WO2023107886A1 - Methods and compositions for discovery of receptor-ligand specificity by engineered cell entry - Google Patents

Methods and compositions for discovery of receptor-ligand specificity by engineered cell entry Download PDF

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Publication number
WO2023107886A1
WO2023107886A1 PCT/US2022/080915 US2022080915W WO2023107886A1 WO 2023107886 A1 WO2023107886 A1 WO 2023107886A1 US 2022080915 W US2022080915 W US 2022080915W WO 2023107886 A1 WO2023107886 A1 WO 2023107886A1
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Prior art keywords
cell
cells
lentivirus
antigen
protein
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PCT/US2022/080915
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English (en)
French (fr)
Inventor
Howard Y. Chang
Ansuman Satpathy
Bingfei YU
Quanming SHI
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Leland Stanford Junior University
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Leland Stanford Junior University
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Priority to CN202280080750.7A priority Critical patent/CN118369426A/zh
Priority to CA3238531A priority patent/CA3238531A1/en
Priority to KR1020247022307A priority patent/KR20240112950A/ko
Priority to US18/716,869 priority patent/US20250027073A1/en
Priority to AU2022405508A priority patent/AU2022405508A1/en
Priority to IL312832A priority patent/IL312832A/en
Priority to JP2024533284A priority patent/JP2024542811A/ja
Priority to EP22905264.2A priority patent/EP4444878A4/en
Publication of WO2023107886A1 publication Critical patent/WO2023107886A1/en
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Definitions

  • the technology relates generally to the field of cell biology. More particularly, the technology relates to methods and compositions for the discovery and identification of ligandreceptor specificity, and for gene and protein delivery. The technology also relates to decoding interactions between T-cell receptors and MHC peptides, antibodies and antigens, or B-cell receptors and B cell antigens, including intracellular/secreted epitopes/cell- surface antigen epitopes, as well as other ligand-receptors.
  • TCR on surface of T cells can recognize and interact with the major histocompatibility complex (MHC)-antigen complexes from the surface of antigen-presenting cells (APC).
  • MHC major histocompatibility complex
  • APC antigen-presenting cells
  • TCR and antibody genes undergo somatic recombination to reach a large and diverse repertoire ( ⁇ 10 16 TCR alpha and beta sequences in humans), which are clonally inherited by daughter cells.
  • T and B cell receptor interactions are highly specific and drive antigen-specific T and B cell expansion and differentiation.
  • TCR-antigen interactions especially linking antigen specificity to TCR sequences and T cell states are essential to understanding how antigen recognition drives T cell fate decisions.
  • Diverse approaches have been developed to decipher the antigen specificity of TCRs including: (1) Cell reporter assay to screen T cell-specific MHC-antigens using artificial APCs such as T-scan, SABR, T cell trogocytosis, and cytokine capturing assay (Joglekar et al., 2019; Kula et al., 2019; Lee and Meyerson, 2021; Li et al., 2019 ); (2) Yeast display platform to screen MHC-antigens for recombinant TCRs (Birnbaum et al., 2012 ); (3) T cell based assay such as cytokine production (ELISpot) upon antigen peptide stimulation (McCutcheon et al., 1997); (4) DNA barcoded MHC-peptide multimer to capture antigen specificity and TCR sequence by single
  • the present disclosure provides a engineered lentivirus comprising a heterologous ligand displayed on a 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, wherein 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.
  • Non-limiting exemplary embodiments of the engineered lentiviruses of the disclosure can include one or more of the following features.
  • the fusogen comprises a modified VSV-G viral envelope protein.
  • the modified VSV-G viral envelope protein comprises one or more amino acid substitutions at any one of the positions H8, K47, Y209, and R354 of the VSV-G polypeptide.
  • the modified VSV- G viral envelope protein comprises a K47Q substitution and a R354A substitution.
  • the ligand is or comprises a protein or an epitope.
  • the ligand is or comprises a MHC peptide, an antibody, an antigen, a secreted protein, a cell- surface protein, or other form of antigen that may be expressed by a cell.
  • the antigen is or comprises an intracellular antigen.
  • the ligand is operably linked, e.g., fused to an optimized transmembrane domain.
  • the optimized transmembrane domain is transmembrane domain derived from HLA-DRA, HLA-DRB, HLA-A2, ICAM1, CD43, CD 162, CD62L, CD49d, or LFA-1.
  • the ligand is operably linked, e.g., fused to an optimized transmembrane domain and a signal peptide.
  • the engineered lentivirus comprises a defective integrase protein.
  • the reporter protein is GFP or mNeon.
  • the structural protein is a nucleocapsid protein.
  • the structural protein is a Gag protein.
  • the barcoded RNA is encapsulated in viral particles.
  • the RNA encodes the ligand, e.g., protein ligand.
  • the RNA encodes a gene of interest to be delivered into target cells, e.g., host cells.
  • target cells e.g., host cells.
  • the RNA is read out by a next-generation sequencing technology.
  • 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, e.g., in a lOx Genomics single-cell sequencing workflow.
  • 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, e.g., in a lOx Genomics single-cell sequencing workflow.
  • the present disclosure further provides a method for identifying a ligand-receptor pair, the method comprising providing at least one engineered lentivirus comprising a heterologous ligand displayed on a 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, wherein the host cell comprises an endogenous receptor, e.g., immune receptor, for the ligand; a reporter protein fused to a lentiviral structural protein; and a barcoded RNA, combining the lentivirus with a population of cells, and sorting the population of cells based on the presence of the reporter gene, thereby identifying a ligand-receptor pair.
  • a method for identifying a ligand-receptor pair comprising providing at least one engineered lentivirus comprising a heterologous ligand displayed on a surface of the lentivirus, a fusogen comprising a
  • Non-limiting exemplary embodiments of the methods for identifying a ligandreceptor pair of the disclosure can include one or more of the following features.
  • the method comprises providing a pool of engineered lentiviruses, the pool displaying different ligands.
  • the method comprises combining the lentivirus with cells and incubating the virus/cell mixture at about 4°C.
  • the method comprises combining the lentivirus with cells, and incubating the virus/cell mixture at room temperature.
  • the method comprises combining the lentivirus with cells and incubating the virus/cell mixture at about 37°C.
  • the method comprises 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.
  • the method further comprises the step of single cell sequencing of the viral RNA to identify the ligand sequence.
  • the method further comprises the step of single cell sequencing of the cell’s transcriptome to identify receptor sequence.
  • the present disclosure further provides a method of delivering a molecule of interest, e.g., a nucleic acid or a protein of interest, to a user-defined target cell, comprising providing an engineered lentivirus comprising a heterologous ligand displayed on a 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, wherein the host cell comprises an endogenous receptor, e.g., immune receptor, for the ligand; a reporter protein fused to a lentiviral structural protein; and a barcoded RNA, contacting the lentivirus with a cell mixture comprising the target cell, and delivering the nucleic acid or protein only to the target cell, wherein the target cell expresses a receptor, e.g., immune receptor, specific to the ligand on the lentivirus surface.
  • a molecule of interest e.g., a nucle
  • Non-limiting exemplary embodiments of the methods for delivering a molecule of interest of the disclosure can include one or more of the following features.
  • the ligand is modified in order to deliver cargo to the user-defined target cell.
  • the nucleic acid of interest is packaged inside the engineered lentiviral particle.
  • the protein of interest is operably linked to, e.g., fused with a gag protein of the lentivirus.
  • the protein of interest replaces the reporter.
  • 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).
  • PBMC primary human blood cell
  • the present disclosure further provides a method for identifying an immunogenic antigen, the method 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, wherein the host cell comprises a native antigen for the receptor, a reporter transgene fused to a lentivirus structural protein, and a barcoded RNA, wherein the RNA encodes antigen information, combining the lentivirus with the population of cells and sorting the population of cells based on the presence of the reporter.
  • the method further comprises sequencing the viral RNA to identify the antigen sequence.
  • the method further comprises a step for sequencing of the cell’s receptor RNA.
  • the present disclosure further provides a method of identifying a T- cell receptor and paired MHC-peptide, the method comprising providing an engineered lentivirus comprising a pMHC displayed on virus surface, a fusogen comprising a modified VSV-G viral envelope protein wherein the fusogen is capable of fusing the lentivirus to a host cell, wherein the host cell comprises a T-cell receptor for the PMHC, a reporter transgene fused to a lentivirus structural protein, and a barcoded RNA, combining the lentivirus with the population of cells and sorting the population of cells based on the presence of the reporter thereby identifying the T-cell receptor.
  • Non-limiting exemplary embodiments of the methods for identifying a T-cell receptor and paired MHC-peptide of the disclosure can include one or more of the following features.
  • the population of cells comprises human primary T-cells.
  • the PMHC is encoded by a RNA comprising a signal peptide, the PMHC, a G4S linker, b2m gene, a G4S linker and a MH allele in tandem.
