incubation with GFP-fusion viruses presenting cognate HLA-A2 peptides, more than 87% of TCR-matched T cells were GFP+, whereas only 1% of these T cells were labeled by the negative control antigen-presenting GFP virus (see, for example, Figure 2B). Highly specific entry of pMHC-presenting viruses into TCR-matched T cells was observed with different antigen peptide lengths and with different HLA alleles, highlighting the versatility of the ENTER platform for presenting diverse pMHC antigens.

[0249] To further test the specificity of pMHC-presenting viruses, CMV pp65 495~503 pp65 antigen-specific TCR Jurkat T cells 495~503 The cells were first incubated with the display virus and then incubated with the widely used commercially available pp65 495~503 As a negative control, these T cells were stained with influenza m1 tetramer. 58~66 Display virus and commercially available m1 58~66 The cells were incubated with tetramer (see FIG. 2H). Flow cytometry results showed that more than 90% of tetramer-positive cells were GFP+, indicating that pp65 495~503 Tetramer staining is pp65 495~503 The binding of the GFP-displayed virus is strongly consistent with that of the negative control influenza m1. 58~66 Tetramers and viruses are pp65 495~503 It did not label TCR T cells (see, e.g., FIG. 2H). 495~503 Tetramer intensity and CD3 surface expression of pp65 495~503A significant decrease was observed after co-incubation with the presented virus (see, e.g., FIG. 21), which is similar to the observed decrease in surface expression of CD19 on Raji B cells after binding of the CD19-CAR presented virus. This result indicates that the pMHC presented virus specifically binds and masks the TCR-CD3 complex, preventing subsequent binding of pMHC tetramers and anti-CD3 antibodies.

[0250] After determining the specificity of pMHC-presenting viruses using pMHC tetramers as a reference, Applicants compared the sensitivity between pMHC-presenting viruses and pMHC tetramers on a moles per reagent basis (see, e.g., General Methods in Example 11). The results showed that 2×10 8 ENTER virus particles can stain 95.7% of TCR-T cells, but 2 × 10 8 pMHC tetramers were shown to be unable to detect TCR-T cells (see, e.g., FIG. 2C). Importantly, 2×10 8 The binding efficiency of ENTER viruses is 8 x 10 9 The results were similar to those of the pMHC tetramer, suggesting that the ENTER virus is more sensitive (approximately 40-fold) than the pMHC tetramer.

[0251] To further determine whether TCR affinity for pMHC influences binding of pMHC-presented viruses to TCR-expressing T cells, we generated a TCR-T cell line (1G4wt) that recognizes NY-ESO-1 antigen variants (Zhang et al., 2021) with different known TCR affinities (Kd ranging from 7 to 85 μM). The NY-ESO-1 TCR T cell line utilized in previous experiments (e.g., see Figures 2A-2C) is highly similar to the 1G4wt TCR T cell line except for some mutations on the TCR, and is highly similar to the NY-ESO-1 TCR T cell line. 157~165 It has a very high binding affinity to the antigen (Robbins et al., 2008). Next, the wild-type peptide (SLLMWITQC), the L3A mutant (SL AMWITQC), and the T7A mutant (SLLMWI A QC) and other different NY-ESO-1 157~165 ENTER was engineered to present antigen peptide variants. Different TCR-T cells were then incubated with the antigen variant-presenting viruses after normalizing for viral titers (indicated by viral p24 protein levels) and the percentage of GFP+ cells was then measured. The results showed that ENTER has the sensitivity to detect TCR affinities as low as 10.8uM when high titers of virus (40ng p24) were added. At very low TCR affinities such as 84.9uM, 25% of on-target cells could still be detected by ENTER under high titers (see Figure 2D), highlighting the broad range of TCR affinities that ENTER can recognize. TCR binding affinity was observed to be positively correlated with ENTER recognition efficiency, suggesting that ENTER can be applied to infer relative TCR affinity by measuring the binding efficiency of pMHC-presenting viruses (see, for example, Figure 2D).

[0252] Furthermore, to determine the specificity and sensitivity of the pMHC viral presentation platform, the applicants administered pMHC-viral-presenting mAbs to on-target T cells (ml 58~66 TCR that recognizes the antigen) and off-target T cells (ny-eso-1 157~165 The TCR that recognizes the antigen is mixed in different ratios, and then the 58~66 The ENTER virus-presenting T cells were incubated with antigen-presenting GFP virus (see, for example, Figures 2E, 2J). To distinguish these two different TCR T cell lines, flu-M1 TCR T cells were labeled with cell trace violet dye. Signal / noise ratios were calculated based on the abundance of on-target GFP+ cells and the abundance of off-target GFP+ cells. Even when the abundance of target T cells was as low as 1:1000, the signal / noise ratio was more than 150-fold, indicating the high specificity and sensitivity of the ENTER virus-presenting platform (see Figures 2F, 2J, and 2K).

[0253] Taken together, the above experimental data demonstrate that ENTER specifically and sensitively captures the interaction between pMHC and TCR.

[0254] Example 3 This example describes the results of experiments performed to demonstrate exemplary elucidation of B cell specificity by ENTER viruses presenting B cell antigens.

[0255] B cells have a wide variety of BCRs that can specifically target foreign antigens from invading viruses and self-antigens. Viral antigen-specific B cells can produce antibodies (secreted from the BCR) that are beneficial for preventing viral infection. In contrast, self-antigen-specific B cells generate harmful autoantibodies that attack the body, which are the cause of autoimmune disorders (Burbelo et al., 2021; Tan, 1989). Therefore, it is important to elucidate the specificity of B cells, which will facilitate the development of highly effective antiviral antibodies, drive rational vaccine design, and provide a better understanding of the formation of autoreactive B cells (Ju et al., 2020). Based on the successful application of ENTER in elucidating T cell specificity, this example describes the results of experiments performed to investigate the feasibility of capturing the interaction between BCR and antigen.

[0256] Unlike TCR recognition of antigenic peptides presented by MHC on the cell surface, BCR can recognize antigenic epitopes derived from cell surface proteins as well as intracellular, extracellular, and secreted proteins. A major challenge for ENTER to elucidate B cell specificity is to present B cell antigens that do not contain a natural transmembrane (TM) domain on the viral surface. To present antigenic epitopes derived from intracellular proteins on the viral surface, applicants attempted to engineer TM domains for optimal surface presentation of B cell antigens. To select candidate TM domains for viral surface presentation, applicants exploited the unique ability of the HIV-1 virus to incorporate host proteins on the viral surface during viral budding. The nascent HIV-1 virus can selectively incorporate specific host TM proteins while excluding other abundant host surface proteins during the viral assembly and budding process (Burnie and Guzzo, 2019). Applicants prioritized a list of highly abundant host TM proteins that are incorporated into the viral surface from previous literature using viral mass spectrometry, immunocapture assays, and flow virometry (Burnie et al., 2020; Cantin et al., 1996; Chertova et al., 2006; Grover et al., 2015; Jalaguier et al., 2015). This list of host TM proteins included MHC class I and II molecules (HLA-DRA, HLA-DRB, HLA-A2), adhesion molecules (ICAM1, CD43, CD162, CD62L), and integrin family members (CD49d, LFA-1) (see, e.g., FIG. 3A).

[0257] To determine the specificity and efficiency of viral presentation of B cell epitopes involving these diverse TM domains, human papillomavirus (HPV) minor capsid antigen L2 (HPV16 L2 residues) was presented. 17~36) and fused to a prioritized list of TM domains. Next, we generated BCR-expressing B cell lines that specifically target the HPV16 L2 B cell epitope (Wang et al., 2015). After incubating the viruses with the fused TM domains and presenting the B cell epitopes with B cells expressing HPV-BCR (on-target) or B cells not expressing BCR (off-target), we quantified the percentage of GFP+ B cells to measure efficiency and specificity (see Figure 3B). In addition to TM domains from host proteins, we further engineered viruses to fuse B cell epitopes with the TM domain from the fusogen VSV-G, a viral envelope protein that can assemble in budding viruses. Results revealed that the ICAM1 TM domain was the most likely candidate, since over 90% of HPV antigen-specific BCR+ B cells were GFP+ (see, e.g., Figures 3B-3C). This is consistent with a previous report showing that ICAM1 is selectively acquired in budding viruses by interaction with viral matrix proteins ( Jalaguier et al., 2015 ).

[0258] To test whether the ICAM1 TM domain could be applied to present other B cell antigens in addition to the linear epitope from HPV16, we engineered the virus to present the receptor binding domain (RBD) from the SARS-CoV-2 spike protein (see Figure 3D). The results showed that 88% of spike RBD BCR+ B cells were labeled by the RBD-presenting virus, indicating that ENTER with the optimized TM domain could target the linear epitope (HPV16 L2 17~36 We show that this can be applied to elucidate B cell specificity for both specific antigens (i.e., single antigens) and complete antigen domains (SARS-CoV-2 spike RBD).

[0259] Beyond the ability of ENTER to present intracellular and extracellular B cell antigens, we performed additional experiments to determine whether ENTER could elucidate B cell specificity for cell surface B cell antigens. ENTER was engineered to present HER2 using its native TM domain. HER2 is an epidermal growth factor receptor that is overexpressed in breast cancer cells (Gutierrez and Schiff, 2011). Experimental data showed that 76% of anti-HER2 BCR B cells were detected by HER2-presenting viruses (see Figures 3G-3H), highlighting the versatility of ENTER to present any B cell antigen derived from intracellular proteins (HPV L2), extracellular proteins (spike RBD), and cell surface proteins (HER2).

[0260] In addition, to further investigate the specificity and sensitivity of ENTER and decipher the interaction between BCR and B cell antigens, on-target B cells (with BCR recognizing SARS-CoV-2 spike RBD antigen) and off-target B cells (with BCR recognizing HPV L2 antigen) were mixed in different ratios and incubated with spike RBD antigen-presenting virus (see Figures 3E and 3I). To distinguish these on-target B cells from off-target B cells, on-target B cells were stained with cell trace violet dye. The signal / noise ratio was calculated based on the abundance ratio of on-target GFP+ cells and the abundance ratio of off-target GFP+ cells. The signal / noise ratio was about 100-200 times (see, e.g., Figure 3F), indicating the great specificity and sensitivity of the TM domain-optimized virus presenting B cell antigen epitopes (see, e.g., Figures 3J-3K).

[0261] Taken together, the above experimental data demonstrate that ENTER is a platform that can successfully capture BCR-antigen interactions with high specificity and sensitivity.

[0262] Example 4 This example describes experiments performed to examine whether ENTER can deplete or expand antigen-specific T cells or antigen-specific B cells by targeted cargo delivery.

[0263] First, to test the antigen specificity of cargo delivery, the GFP transgene was used as the cargo to measure delivery efficiency and specificity. When lentiviruses pseudotyped with wild-type VSV-G were infected, comparable transduction efficiency was observed regardless of TCR or BCR specificity (see, for example, FIG. 3L). Then, pp65 495~503 Additional experiments were performed to engineer viruses displaying pMHC ligands and VSV-G mutant fusogens carrying a GFP transgene on the viral RNA, as well as other viral components, including wild-type integrase (see, e.g., General Methods, Example 11). 495~503 pMHC virus to pp65 495~503 Following infection of CMV pp65 TCR+ T cells targeting HER2 and NY-ESO-1 TCR+ T cells targeting an unrelated antigen, it was observed that 82% of on-target TCR- T cells expressed GFP, whereas only 0.22% of off-target TCR- T cells were GFP+ (see, e.g., Figures 4A-4B). Similarly, additional experiments were performed to engineer viruses presenting the B cell antigen HER2 and carrying a GFP transgene as cargo (see, e.g., Figure 4C). Experimental data demonstrated specific delivery of the GFP transgene in HER2 BCR+ B cells, but not in RBD BCR+ B cells (see, e.g., Figure 4D).

[0264] Next, to examine whether ENTER-mediated targeted gene delivery could exert its correct function in antigen-specific T or B cells, we performed additional experiments in which we engineered pMHC-presenting viruses to carry the herpes simplex virus thymidine kinase (HSV-TK) gene, a well-established suicide gene that responds to the drug ganciclovir (GCV) (Beltinger et al., 1999). CMV-pp65 TCR+ T cells were mixed at a 1:1 ratio with NY-ESO-1 TCR+ T cells expressing mScarlet, and then transfected with pp65 TCR+ T cells carrying the suicide gene. 495~503 The presented virus was added. Three days after infection, GCV drug was added to kill cells expressing HSV-TK, and cell survival was monitored for four days (see Figure 4E). After virus infection, specific cargo delivery was observed in on-target T cells among the mixed T cell pool (see, e.g., Figure 3M). Four days after GCV treatment, a specific reduction in on-target T cells (CMV-pp65 TCR+) was observed without affecting off-target T cells (see, e.g., Figure 4F). To determine whether targeted killing was not induced by virus infection, a virus carrying the GFP gene was generated as a negative control. HSV-TK gene delivery was found to result in a significant reduction of on-target T cells by approximately 8-fold compared to GFP gene delivery (see Figure 4G), further validating that selective depletion of one T cell clone is achieved by delivery of an antigen-specific suicide gene. To test whether antigen-specific gene delivery can be applied to B cells, HER2 BCR+ B cells were mixed with RBD BCR+ B cells expressing mScarlet, followed by the addition of a HER2-presenting virus carrying a suicide gene (see Figures 4H and 3N). Similarly, after treatment with GCV drugs, the results showed a significant reduction in on-target B cells by suicide gene delivery compared to the control GFP gene (see Figures 4I-4J), suggesting that ENTER allows selective depletion of one B cell clone of the B cell pool by delivery of an antigen-specific suicide gene.