  • the method further comprises a step for single cell sequencing of the viral RNA to identify the MHC peptide sequence.
  • the method further comprises a step for sequencing of the cell’s receptor sequence to identify the MHC peptide and T-cell receptor sequence.
  • the present disclosure also provides a method of identifying a B-cell receptor or antibody, the method comprising providing an engineered lentivirus comprising an epitope displayed on lentivirus surface wherein the epitope is operably linked to, e.g., fused with 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, wherein the host cell comprises a B-cell receptor for the intracellular epitope, a reporter transgene fused to a lentivirus structural protein, and a barcoded RNA, combining the lentivirus with the population of B cells and sorting the population of cells based on the presence of the reporter thereby identifying the B-cell receptor or antibody.
  • Non-limiting exemplary embodiments of the methods for identifying a B-cell receptor or antibody of the disclosure can include one or more of the following features.
  • the antigen is a cell surface membrane protein, an intracellular protein, a secreted protein, or glycosylated protein.
  • the method further comprises the step of single cell sequencing of the viral RNA to identify the antigen and matching B-cell receptor sequences.
  • the present disclosure further provides at least one embodiment of a method of identifying an antigen for a B-cell receptor, the method comprising providing an engineered lentivirus comprising a heterologous ligand displayed on a 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, wherein the host cell comprises an endogenous receptor for the ligand; a reporter protein fused to a lentiviral structural protein; and a barcoded RNA, combining the lentivirus with the population of B cells and sorting the population of cells based on the presence of the reporter thereby identifying the B-cell antigen.
  • the present disclosure also provides at least one embodiment of a method of single cell multiomics, 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, wherein the host cell comprises a an endogenous receptor for the ligand, a reporter transgene fused to a lentivirus structural protein, and a RNA, wherein the RNA comprises an antigen sequence and a capture tag for single cell sequencing and retrieving transcriptome and phenotype information simultaneously at the single cell level.
  • the single cells sequencing is a droplet based platform.
  • the cell’s phenotype comprise surface markers by CITE-seq.
  • the information comprises ligand and receptor sequences.
  • the single cells multiomics use whole cell as input.
  • the single cells multiomics use whole cell as input and comprises a step of reverse transcription.
  • the present disclosure provide a method for selectively depleting or enriching a target cell population in a cell mixture, the method including: providing (a) an engineered lentivirus as described herein, and (b) a cell mixture comprising (i) a target cell population expressing a receptor specific for the ligand displayed on the surface of the engineered lentivirus, and (ii) a non-target cell population that does not express the receptor specific for the ligand displayed on the surface of the engineered lentivirus; contacting the engineered lentivirus with the cell population, and delivering the nucleic acid or protein only to the target cell; adding a reagent that specifically inhibits growth of the target cell population or inhibits growth of the non-target cell population, thereby selectively depleting or enriching the target cell population.
  • the receptor is an immune receptor.
  • the immune receptor is a B-cell receptor.
  • immune receptor is a T-cell receptor.
  • Non-limiting exemplary embodiments of the methods for selectively depleting or enriching a target cell population of the disclosure can include one or more of the following features.
  • the target cell expresses a herpes simplex virus thymidine kinase (HSV-TK) transgene, and the added reagent comprises or is ganciclovir (GCV).
  • the target cell expresses shRNA to decrease expression of cell death receptor FAS to prevent cell death of the target population.
  • the target cell population comprises immune cells.
  • the immune cells comprise a T cell.
  • the immune cells comprise a B cell.
  • the immune cells are autoreactive immune cells.
  • the immune cells are specific for an antigen associated with a health condition.
  • the health condition is a proliferative disorder, inflammatory disorder, autoimmune disorder, or a microbial infection.
  • the proliferative disorder is a cancer.
  • the microbial infection is a bacterial infection, viral infection, or microfungal infection.
  • FIGS. 1A-1G illustrate a viral display platform to display ligand proteins and fusogen on viral surface, deliver fluorescent proteins, and record ligand-receptor interaction by cell entry.
  • FIG. 1A shows a schematic view of an exemplary all-in-one platform.
  • the lentiviruses are engineered in diverse components including: (1) user-defined ligand proteins displayed on viral surface; (2) modified fusogen with intact fusion ability and defective binding to natural receptors; (3) cargo proteins fused with viral structure protein; and (4) barcoded viral RNA for tracing and gene delivery.
  • FIG. IB shows a schematic view of an experimental set up and flow cytometry analysis of GFP expression after 3 days of viral infection.
  • Raji and Jurkat cells are infected by three groups of lentiviruses encoding GFP in the viral RNA: (1) viruses with wild-type VSV-G (left); (2) viruses with receptor-binding mutated VSV-G (middle); (3) viruses with VSV-G mutant and anti-CD19 single-chain antibody variable fragment (scFv).
  • FIG. 1C shows a schematic view (top) of an experimental set up.
  • GFP protein are fused with matrix protein (MA-GFP) or Nucleocapsid protein (NC-GFP), or viral protein R (VPR-GFP).
  • MA-GFP matrix protein
  • NC-GFP Nucleocapsid protein
  • VPR-GFP viral protein R
  • scFv-CD19 displayed viruses carrying GFP protein fused with different viral proteins were incubated with Raji (CD 19+) or Jurkat (CD19-) cells for 2 hours and then subjected to flow cytometry.
  • Bar plot (bottom) showing the percentage of GFP+ cells upon incubation of viruses with different GFP fusion viral proteins.
  • FIG. ID shows exemplary flow cytometry plots of GFP signal after transient viral incubation as in FIG. 1C.
  • FIG. IE shows schematic view of experimental set up and flow cytometry analysis of GFP signal in primary human B cells with or without viral incubation.
  • Naive and activated human primary B cells were incubated with NC-GFP fused and scFv-CD19 displayed viruses for 2 hours and then subject to flow cytometry analysis.
  • B cells were gated on live CD20+ cells.
  • FIG. IF is a histogram analysis of surface CD19 expression of groups from FIG. IE.
  • FIG. 1G depicts bar plots showing scFv-CD19 virus binding and CD19 surface expression in naive and activated human B cells. P-values in FIGS. 1C and IF are calculated by unpaired t-test. **** P ⁇ 0.0001 *** P ⁇ 0.001.
  • FIGS. 1H - IN illustrate how to decipher a specific interaction of ligand-receptor for co-stimulatory molecules by ENTER.
  • FIG. 1H depicts a Flow cytometry analysis of the viral binding and fusion of scFv- CD19 displayed viruses on Raji B cells at different temperature before and after proteinase K treatment.
  • FIG. II is a Bar plot showing the percentage of GFP+ cells from FIG. 1H.
  • FIG. 1J shows a schematic view of an experimental set up.
  • CD40 expressing Raji B cells are incubated with GFP viruses displaying either wild-type CD40 ligand (CD40L) or CD40L mutant (K142E, R202E) with decreased binding to its cognate receptor CD40.
  • CD40L wild-type CD40 ligand
  • K142E, R202E CD40L mutant
  • FIG. IK shows flow cytometry analysis of GFP signal in Raji B cells upon incubation with either wild-type CD40E or mutant CD40E displayed GFP viruses.
  • FIG. IL is a Bar plot showing the percentage of GFP+ cells from FIG. IK. P value was calculated by unpaired t-test. *** P ⁇ 0.001.
  • FIG. IM shows a schematic view of immunocapture assay.
  • magnetic beads were conjugated with anti-CD40L, anti-VSV-G, and IgG antibodies and then incubated with CD40L displayed viruses.
  • Viruses that were immunocaptured were subjected to viral RNA isolation and qRT-PCR of CD40L.
  • FIG. IN depicts a Bar plot showing the qRT-PCR result of enrichment of ENTER viruses by different antibody conjugated beads as in FIG. IM. All p values were calculated by unpaired t-test. n.s. not significant p>0.05; * p ⁇ 0.05; *** p ⁇ 0.001.
  • FIGS. 2A-2F show how ENTER decodes interaction between MHC-peptides (pMHCs) with TCR.
  • FIG. 2A shows a schematic view of pMHC displayed viruses and flow cytometry analysis of GFP signal in specific TCR expressing Jurkat T cells upon incubation of pMHC displayed viruses.
  • GFP fused viruses displaying either a 9-mer peptide (NY-ESO-I157-I65) presented by HLA-A0201(A2) allele or an 11-mer CMV peptide (pp65363-373) presented by HLA- A0101(Al) allele are incubated with T cells expressing specific TCRs (e.g., NY-ESO-I157-165- TCR or CMV-pp65 3 63-373-TCR) that recognize the cognate antigens.
  • SP signal peptide
  • Peptide antigen peptide
  • B2M Beta-2-Microglobulin.
  • FIG. 2B shows flow cytometry analysis of GFP signal in Jurkat T cells expressing specific TCRs (e.g., NY-ESO-I157-165-TCR, CMV pp65495-5033-TCR, or Flu m l(58-66)-TCR) upon incubation of viruses displaying various HLA-A2 presented peptides.