[0265] In contrast, additional experiments were also performed to examine whether ENTER allows selective survival and retention of antigen-specific T cells. The purpose of these experiments was to deliver short hairpin RNA (shRNA) against the death receptor FAS in antigen-specific T cells to prevent FAS-induced programmed cell death (Yonehara et al., 1989). After screening multiple shRNAs targeting FAS, shFAS#2 was selected, which showed the highest knockdown efficiency, reflected by dramatically reduced surface expression of FAS compared to the control shRNA (shCtrl) group (see Figure 30). Furthermore, pp65 495~503 We generated the presented shFAS#2 or shCtrl and infected a mixed pool of on-target (CMV-pp65 TCR+) and off-target (NY-ESO-1 TCR+) T cells with these viruses (see Figure 4K). The results showed a significant reduction in FAS protein surface expression in on-target T cells infected with shFAS#2 compared to the shCtrl group or off-target uninfected T cells, indicating targeted shRNA delivery (see Figure 4L). These cells were then treated with anti-FAS antibody to induce FAS-mediated cell death as revealed by Annexin V and 7-AAD staining (see e.g. Figures 4K and 3P). After normalization of on-target versus off-target populations, a significant increase in on-target T cells was observed among surviving cells after FAS knockdown compared to the shCtrl group (see e.g. Figure 4M). Taken together, the data described herein demonstrate that ENTER allows for the engineering of complex cell populations with ligand-receptor specificity by delivery of targeted cargo.

[0266] Example 5 This example describes the results of experiments that combined the viral display platform ENTER with droplet-based single-cell RNA-seq to develop ENTER-seq, a technology that captures ligand-receptor interactions and molecular blueprints at single-cell resolution. Thus, ENTER-seq captures MHC peptide antigen specificity, TCR repertoire, and gene expression profiles at single-cell resolution. In some embodiments, the ENTER-seq workflow includes: (1) generation of pooled pMHC-presenting GFP viruses; (2) incubation of these viruses with human T cells; (3) sorting of virus-labeled GFP+ cells for droplet-based single-cell profiling (e.g., 5-prime single-cell RNA-seq / V(D)J-seq by 10x Genomics); and (4) generation and sequencing of three single-cell libraries containing gene expression, V(D)J TCR repertoire, and antigen peptide sequences (see, e.g., FIG. 5A).

[0267] Single-stranded PMHC information is stored in viral single-stranded RNA (ssRNA) packaged into lentiviral particles. The viral ssRNA is approximately 4.6 kb, which makes it difficult to reverse transcribe (RT) into full-length cDNA in the droplets. To efficiently capture the pMHC information on the viral RNA during the RT step of each droplet, a capture tag was inserted in the linker region between B2M and MHC and another PCR handle was inserted next to the CMV promoter (see, for example, Figure 5B). This capture tag allows capture by commercially available 5'GEM beads by hybridizing with a template switch oligo (TSO) sequence bound on the beads. The PCR handle allows the targeted peptide sequence to be conveniently amplified without additional primer spiking during the cDNA amplification step (see Figure 5B). Furthermore, nested PCR and index PCR allow targeted enrichment of antigen peptide sequences to generate the final antigen library for deep sequencing. Insertion of the capture tag and PCR handle does not affect the presentation of pMHC on the virus and its specific interaction with TCR-expressing T cells (see, e.g., Figures 4N-4O).

[0268] To evaluate ENTER-seq for single-cell profiling of antigen specificity and TCR repertoire, we performed ENTER-seq on TCR-expressing T cells mixed with pooled pMHC-presenting viruses. To mimic real T cell populations, 10% of T cells were mixed with 90% of T cells bearing a TCR recognizing the ny-eso-1157-165 antigen and a TCR recognizing the CMV pp65495-503 antigen, then incubated with pooled viruses presenting the ny-eso-1157-165 antigen or the pp65495-503 antigen (see, e.g., Figure 5C). Analysis of unique TCR sequences after excluding doublets confirmed a T cell mixture ratio of 9.4% (ny-eso-1157~165-TCR+) vs. 90.6% (CMV pp65495~503-TCR+), which is similar to the input mixture ratio (10% vs. 90%) (see Figure 5D). After excluding counts of molecular barcodes (UMIs) of TCRs and antigenic peptides (Methods), a total of 4198 T cells with reliable antigenic peptide information and TCR sequences were further recovered. The ratio of UMIs of the predominant antigenic peptides among all peptides was calculated. A high concordance between the antigenic peptides and the paired TCRs was observed (see, for example, Figure 5E). Matching of TCR sequences to antigen peptides at the single-cell level showed that 99.8% of pp65495~503+ cells and 97.4% of ny-eso-1157~165+ cells matched the corresponding TCR sequences, respectively (see, for example, Figure 5F).

[0269] Thus, the above experimental data demonstrate that ENTER-seq can sensitively and robustly capture TCR repertoire and cognate HLA antigen-peptide interactions at single-cell resolution.

[0270] Example 6 This example describes the results of experiments performed to demonstrate that the optimized ENTER-seq detects rare antigen-specific primary human T cells. In particular, ENTER-seq can be applied to rare antigen-specific primary T cells isolated directly from human blood.

[0271] We first validated the sensitivity of the ENTER-seq system using GFP virus presenting the CMV-pp65 antigen epitope on the HLA-A2 allele and primary T cells from HLA-A2+ patients with a history of CMV infection. 495~503 Incubate with the antigen-presenting virus and then use the widely used CMV pp65, which serves as a positive control. 495~503 Tetramer staining analysis revealed that 1% of T cells were pp65 495~503 It was shown to be antigen-specific (see Figure 4Q). 495~503 83% of tetramer-positive T cells were labeled by the GFP virus. To further increase the sensitivity of detection by flow cytometry, GFP was replaced by mNeon, a monomeric green fluorescent protein that is significantly brighter than GFP (see Figures 4P-4Q). Indeed, pp65 495~503 98% of tetramer-positive T cells are pp65 495~503 The epitope was recovered by the mNeon virus displaying the epitope, but not by the negative control virus, indicating significantly higher efficiency than the GFP virus (see, e.g., Figures 4Q-4S).

[0272] Example 7 This example describes the results of experiments performed to demonstrate that ENTER-seq of peptide-enriched CMV-specific T cells can reveal donor-specific immunogenic CMV epitopes and antigen-specific molecular phenotypes.

[0273] Antiviral T cells are essential for controlling viral replication and spread. Adoptive transfer of in vitro expanded CMV-specific T cells has shown great efficacy in controlling CMV infection in transplant recipients. However, little has been studied about how in vitro antigen-specific proliferation induced by CMV peptides affects the molecular phenotype, clonal expansion, and potential function of CMV-specific T cells.

[0274] In these experiments, ENTER-seq was used to characterize the transcriptional program, antigen specificity, and TCR clonality of CMV-specific T cells expanded by CMV antigen peptide stimulation. To enrich and expand CMV-specific T cells, human peripheral blood mononuclear cells (PBMCs) from CMV seropositive donors were first cultured for 10 days with a pool of 12 CMV antigen peptides (Lehmann et al., 2020; Liibke et al., 2020; Solache et al., 1999) (see, e.g., Figure 4T). The peptides were processed and presented by autoantigen-presenting cells, which then stimulated CMV antigen-specific T cells for subsequent expansion. To test the specificity of the ENTER virus of the present disclosure for peptide-enriched T cells, peptide pp65 495~503 T cells were expanded using and then pp65 495~503 Incubation with antigen-presenting mNeon virus followed by pp65 495~503 Stained with tetramer. Flow cytometry analysis showed that approximately 99% of tetramer+ T cells were labeled by virus (see, e.g., FIG. 4U), indicating the high specificity and sensitivity of ENTER for detecting peptide-enriched antigen-specific T cells.

[0275] A pool of ENTER viruses presenting these 12 CMV antigen epitopes was prepared and incubated with expanded T cells from four different CMV seropositive HLA-A2 positive donors (see Figure S4G). A dramatic expansion of CMV antigen-specific T cells was observed in two of the four donors (19.4% in donor #1 and 8.78% in donor #2) (see, e.g., Figure 4V). We then performed ENTER-seq on expanded T cells from these two donors, labeling each donor sample with a unique hashtag antibody. To further investigate the phenotype of antigen-specific T cells, we combined ENTER-seq with CfTE-seq by staining cells with DNA barcoded antibodies targeting the cell surface proteins CD45RA, CD45RO, and IL7R (see, e.g., Figure 6A).

[0276] After sorting GFP+ (CMV antigen-specific T cells) and GFP- (bystander T cells) CD8+ T cells, followed by droplet-based single-cell capture, we generated libraries to profile gene expression programs, CMV antigen peptides, TCR repertoires, and surface proteins (including CITE-seq and hashtag proteins) in individual cells. Results showed that cells bound to CMV antigen-presenting virus (ENTER+) were phenotypically distinct from cells without virus binding (ENTER-) (see, for example, Figure 6B). To test whether such phenotypic differences were induced by ENTER virus binding, we used RNA-seq data from CMV pp65TCR- T cells to profile pp65 495~503 Presenting ENTER virus or pp65 495~503 The results were compared with tetramer incubation. In summary, 28 genes with differential expression between the ENTER and tetramer groups were identified (2-fold change and adjusted P value < 0.01) (see FIG. 4W). Notably, all 28 genes were significantly upregulated in ENTER virus-treated cells, mainly related to TCR activation (CD69) and transcription factors induced by TCR signaling (FOS, NR4A1, NR4A3, EGR1, etc.) (see, e.g., FIG. 4W). This result suggested that pMHC-presenting virus binding may weakly induce TCR activation of resting / naive CMV pp65TCR-T cells compared with tetramer binding. To further determine whether this ENTER-induced gene upregulation leads to phenotypic differences between CMV-specific and bystander T cells, Leiden clustering with the presence or absence of ENTER-induced gene signatures was performed (see, e.g., General Methods in Example 11). A clear distinction was found between CMV-specific and bystander T cells after deletion of these 28 genes, suggesting that the phenotypic differences were not caused by ENTER virus binding (see Fig. 5G ).

[0277] After integrating surface protein landscape and gene expression, we observed that peptide-enriched CMV-specific T cells (ENTER+) were primarily effector memory T (TEM) cells (CD45RO+CD45RA-), whereas ENTER- cells were a mixture of naive and central memory T (TCM) cells (e.g., see Figures 6C and 5H-5I). Compared to ENTER- cells, ENTER+ cells were potentially protective T cells based on their high expression of effector molecules such as IFN-G, TNF, and cytotoxic molecules including granzymes and perforin (e.g., see Figure 5J). Single-cell RNA-seq data clustered all T cells into 10 clusters, including: (1) naïve T cells: CD45RA+CCR7+, (2) TCM: CD45RA-CCR7+, (3) terminally differentiated effector T cells (TEMRA): CD45RA+CCR7-, (4) mucosal-associated invariant T cells (MAIT): CD45RA-CD161+CXCR6+, (5) proliferative T cells: CD45RA+KI67+, (6) IL4+TEM: CD45RO+IL4+, (7) KLRC2+TEM: CD45RO+KLRC2+, (8) CST7+TEM: CD45RA+KLRC2+, (9) TEM: CD45RA+KLRC2+, and (10) TEM: CD45RA+KLRC2+. D45RO+CST7+, (9):HSP+TEM:CD45RO+heat shock protein (e.g., HSPA1A)+, (10):Proliferative TEM:CD45RO+KI67+ (see, e.g., Figures 6D and 5H-5L). Comparison of subset abundance ratios between donors showed that ENTER-bystander T cells were relatively similar between the two donors, whereas ENTER+CMV antigen-specific T cells were phenotypically distinct between the two donors, suggesting that the two donors may have different immune responses to CMV antigens (see, e.g., Figures 5M-5N).

[0278] To determine whether there was a donor-specific immune response to CMV antigens, we measured the number of T cells that recognized specific CMV antigen epitopes in each donor. 495~503 pp65 specific T cells were the most predominant antigen-specific T cells in both donors, which 495~503This suggests that CMV pp65 is the most common and immunogenic CMV antigen (see, for example, Figure 6E ). 495~503 This is consistent with previous reports showing a high abundance of specific T cells (Elkington et al., 2003; Gillespie et al., 2000; Wills et al., 1996). In addition, donor #2 had a higher proportion of US8 T cells than donor #1. 74~82 and UL100 200~208 A high abundance of specific T cells was observed, indicating donor-specific viral epitope immunogenicity. Interestingly, three distinct clusters were observed when the top three antigenic epitopes were reflected in a gene expression UMAP plot, suggesting that distinct epitopes may drive distinct genetic CD8+ T cell fate and expression programs (see Figure 6F). Indeed, pp65 495~503 In specific T cells, the expression of effector cytokines (e.g., IFNG, FASLG, PRF1, etc.) and transcription factors essential for effector T cells (e.g., ZEB2) is high (see, e.g., FIG. 6G). 200~208 Specific T cells express high levels of FOXP3, IL2RA (CD25), and CTLA4, which are characteristic of regulatory T (Treg) cells (Billerbeck et al., 2007; Churlaud et al., 2015; Fontenot et al., 2003; Wing et al., 2008), which is related to the expression of UL100. 200~208 These results suggest that donor-specific T cells resemble CD8+ Treg cells (see, for example, Figure 6G) (Vieyra-Lobato et al., 2018). Thus, ENTER-seq not only reveals donor-specific viral epitopes, but also reveals distinct molecular blueprints of antigen-specific T cells when recognizing different antigenic epitopes from the same virus.