  • TCRs e.g., NY-ESO-I157-165-TCR, CMV pp65495-5033-TCR, or Flu m l(58-66)-TCR
  • FIG. 2C shows flow cytometry plots of NY-ESO-1 TCR-T cells upon incubation of 2X10 8 ENTER viral particles displaying ny-eso-1157-166 antigen (left) or with different amount of ny-eso-1157-166 pMHC tetramers.
  • FIG. 2D shows a comparison of binding efficiency (GFP+%) of viruses displaying antigen variants with different TCR affinity.
  • lG4wt-TCR T cells were incubated with ENTER displaying wild-type of mutant ny-eso-1157-166 antigen variants for 2 hours.
  • CMV-pp65495-503- TCR T cells were used as negative control.
  • the viral titers were normalized using the p24 protein level.
  • FIG. 2E shows a schematic view of experimental set up (top) and flow cytometry analysis (bottom) of TCR-T cell mixing experiment.
  • Flu-ml58-66-TCR T cells were labeled by CellTrace Violet dye and then mixed with CMV-pp65495-503-TCR T cells at different ratio.
  • Mixed T cell population was incubated with HLA-A2:ml displayed GFP viruses for 2h and then subjected to flow cytometry.
  • Representative flow cytometry plot showing 1:1000 mixing of two T cell population and GFP signal of T cells gated on Violet-i- and Violet- population.
  • FIG. 2F depicts a bar plot showing the signal-to-noise ratio of ENTER in FIGS. 2Eand 2J.
  • FIG. 2G shows a flow cytometry analysis of HLA-A2 and B2M surface expression in wild-type HEK293T, HLA-KO HEK293T, and HLA-A2 reconstituted HLA-KO HEK293T cells.
  • FIG. 2H shows flow cytometry plots of CMV-pp65495-503-TCR T cells upon 2-hour incubation of HLA-A2 -peptide displayed GFP viruses followed by staining of PE-tetramers.
  • Ml (58-66) (flu antigen) displayed viruses and Ml (58-66) tetramers are negative controls.
  • FIG. 21 shows Histogram plots (left) and Bar plots (right) showing tetramer intensity and CD3 surface expression of CMV-pp65495-503-TCR T cells.
  • FIG. 2J shows a schematic view of experimental design (top) and flow cytometry analysis (bottom) of T cell mixing experiments.
  • Flu-ml58-66-TCR T cells were labeled by Cell Trace Violet dye and then mixed with NY-ESO-1157-165-TCR T cells at different ratio.
  • Mixed T cell population was incubated with HLA-A2:ml displayed GFP viruses for 2h and then subjected to flow cytometry.
  • Representative flow cytometry plot showing mixing of two T cell population and GFP signal of T cells gated on Violet+ and Violet- population.
  • FIG. 2K depict Bar plots showing sensitivity (left) and specificity (right) of ENTER from FIG. 2J.
  • FIGS. 3A-3F illustrate the optimization of ENTER to present intracellular antigens on viral surface and decode interaction between BCR with antigens.
  • FIG. 3A shows a schematic view of experimental design. During the assembly and budding of lentiviruses, certain host cell surface proteins can be incorporated into the surface of viruses. TM domains of host proteins selected from literature are fused with a B cell epitope derived from intracellular antigen HPV16 L2. These HPV-epitope displayed GFP viruses are incubated with B cells expressing BCR targeting this HPV epitope (on-target) or B cells without any BCR expression (off-target).
  • FIG. 3B shows flow cytometry analysis of GFP signal in B cells incubated with GFP viruses displaying an HPV epitope fused with different TM domains. B cells without BCR expression serve as a negative control.
  • FIG. 3C depicts Bar plots showing the percentage of GFP+ B cells from FIG. 3B.
  • FIG. 3D shows flow cytometry analysis of GFP signal in RBD-BCR+ B cells upon incubation of ENTER viruses displaying SARS-CoV-2 Spike RBD antigen or HPV L2 antigen as a negative control (left). Bar plot showing the frequency of GFP+ cells upon incubation of on- target or off-target ENTER viruses (right).
  • FIG. 3E shows a schematic view of experimental set up (top) and flow cytometry analysis (bottom) of B cell mixing experiment.
  • RBD-BCR+ B cells were labeled by cell trace violet dye and then mixed with HPV-BCR+ B cells at different ratio.
  • Mixed B cell population was incubated with GFP viruses displaying RBD antigen fused with ICAM1 TM domain and then subjected to flow cytometry.
  • Representative flow cytometry plot showing 1:1000 mixing of two B cell population and GFP signal of B cells gated on violet-i- and violet- population.
  • FIG. 3F depicts a Bar plot showing the signal-to-noise ratio of ENTER from FIGS. 3E and 31.
  • FIGS. 3G-3P schematically summarize the results of experiments performed to decode antigen specificity of B cells and characterizing cargo delivery efficiency and specificity by ENTER.
  • FIG. 3G depicts a flow cytometry analysis of GFP+ cells among HER2-BCR+ B cells that were incubated with HER2 displayed viruses or RBD displayed viruses.
  • FIG. 3H depicts a Bar plot showing the percentage of GFP+ cells as in FIG. 3G.
  • FIG. 31 shows a schematic view of experimental design (top) and flow cytometry analysis (bottom) of B cell mixing experiments.
  • RBD-BCR+ B cells were labeled by Cell Trace violet and mixed with HPV-BCR+ B cells at different ratios.
  • Mixed B cell population was incubated with GFP viruses displaying RBD antigen fused with ICAM1 TM domain and then subjected to flow cytometry.
  • Representative flow cytometry plot showing mixing of two B cell population and GFP signal of B cells gated on Violet+ and Violet- population.
  • FIG. 3 J depicts Bar plot showing sensitivity of ENTER from FIG. 31.
  • FIG. 3K depicts Bar plot showing sensitivity of ENTER from FIG. 31.
  • FIG. 3L depicts Bar plot showing the delivery efficiency of viruses with wild-type VSV-G in cell types with different antigen specificity. The p values were calculated by unpaired t-test. n.s. not significant P>0.05.
  • FIG. 3M depicts a flow cytometry analysis of cargo delivery in T cell mixing experiment.
  • mScarlet expressed NY-ESO-1 TCR+ T cells were mixed with CMV-pp65 TCR T cells and then infected with ENTER viruses displaying pp65495-503 antigen and carrying transgene containing HSV-TK and GFP.
  • FIG. 3N depicts a flow cytometry analysis of cargo delivery in B cell mixing experiment.
  • mScarlet expressed RBD BCR+ B cells were mixed with HER2 BCR+ B cells and then infected with ENTER viruses displaying HER2 antigen and carrying transgene containing HSV-TK and GFP.
  • FIG. 30 depicts a Histogram plot showing the surface expression of FAS in T cells transduced with diverse FAS shRNAs or control shRNA.
  • FIG. 3P depicts a representative flow plot showing the Annexin V and 7-AAD gating of T cells upon anti-FAS induced apoptosis and cell death.
  • FIGS. 4A-4M schematically summarize the results of experiments performed to demonstrate that ENTER permits selective depletion or expansion of antigen- specific T or antigen-specific B cells.
  • FIG. 4A is a schematic view 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 viruses that display pp65495-503 and carry GFP transgene as a cargo. Representative histogram plot showing GFP expression between CMV-pp65 TCR+ T cells and NY-ESO-1 TCR+ T cells after 2 days infection.
  • FIG. 4B is a Bar plot showing the percentage of GFP+ cells as in FIG. 4A.
  • FIG. 4C is a schematic view of cargo delivery in antigen- specific B cells (left). HER2 BCR+ B cells or RBD BCR+ B cells were individually infected with ENTER viruses that display HER2 and carry GFP transgene as a cargo. Representative histogram plot showing GFP expression between HER2 BCR+ B cells and RBD BCR+ B cells after 2 days infection.
  • FIG. 4D is a Bar plot showing the percentage of GFP+ cells as in FIG. 4C.
  • FIG. 4E is a schematic view of suicide gene delivery in a pool of different antigenspecific T cells.
  • CMV-pp65 TCR+ T cells and NY-ESO-1 TCR+ T cells expressing mScarlet were mixed together and then infected of ENTER viruses that display pp65495-503 and carry a herpes simplex virus thymidine kinase (HSV-TK) transgene. After 2 days of infection, ganciclovir (GCV) drug was added to kill HSV-TK expressing cells and cell survival was monitored for 4 days.
  • HSV-TK herpes simplex virus thymidine kinase
  • FIG. 4F is a Bar plot showing the number of live T cells at day 4 post GCV treatment.
  • FIG. 4G schematically summarizes the results of a kinetics analysis of fold enrichment between the number of CMV-pp65 TCR+ T cells versus that of NY-ESO-1 TCR+ T cells that were either infected with ENTER carrying GFP gene or ENTER carrying HSV-TK gene upon GCV drug treatment.