[0279] Example 8 Interclonal phenotypic diversity underlying the same antigen specificity This example describes the results of experiments performed to demonstrate the interclonal phenotypic diversity underlying the same antigen specificity.To investigate the clonal expansion of CMV antigen-specific T cells, we performed an integrated analysis of TCR repertoire, antigen specificity, and gene expression at the single-cell level.

[0280] In these experiments, TCR clonotypes were defined by the identity of the CDR3 nucleotide sequence (Yassai et al., 2009). Peptide-enriched CMV-specific T cells (ENTER+) showed higher clonal expansion (up to 3856 cells per TCR clone) compared to bystander T cells (ENTER-, up to 174 cells per TCR clone) (see, e.g., FIG. 6J). Using the predominant TCR clonotype as a reference, low false negative (FNR<3%) and false positive (FPR<1%) rates were calculated and observed, highlighting the high sensitivity and specificity of ENTER-seq for primary T cells (see General Methods in Example 11). Additional experiments were then performed to examine whether there was an overlap of CMV antigen-specific TCR clones between the two donors. The results of these experiments showed that the two donors had unique expanded TCR clones with no overlap (see, e.g., Figure 6K), which is consistent with previous studies showing that antigen-specific TCR clonotypes are usually unique to each individual due to the high diversity of the TCR repertoire (Dupic et al., 2021; Robins et al., 2009). Thus, the specificity of the shared TCR could not be predicted from the TCR sequence alone, and pMHC binding data was required. Further integrated analysis of antigen specificity and TCR clonal expansion showed that different CMV antigen epitope-specific T cells exhibited different behaviors of TCR clonal expansion (see, e.g., Figure 6H). pp65 495~503 The size of the clonal expansion of specific T cells is 200~208 The size of the clonal expansion of specific T cells was significantly larger than that of the clonal expansion of specific T cells (see, e.g., FIG. 6H). 495~503 Specific T cells and UL100 200~208Compared to specific T cells, high TCR clonal expansion was associated with high expression of cytotoxicity genes. Indeed, in all clonotypes, expansion of genes related to cytotoxicity was significantly correlated with TCR clonal expansion (Pearson correlation r = 0.48, p = 0.0, Figure 6L). In contrast, correlations between clonal expansion and other gene signatures were relatively weak (r = 0.13 for T cell exhaustion genes and r = 0.09 for T cell activation genes) (see, e.g., Figure 6M).

[0281] Because multiple TCR nucleotide sequences can encode the same CDR3 amino acid sequence targeting the same antigenic epitope, we then pooled clonotypes based on identical CDR3 amino acid sequences for each CMV antigenic epitope (see Figures 6N-6O). 495~503 Three predominant CDR3 clonotypes were identified, of which two TCR beta chain sequences (CASSFQGYTEAFF; SEQ ID NO: 54 and CASSYQTGASYGYTF; SEQ ID NO: 55) matched the pp65 TCR beta chain sequences published in the IEDB database. 495~503 Specific TCRs were identified, further validating the specificity of the ENTER platform disclosed herein (see, e.g., FIG. 6O). 495~503 When combining CDR3 clonotypes with gene expression profiles in specific T cells, it was discovered that different clonotypes exhibit distinct gene expression phenotypes, including distinct cytokine profiles, cytolytic enzymes, and transcription factor expression, indicating phenotypic diversity among clones targeting the same antigen epitope (see, e.g., Figures 6I and 6P). Thus, ENTER-seq can functionally characterize both TCR binding specificity and TCR-associated cellular states.

[0282] Example 9 Here we describe the results of experiments demonstrating that ENTER-seq of primary CMV-specific T cells from patients reveals intraclonal diversity of genes associated with cytotoxicity and type I IFN responses.

[0283] To elucidate antiviral T cell memory in CMV seropositive patients, we engineered the ENTER virus to present the top three previously identified CMV antigenic epitopes and performed ENTER-seq on primary T cells isolated directly from the patient's blood without in vitro expansion (see Figure 7A). Integrated analysis of CITE-seq and gene expression profiles showed that the patient's CMV-specific T cells (ENTER+) were predominantly terminally differentiated effector memory T cells (TEMRA, CD45RO-CD45RA+CCR7-) (see, e.g., Figures 7B-7C). This observation is consistent with previous studies that showed accumulation of TEMRA CMV-specific T cells in CMV seropositive patients (Appay et al., 2002; Derhovanessian et al., 2011).

[0284] After subset clustering, TEMRA population heterogeneity was observed in CMV-specific T cells with diverse patterns of gene expression related to cytotoxic function, chemokines, costimulatory / co-inhibitory molecules, and type I IFN response (see, e.g., Figures 7D and 7L). For example, in CMV-specific T cells, the TEMRA#1 cluster contains high expression of cytotoxic genes such as IFNG, TNF, and PRF1 but not GZMK, and the TEMRA#4 cluster is IFNG-TNF-PRF1+GZMK+ (see, e.g., Figures 7C and 7L). Notably, the TEMRA#2 cluster in CMV-specific T cells contains low expression of all cytotoxic genes but high expression of type I IFN-stimulated genes (ISGs), such as ISG15, ISG20, IFIT1, and OASL (see, e.g., Figures 7C and 7L). Such upregulation of ISG genes reflects the specific induction of type I IFN responses in a small subset of CMV-specific T cells, which could be stimulated by local production of type I IFN in response to the CMV virus or bystander production of type I IFN from other pathogens in the patient. To test whether ENTER virus binding affects T cell status (such as type I IFN response), we compared Leiden clustering with and without ENTER-induced genes and observed highly concordant clusters. This result suggests that ENTER virus binding has minimal impact on the T cell status of primary T cells isolated from the patient's blood (see Figure 7M).

[0285] We then synthesized the antigen specificity, TCR repertoire, and gene expression of CMV-specific T cells in patients. As expected, CMV-specific T cells were predominantly pp65 T cells associated with extensive clonal expansion. 495~503 It was found that the specific T cells express pp65 495~503 It was confirmed that US8 is a highly immunogenic CMV epitope (see, e.g., Figures 7E-7F). 74~82A rare population of T cells (43) was observed that was labeled by the pMHC ENTER virus (see Figure 7N). TCR sequencing identified resting US8 74~82 Up to 60% of specific cells express peptide US8 74~82 These clones were found to share TCRs with specific T cells, confirming their clonal identity (see, for example, Figure 7O). Interestingly, by tracking the predominant TCR clones in each donor with expansion, it was found that donor #2 contained the most expanded TCR clones, whereas the TCR clones in donor #1 hardly expanded (see Figures 7P-7Q). These experimental data indicate that ENTER-seq can detect multiple antigen specificities in the presence of highly dominant epitopes from fresh PBMCs without expansion.

[0286] To measure the strength of binding of pMHC-presenting ENTER virus, the number of pMHC bound per cell was also quantified. Integrating the strength of pMHC binding and TCR clonal expansion of CMV pp65-specific T cells in patients showed that pMHC binding was significantly higher in highly proliferating T cell clones (clone size >50) than in less proliferating T cells (see, e.g., Figure 7G). Because ENTER pMHC binding is positively correlated with TCR affinity (see, e.g., Figure 2F), the experimental data described herein suggest that high TCR affinity is likely associated with and drives greater T cell clonal expansion.

[0287] p65 495~503Among the specific T cells, three predominant TCR clones were found that had the same TCR sequence as peptide-enriched pp65-specific T cells. Similar to in vitro peptide-expanded T cells, these pp65-specific TCR clones display phenotypic differences despite targeting the same antigen epitope (see, e.g., Figure 7R). Surprisingly, phenotypic heterogeneity was observed within the same TCR clones (see, e.g., Figures 7R-7S). For example, TCR clone #2 is composed of type I IFN ISG+TEMRA, stressed high IFNG·low FASLG TEMRA, and low IFNG·high FASLG TEMRA (see, e.g., Figures 7R-7S). This data revealed the underlying intraclonal phenotypic diversity of the same TCR clones, suggesting that the T cell state is influenced by both TCR binding specificity and the local microenvironment.

[0288] Example 10 This example describes the results of experiments performed to demonstrate phenotypic transition and clonal diversity of CMV-specific T cells upon ex vivo antigenic peptide-induced expansion.

[0289] To understand how antigen-specific proliferation affects the molecular phenotype of antiviral T cells, we performed a comparison of ENTER-seq of CMV-specific T cells before and after peptide-induced proliferation. Combining antigen specificity, CITE-seq, and transcriptional programs, we observed that a phenotypic transition of pp65-specific T cells occurs upon antigen-specific proliferation (see, e.g., Figure 7H). pp65-specific T cells isolated directly from the patient's blood were mostly TEMRA T cells, i.e., a terminally differentiated effector memory subset. After antigen-induced proliferation, these T cells lost CD45RA expression and gained CD45RO expression, suggesting that these TEMRA T cells can further differentiate into effector memory T cells (TEM, CD45RA-CD45RO+) (see, e.g., Figure 7H). This observation was validated in both donors by flow cytometry (see, e.g., Figure 7I).

[0290] Additional experiments were performed to determine whether the phenotypic changes upon expansion were driven by a cell state transition of the entire antiviral repertoire or biased by selective expansion of certain T cell clones. In these experiments, TCR sequences were utilized as a "natural" barcode to track the cell state of each dominant T cell clone before and after expansion. IFN-I ISG gene scores and cytotoxicity gene scores were calculated to reflect the cell state and cytotoxic function of individual T cells upon type I IFN stimulation (see, for example, general methods in Example 11). IFN-I ISG and cytotoxicity scores were found to be highly heterogeneous at rest in each T cell clone of the patient (see, for example, Figure 7J). After ex vivo expansion, IFN-I ISG and cytotoxicity scores were significantly concentrated across T cell clones, and loss of type I IFN ISG gene expression and upregulation of cytotoxicity genes were found in all three clones (see, for example, Figure 7J). This result suggested that T cell effector function could be further enhanced by peptide-induced expansion, but the type I IFN responses observed in patients could not be sustained upon ex vivo expansion.

[0291] In contrast, antigen activation can also generate interclonal phenotypic diversity. 495~503 All three of the specific T cell clones had low expression of the T helper 2 (Th2) cytokine gene IL13, but widespread expression of the memory T cell transcription factor EOMES was found during resting (see, e.g., FIG. 7J). Upon antigen activation and expansion, clone 1 generated cells expressing either IL13, EOMES, or both, clone 3 generated cells expressing IL13 or EOMES mutually exclusively, and clone 2 increased the frequency of EOMES but not IL13 (see FIG. 7J). Consistently, upon expansion, pp65 495~503Specific T cell clones showed further clonal diversity in the expression of two Th2 cytokines, IL4 and IL13 (see, e.g., FIG. 7T). Thus, each TCR may recognize the same antigen in a different way, driving diverse transcriptional programs and cellular states.

[0292] Taken together, ENTER-seq enabled systematic dissection of T cell specificity, resting cell state, and possible antigen-induced cell fates following viral infection in patients. Transition of anti-CMVT cells from TEMRA T cells to TEMT cells in cytotoxic and type I IFN responses, accompanied by upregulation of Th2 cytokine genes in specific T cell clones following peptide-induced antigen-specific proliferation (see, e.g., Figure 7K).

[0293] Example 11 General Materials and Methods for Examples 1-10 Plasmid cloning and construction Primers were ordered from IDT DNA Technologies, and gene fragments were synthesized by twist bioscience and IDT. Table 3 shows a list of vector designs used in this study. In general, all constructs were made by Gibson assembly (New England Biolabs). Briefly, pMD2.G (addgene#12259) was digested with EcoRI to remove the wild type VSV-g gene fragment. It was assembled with the mutation VSV-g (K37Q and R354Q) introduced by PCR primers to generate the VSV-g double mutant. psPax2 (addgene#12260) was digested with BsiWI and SphI, and eGFP was fused after MA. To generate the packaging vector with NC-eGFP / NC-mNeon fusion, psPAX2-D64V-NC-MS2 (addgene#122944) was digested sequentially with SphI and BspEI. Then, a part of gag and eGFP or mNeon were assembled together with the backbone. GFP-VPR was obtained from Addgene (#83374).

[0294] To generate HPV16_L2 antigen-specific BCR, the light and heavy chains were amplified separately from vector JWW-1 (addgene#66748) and connected by 2A peptide. This was then inserted into a piggybac vector behind the CMV promoter (PB-CMV), after which the PDGFR transmembrane (TM) domain and 2A-mCherry were added to express this antibody on the cell surface. Anti-Her2 BCR was cloned in the same way from the source trastuzumab vector (addgene#61883). To generate anti-SAR2-RBD BCR, DNA fragments encoding the light and heavy chains of the RBD antibody (Protein Data Bank, accession number 7BWJ, (Ju et al., 2020)) were codon-optimized and synthesized (Twist Bio). A signal peptide was then added to each chain, and the heavy chain was further extended to full length using human IgG1Fc and PDGFR TM sequences. The BCR was then inserted into a lentiviral vector driven by the SFFV promoter with hygromycin resistance.