  • FIG. 4H is a schematic view of suicide gene delivery in a pool of different antigenspecific B cells.
  • HER2 BCR+ B cells and RBD BCR+ B cells expressing mScarlet were mixed together and then infected of ENTER viruses that display pp65495-503 and carry HSV-TK transgene. After 2 days of infection, GCV drug was added to kill HSV-TK expressing cells and cell survival was monitored for 4 days.
  • FIG. 41 is a Bar plot showing the number of live B cells at day 4 post GCV treatment.
  • FIG. 4J schematically summarizes the results of a kinetics analysis of fold enrichment between the number of HER2 BCR+ B cells versus that of RBD BCR+ B cells that were either infected with ENTER carrying GFP gene or ENTER carrying HSV-TK gene upon GCV drug treatment.
  • FIG. 4K is a schematic view of shRNA delivery in a pool of different antigenspecific T cells.
  • CMV-pp65 TCR+ T cells and NY-ESO-1 TCR+ T cells expressing mScarlet were mixed together and then infected of ENTER viruses that display pp65495-503 and carry FAS shRNA or control shRNA.
  • Anti-FAS antibody was added to induce apoptosis.
  • FIG. 4L is a Bar plot showing the 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.
  • FIG. 4M is a Bar plot showing the CMV-pp65 TCR+ T cells/ NY-ESO- 1 TCR+ T cells fold enrichment that is normalized by shCtrl group for the live cells (gated on Annexin V and 7-AAD double negative) as in FIG. 4K. P-values are calculated by unpaired t-test. **** P ⁇ 0.0001; *** P ⁇ 0.001; **P ⁇ 0.01; * P ⁇ 0.05; n.s. P>0.05.
  • FIGS. 4N-4W schematically summarize the results of experiments performed to optimize ENTER to detect antigen-specific primary human T cells.
  • FIG. 4N is a flow cytometry analysis of GFP signal in T cells expressing TCRs (NY- ESO-1 TCR or CMV pp65-TCR) upon incubation with ny-eso-1157-165 antigen peptide displayed GFP viruses whose viral RNA are either intact or inserted with sequencing capture tag.
  • FIG. 40 is a Bar plot showing the percentage of GFP+ cells from FIG. 4N. P value was calculated by unpaired t-test. n.s. P>0.05.
  • FIG. 4P is a schematic view of experimental design.
  • Donor isolated T cells were incubated with pp65495-503 displayed viruses carrying either GFP or mNeon fluorescence proteins and then stained with pp65495-503 tetramer and other antibodies followed by flow cytometry.
  • FIG. 4Q is a flow cytometry analysis of tetramer and GFP signal in primary human T cells from donors with CMV infection as in FIG. 4P.
  • FIG. 4R is a Bar plot showing the percentage of GFP+ and GFP- cells among pp65 tetramer+ T cells from FIG. 4Q. P value was calculated by unpaired t-test. * p ⁇ 0.05.
  • FIG. 4S is a representative flow cytometry plot showing the GFP+ cells among pp65 tetramer+ T cells after incubation with pp65495-503 viruses or negative control viruses (ny-eso- 1157-165).
  • FIG. 4T is a schematic view of experimental design.
  • PBMCs were isolated from CMV seropositive HLA-A2+ donors and cultured with a pool of twelve different CMV antigen peptides (10 pg/mL) for 10 days.
  • Primary T cells prior or post peptide-induced expansion were incubated with pp65 antigen displayed mNeon viruses for 2 hours and then stained with antibodies followed by flow cytometry.
  • FIG. 4U is a representative flow cytometry plot showing co-staining of pp65495-503 tetramer and pp65495-503 displayed mNeon viruses in pp65495-503 peptide enriched T cells post 15 days expansion. These T cells are gated on live CD8+ CD3+ T cells.
  • FIG. 4V is a representative flow cytometry plot showing the percentage of GFP+ T cells (stained by 12 pooled HLA-A2:CMV-antigen mNeon viruses) from 4 different CMV seropositive donors prior or post pooled CMV peptide-induced expansion as in FIG. 4T.
  • FIG. 4W is a MA plot showing the bulk RNA-seq analysis of CMV-pp65 TCR T cells incubated with pp65495-503 tetramer or pp65495-503 displayed ENTER viruses. Genes with log2 fold change and adjusted p value ⁇ 0.01 are highlighted in red.
  • FIGS. 5A-5F schematically illustrate an ENTER-seq for massively parallel measurement of antigen peptide sequence, TCR sequence, and transcriptome.
  • FIG. 5A shows a schematic view of ENTER-seq workflow.
  • a library of pooled viruses displaying individual pMHCs was incubated with T cells for 2 hours.
  • GFP+ T cells are sorted for droplet-based single cell genomics profiling (e.g. lOx Genomics 5’ kit for gene expression and V(D)J immune profiling).
  • FIG. 5B shows a viral RNA engineering strategy for droplet-based single cell capture.
  • lOx Genomics capture tag is inserted in the linker region between B2M and HLA-A2.
  • lOx Genomics PCR handle is inserted after CMV promoter.
  • CMV CMV promoter
  • SP signal peptide sequence
  • Peptide antigen peptide
  • B2M Beta-2-Microglobulin
  • MHC Class I HLA- A0201 allele
  • LTR long terminal repeat
  • TSO template switching oligo sequence.
  • FIG. 5C shows a schematic view of T cell mixing experiment for ENTER-seq.
  • FIG. 5D shows number of Pp65(495-503)-TCR T cells UMI counts (x-axis) and NY- ESO-1157-165-TCR UMI counts (y-axis) associated with each cell barcode (dot).
  • the colors are assigned as NY-ESO-I157-165-TCR+ T cells (light blue), Pp65(495-503)-TCR+ T cells (red), and doublets (green, with both NY-ESO-I157-165-TCR and Pp65(495-503)-TCR).
  • FIG. 5E shows scatter plots of TCR UMI counts after doublet removal, colored by enrichment ratio of pp65(495-503)-antigen UMI count among total UMI counts (left), and enrichment ratio of nyesoi57-i65-antigen UMI count among total UMI counts (right).
  • FIG 5F shows number of pp65(495-503)-antigen UMI counts (x-axis) and nyesoi57-i65- antigen UMI counts (y-axis) associated with each cell barcode (dot) after doublet removal.
  • the colors are assigned as NY-ESO-I157-165-TCR + T cells (light blue) and Pp65(495-503)-TCR + T cells (red).
  • FIGS. 5G-5N schematically summarize the results of experiments performed for the characterization of T cell subsets by ENTER-seq.
  • FIG. 5G is a UMAP plots showing CMV- specific T cells (yellow) and bystander T cells (blue) via clustering before or after removal of ENTER-induced gene signature.
  • FIG. 5H is a UMAP plot showing 10 clusters of human CD8 T cell subsets.
  • FIG. 51 depicts UMAP plots showing amount of surface protein CD 127 from CITE- seq, and expression of genes for naive T cells (CCR7, SELL, and LEF1).
  • FIG. 5J depicts UMAP plots showing gene expression of cytolytic molecules.
  • FIG. 5K depicts UMAP plots showing gene expression of markers for MAIT cells.
  • FIG. 5L depicts UMAP plots showing expression of marker genes for each subset/cluster.
  • FIG. 5M is a UMAP plot showing donor origin.
  • FIG. 5N shows fraction of T cell subsets from 10 clusters in CMV antigen-specific T cells (ENTER+) and bystander T cells (ENTER-), separated by donor origin.
  • FIGS. 6A-6I schematically summarize the results of ENTER-seq of ex vivo expanded CMV-specific primary T cells.
  • FIG. 6A shows a schematic view of CMV antigen peptide induced T cell expansion and ENTER-seq workflow.
  • FIG. 6B is a UMAP plot showing cells with (ENTER+, colored in yellow) or without (ENTER-, colored in blue) CMV antigen displayed viruses binding.
  • FIG. 6C depicts UMAP plots showing CITE-seq of surface protein expression of CD45RA (naive marker) and CD45RO (memory marker).
  • FIG. 6D is a UMAP plot showing 10 clusters of human CD8+ T cell subsets labeled in different colors.
  • FIG. 6E is a Bar plot showing the number of CMV antigen- specific T cells that recognize specific CMV antigen epitope in donor #1 (labeled in black) and donor #2 (labeled in gray).
  • FIG. 6F depicts UMAP plots showing the amount of CMV antigen epitopes per cell for the top 3 CMV antigen epitopes identified from FIG. 6E.
  • 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.
  • FIG. 6H is a UMAP plot (left) showing the clonal expansion size of CMV antigenspecific T cells colored by the number of cells in each clonotype. Violin plot (right) showing the distribution of clone size in different CMV antigen-specific T cells colored by antigen epitopes.
  • FIG. 61 is a Violin plots showing expression of cytokines and transcription factors in pp65495-503-specific TCR clones, separated and colored by CDR3 clones. A simplified model (right).