[0295] NY-ESO-1 TCR (Roth et al., Nature 2018; clone 1G4, wild type (1G4wt) and its mutant form (a95:LY) (with high affinity), alpha and beta chains linked in tandem by 2A peptide (Robbins et al., 2008) were synthesized in single-strand format and inserted into a lentiviral vector with hygromycin resistance. TCR5 binding to p5 peptide derived from CMV virus was amplified from alpha chain (addgene#164999) and beta chain (addgene#165000) and made into single-strand format similar to NY-ESO-1 TCR described above.

[0296] To present antigen and HLA peptide complexes on the viral surface, we first generated a cloning lentiviral vector with a strong CMV promoter, multiple cloning sites, and a WPRE element to enhance expression. The CD19-CAR vector was generated by inserting scFv CD19 (provided by Macall lab) with CD8 stalking linker and TM into the lentiviral plasmid, followed by 2A-puromycin and 2A-eGFP. The scFv CD19 and TM were replaced to generate other antigen candidates, including HPV-L2 antigen, CD40L (addgene#125795), and CD40L mutant (addgene#125796). For screening of the TM domain, the TM was replaced with 10 alternatives in the HPV-L2 antigen viral vector (Table 4). The DNA fragment of the SAR2 spike RBD domain was synthesized and inserted into the lentiviral expression vector as described above, followed by the insertion of the CD8 stalking linker and TM domain. For Her2 presentation, a truncated Her2 (a1-a700) fragment containing the native TM and an additional 55 aa cytoplasmic tail was amplified from WTHER2 (addgene#16257) and inserted into the above vector.

[0297] To present the pMHC complex, a single-stranded vector was constructed that contained a signal peptide, an antigen peptide, a G4S linker, beta 2 myeloglobulin (B2M), a second G4S linker, and an HLA allele in tandem. DNA encoding the human growth hormone signal peptide through beta 2 myeloglobulin was synthesized and inserted into a lentiviral vector along with the HLA allele. Here, the HLA allele A0201 was amplified from addgene vector #119052, and the allele A0101 was amplified from addgene #165009. Two cysteine ​​mutations were introduced to stabilize the peptide bond by a disulfide bond between Y84C of the HLA allele and G2C present in the post-peptide G4S linker. To make it compatible with the 10x Genomics sequencing platform, the 10x TSO sequence (Table 6) was further inserted into the linker between B2M and HLA encoding amino acid SHIRN, and the 10x PCR handle in the 5'UTR after the CMV promoter (Table 6). A cloning vector was constructed by replacing the antigen peptide into two esp3I sites, in which various HLA peptides (Table 5) can be suitably inserted.

[0298] Various vectors were generated for delivery purposes. First, in the same approach as for the VSV-g mutant, VSV-G in the pMD vector was replaced with different envelope proteins such as RBD, HER2, pp65-HLA-A2. Then, cargo delivery vectors were constructed in which only the cargo, e.g., HSV-TK-2A-egfp (HSV-TK from addgene#33308) and eGFP were driven by the Efla promoter in a lentiviral vector. For delivery of shRNA, different shRNAs were placed under the human U6 promoter in a lentiviral vector containing eGFP and puromycin as fluorescent and selection markers. To label cells with red fluorescent protein, the mScarlet transgene was inserted after the EF1 short promoter in a lentiviral vector with puromycin resistance.

[0299] Transfection and lentivirus production To generate regular lentivirus for cell line infection and production, HEK293T were transfected with the virus expression vector (2 μg), pMD2.G (VSV-G wild type) (1 μg), and psPax2 (2 μg) using Lipofectamine 3000 per 6 wells. The medium was changed the next day, and the virus supernatant was collected twice at 48 and 72 hours, respectively. The virus was concentrated with 4× Lenti-X according to the manufacturer's protocol and stored at 20-fold concentration at -80°C. To generate viruses that target specific receptors and integrate, VSV-G mutants were used instead. To generate antigen-presenting viruses that can be fluorescently detected without integration, fluorescent protein fusions (NC-eGFP or NC-mNeon) of VSV-G mutants and psPAX2-D64V (D64V mutation on integrase) vectors were mixed with the antigen expression vector according to the above ratio and transfected HEK293T cells. For pMHC-presenting viruses, HLA-KO HEK293T cells were used for transfection. Viruses were collected, concentrated 40-fold, and stored at -80 °C. To generate lentivirus for cargo delivery, HEK293T were transfected with cargo expression vector (1.6 μg), pMD2.G VSV-g mutant (0.8 μg), psPax2 (1.6 μg), and envelope plasmid (1 μg) per 6 wells using Lipofectamine 3000. Viruses were collected as above, concentrated 40-fold, and stored at -80 °C before use. Lentivirus titers were measured by Lenti-X GoStix Plus kit (Takarabio) according to the manufacturer's protocol.

[0300] Cell culture and cell line production Raji, Ramos, and Jurkat related cell lines were cultured in RPMI supplemented with 10% FBS (Invitrogen) and 1x penicillin / streptomycin. HEK293T related cells were maintained in DMEM supplemented with 10% FBS and 1x penicillin / streptomycin. HLA-KO HEK293T cells were generated by electroporation of Cas9RNP targeting HLA-A, HLA-B, and HLA-C alleles, and HLA-KO cells were further sorted based on surface expression of HLA-A / B / C. Ramos cells were obtained. Jurkat TCR-negative-76 cells, as well as Jurkat expressing CMV pp65TCR and flu-mlTCR were obtained. To generate stable cell lines containing BCR and TCR expressing cells, Ramos or Jurkat cells were infected with virus and selected after 4-5 days by sorting or with drugs. NYESO-TCR Jurkat cells and RBD-BCR Ramos cells were infected with red mScarlet virus and selected with puromycin to generate mScarlet red fluorescently labeled cell lines.

[0301] Lentiviral infection and viral incubation assays In Figure 1B, 30 μL of concentrated lentivirus was added to 250K Raji or Jurkat cells in a 12-well plate. After 3 days, GFP signals were measured by flow cytometry. In the figures after 1B, 200K target cells were collected in a tube and the supernatant was removed after centrifugation. The cell pellet was resuspended in 30 μL of concentrated GFP-fusion lentivirus and incubated at 37°C. After 2 hours of incubation, the cells were stained with flow cytometry antibodies at 4°C for 10 minutes (if necessary), washed twice with RPMI medium, and finally subjected to flow cytometry. In Figure 2D, to quantify binding of ENTER viruses presenting pMHC antigen variants with different TCR affinities, virus titers were normalized and 100K NY-ESO-1 TCR T cells (1G4 wild type or a95:LY mutant) or off-target CMV-pp65 TCR+ T cells were incubated with titrations of ENTER viruses presenting antigen variants (gp24 levels of 4ng, 20ng, 40ng). After 2 hours of incubation, cells were washed and subjected to flow cytometry to quantify GFP+ cells. In Figure 2C, to compare the sensitivity of pMHC-presenting ENTER viruses and pMHC tetramers on a per mole basis of each reagent, 100K NY-ESO-1 TCR+ T cells were incubated with NY-ESO-1 157~165 2 x 10 antigen-presenting 8 ENTER virus (20 ng p24) or various NY-ESO-1 157~165 pMHC tetramer (2 × 10 8 ~8×10 9 The moles of each reagent were calculated as follows: 10 4 Since each virus particle contains 1 pg of p24 protein, a virus with 20 ng of p24 = 20 * 1000 * 10 4 =2×10 8 In the case of pMHC tetramer, the molecular weight is about 500KD (PE-streptavidin: about 300KD, pMHC tetramer: about 200KD (50KD monomer *4) Therefore, 1 μg of pMHC tetramer = 1 μg * 10 6* (6.02×10 23 ) / 500 / 1000=1.2×10 12 This results in tetramers.

[0302] Viral binding and fusion assays 20 μL of CD19-scFv-displaying GFP virus was incubated with 200K Raji B cells for 2 h at 4° C. or 37° C. Cells were washed twice and subjected to 0.5 mg / mL proteinase K treatment to digest cell surface-bound virus for 15 min at 37° C. Cells before and after proteinase K treatment were subjected to flow cytometry to quantitate the percentage of GFP-positive cells.

[0303] Immunocapture assay 10 μL of Protein G Dyna beads were incubated in 1 mL of blocking buffer (PBS with 0.1% BSA) at room temperature for 20 min. 2 μg of anti-CD40L antibody (Cat#157009, Biolegend), anti-VSV-G antibody (clone 8G5F11, Millipore sigma), or IgG antibody was added to the beads with 100 μL of blocking buffer and rotated at 4 °C for 30 min. The antibody-bound beads were washed three times and the supernatant was removed. 30 μL of CD40L-displaying virus was added to the beads with 30 μL of blocking buffer and rotated at room temperature for 1 h. 5 μL of CD40L-displaying virus from the same batch was prepared as an input sample. The beads were washed three times and the supernatant was removed. 100 μL of Trizol was added to the beads or input sample and subjected to RNA extraction with Zymo Quick-RNA Miniprep Kit. RT-qPCR was performed using Stratagene Brilliant II SYBR Green QRT-PCR Master Mix (Agilent).

[0304] Lentiviral targeted cargo delivery Cells were incubated with virus in medium containing 6 μg / ml polybrene as described above. Delivery efficiency and specificity were assessed after 3 days using flow cytometry (Attune NxT). When necessary, cells were first stained with PeCy7 anti-human IgG (for B cells, clone G18-145, BD bioscience) or APC anti-human CD3 (for T cells, clone HIT3a, Biolegend) before flow cytometry analysis. For HSV-TK cell killing assays, two cell populations, one of which was labeled by mScarlet (off-target) and the other was non-fluorescent (on-target), were mixed in a 1:1 ratio and incubated with virus. After 3 days, ganciclovir (GCV, Invivogen) was added to a final concentration of 0.1 g / ml, which was counted as day 0. Cell medium and drugs were refreshed every 3 days. After 2 days, 300 pL of cell culture was harvested daily and analyzed by flow cytometry after staining with IgG or CD3. The percentage of viable on-target and off-target cells was calculated and plotted against the days (normalized to day 0). Alternatively, the raw count of viable cells in the targeted or NT populations on day 4 of treatment was also compared between TK delivery and eGFP alone delivery.

[0305] For the apoptosis assay with FAS shRNA delivery, Jurkat T cells were infected with different shRNAs and stained with PE-FAS / CD95 (Biolegend) to compare the effect of shRNA knockdown. A mixture of CMV-Jurkat (on-target) and mScarlet+NY-ESO-Jurkat (off-target) was incubated with the shRNA virus. After 5 days, anti-FAS antibody (clone CH11, Millipore Sigma) was added at 0.25 μg / ml to induce apoptosis. Cells were harvested 14 hours later and stained with APC anti-annexin V (Biolegend) and 7-AAD according to the manufacturer's protocol. Samples were then analyzed by flow cytometry (BDLSR II), first gating on low 7-AAD and low annexin V populations. The ratio of transduced on-target and off-target cells was then compared between FAS shRNA and control shRNA, and normalized bar graphs were generated.

[0306] Incubation of lentivirus with mixed cell populations For T cell mixing experiments, Jurkat T cells expressing Flu-TCR were labeled by CellTrace Violet dye (#C34571, ThermoFisher) according to the manufacturer's protocol. Violet-labeled Flu-m1 TCR+ T cells were mixed with NY-ESO-1-TCR+ T cells at various ratios including 1:1, 1:10, 1:100, and 1:1000. Mixed T cells were incubated with 40 μL of concentrated HLA-A2-Flu antigen-presenting GFP virus for 2 h at 37 °C. T cells were stained with CD3-APC (clone HIT3a, BioLegend) antibody, washed twice, and subjected to flow cytometry. For B cell mixing experiments, Ramos B cells expressing HPV-BCR were labeled with CellTrace Violet dye and mixed with Ramos B cells expressing HPV-L2 BCR at various ratios including 1:1, 1:10, 1:100, and 1:1000. The mixed cells were incubated with 40 μL of concentrated RBD antigen-presenting GFP virus for 2 h at 37°C. B cells were stained with IgG-PE-Cy7 antibody (clone G18-145, BD Biosciences), washed twice, and subjected to flow cytometry. Metrics were calculated as follows:

[0307] Sensitivity = percentage of GFP+ on-target cells out of total on-target cells

[0308] Specificity = 1-(percentage of GFP+ off-target cells among total off-target cells)

[0309] Signal / noise ratio = (percentage of GFP+ on-target cells among total on-target cells) / (percentage of GFP+ off-target cells among total off-target cells).

[0310] Isolation and activation of human primary immune cells Buffy coats from healthy donors were obtained from the Stanford Blood Center with written consent. Peripheral blood mononuclear cells (PBMCs) were isolated using Lymphoprep (Cat#07811, STEMCELL Technologies) density gradient centrifugation, cryopreserved, and stored at -80°C. B cells were purified by negative selection from thawed PBMCs using the EasySep Human B Cell Enrichment Kit (Cat#19844, STEMCELL Technologies) according to the manufacturer's protocol. Isolated B cells were purified at 1 × 10 6 The cells were incubated at 10000 cells / mL in IMDM medium supplemented with 10% FBS and 55 mM β-mercaptoethanol and activated for 2 days with a CellXVivo Human B cell expander (1:250 dilution, R&D Systems) and 50 ng / mL IL2 (Cat#200-02-10ug, PeproTech). LRS chambers from CMV-infected (CMV seropositive) HLA-A2+ donors were obtained from Stanford Blood Center with written consent. PBMCs were isolated and stored as described above. CD8+ T cells were purified by negative selection from thawed PBMCs using the EasySep Human CD8+ T Cell Enrichment Kit (Cat#19053, STEMCELL Technologies) according to the manufacturer's protocol.

[0311] Peptide enrichment of antigen-specific T cells Short 9-mer peptides encoding CMV epitopes (compatible with HLA-A2 alleles, Table S3) were synthesized as lyophilized powders by Elimbio. Peptides were dissolved in DMSO at 10 mg / mL. PBMCs were isolated from donor blood as described above. PBMCs were incubated in T cell medium (RPMI medium supplemented with 10% FBS, 1x penicillin / streptomycin, 100 mM HEPES, 55 mM beta-mercaptoethanol). Individual peptides (10 μg / mL) or pooled peptides (1 μg / mL for each peptide) were added to PBMCs and incubated in T cell medium for 10 days. 50 ng / mL IL-2 was added every 2 days. After peptide enrichment, PBMCs were incubated with virus and / or PE tetramers and then analyzed by flow cytometry.

[0312] Flow cytometry In Figure 1D, B cells were incubated with virus for 2 hours and then stained with Human TruStain FcXTM (FcBlock, BioLegend), CD19-APC (clone HIB19), and CD20-V450 (clone L27) antibodies in cell staining buffer (BioLegend) for 10 minutes at 4°C. In Figure 2, Jurkat T cells were incubated with virus for 2 hours and then stained with CD3-APC (clone HIT3a) and PE-labeled tetramer loaded with peptide (NIH tetramer core) for 30 minutes at 4°C. In Figure 5, cells were incubated with virus for 2 hours and then stained with human Fc block, CD3-APC, CD8-BV711 (clone SK1), tetramer-PE as required, and viability dye for 30 minutes at 4°C. After staining, cells were washed twice with cell staining buffer and analyzed by flow cytometry (Attune, Thermo Fisher). Unless otherwise specified, all antibodies were obtained from BioLegend. Tetramers were obtained from the NIH tetramer core.

[0313] RNA-seq experiments and analysis CMV pp65-TCR+ T cells were incubated in 30 μL of pp65 495~503 Presented ENTER virus or 1 μg pp65 495~503 The cells were incubated with tetramers for 2 hours. Cells were washed twice and subjected to RNA extraction. RNA was extracted using a Quick-RNA Miniprep Kit (Zymo Research) with on-column Dnase digestion. RNA-seq libraries were prepared for each sample using the TruSeq® Stranded mRNA Library Prep Kit (Cat#20020594, Illumina) according to the manufacturer's instructions using at least 100 ng of RNA. Libraries were sequenced with Illumina Nextseq, generating 2 × 150 paired-end reads. RNA-seq reads were mapped to the human genome (hg19) by STAR using default parameters (--outFilterMultimapNmax 1--alignEndsType EndToEnd--outSAMattributes NH HI NM MD). Quantification of aligned reads at gene level was performed by HTseq counts with default parameters (--stranded=reverse-additional-attr=gene_name). Raw counts were used to identify differentially expressed genes (DEGs) using DESeq2 with size factor normalization, and DEGs were identified if the Benjamini & Hochberg adjusted p-value was <0.01 and the difference in gene expression change was >2-fold.

[0314] ENTER-seq workflow for mixed TCR expressing Jurkat T cells All primers were synthesized and ordered from IDT (Table 6). Jurkat cells expressing different TCRs were mixed together and the virus mixture was stained as described above. GFP+ cells were then sorted on a BD Aria II. A commercial 10x Genomics 5'RNA kit was customized to simultaneously read out HLA peptides, TCR, and transcriptome per single cell. Immediately after sorting, cells were washed once with PBS+0.4% BSA at 4°C, mixed with RT (reverse transcription) mixture spiked with 0.1 μM customized TCRalpha RT primers (mixture of hs_TRAc_RT and NYESO_TRAc_RT) and loaded into 10x Chromium. cDNA was amplified and cleaned up according to the manufacturer's protocol to generate the transcriptome.

[0315] During cDNA cleanup, the supernatant containing shorter fragments of HLA peptides and TCR information was further mixed with SPRISelect beads to 0.9x and cleaned up. HLA peptide-encoding libraries were generated by two rounds of nested PCR and a final round of index PCR. First, HLA peptide cDNA was enriched by eight cycles of PCR (98°C for 45 min, followed by eight cycles of 98°C for 20 sec, 59°C for 20 sec, and 72°C for 30 sec) using 0.5uM of 10x_5pRNA_Fw and HLA_nested_fw. After cleanup, 5ul of the eluate was used for a second round of PCR using 0.5um of the nested primers mentioned above and Illumina adapters P7_Tru_HLA_fw and P5_adapter primers. Finally, 5ul of the eluate was taken and the final library was generated using Illumina Truseq-based index primers. The above primers are designed in a manner compatible with dual indexes, therefore either customized index primers or 10x dual index primers can be used here.

[0316] To read out TCR information in Jurkat cell lines, we generated a library covering the VDJ portion of TCR alpha to infer the TCR identity of cells. First, TCR DNA was enriched by nested PCR (specifically, 45 min at 98°C, followed by 8 rounds of 20 sec at 98°C, 20 sec at 59°C, and 30 sec at 72°C) using 0.5 μM 10×_5pRNA_Fw and a 0.5 uM mixture of nyeso_TRAc_rev and hs_TRAc_rev targeting two different TCRs. Then, 5 ul of eluate was taken and a second round of nested PCR was performed using Illumina adapters (a mixture of P7_TRAc_nyeso_Rev and P7_TRAc_hs_Rev) and P5_adapter for 8 cycles, followed by a final index PCR in the same way as for the HLA library.

[0317] ENTER-seq analysis of mixed TCR-expressing Jurkat T cells Libraries were sequenced using Illumina's Novaseq and Miseq platforms. Transcriptome fastq files were analyzed using 10x CellRanger to provide single-cell barcodes. TCR library fastq files were mapped to the TCR alpha chain using a custom Python script. UMI counts of each TCR species per cell barcode were calculated. To exclude doublets, the UMI count of the dominant TCR was set to be at least 10 times higher than the UMI count of the non-dominant TCR species per gem barcode. HLA peptide readouts were then processed using CellRanger counts using peptide sequences as feature references. Downstream analysis and plots were generated using the matplotlib package in python.

[0318] ENTER-seq workflow for primary T cells before or after ex vivo expansion with CMV antigen peptide induction Peptide-stimulated donor PBMCs were harvested and IE1 81~89 , IE1 316~324, US150A 152~161 , US8 74~82 , UL100 200~208 , UL46 100~108 , pp65 417~425 , pp65 325~333 , pp65 188~196 , pp65 120~128 , pp65 495~503 , and pp65 14~22 The donors were stained with a mixture of 12 viruses that display CMV antigenic epitopes, including IL7R, CD45RA, and IL7R. The peptide sequences of the CMV antigens are shown in Table 5. After 2 hours, the cells were stained with barcoded antibodies CD45RA, CD45RO, and IL7R (Biolegend totalseq-C, Cat#304163, Cat#304259, Cat#351356), live dead stain, CD3-APC, and CD8-BV711 for 20 minutes on ice. Samples from each donor were also stained with unique hashtag barcoded antibodies (Biolegend totalseq-C, Cat#394661, Cat#394663). After two washes, CD8+CD3+GFP+ cells were sorted and run on the 10x Genomics platform using the 5'RNA and VDJ kits according to the manufacturer's protocol. Here, the following libraries were obtained for each sample: 10x gene expression library, VDJ library, and feature barcoded CITE-seq library, according to the manufacturer's protocol. In addition, HLA peptide libraries were also generated in the same manner as above. The final libraries were sequenced on either Illumina Miseq, Nextseq550, or Novaseq6000. For ENTER-seq of primary T cells directly isolated from patient blood samples without peptide stimulation / expansion, total CD8+ T cells were first purified from cryopreserved patient PBMC samples using EasySep Human CD8+ T Cell Isolation Kit (Cat#17953, STEMCELL Technologies) according to the user's protocol. Then, the top three ENTER pMHC viral (pp65 495~503 , US8 74~82 , and UL100 200~208) mixture was prepared for 2 h at 37° C. Subsequent steps of antibody staining, flow cytometry sorting, and 10× Genomics library generation were the same as for ex vivo expanded T cells described above.

[0319] ENTER-seq analysis of primary cells from CMV seropositive donors scRNA-seq reads were aligned to the GRCh38 genome and quantified using CellRanger counts (10x Genomics). CITE-seq reads were processed using CellRanger counts, using antibody oligo barcodes as feature references. TCR-seq reads were mapped to a VDJ compatible reference (refdata-CellRanger-vdj-GRCh38-alts-ensembl-5.0.0) using CellRanger vdj (10x Genomics). HLA peptide reads were processed using CellRanger counts, using peptide sequences as feature references.

[0320] Subsequent analysis of single-cell RNA-seq and CITE-seq was performed using SCANPY (Wolf et al., 2018). Cells with fewer than 200 detected genes or more than 10% mitochondrial RNA readouts were excluded from the analysis. Doublet cells were excluded using CITE-seq analysis of barcoded hashtag antibodies labeling donor origin. For cell clustering, raw UMI counts were first normalized by total counts to correct for library size and then log-normalized. Variable genes were called using scanpy.pp.highly_variable_genes() with default parameters. Variable TCR genes were removed prior to principal component analysis (PCA) to prevent clustering bias from variable TCR transcripts. The effects of total counts and proportion of mitochondrial genes per cell were then removed and the data scaled to unit variance. The scaled data was used as input to PCA analysis based on variable genes (without TCR genes). Clusters were identified using the Leiden graph clustering method using the first 40 principal components. To determine whether ENTER virus-induced gene expression influences T cell status and clustering, Leiden graph clustering was performed before and after removal of 28 ENTER virus-induced genes identified from bulk RNA-seq data. UMAP plots were generated using scanpy.tl.umap() and scanpy.pl.umap() with default parameters. Heatmap plots were generated using scanpy.tl.heatmap() using raw value or z-score scaled gene expression.

[0321] Initial clusters were annotated using expression of known markers such as CD3E, CD4, CD8A, CD45RA, CD45RO, CCR7, GZMB, and KLRB1. All CD8+ T cells were CD3E+CD8A+CD4-. Naive T cells were CD45RA+CCR7+. Central memory T cells (TCM) were CD45RA-CCR7+. Effector memory T (TEM) cells were CD45RO+CCR7-. Terminally differentiated effector cells that re-express CD45RA (TEMRA) were CD45RA+CCR7-CD45RO- and MAIT cells were KLRB1+CXCR6+TRAV1-2+. Gene scores were calculated using scanpy.tl.score_genes() with ctrl_size=500, use_raw=True. A gene set of cytotoxic genes was curated from well-established cytotoxic molecules. T cell exhaustion genes, T cell activation genes, and type I IFN response genes were selected from previous literature ( Yost et al., 2019 ).

[0322] TCR association analysis was performed using Scirpy (Sturm et al., 2020). Contiguannotation files generated by CellRanger vdj were used as input for TCR analysis. TCR quality was analyzed using scirpy.tl.chain_qc(). TCR clonotypes were defined using scirpy.pp.ir_dist() and scirpy.tl.define_clonotypes() with default parameters based on CDR3 nucleotide sequence similarity. TCR clonotypes were visualized on a network using scirpy.tl.clonotype_network() with min_cells=3. CDR3 amino acid composition was generated using weblogo (Crooksetal., 2004). Cells with raw counts of 6 or more HLA peptides against any individual antigen were labeled as antigen-specific T cells. The number of antigen peptides per cell was quantified using log(counts+l) transformation. pp65-specific T cells were separated into various clonally expanded cells with clone sizes of >50, >10, or >1. The distribution of the number of antigen peptides bound per cell in the different clonally expanded T cells was shown by violin plots. Using the predominant TCR clonotype as a barcode, 2D density plots were generated using the kdeplotQ function to show the cytotoxicity and type I IFN gene scores of T cells with the same TCR sequence before and after antigen-induced expansion. All plots (including violin plots, scatter plots, density plots, and bar graphs) were generated by Python matplotlib and seaborn.

[0323] Using the TCR as a barcode, false-negative T cells were identified in the ENTER-negative population with the same TCR sequence as the predominant antigen-specific T cells. Similarly, false-positive antigen-specific T cells, such as pp65-specific T cells, could be generated by tracing the TCR sequence between the pp65-specific T cells with the predominant clone and other CMV antigen-specific T cells or ENTER-negative T cells. Furthermore, the false-negative rate (FNR) and false-positive rate (FPR) were calculated for the top three antigen epitopes. pp65 495~503 For specific T cells, the FNR was 0.19% and the FDR was 0.36%. 74~82 For specific T cells, the FNR was 2.73% and the FDR was 0.18%. 200~208 For specific T cells, the FNR was 0% and the FDR was 0.07%.