  • FIGS. 6J-6P summarize the results of a TCR clonotype analysis of ex vivo expanded CMV-specific T cells.
  • FIG. 6J depicts UMAP plots showing the clonal expansion size of CMV antigen-specific T cells (ENTER+) and bystander T cells (ENTER-).
  • FIG. 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 circle represents the number of cells in each clonotype.
  • FIG. 6L depicts scatter plots showing the correlation of clonal expansion size with cytotoxic gene score.
  • FIG. 6M depicts scatter plots showing the correlation of clonal expansion size with exhaustion gene score or activation gene score.
  • FIG. 6N is a summary table of pp65495-503-specific TCR clones showing convergent TCR clonotypes with identical CDR3 amino acid sequences.
  • FIG. 60 is a summary tables of US874-82 - and ULIOO2OO-2O8 -specific TCR clones.
  • FIG. 6P depicts fraction of T cell subsets in pp65495-503-specific TCR clones, separated by CDR3 clones and colored by 10 clusters. Coefficient r and p values in FIGS. 6L- 6M are calculated by Pearson correlation.
  • FIGS. 7A-7K schematically summarize the results of ENTER-seq of primary CMV- specific T cells isolated directly (e.g., without in vitro expansion) from CMV seropositive patient blood.
  • FIG. 7A depicts a schematic view of isolation of primary T cells from patient blood and ENTER-seq workflow.
  • FIG. 7B is a UMAP plot (left) showing cells with (ENTER+, colored in yellow) or without (ENTER-, colored in blue) CMV antigen displayed viruses binding.
  • UMAP plot (right) showing 13 clusters of human CD8+ T cell subsets labeled in different colors.
  • FIG. 7C depicts UMAP plots showing the surface protein expression of CD45RA and CD45RO from CITE-seq and expression of representative genes.
  • FIG. 7D is a heatmap showing the scaled z score of expression of genes associated diverse functions (e.g.. type-I IFN, cytotoxicity, etc.) across different clusters.
  • FIG. 7E is a UMAP plot showing the CMV antigen specificity colored by antigen epitope.
  • FIG. 7F is a UMAP plot (left) showing the clonal expansion size of CMV antigenspecific T cells colored by the number of cells in each clonotype.
  • FIG. 7G is a Violin plot showing the number of pMHC bound per cell in T cells with different clone size. P value was calculated by Mann-Whitney test. n.s. p>0.05; * p ⁇ 0.05; ** p ⁇ 0.01, *** p ⁇ 0.001, **** p ⁇ 0.0001.
  • FIG. 7H is a CITE-seq density plot showing surface expression of CD45RA and CD45RO in pp65-specific T cells from donor #1 (colored by orange) and donor #2 (colored by blue) before and after peptide-induced expansion.
  • FIG. 71 depicts flow cytometry plots showing CD45RA and CD45RO in pp65- specific T cells from donor #1 and donor #2 before and after peptide-induced expansion.
  • FIG. 7J is a density plot showing the distribution of type-I IFN ISG gene score and cytotoxicity gene score prior and post peptide-induced expansion in top 3 TCR clones of pp65- specific T cells (left). Density plot showing the expression of IL13 and EOMES prior and post peptide-induced expansion in top 3 TCR clones of pp65-specific T cells (right).
  • FIG. 7K is a proposed model of phenotypic transition of CMV-specific T cells upon ex vivo expansion.
  • FIGS. 7L-7T schematically summarize the results of intra-clonal phenotypic diversity of CMV-specific T cells isolated directly from patient blood.
  • FIG. 7L depicts UMAP plots showing the expression of key genes associated with diverse functions (type-I IFN ISG, cytotoxicity, chemokine, and transcription factors).
  • FIG. 7M depicts UMAP plots showing the subset clustering of primary T cells directly isolated from patient blood sample before and after removal of ENTER- induced gene signature.
  • FIG. 7N depicts UMAP embedding density plots showing the density of CMV antigen-specific T cells for different CMV antigen epitopes.
  • FIG. 70 is a Bar plot showing the percentage of US8?4-82-specific T cells with shared TCR before and after peptide induced expansion.
  • FIG. 7P depicts the number of US 874-82- specific T cells isolated from fresh PBMC before expansion, separated by donor and colored by TCR CDR3 sequence. TCR sequence shared before and after expansion are circled in black.
  • FIG. 7Q is a summary table of top TCR clones of US874-82-specific T cells (identified from E) with cell number and frequency metrics before and after expansion.
  • FIG. 7R depicts the number of T cell subsets in pp65495-503-specific TCR clones, separated by CDR3 clones and colored by 13 clusters.
  • FIG. 7S is a heatmap showing the expression of genes associated with diverse functions among different TCR clone-specific T cells.
  • FIG. 7T depicts density plots showing the expression of ILL 3 and ILA in different TCR clone- specific T cells before and after peptide-induced expansion.
  • the present disclosure generally relates to systems, compositions and methods for the identification of receptor-ligand pairing including MHC-peptides/T-cell receptors and antigen/B- cell receptor (antibody) pairs and for decoding, e.g., displaying ligand proteins, delivering payloads, and recording receptor specificity.
  • Some embodiments of the disclosure relate to decoding 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, as well as other ligand-receptors (e.g., CD40 ligand vs CD40).
  • the disclosure also relates to compositions and methods for protein or nucleic acid delivery into user-defined target cells.
  • some embodiments of the disclosure relate to a modular viral display and delivery platform to decode ligand-receptor interactions, deliver cargos in target cells, and connect ligand-receptor interactions with cellular state.
  • lentiviruses can be engineered at multiple levels including (1) displaying user- defined ligand proteins on the viral surface; (2) engineering a fusogen to achieve receptorspecific cell entry of cognate ligand displayed viruses; (3) carrying fluorescent proteins to track engineered viruses; (4) delivering cargos upon paired ligand-receptor recognition; and (5) modifying viral RNA to record ligand information by sequencing.
  • This technology is termed “ENTER” for “lentiviral-mediated cell entry by engineered ligand-receptor interaction), which can systematically deorphanize pairs of interactions including TCR-pMHC, antibody-antigen, costimulatory ligand-receptors, and B cell antigen-BCR.
  • ENTER can permit gene delivery in a receptor- specific manner, allowing for the selective manipulation of cellular behavior in antigen- specific T and B cells.
  • ENTER can be combined ENTER with droplet-based single-cell genomics profiling (ENTER-seq) to measure antigen specificity, TCR repertoire, gene expression and surface protein landscape in individual human primary T cells.
  • ENTER-seq droplet-based single-cell genomics profiling
  • a “lentivirus” as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.
  • cell refers not only to the 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, without regard to the number of transfers or passages in culture. It should be understood that not all progeny are exactly identical to the parental cell.
  • progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein, so long as the progeny retain the same functionality as that of the original cell, cell culture, or cell line.
  • exogenous refers to any material introduced from or produced outside an organism, cell, tissue or system.
  • linker also referred to as a “spacer” or “spacer domain” as used herein, refers to an amino acid or sequence of amino acids that that is optionally located between two amino acid sequences in a fusion protein of the invention.
  • biological sample refers to any solid or liquid sample isolated from an individual or a subject.
  • tissue sample or liquid sample (e.g., blood) isolated from an animal (e.g., human), such as, without limitations, a biopsy material (e.g., solid tissue sample), or blood (e.g., whole blood).
  • sample can be, for example, fresh, fixed (e.g., formalin-, alcohol- or acetone-fixed), paraffin-embedded or frozen prior to an analysis.
  • the biological sample is obtained from a tumor (e.g., a pancreatic cancer).
  • a “test biological sample” is the biological sample that has been the subject of analysis, monitoring, or observation.
  • a “reference biological sample,” containing the same type of biological sample e.g., the same type of tissues or cells
  • operably linked denotes a physical or functional linkage between two or more elements, e.g., polypeptide sequences or polynucleotide sequences, which permits them to operate in their intended fashion.
  • operably linked when used in context of the nucleic acid constructs described herein (e.g., lentiviral vectors) or the coding sequences and promoter sequences in a nucleic acid molecule means that the coding sequences and promoter sequences are in-frame and in proper spatial and distance away to permit the effects of the respective binding by transcription factors or RNA polymerase on transcription.
  • operably linked elements may be contiguous or noncontiguous (e.g., linked to one another through a linker).
  • “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 for a described activity of the constructs.
  • Operably linked segments, portions, regions, and domains of the polypeptides or nucleic acid molecules disclosed herein may be contiguous or non-contiguous (e.g., linked to one another through a linker).
  • the operably linked segments, portions, regions, and domains of the polypeptides described herein are fused in-frame to one another.
  • genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable.
  • the terms include, but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates.
  • the genes or gene products disclosed herein which in some embodiments relate 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.
  • the genes, nucleic acid sequences, amino acid sequences, peptides, polypeptides and proteins are human.
  • the term “gene” is also intended to include variants thereof.
  • a population of cells as described herein may be any mammalian cell population.
  • a population of cells is a population of human, mouse, rat, or non-human primate cells.
  • a population of cells is a somatic cell population or a reproductive cell population.
  • a population of cells comprises antigenspecific cells (e.g., cells that binds to a specific antigen).
  • a population of antigen-specific cells comprises immune cells.
  • a population of antigenspecific cells comprises B cells and/or T cells.
  • a population of cells comprises a homogenous population of cells.
  • a population of cells comprises a heterogeneous population of cells.
  • a population of cells is a population of cells isolated from a subject.
  • a 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.
  • a population of cells is isolated from the blood or a tumor of a subject.
  • aspects and embodiments of the disclosure described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments.
  • “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.
  • “consisting of’ excludes any elements, steps, or ingredients not specified in the claimed composition or method.
  • “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.
  • compositions comprising a lentivirus engineered so that it can a) display a specific ligand on the cell surface, b) have a mutated fusogen that allows the virus to fuse with and enter only host cells having a receptor that naturally pairs with the ligand, c) deliver a reporter to the host cells and d) be tagged, on the viral RNA, to allow single cell sequencing
  • the lentivirus can be further engineered to comprise a defective integrase so that the viral RNA cannot integrate into the genome of the host cell, thereby avoiding integration-induced mutagenesis of the host genome.
  • the engineered lentiviruses of the present disclosure comprise one or more user-defined ligands displayed on the viral cell surface.
  • the ligand is heterologous relative to the lentivirus displaying the ligand on its surface, e.g., the ligand is from a heterologous source, such as from another cell or another virus.
  • suitable ligand types include cell surface receptors, adhesion proteins, glycoproteins, carbohydrates, lipids, glycolipids, lipoproteins, and lipopolysaccharides that are surface-bound, integrins, mucins, and lectins.
  • the ligands can be or comprise proteins.
  • the ligands can be or comprise epitopes.
  • epitope refers to an antigenic determinant that interacts with a specific antigen-binding site of an antigen-binding polypeptide, e.g., a variable region of an antibody molecule, known as a paratope.
  • a single antigen can have more than one epitope.
  • different antibodies may bind to different areas on an antigen and may have different biological effects.
  • epitopes also refers to a site on an antigen to which B cells respond. It also refers to a region of an antigen that is bound by an antibody. Epitopes can be defined as structural or functional.
  • Epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction.
  • Epitopes can be linear or conformational, that is, composed of non-linear amino acids.
  • epitopes can include determinants that are chemically active surface groupings 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.
  • the ligands can be or comprise cell surface proteins or intracellular proteins or parts thereof.
  • the ligands can be or comprise MHC peptides, antibodies, intracellular antigens, secreted proteins, or other forms of proteins or peptides.
  • the transmembrane domain is operably linked to a transmembrane domain.
  • the TM domains may facilitate the efficiency in virus carry-over.
  • the transmembrane domain is a heterologous transmembrane domain.
  • the transmembrane domain is a heterologous transmembrane derived from HLA- DRA, HLA-DRB, HLA-A2, ICAM1, CD43, CD 162, CD62L, CD49d, or LFA-1.
  • the TM is replaced with an optimized TM (such as, for example, TM from ICAM1, PDGFR).
  • the ligand can be engineered to first be displayed on the surface of a cell line that is suitable to produce lentivirus (for example, HEK 293T, a common type of cell to produce lentivirus).
  • lentivirus for example, HEK 293T, a common type of cell to produce lentivirus.
  • the ligand can get carried over on to the virus surface during virus budding to produce virus particles.
  • the engineered lentivirus of the disclosure comprises a defective integrase protein.
  • the lentiviruses described herein may comprise a reporter (e.g., a reporter protein).
  • the lentiviruses comprises a nucleic acid encoding a reporter (e.g., a reporter protein).
  • a reporter is generally a protein or gene that can be detected when expressed in a retrovirus and/or target cell.
  • the presence or absence of a reporter in a target cell or a subset of a target cells in a population of cells allows for the ability to sort cells (e.g., using flow cytometry and/or fluorescence-activated cell sorting).
  • a reporter is a fluorescent protein.
  • a fluorescent protein may be a green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP).
  • GFP green fluorescent protein
  • YFP yellow fluorescent protein
  • RFP red fluorescent protein
  • Exemplary fluorescent proteins may be as described in U.S. Pat. No. 7,060,869.
  • the engineered lentiviral particles displaying specific ligands deliver fluorescent protein into target cells upon cognate receptor-ligand interaction.
  • the reporter is mNeon, a monomeric green fluorescence protein that is substantially brighter than GFP.
  • the reporter can be operably linked, e.g., fused to a viral structural gene.
  • the structural gene can be a nucleocapsid protein (NC) or a Gag protein.
  • the barcoded RNA is encapsulated in viral particles, e.g., those produced by the engineered lentiviruses.
  • the RNA encodes the ligand.
  • the RNA encodes a gene of interest to be delivered into target cells, e.g., host cells.
  • the RNA is read out by a next-generation sequencing technology.
  • the RNA comprises a capture sequence.
  • fusogen or “fusogenic molecule” as used herein generally refers to any molecule that can facilitate, catalyze, or trigger membrane fusion when present on the surface of a virus.
  • fusogens suitable for the compositions and methods of the disclosure include fusogens derived from viruses or fusogens endogenously expressed in a host cell, e.g., mammalian cell (e.g., human cell).
  • the fusogens of the disclosure are glycoproteins.
  • the fusogens of the disclosure are viral glycoproteins.
  • Exemplary viral fusogens suitable for the compositions and methods disclosed herein include those belonging to Classes I, II, and III of viral fusion proteins, which are produced by enveloped viruses to facilitate virus-host membrane fusion. Addition information in this regard can be found in, e.g., Vance T.D.R and Lee J.E. (Curr. Biol. Jul 6, 30(13), R750- R754, 2020), which is incorporated herein by reference.
  • the fusogens of the disclosure belong to Class I viral fusion proteins, i.e., those capable of forming form coiled- coil trimers, which include but are not limited to, from influenza viruses, coronaviruses, HIV, and Ebola virus.
  • the fusogens of the disclosure belong to Class II viral fusion proteins, i.e., those capable of transitioning from dimers to trimers during fusion, producing an elongated ectodomain heavily composed of P sheets that settles into a hairpin trimer after fusion.
  • Suitable Class II viral fusion proteins include but are not limited to, those from Dengue fever virus, West Nile virus, Zika virus, and tick-bome encephalitis virus.
  • the fusogens of the disclosure belong to Class III viral fusion proteins, those capable of combining elements from the former two classes, taking on a post-fusion conformation that contains both a coiled-coil trimerization region similar to Class I, and an elongated trimer of hairpins as in Class II.
  • Suitable Class III viral fusion proteins include but are not limited to, those from vesicular stomatitis virus (VSV), herpes simplex virus 1 (HSV1), rabies virus.
  • VSV-G vesicular stomatitis virus G
  • the fusogen of the compositions and methods disclosure herein is or comprises a viral fusion protein from 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 human endogenous retrovirus (HERV) such as ERVW-1 (e.g., Syncytin-1).
  • a viral fusion protein from 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 human endogenous retrovirus (HERV) such as ERVW-1 (e.g., Syncytin-1).
  • the engineered lentiviruses can comprise modified fusogens to, e.g., facilitate the fusion of the virus with cell membranes.
  • the fusogen can be modified so that it facilitates fusion of the virus to cell membranes without the need for a viral surface glycoprotein.
  • the fusogen comprises a modified vesicular stomatitis virus G (VSV-G) viral envelope protein.
  • VSV-G vesicular stomatitis virus G
  • the VSV-G polypeptide is or comprises the sequence of SEQ ID NO: 56.
  • the modified VSV-G viral envelope protein comprises one or more substitutions, for example, substitutions that abolish the binding of VSV-G with the cellular receptor.
  • the VSV-G envelope protein may include one or more amino acid substitutions at a position corresponding to any one of the positions H8, K47, Y209, and R354 of the VSV-G polypeptide. It is within the knowledge of the skilled person to know how to align amino acid sequences, e.g., sequences of multiple VSV-G polypeptides, in order to determine which amino acid in a particular position referred to herein “corresponds to” in another VSV-G amino acid sequence not listed herein. Thus, the term “position corresponding to” as used herein, is well-known within the art.
  • the modified VSV-G viral envelope protein disclosed herein can also include conservative modifications and substitutions at other positions of VSV-G (e.g., those that abolish the binding of VSV-G with the cellular receptor).
  • conservative substitutions include those described by Dayhoff 1978, supra, and by Argos 1989, supra.