[0324] While the present disclosure has been particularly shown and described with reference to certain embodiments, some of which are preferred embodiments, it should be understood by those skilled in the art that various changes in form and detail can be made therein without departing from the spirit and scope of the present disclosure disclosed herein. Accordingly, it is not intended to be limited to the precise abstract and disclosure presented herein.

[0325] [Table 3]

[0326] [Table 4]

[0327] [Table 5]

[0328] [Table 6]

[0329] References 1. Appay, V., Dunbar, P.R., Callan, M., Klenerman, P., Gillespie, G.M.A., Papagno, L., Ogg, G.S., King, A., Lechner, F., Spina, C.A., et al. (2002). Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections. Nat Med 8, 379-385. doi.org / 10.1038 / nm0402-379. 2. Armingol, E., Officer, A., Harismendy, O., and Lewis, N.E. (2021). Deciphering cell-cell interactions and communication from gene expression. Nat Rev Genet 22, 71-88. doi.org / 10.1038 / s41576-020-00292-x. 3. Bai, Z., Woodhouse, S., Zhao, Z., Arya, R., Govek, K., Kim, D., Lundh, S., Baysoy, A., Sun, H., Deng, Y., et al. (2022). Single-cell antigen-specific landscape of CAR T infusion product identifies determinants of CD19-positive relapse in patients with ALL. Sci Adv 8, eabj2820. doi.org / 10.1126 / sciadv.abj2820. 4. Beltinger, C., Fulda, S., Kammertoens, T., Meyer, E., Uckert, W., and Debatin, K.-M. (1999). Herpes simplex virus thymidine kinase / ganciclovir-induced apoptosis involves ligand-independent death receptor aggregation and activation of caspases. Proceedings of the National Academy of Sciences 96, 8699-8704. doi.org / 10.1073 / pnas.96.15.8699. 5. Bentzen, A.K., and Hadrup, S.R. (2017). Evolution of MHC-based technologies used for detection of antigen-responsive T cells. Cancer Immunol Immunother 66, 657-666. doi.org / 10.1007 / s00262-017-1971-5. 6. Bentzen, A.K., Marquard, A.M., Lyngaa, R., Saini, S.K., Ramskov, S., Donia, M., Such, L., Furness, A.J.S., McGranahan, N., Rosenthal, R., et al. (2016). Large-scale detection of antigen-specific T cells using peptide-MHC-I multimers labeled with DNA barcodes. Nat Biotechnol 34, 1037-1045. doi.org / 10.1038 / nbt.3662. 7. Billerbeck, E., Blum, H.E., and Thimme, R. (2007). Parallel expansion of human virus-specific FoxP3- effector memory and de novo-generated FoxP3+ regulatory CD8+ T cells upon antigen recognition in vitro. J Immunol 179, 1039-1048. doi.org / 10.4049 / jimmunol.179.2.1039. 8. Birnbaum, M.E., Dong, S., and Garcia, K.C. (2012). Diversity-oriented approaches for interrogating T-cell receptor repertoire, ligand recognition, and function. Immunol Rev 250, 82-101. doi.org / 10.1111 / imr.12006. 9. Bollard, C.M., Kuehnle, I., Leen, A., Rooney, C.M., and Heslop, H.E. (2004). Adoptive immunotherapy for posttransplantation viral infections. Biol Blood Marrow Transplant 10, 143-155. doi.org / 10.1016 / j.bbmt.2003.09.017. 10. Briggs, J.A.G., Simon, M.N., Gross, I., Krausslich, H.-G., Fuller, S.D., Vogt, V.M., and Johnson, M.C. (2004). The stoichiometry of Gag protein in HIV-1. Nat Struct Mol Biol 11, 672-675. doi.org / 10.1038 / nsmb785. 11. Briney, B., Inderbitzin, A., Joyce, C., and Burton, D.R. (2019). Commonality despite exceptional diversity in the baseline human antibody repertoire. Nature 566, 393-397. doi.org / 10.1038 / s41586-019-0879-y. 12. Buchholz, C.J., Muhlebach, M.D., and Cichutek, K. (2009). Lentiviral vectors with measles virus glycoproteins - dream team for gene transfer? Trends Biotechnol 27, 259-265. doi.org / 10.1016 / j.tibtech.2009.02.002. 13. Buchholz, C.J., Friedel, T., and Buning, H. (2015). Surface-Engineered Viral Vectors for Selective and Cell Type-Specific Gene Delivery. Trends Biotechnol 33, 777-790. doi.org / 10.1016 / j.tibtech.2015.09.008. 14. Buenrostro, J.D., Wu, B., Litzenburger, U.M., Ruff, D., Gonzales, M.L., Snyder, M.P., Chang, H.Y., and Greenleaf, W.J. (2015). Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-490. doi.org / 10.1038 / nature14590. 15. Burbelo, P.D., Iadarola, M.J., Keller, J.M., and Warner, B.M. (2021). Autoantibodies Targeting Intracellular and Extracellular Proteins in Autoimmunity. Front Immunol 12, 548469. doi.org / 10.3389 / fimmu.2021.548469. 16. Burnie, J., and Guzzo, C. (2019). The Incorporation of Host Proteins into the External HIV-1 Envelope. Viruses 11, E85. doi.org / 10.3390 / v11010085. 17. Burnie, J., Tang, V.A., Welsh, J.A., Persaud, A.T., Thaya, L., Jones, J.C., and Guzzo, C. (2020). Flow Virometry Quantification of Host Proteins on the Surface of HIV-1 Pseudovirus Particles. Viruses 12, E1296. doi.org / 10.3390 / v12111296. 18. Burns, J.C., Friedmann, T., Driever, W., Burrascano, M., and Yee, J.K. (1993). Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc Natl Acad Sci U S A 90, 8033-8037. doi.org / 10.1073 / pnas.90.17.8033. 19. Cantin, R., Fortin, J.F., and Tremblay, M. (1996). The amount of host HLA-DR proteins acquired by HIV-1 is virus strain- and cell type-specific. Virology 218, 372-381. doi.org / 10.1006 / viro.1996.0206. 20. Certo, M.T., Ryu, B.Y., Annis, J.E., Garibov, M., Jarjour, J., Rawlings, D.J., and Scharenberg, A.M. (2011). Tracking genome engineering outcome at individual DNA breakpoints. Nat Methods 8, 671-676. doi.org / 10.1038 / nmeth.1648. 21. Chandran, S.S., and Klebanoff, C.A. (2019). T cell receptor-based cancer immunotherapy: Emerging efficacy and pathways of resistance. Immunol Rev 290, 127-147. doi.org / 10.1111 / imr.12772. 22. Chertova, E., Chertov, O., Coren, L.V., Roser, J.D., Trubey, C.M., Bess, J.W., Sowder, R.C., Barsov, E., Hood, B.L., Fisher, R.J., et al. (2006). Proteomic and biochemical analysis of purified human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages. J Virol 80, 9039-9052. doi.org / 10.1128 / JVI.01013-06. 23. Churlaud, G., Pitoiset, F., Jebbawi, F., Lorenzon, R., Bellier, B., Rosenzwajg, M., and Klatzmann, D. (2015). Human and Mouse CD8(+)CD25(+)FOXP3(+) Regulatory T Cells at Steady State and during Interleukin-2 Therapy. Front Immunol 6, 171. doi.org / 10.3389 / fimmu.2015.00171. 24. Crooks, G.E., Hon, G., Chandonia, J.-M., and Brenner, S.E. (2004). WebLogo: a sequence logo generator. Genome Res 14, 1188-1190. doi.org / 10.1101 / gr.849004. 25. Davis, M.M., and Bjorkman, P.J. (1988). T-cell antigen receptor genes and T-cell recognition. Nature 334, 395-402. doi.org / 10.1038 / 334395a0. 26. Davis, M.M., and Boyd, S.D. (2019). Recent progress in the analysis of αβT cell and B cell receptor repertoires. Curr Opin Immunol 59, 109-114. doi.org / 10.1016 / j.coi.2019.05.012. 27. De Guzman, R.N., Wu, Z.R., Stalling, C.C., Pappalardo, L., Borer, P.N., and Summers, M.F. (1998). Structure of the HIV-1 nucleocapsid protein bound to the SL3 psi-RNA recognition element. Science 279, 384-388. doi.org / 10.1126 / science.279.5349.384. 28. Derhovanessian, E., Maier, A.B., Hahnel, K., Beck, R., de Craen, A.J.M., Slagboom, E.P., Westendorp, R.G.J., and Pawelec, G. (2011). Infection with cytomegalovirus but not herpes simplex virus induces the accumulation of late-differentiated CD4+ and CD8+ T-cells in humans. J Gen Virol 92, 2746-2756. doi.org / 10.1099 / vir.0.036004-0. 29. Dobson, C.S., Reich, A.N., Gaglione, S., Smith, B.E., Kim, E.J., Dong, J., Ronsard, L., Okonkwo, V., Lingwood, D., Dougan, M., et al. (2022). Antigen identification and high-throughput interaction mapping by reprogramming viral entry. Nat Methods 19, 449-460. doi.org / 10.1038 / s41592-022-01436-z. 30. Dupic, T., Bensouda Koraichi, M., Minervina, A.A., Pogorelyy, M.V., Mora, T., and Walczak, A.M. (2021). Immune fingerprinting through repertoire similarity. PLoS Genet 17, e1009301. doi.org / 10.1371 / journal.pgen.1009301. 31. Einsele, H., Kapp, M., and Grigoleit, G.U. (2008). CMV-specific T cell therapy. Blood Cells Mol Dis 40, 71-75. doi.org / 10.1016 / j.bcmd.2007.07.002. 32. Elkington, R., Walker, S., Crough, T., Menzies, M., Tellam, J., Bharadwaj, M., and Khanna, R. (2003). Ex vivo profiling of CD8+-T-cell responses to human cytomegalovirus reveals broad and multispecific reactivities in healthy virus carriers. J Virol 77, 5226-5240. doi.org / 10.1128 / jvi.77.9.5226-5240.2003. 33. Finkelshtein, D., Werman, A., Novick, D., Barak, S., and Rubinstein, M. (2013). LDL receptor and its family members serve as the cellular receptors for vesicular stomatitis virus. Proc Natl Acad Sci U S A 110, 7306-7311. doi.org / 10.1073 / pnas.1214441110. 34. Fontenot, J.D., Gavin, M.A., and Rudensky, A.Y. (2003). Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol 4, 330-336. doi.org / 10.1038 / ni904. 35. Garboczi, D.N., Hung, D.T., and Wiley, D.C. (1992). HLA-A2-peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides. Proc Natl Acad Sci U S A 89, 3429-3433. doi.org / 10.1073 / pnas.89.8.3429. 36. Gerdemann, U., Katari, U.L., Papadopoulou, A., Keirnan, J.M., Craddock, J.A., Liu, H., Martinez, C.A., Kennedy-Nasser, A., Leung, K.S., Gottschalk, S.M., et al. (2013). Safety and clinical efficacy of rapidly-generated trivirus-directed T cells as treatment for adenovirus, EBV, and CMV infections after allogeneic hematopoietic stem cell transplant. Mol Ther 21, 2113-2121. doi.org / 10.1038 / mt.2013.151. 37. Gillespie, G.M., Wills, M.R., Appay, V., O’Callaghan, C., Murphy, M., Smith, N., Sissons, P., Rowland-Jones, S., Bell, J.I., and Moss, P.A. (2000). Functional heterogeneity and high frequencies of cytomegalovirus-specific CD8(+) T lymphocytes in healthy seropositive donors. J Virol 74, 8140-8150. doi.org / 10.1128 / jvi.74.17.8140-8150.2000. 38. Gotch, F., Rothbard, J., Howland, K., Townsend, A., and McMichael, A. (1987). Cytotoxic T lymphocytes recognize a fragment of influenza virus matrix protein in association with HLA-A2. Nature 326, 881-882. doi.org / 10.1038 / 326881a0. 39. Grover, J.R., Veatch, S.L., and Ono, A. (2015). Basic motifs target PSGL-1, CD43, and CD44 to plasma membrane sites where HIV-1 assembles. J Virol 89, 454-467. doi.org / 10.1128 / JVI.02178-14. 40. Guo, X.-Z.J., and Elledge, S.J. (2022). V-CARMA: A tool for the detection and modification of antigen-specific T cells. Proc Natl Acad Sci U S A 119, e2116277119. doi.org / 10.1073 / pnas.2116277119. 41. Gutierrez, C., and Schiff, R. (2011). HER2: biology, detection, and clinical implications. Arch Pathol Lab Med 135, 55-62. doi.org / 10.5858 / 2010-0454-RAR.1. 42. Hinrichs, C.S., and Rosenberg, S.A. (2014). Exploiting the curative potential of adoptive T-cell therapy for cancer. Immunol Rev 257, 56-71. doi.org / 10.1111 / imr.12132. 43. Hu, Z., Ott, P.A., and Wu, C.J. (2018). Towards personalized, tumour-specific, therapeutic vaccines for cancer. Nat Rev Immunol 18, 168-182. doi.org / 10.1038 / nri.2017.131. 44. Jager, E., Chen, Y.T., Drijfhout, J.W., Karbach, J., Ringhoffer, M., Jager, D., Arand, M., Wada, H., Noguchi, Y., Stockert, E., et al. (1998). Simultaneous humoral and cellular immune response against cancer-testis antigen NY-ESO-1: definition of human histocompatibility leukocyte antigen (HLA)-A2-binding peptide epitopes. J Exp Med 187, 265-270. doi.org / 10.1084 / jem.187.2.265. 45. Jalaguier, P., Cantin, R., Maaroufi, H., and Tremblay, M.J. (2015). Selective acquisition of host-derived ICAM-1 by HIV-1 is a matrix-dependent process. J Virol 89, 323-336. doi.org / 10.1128 / JVI.02701-14. 46. Joglekar, A.V., Leonard, M.T., Jeppson, J.D., Swift, M., Li, G., Wong, S., Peng, S., Zaretsky, J.M., Heath, J.R., Ribas, A., et al. (2019). T cell antigen discovery via signaling and antigen-presenting bifunctional receptors. Nat Methods 16, 191-198. doi.org / 10.1038 / s41592-018-0304-8. 47. Ju, B., Zhang, Q., Ge, J., Wang, R., Sun, J., Ge, X., Yu, J., Shan, S., Zhou, B., Song, S., et al. (2020). Human neutralizing antibodies elicited by SARS-CoV-2 infection. Nature 584, 115-119. doi.org / 10.1038 / s41586-020-2380-z. 48. Kaczmarczyk, S.J., Sitaraman, K., Young, H.A., Hughes, S.H., and Chatterjee, D.K. (2011). Protein delivery using engineered virus-like particles. Proc Natl Acad Sci U S A 108, 16998-17003. doi.org / 10.1073 / pnas.1101874108. 49. Kaya-Okur, H.S., Wu, S.J., Codomo, C.A., Pledger, E.S., Bryson, T.D., Henikoff, J.G., Ahmad, K., and Henikoff, S. (2019). CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat Commun 10, 1930. doi.org / 10.1038 / s41467-019-09982-5. 