  • amino acids belonging to one of the following groups represent conservative changes: Group I: Ala, Pro, Gly, Gin, Asn, Ser, Thr; Group II: Cys, Ser, Tyr, Thr; Group III: Vai, He, Leu, Met, Ala, Phe; Group IV: Lys, Arg, His; Group V: Phe, Tyr, Trp, His; and Group VI: Asp, Glu.
  • the amino acid substitution(s) in the amino acid sequence of the modified VSV-G viral envelope protein disclosed herein is independently selected from the group consisting of an alanine (A) substitution, an arginine (R) substitution, an asparagine (N) substitution, an aspartic acid (D) substitution, a leucine (L) substitution, a lysine (K) substitution, a phenylalanine (F) substitution, a lysine substitution, a glutamine (Q) substitution, a glutamic acid (E) substitution, a serine (S) substitution, and a threonine (T) substitution, and combinations of any thereof.
  • the amino acid substitutions(s) in the amino acid sequence of the modified VSV-G viral envelope protein disclosed herein includes an alanine substitution.
  • the modified VSV-G viral envelope protein comprises an amino acid substitution corresponding to K47Q substitution or a R354A substitution of the sequence of SEQ ID NO: 56. In some embodiments, the modified VSV-G viral envelope protein comprises a K47Q and a R354A substitution. As described above, other viral fusogens that are able to fuse viral particles with cell membrane may also be suitably used with ENTER.
  • ENTER can be adapted to couple with any single cell methods that use whole cell as input and contains a step of reverse transcription.
  • any single cell RNA-seq (lOx Genomics or others) that include 5’ or 3’ approach
  • scTCR/BCR-seq lOx Genomics
  • CITE-seq /ECCITE to identify surface markers with barcoded antibodies
  • single cell CRISPR perturb-seq to perturb genes with CRISPR coupled with single cell’s transcriptome.
  • identifying a ligand-receptor pair by (i) providing at least one engineered lentivirus as disclosed herein, (ii) combining the lentivirus with a population of cells; and (iii) sorting the population of cells based on the presence of the reporter gene, thereby identifying a ligand-receptor pair.
  • the engineered lentivirus comprises a ligand displayed on a surface of the lentivirus wherein the ligand is heterologous relative 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 facilitating, catalyzing, or triggering fusion of the lentivirus with the host cell), wherein 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.
  • a host cell e.g., capable of facilitating, catalyzing, or triggering fusion of the lentivirus with the host cell
  • 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.
  • the lentivirus and a population of cells can be combined for a defined period of time.
  • a period of time may be measured in seconds, minutes, hours or days.
  • period of time is 0-30 seconds, 15-45 seconds, 30-60 seconds, 45-90 seconds, 60-90 seconds, or 60-120 seconds.
  • the virus and a population of cells are combined and in contact for 0-30 seconds, 15-45 seconds, 30-60 seconds, 45-90 seconds, 60-90 seconds, or 60-120 seconds.
  • period of time 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.
  • a retrovirus and a population of cells are combined and in contact 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, SO- VO minutes, or 60-120 minutes.
  • a period of time 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.
  • a virus and a population of cells are combined and in contact 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.
  • a period of time is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 5-15 days.
  • a virus and a population of cells are combined and in contact for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 5-15 days.
  • the method comprises combining the lentivirus with cells, for 2 hours to identify ligand-receptor pairs.
  • the lentivirus and the population of cells can be combined 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.
  • the population of cells and the virus can be incubated at 37°C for about 2 hours to identify ligand-receptor pairs.
  • modifications are within the scope of the disclosure.
  • identifying a T-cell receptor and paired pMHC comprising (i) providing an engineered lentivirus as disclosed herein, (ii) combining the lentivirus with the population of cells; and (iii) sorting the population of cells based on the presence of the reporter thereby identifying the T-cell receptor.
  • the engineered lentivirus comprises a pMHC displayed on virus surface, a fusogen comprising a modified VSV-G viral envelope protein wherein the fusogen is capable of fusing the lentivirus to a host cell, wherein the host cell comprises a T-cell receptor for the pMHC, a reporter transgene operably linked, e.g., fused to a lentivirus structural protein, and a barcoded RNA.
  • the method provides a mixture of virus displaying different MHC peptide, (e.g., a pool of MHC peptides).
  • the T-cells can be a population of cell lines. In another embodiment the cells are human primary T-cells.
  • the displayed pMHC can be engineered as a single chain format is used (FIG. 2A), i.e. the RNA can encode a signal peptide, an antigen peptide, a G4S linker, b2m gene, a G4S linker and MH allele in tandem.
  • the method can further comprise the step of single-cell sequencing of the viral RNA and cell’s receptor sequence to identify the MHC peptide sequence and TCR receptor information.
  • the methods that are provided herein can be used to identify matching MHC peptides for known T-cell receptors. For example, when the alpha and beta chains of known T-cell receptors are expressed on TCR negative cell lines such as Jurkat -76, a pool of virus with pMHCs candidates can be mixed with cells, as in described in the examples below.
  • Also provided herein are methods for identifying a B-cell receptor or antibody comprising providing engineered lentivirus as disclosed herein, (ii) combining the lentivirus with the population of B cells; and (iii) sorting the population of cells based on the presence of the reporter thereby identifying the B-cell antigen.
  • the engineered lentivirus comprises an epitope displayed on lentivirus surface wherein the epitope is operably linked, e.g., fused with 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 facilitating, catalyzing, or triggering fusion of the lentivirus with the host cell), wherein the host cell comprises a B-cell receptor for the intracellular epitope, a reporter transgene operably linked, e.g., fused to a lentivirus structural protein, and a barcoded RNA.
  • the antigen are cell surface membrane protein, intracellular protein, secreted protein, or other forms (e.g. glycosylated molecules) that can be expressed on cell surface.
  • the method can further comprise the step of single-cell sequencing of the viral RNA and cell’s receptor sequence to identify the antigen and matching B-cell receptor (BCR, antibody) information.
  • the method can identify novel antibody/BCR and antigen pairs.
  • the present disclosure further provides a method of delivering a molecule of interest, e.g., a nucleic acid or a protein of interest, to a user-defined target cell, comprising providing an engineered lentivirus comprising a heterologous ligand displayed on a 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 facilitating, catalyzing, or triggering fusion of the lentivirus with the host cell), wherein 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, contacting the lentivirus with a cell mixture comprising the target cell, and delivering the nucleic acid or protein only to the target cell, wherein the target cell expresses a receptor specific to the
  • the ligand is modified in order to deliver cargo to the user-defined target cell.
  • the nucleic acid of interest is packaged inside the engineered lentiviral particle.
  • the protein of interest is operably linked, e.g., fused with a gag protein of the lentivirus.
  • the protein of interest replaces the reporter.
  • the target cell is in vivo.
  • the target cell is ex vivo.
  • the target cell is in vitro.
  • the target cell is a mammalian cell.
  • the mammalian cell is a human cell.
  • the target cell is an immune cell.
  • 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).
  • PBMC primary human blood cell
  • one aspect of the present disclosure relates to a method for selectively depleting or enriching a target cell population in a cell mixture, the method including: providing (a) an engineered lentivirus according to any one of claims 1-17, and (b) a cell mixture comprising (i) a target cell population expressing a receptor specific for the 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, and delivering the nucleic acid or protein only to the target cell; adding an reagent that specifically inhibits growth of the target cell population or inhibits growth of the non-target cell population, thereby selectively depleting or enriching the target cell population.
  • the receptor is an immune receptor.
  • the immune receptor is a B-cell receptor.
  • immune receptor is a T-cell receptor.
  • Non-limiting exemplary embodiments of the methods for selectively depleting or enriching a target cell population of the disclosure can include one or more of the following features.
  • the target cell expresses a herpes simplex virus thymidine kinase (HSV-TK) transgene, and the added reagent comprises or is ganciclovir (GCV).
  • the target cell population comprises immune cells.
  • the target cell expresses shRNA to decrease expression of cell death receptor FAS to prevent cell death of the target population.
  • the immune cells comprise a T cell.
  • the immune cells comprise a B cell.
  • the immune cells are autoreactive immune cells.
  • the immune cells are specific for an antigen associated with a health condition.
  • the health condition is a proliferative disorder, inflammatory disorder, autoimmune disorder, or a microbial infection.
  • the proliferative disorder is a cancer.
  • the microbial infection is a bacterial infection, viral infection, or microfungal infection.
  • ENTER lentiviral- mediated cell entry by engineered receptor- ligand interaction
  • exemplary applications of ENTER include decoding ligandreceptor interactions, linking receptor interaction with cell state at the single-cell level, and deliver cargos in a receptor- specific manner.
  • ENTER offers the user one platform that solves many important problems.
  • lentivirus was engineered to enable the display of heterologous cell surface proteins, intracellular and extracellular epitopes, including pMHC complexes, antibodies, co- stimulatory molecules and B cell antigens.
  • ENTER has several advantages compared to yeast or phage display platforms (see, e.g., Table 1).