50. Kneissl, S., Zhou, Q., Schwenkert, M., Cosset, F.-L., Verhoeyen, E., and Buchholz, C.J. (2013). CD19 and CD20 targeted vectors induce minimal activation of resting B lymphocytes. PLoS One 8, e79047. doi.org / 10.1371 / journal.pone.0079047. 51. Kula, T., Dezfulian, M.H., Wang, C.I., Abdelfattah, N.S., Hartman, Z.C., Wucherpfennig, K.W., Lyerly, H.K., and Elledge, S.J. (2019). T-Scan: A Genome-wide Method for the Systematic Discovery of T Cell Epitopes. Cell 178, 1016-1028.e13. doi.org / 10.1016 / j.cell.2019.07.009. 52. Kutluay, S.B., Zang, T., Blanco-Melo, D., Powell, C., Jannain, D., Errando, M., and Bieniasz, P.D. (2014). Global changes in the RNA binding specificity of HIV-1 gag regulate virion genesis. Cell 159, 1096-1109. doi.org / 10.1016 / j.cell.2014.09.057. 53. Lee, M.N., and Meyerson, M. (2021). Antigen identification for HLA class I- and HLA class II-restricted T cell receptors using cytokine-capturing antigen-presenting cells. Sci Immunol 6, eabf4001. doi.org / 10.1126 / sciimmunol.abf4001. 54. Lee, D.S.W., Rojas, O.L., and Gommerman, J.L. (2021). B cell depletion therapies in autoimmune disease: advances and mechanistic insights. Nat Rev Drug Discov 20, 179-199. doi.org / 10.1038 / s41573-020-00092-2. 55. Lehmann, A.A., Zhang, T., Reche, P.A., and Lehmann, P.V. (2020). Discordance Between the Predicted Versus the Actually Recognized CD8+ T Cell Epitopes of HCMV pp65 Antigen and Aleatory Epitope Dominance. Front Immunol 11, 618428. doi.org / 10.3389 / fimmu.2020.618428. 56. Li, G., Bethune, M.T., Wong, S., Joglekar, A.V., Leonard, M.T., Wang, J.K., Kim, J.T., Cheng, D., Peng, S., Zaretsky, J.M., et al. (2019). T cell antigen discovery via trogocytosis. Nat Methods 16, 183-190. doi.org / 10.1038 / s41592-018-0305-7. 57. Lubke, M., Spalt, S., Kowalewski, D.J., Zimmermann, C., Bauersfeld, L., Nelde, A., Bichmann, L., Marcu, A., Peper, J.K., Kohlbacher, O., et al. (2020). Identification of HCMV-derived T cell epitopes in seropositive individuals through viral deletion models. J Exp Med 217, e20191164. doi.org / 10.1084 / jem.20191164. 58. McCutcheon, M., Wehner, N., Wensky, A., Kushner, M., Doan, S., Hsiao, L., Calabresi, P., Ha, T., Tran, T.V., Tate, K.M., et al. (1997). A sensitive ELISPOT assay to detect low-frequency human T lymphocytes. J Immunol Methods 210, 149-166. doi.org / 10.1016 / s0022-1759(97)00182-8. 59. Mimitou, E.P., Cheng, A., Montalbano, A., Hao, S., Stoeckius, M., Legut, M., Roush, T., Herrera, A., Papalexi, E., Ouyang, Z., et al. (2019). Multiplexed detection of proteins, transcriptomes, clonotypes and CRISPR perturbations in single cells. Nat Methods 16, 409-412. doi.org / 10.1038 / s41592-019-0392-0. 60. Moore, M.D., and Hu, W.S. (2009). HIV-1 RNA dimerization: It takes two to tango. AIDS Rev 11, 91-102. 61. Nikolic, J., Belot, L., Raux, H., Legrand, P., Gaudin, Y., and A Albertini, A. (2018). Structural basis for the recognition of LDL-receptor family members by VSV glycoprotein. Nat Commun 9, 1029. doi.org / 10.1038 / s41467-018-03432-4. 62. Pai, J.A., and Satpathy, A.T. (2021). High-throughput and single-cell T cell receptor sequencing technologies. Nat Methods 18, 881-892. doi.org / 10.1038 / s41592-021-01201-8. 63. Pasqual, G., Chudnovskiy, A., Tas, J.M.J., Agudelo, M., Schweitzer, L.D., Cui, A., Hacohen, N., and Victora, G.D. (2018). Monitoring T cell-dendritic cell interactions in vivo by intercellular enzymatic labelling. Nature 553, 496-500. doi.org / 10.1038 / nature25442. 64. Purcell, A.W., Ramarathinam, S.H., and Ternette, N. (2019). Mass spectrometry-based identification of MHC-bound peptides for immunopeptidomics. Nat Protoc 14, 1687-1707. doi.org / 10.1038 / s41596-019-0133-y. 65. Ranzani, M., Cesana, D., Bartholomae, C.C., Sanvito, F., Pala, M., Benedicenti, F., Gallina, P., Sergi, L.S., Merella, S., Bulfone, A., et al. (2013). Lentiviral vector-based insertional mutagenesis identifies genes associated with liver cancer. Nat Methods 10, 155-161. doi.org / 10.1038 / nmeth.2331. 66. Rasmussen, A.-M., Borelli, G., Hoel, H.J., Lislerud, K., Gaudernack, G., Kvalheim, G., and Aarvak, T. (2010). Ex vivo expansion protocol for human tumor specific T cells for adoptive T cell therapy. J Immunol Methods 355, 52-60. doi.org / 10.1016 / j.jim.2010.02.004. 67. Reynisson, B., Alvarez, B., Paul, S., Peters, B., and Nielsen, M. (2020). NetMHCpan-4.1 and NetMHCIIpan-4.0: improved predictions of MHC antigen presentation by concurrent motif deconvolution and integration of MS MHC eluted ligand data. Nucleic Acids Res 48, W449-W454. doi.org / 10.1093 / nar / gkaa379. 68. Robbins, P.F., Li, Y.F., El-Gamil, M., Zhao, Y., Wargo, J.A., Zheng, Z., Xu, H., Morgan, R.A., Feldman, S.A., Johnson, L.A., et al. (2008). Single and dual amino acid substitutions in TCR CDRs can enhance antigen-specific T cell functions. J Immunol 180, 6116-6131. doi.org / 10.4049 / jimmunol.180.9.6116. 69. Robins, H.S., Campregher, P.V., Srivastava, S.K., Wacher, A., Turtle, C.J., Kahsai, O., Riddell, S.R., Warren, E.H., and Carlson, C.S. (2009). Comprehensive assessment of T-cell receptor beta-chain diversity in alphabeta T cells. Blood 114, 4099-4107. doi.org / 10.1182 / blood-2009-04-217604. 70. Roth et al., Reprogramming human T cell function and specificity with non-viral genome targeting. Nature. 2018 Jul; 559(7714): 405-409. 71. Rubin, A.J., Parker, K.R., Satpathy, A.T., Qi, Y., Wu, B., Ong, A.J., Mumbach, M.R., Ji, A.L., Kim, D.S., Cho, S.W., et al. (2019). Coupled Single-Cell CRISPR Screening and Epigenomic Profiling Reveals Causal Gene Regulatory Networks. Cell 176, 361-376.e17. doi.org / 10.1016 / j.cell.2018.11.022. 72. Satpathy, A.T., Saligrama, N., Buenrostro, J.D., Wei, Y., Wu, B., Rubin, A.J., Granja, J.M., Lareau, C.A., Li, R., Qi, Y., et al. (2018). Transcript-indexed ATAC-seq for precision immune profiling. Nat Med 24, 580-590. doi.org / 10.1038 / s41591-018-0008-8. 73. Solache, A., Morgan, C.L., Dodi, A.I., Morte, C., Scott, I., Baboonian, C., Zal, B., Goldman, J., Grundy, J.E., and Madrigal, J.A. (1999). Identification of three HLA-A*0201-restricted cytotoxic T cell epitopes in the cytomegalovirus protein pp65 that are conserved between eight strains of the virus. J Immunol 163, 5512-5518. 74. Srivastava, A. (2016). In vivo tissue-tropism of adeno-associated viral vectors. Curr Opin Virol 21, 75-80. doi.org / 10.1016 / j.coviro.2016.08.003. 75. Stano, A., Leaman, D.P., Kim, A.S., Zhang, L., Autin, L., Ingale, J., Gift, S.K., Truong, J., Wyatt, R.T., Olson, A.J., et al. (2017). Dense Array of Spikes on HIV-1 Virion Particles. J Virol 91, e00415-17. doi.org / 10.1128 / JVI.00415-17. 76. Stoeckius, M., Hafemeister, C., Stephenson, W., Houck-Loomis, B., Chattopadhyay, P.K., Swerdlow, H., Satija, R., and Smibert, P. (2017). Simultaneous epitope and transcriptome measurement in single cells. Nat Methods 14, 865-868. doi.org / 10.1038 / nmeth.4380. 77. Sturm, G., Szabo, T., Fotakis, G., Haider, M., Rieder, D., Trajanoski, Z., and Finotello, F. (2020). Scirpy: a Scanpy extension for analyzing single-cell T-cell receptor-sequencing data. Bioinformatics 36, 4817-4818. doi.org / 10.1093 / bioinformatics / btaa611. 78. Su, F.-Y., Zhao, Q.H., Dahotre, S.N., Gamboa, L., Bawage, S.S., Silva Trenkle, A.D., Zamat, A., Phuengkham, H., Ahmed, R., Santangelo, P.J., et al. (2022). In vivo mRNA delivery to virus-specific T cells by light-induced ligand exchange of MHC class I antigen-presenting nanoparticles. Sci Adv 8, eabm7950. doi.org / 10.1126 / sciadv.abm7950. 79. Tan, E.M. (1989). Antinuclear antibodies: diagnostic markers for autoimmune diseases and probes for cell biology. Adv Immunol 44, 93-151. doi.org / 10.1016 / s0065-2776(08)60641-0. 80. Vance, T.D.R and Lee, J.E. (2020) Virus and eukaryote fusogen superfamilies. Curr. Biol. Jul 6; 30(13): R750-R754. 81. Verma, I.M., and Somia, N. (1997). Gene therapy -- promises, problems and prospects. Nature 389, 239-242. doi.org / 10.1038 / 38410. 82. Vieyra-Lobato, M.R., Vela-Ojeda, J., Montiel-Cervantes, L., Lopez-Santiago, R., and Moreno-Lafont, M.C. (2018). Description of CD8+ Regulatory T Lymphocytes and Their Specific Intervention in Graft-versus-Host and Infectious Diseases, Autoimmunity, and Cancer. J Immunol Res 2018, 3758713. doi.org / 10.1155 / 2018 / 3758713. 83. Vita, R., Mahajan, S., Overton, J.A., Dhanda, S.K., Martini, S., Cantrell, J.R., Wheeler, D.K., Sette, A., and Peters, B. (2019). The Immune Epitope Database (IEDB): 2018 update. Nucleic Acids Res 47, D339-D343. doi.org / 10.1093 / nar / gky1006. 84. Waldman, A.D., Fritz, J.M., and Lenardo, M.J. (2020). A guide to cancer immunotherapy: from T cell basic science to clinical practice. Nat Rev Immunol 20, 651-668. doi.org / 10.1038 / s41577-020-0306-5. 85. Wang, J.W., Jagu, S., Wu, W.-H., Viscidi, R.P., Macgregor-Das, A., Fogel, J.M., Kwak, K., Daayana, S., Kitchener, H., Stern, P.L., et al. (2015). Seroepidemiology of Human Papillomavirus 16 (HPV16) L2 and Generation of L2-Specific Human Chimeric Monoclonal Antibodies. Clin Vaccine Immunol 22, 806-816. doi.org / 10.1128 / CVI.00799-14. 86. Wildt, S., and Gerngross, T.U. (2005). The humanization of N-glycosylation pathways in yeast. Nat Rev Microbiol 3, 119-128. doi.org / 10.1038 / nrmicro1087. 87. Wills, M.R., Carmichael, A.J., Mynard, K., Jin, X., Weekes, M.P., Plachter, B., and Sissons, J.G. (1996). The human cytotoxic T-lymphocyte (CTL) response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity, and T-cell receptor usage of pp65-specific CTL. J Virol 70, 7569-7579. doi.org / 10.1128 / JVI.70.11.7569-7579.1996. 88. Wing, K., Onishi, Y., Prieto-Martin, P., Yamaguchi, T., Miyara, M., Fehervari, Z., Nomura, T., and Sakaguchi, S. (2008). CTLA-4 control over Foxp3+ regulatory T cell function. Science 322, 271-275. doi.org / 10.1126 / science.1160062. 89. Wolf, F.A., Angerer, P., and Theis, F.J. (2018). SCANPY: large-scale single-cell gene expression data analysis. Genome Biol 19, 15. doi.org / 10.1186 / s13059-017-1382-0. 90. Wolfert, M.A., and Boons, G.-J. (2013). Adaptive immune activation: glycosylation does matter. Nat Chem Biol 9, 776-784. doi.org / 10.1038 / nchembio.1403. 91. Wu, X., Liu, H., Xiao, H., Kim, J., Seshaiah, P., Natsoulis, G., Boeke, J.D., Hahn, B.H., and Kappes, J.C. (1995). Targeting foreign proteins to human immunodeficiency virus particles via fusion with Vpr and Vpx. J Virol 69, 3389-3398. doi.org / 10.1128 / JVI.69.6.3389-3398.1995. 92. Yang, L., Bailey, L., Baltimore, D., and Wang, P. (2006). Targeting lentiviral vectors to specific cell types in vivo. Proc Natl Acad Sci U S A 103, 11479-11484. doi.org / 10.1073 / pnas.0604993103. 93. Yassai, M.B., Naumov, Y.N., Naumova, E.N., and Gorski, J. (2009). A clonotype nomenclature for T cell receptors. Immunogenetics 61, 493-502. doi.org / 10.1007 / s00251-009-0383-x. 94. Yonehara, S., Ishii, A., and Yonehara, M. (1989). A cell-killing monoclonal antibody (anti-Fas) to a cell surface antigen co-downregulated with the receptor of tumor necrosis factor. J Exp Med 169, 1747-1756. doi.org / 10.1084 / jem.169.5.1747. 95. Yost, K.E., Satpathy, A.T., Wells, D.K., Qi, Y., Wang, C., Kageyama, R., McNamara, K.L., Granja, J.M., Sarin, K.Y., Brown, R.A., et al. (2019). Clonal replacement of tumor-specific T cells following PD-1 blockade. Nat Med 25, 1251-1259. doi.org / 10.1038 / s41591-019-0522-3. 96. Zhang, H., Sun, M., Wang, J., Zeng, B., Cao, X., Han, Y., Tan, S., and Gao, G.F. (2021). Identification of NY-ESO-1157-165 Specific Murine T Cell Receptors With Distinct Recognition Pattern for Tumor Immunotherapy. Front Immunol 12, 644520. doi.org / 10.3389 / fimmu.2021.644520. 97. Zhang, J., Medaer, R., Stinissen, P., Hafler, D., and Raus, J. (1993). MHC-restricted depletion of human myelin basic protein-reactive T cells by T cell vaccination. Science 261, 1451-1454. doi.org / 10.1126 / science.7690157. 98. Zhang, S.-Q., Ma, K.-Y., Schonnesen, A.A., Zhang, M., He, C., Sun, E., Williams, C.M., Jia, W., and Jiang, N. (2018). High-throughput determination of the antigen specificities of T cell receptors in single cells. Nat Biotechnol doi.org / 10.1038 / nbt.4282.

Claims

1. 1. An engineered lentivirus comprising: a heterologous ligand displayed on the surface of the lentivirus; a fusogen comprising a modified viral envelope protein, wherein the fusogen is capable of fusing the lentivirus to a host cell, the host cell comprising an endogenous receptor for the ligand; and Barcoded RNA An engineered lentivirus, including

2. 2. The engineered lentivirus of claim 1, wherein the fusogen is or comprises a modified VSV-G viral envelope protein.

3. 3. The engineered lentivirus of claim 2, wherein the modified VSV-G viral envelope protein comprises one or more amino acid substitutions at any one of positions H8, K47, Y209, and R354 of the VSV-G polypeptide.

4. 4. The engineered lentivirus of claim 3, wherein the modified VSV-G viral envelope protein comprises a K47Q substitution and a R354A substitution.

5. 2. The engineered lentivirus of claim 1, wherein the ligand (i) is or comprises a protein or epitope; (ii) is or comprises an MHC peptide, antibody, antigen, secreted protein, cell surface protein, or other form of antigen expressed by a cell; (iii) is operably linked to an optimized transmembrane domain; or (iv) is operably linked to an optimized transmembrane domain and a signal peptide.

6. 6. The engineered lentivirus of claim 5, wherein the antigen is an intracellular antigen.

7. 6. The engineered lentivirus of claim 5, wherein the optimized transmembrane domain is a transmembrane domain derived from HLA-DRA, HLA-DRB, HLA-A2, ICAM1, CD43, CD162, CD62L, CD49d, or LFA-1.

8. 10. The engineered lentivirus of claim 1, comprising a defective integrase protein.

9. The engineered lentivirus of claim 1, further comprising a reporter protein operably linked to the lentiviral structural protein.

10. 10. The engineered lentivirus of claim 9, wherein the structural protein is (i) a nucleocapsid protein or (ii) a Gag protein.

11. 2. The engineered lentivirus of Claim 1, wherein the barcoded RNA (i) is encapsulated in a viral particle; (ii) encodes the ligand protein; (iii) encodes a gene of interest to be delivered to the host cell; (iv) is read out by next-generation sequencing technology; and / or (v) comprises a capture sequence.

12. 1. A method for identifying a ligand-receptor pair, comprising: Providing at least one engineered lentivirus according to claim 1; combining the lentivirus with a cell population; sorting said cell population, thereby identifying ligand-receptor pairs; A method comprising:

13. 13. The method of claim 12, wherein the method comprises providing a pool of the engineered lentiviruses, the pools displaying different ligands.

14. 13. The method of claim 12, comprising combining the lentivirus with the cells and incubating the virus / cell mixture (i) at about 4°C; (ii) at room temperature; or (iii) at about 37°C.

15. 13. The method of claim 12, comprising incubating the virus / cell mixture for about 30 minutes, or about 1 hour, or about 2 hours, or any period from about 0.5 hours to about 2.5 hours.

16. The method of claim 12, further comprising the step of performing single-cell sequencing of (i) the viral RNA; and / or (ii) the transcriptome of the cell to identify the sequence of the ligand.

17. 1. A method for delivering a nucleic acid or protein of interest to a user-defined target cell, comprising: providing an engineered lentivirus of claim 1; contacting the lentivirus with a cell mixture comprising the target cells; delivering the nucleic acid or protein only to the target cells, wherein the target cells express a receptor specific for the ligand on the surface of the lentivirus; A method characterized by:

18. The method of claim 17, wherein (a) the ligand is modified to deliver a cargo to the target cell; (b) the nucleic acid of interest is packaged within the engineered lentiviral particle; and / or (c) the protein of interest is operably linked to (i) a lentiviral structural protein or (ii) a gag protein of the lentivirus.

19. 20. The method of claim 18, wherein the target cell is a mammalian cell.

20. 19. The method of claim 18, wherein the mammalian cell is a human cell.

21. 19. The method of claim 18, wherein the target cell is an immune cell.

22. 1. A method for identifying an immunogenic antigen, comprising: below: a heterologous receptor protein displayed on the surface of the lentivirus; a fusogen comprising a modified VSV-G viral envelope protein, wherein said fusogen is capable of fusing said lentivirus to a host cell, said host cell containing a native antigen for said receptor; a transgene operably linked to a lentiviral structural protein; and barcoded RNA, wherein the RNA encodes antigen information; providing an engineered lentivirus comprising: combining the lentivirus with a cell population; selecting said cell population based on the transgene; A method comprising:

23. The method of claim 22, further comprising: (i) sequencing viral RNA to identify the sequence of the antigen; or (ii) sequencing the RNA of the cellular receptor.

24. 1. A method for identifying a T cell receptor and paired pMHC, comprising: below: pMHC displayed on the surface of the virus, a fusogen comprising a modified VSV-G viral envelope protein, wherein the fusogen is capable of fusing the lentivirus to a host cell, the host cell comprising a T cell receptor for the PMHC; a transgene operably linked to a lentiviral structural protein; and barcoded RNA, providing an engineered lentivirus comprising: combining the lentivirus with a cell population; sorting said cell population based on the transgene, thereby identifying said T cell receptor; A method comprising:

25. 25. The method of claim 24, wherein the pMHC is encoded by RNA comprising, in tandem, a signal peptide, a PMHC, a G4S linker, a b2m gene, a G4S linker, and an MH allele.

26. The method described in claim 24, further comprising the steps of: (i) performing single-cell sequencing of viral RNA to identify the sequence of the MHC peptide; or (ii) performing sequencing of the cell receptor to identify the sequences of the MHC peptide and the T cell receptor.

27. 25. The method of claim 24, further comprising sequencing the cell receptor sequence to identify the MHC peptide and the T cell receptor sequence.

28. 1. A method for identifying a B cell receptor or antibody, comprising: below: an epitope displayed on the surface of a lentivirus, wherein said epitope is operably linked to an ICAM1 transmembrane domain; a fusogen comprising a modified VSV-G viral envelope protein, wherein the fusogen is capable of fusing the lentivirus to a host cell, the host cell containing a B-cell receptor for an intracellular epitope; a transgene operably linked to a lentiviral structural protein; and barcoded RNA, providing an engineered lentivirus comprising: combining the lentivirus with a population of B cells; selecting said cell population based on the presence of the transgene, thereby identifying said B cell receptor or antibody; A method comprising:

29. 29. The method of claim 28, wherein the antigen is a cell surface membrane protein, an intracellular protein, a secreted protein, or a glycosylated protein.

30. 29. The method of claim 28, further comprising single-cell sequencing of viral RNA to identify the antigen and matching B-cell receptor sequence.

31. 1. A method for identifying an antigen for a B cell receptor, comprising: providing an engineered lentivirus of claim 1; combining the lentivirus with a population of B cells; sorting said cell population, thereby identifying said B cell antigen; A method comprising:

32. A method for single cell multiomics, comprising: below: a heterologous ligand displayed on the surface of the lentivirus; a fusogen comprising a modified VSV-G viral envelope protein, wherein said fusogen is capable of fusing said lentivirus to a host cell, said host cell comprising an endogenous receptor for said ligand; a transgene operably linked to a lentiviral structural protein; and RNA, wherein the RNA comprises an antigen sequence and a capture tag for single-cell sequencing; providing an engineered lentivirus comprising: Simultaneous acquisition of transcriptome and phenotype information at the single-cell level A method comprising:

33. The method of claim 32, wherein: (i) the single-cell sequencing is a droplet-based platform; (ii) the cell phenotype includes surface markers by CITE-seq; (iii) the information includes the ligand and receptor sequences; (iv) the single-cell multi-omics uses whole cells as input; and (v) the single-cell multi-omics uses whole cells as input and includes a reverse transcription step.

34. 1. A method for selectively depleting or enriching a target cell population in a cell mixture, comprising: (a) the engineered lentivirus of claim 1, and (b) a cell mixture, (i) a target cell population that expresses a receptor specific for a ligand displayed on the surface of the engineered lentivirus; and (ii) a cell mixture comprising a non-target cell population that does not express the receptor; and contacting the engineered lentivirus with the cell population to deliver nucleic acid or protein only to the target cells; adding a reagent that specifically inhibits proliferation of the target cell population or inhibits proliferation of the non-target cell population, thereby selectively depleting or enriching the target cell population; A method comprising:

35. 35. The method of claim 34, wherein the target cells express a herpes simplex virus thymidine kinase (HSV-TK) transgene and the added reagent comprises or is ganciclovir (GCV).

36. 35. The method of claim 34, wherein the target cells express an shRNA to reduce expression of the death receptor FAS, thereby preventing cell death in the target population.

37. 35. The method of claim 34, wherein the target cell population comprises immune cells, wherein the immune cells are autoreactive immune cells.

38. 35. The method of claim 34, wherein the immune cells are specific for an antigen associated with a condition, wherein the condition is a proliferative disease, an inflammatory disease, an autoimmune disease, or a microbial infection.

39. 39. The method of claim 38, wherein the proliferative disease is cancer.

40. 39. The method of claim 38, wherein the microbial infection is a bacterial infection, a viral infection, or a microfungal infection.

41. A pharmaceutical composition for delivering a target nucleic acid or a target protein to a user-defined target cell, comprising the engineered lentivirus described in claim 1.

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