  • the glycosylation pattern in yeast/phage display platform is different from mammalian system, which may interfere with the correct MHC presentation and recognition of paired TCRs.
  • yeast or phage display requires substantial optimization to achieve proper folding, stability, and presentation of MHC.
  • ENTER is built in human cells and enables human glycosylation and protein folding patterns, as evidenced by Applicants’ ability to present multiple HLA-peptide combinations.
  • soluble recombinant TCR is required for screening in yeast and phage display platforms, thus making it challenging to test diverse TCRs in parallel.
  • ENTER allows investigators to screen primary T cell samples, opening the door to examine the vast diversity of human TCR repertoire (see, e.g., Table 1).
  • ENTER also has advantages over cytolytic T cell reporter assay such as T-scan for decoding pMHC-TCR interaction because the latter cannot record the pairing of pMHC vs. TCR at single cell level.
  • ENTER can be engineered to deliver cargos in a receptor- specific manner.
  • the lentivirus was engineered such that receptor-ligand interaction drives viral fusion and infection.
  • the system was further engineered so that investigators may choose to transiently or stably deliver cargos with the flexibility of using integration-defective machinery.
  • ENTER may have applications in gene therapy or RNA medicine as ENTER can achieveaji cell type specificity compared to existing modalities like AAV.
  • ENTER can selectively deplete or expand antigen- specific T cells, based on specific delivery of gene cargos that induce or protect from cell death (see, e.g., FIG. 4).
  • ENTER enabled the depletion of antigen- specific B cells, which can be applied to eradicate pathogenic autoantigen-specific B cells to potentially treat autoimmune disorders. TABLE 1. Comparison of ENTER with other platform n.d.: not done n.a.: not available
  • ENTER as described herein can be used for linking ligandreceptor interaction with molecular blueprints at the single-cell level.
  • ENTER-seq combines the ability to decode ligand-receptor interactions with the power of single-cell genomics to resolve cell-cell communication and cell states at a massively parallel scale.
  • ENTER-seq for pMHC is conceptually similar to a DNA-barcoded library of pMHC tetramer molecules but with several potential advantages.
  • ENTER can be lower cost compared to commercial DNA barcoded pMHC tetramers.
  • ENTER can be easily implemented in any laboratories compared to in house generation of pMHC tetramers with DNA barcodes (see, e.g., Table 2).
  • DNA conjugation to pMHC tetramers may suffer from unequal barcode oligonucleotide loading during the conjugation reaction, whereas ENTER-seq library leverages lentiviral biology that ensures 2 copies of barcoded viral RNA for each viral particle.
  • the uniform distribution of DNA barcode per virus like particle enabled ENTER-seq to quantify pMHC binding strength, which has not been investigated in studies using DNA barcoded pMHC tetramers.
  • ENTER is more sensitive than pMHC tetramer on a molar basis per reagent (see, e.g., Figure 2C).
  • the superior sensitivity may arise from a high number of pMHCs that are displayed on ENTER.
  • HIV-based lentiviral particle displays 14-100 molecules of envelope protein per viral particle whereas pMHC tetramers are 4 linked molecules by definition.
  • ENTER-seq allows investigators to record ligand-receptor specificity and read out the biological consequences of this interaction, such as antigen-dependent T cell fates including naive cell activation, effector cell expansion, memory cell formation, or T cell exhaustion.
  • ENTER-seq may be used to understand the molecular programs of antigen- specific B cells in the context of infectious diseases and autoimmunity.
  • ENTER-seq analysis of primary CMV-specific T cells demonstrates the power of the platform to connect the landscape of antigen epitope, TCR repertoire, gene expression program, and surface protein phenotypes across tens of thousands of primary T cells in a single experiment. Such massively parallel profiling of diverse modalities uncovered donor- specific antigen specificity and immunogenicity of viral epitopes.
  • ENTER-seq of T cells pre- and post-peptide stimulation unveiled transcriptional alteration upon expansion and inter-clonal phenotypic diversity in response to the same antigen.
  • Th2 cytokine expression might be impacted by the different TCR affinity/avidity/density to the same pMHC antigen, or different priming environment from antigen-presenting cells.
  • a recent study of single cell profiling CD19-CAR T cells in acute lymphoblastic leukemia patients showed that an induction of Th2 expression is positively associated with clinical efficacy in durable responders compared to relapsed patients (Bai et al., 2022).
  • Th2 cytokines can boost CD8 T cell effector function to achieve long-term remission and if such benefit can be generalizable to infectious disease.
  • TCR clone-specific induction of Th2 cytokine expression might inform the selection of TCR to engineer TCR-T cells for adoptive T cell therapy.
  • ENTER-seq as described herein provides insights into a comprehensive understanding of how T cell clonality and specificity influence the molecular phenotypes and physiological function of antigen-specific T cells.
  • ENTER as described herein may be used to isolate and enrich tumor antigen-reactive T cells to infuse back into patients.
  • ENTER as described herein may be further used in a discovery context to screen immunogenic antigen or elite TCRs for the rational design of vaccine development or cancer immunotherapy.
  • ENTER as described herein may also be applied to screen BCRs that target viral antigens, facilitating the development of therapeutic antibodies to prevent viral infections.
  • ENTER as described herein enables antigen- specific delivery of cargos such as genes and shRNAs, allowing perturbation and manipulation of antigen- specific T and B cells.
  • ENTER may be extended to additional receptor- ligand pairs, such as G-protein coupled receptors, adhesion molecules, or protocadherins. Therefore, ENTER may be used to address cell-cell connectivity beyond the immune system.
  • ENTER-seq can combine the ability to decode ligand-receptor interactions with the power of single-cell genomics to resolve cell type and cell states at a massively parallel scale.
  • ENTER-seq for pMHC is conceptually similar to a DNA-barcoded library of MHC tetramer molecules but with several potential advantages. MHC tetramer libraries require individual peptide synthesis and then loading into MHC tetramers, leading to high cost, long lead time, and lower throughput compared to ENTER-seq library that can be prepared by massively parallel DNA synthesis.
  • DNA conjugation to MHC tetramers may suffer from unequal barcode oligonucleotide loading during the conjugation reaction, whereas ENTER-seq library leverages lentiviral biology that ensures 2 copies of barcoded viral RNA for each viral particle.
  • ENTER-seq maybe more sensitive compared to MHC tetramers. HIV-based lentiviral particle displays 14-100 molecules of Env protein per viral particle whereas MHC tetramers are four linked molecules by definition.
  • ENTER-seq allows investigators to record ligand-receptor specificity and readout the biological consequences of this interaction, such as antigen-dependent T cell fates such as naive cell activation, effector cell expansion, memory cell formation, or T cell exhaustion. Similarly, ENTER-seq may be used to understand the molecular programs of autoantibody producing B cells in autoimmunity.
  • ENTER links ligand-receptor interaction with molecular blueprints at single-cell resolution.
  • ENTER has advantages over cytolytic T cell reporter assay such as T-scan because the latter cannot record the pairing of peptide-MHC vs. TCR at single cell level, which precludes pooled analyses.
  • ENTER may have translational application in immunology and beyond. ENTER may be used to isolate and enrich tumor antigen-reactive T cells to infuse back into patients. The non- integrative nature of ENTER facilitates adoptive T cell therapy. ENTER may be furthered used in a discovery context to screen immunogenic antigen or elite TCRs for rational design of vaccine development or cancer immunotherapy.
  • This Example describes the results of experiments performed to illustrate an exemplary ENTER in accordance with some embodiments of the disclosure where ENTER is engineered to be a modular viral display and delivery platform to capture and decode ligandreceptor interactions, deliver cargos in target cells, and connect ligand-receptor interactions with cellular state.
  • Lentivirus was engineered at multiple levels including (i) ligand proteins displayed on viral surface, (ii) host receptor-targeted viral entry by displayed ligand and modified fusogen, (iii) fluorescent protein delivery via fusion of viral capsid, and (iv) tagged viral RNA for single cell sequencing (see, e.g., FIG. 1A).
  • a viral envelope protein with disrupted native receptor binding while maintaining intact fusion ability (termed as fusogen) is designed to cooperate with user-defined ligand proteins displayed on viral surface.
  • the cooperation of two separate modules (ligand protein + fusogen) allows the interaction between viral-displayed ligand and host receptor to further facilitate viral fusion to host cells by fusogen (see, e.g., FIG. 1A).
  • the vesicular stomatitis virus G protein (VSV-G) a viral envelope protein is used to pseudodype lentiviruses.
  • VSV-G pseudotyped viruses have a broad tropism since VSV-G can recognize and interact with Low Density Lipoprotein Receptor (LDLR), which is expressed in many cell types.
  • LDLR Low Density Lipoprotein Receptor
  • VSV-G mutant was engineered which harbors two point- mutations (K47Q, R354A) to prevent its recognition and interaction with LDLR on host cells (Nikolic et al., 2018)
  • a minimal GFP expression in Raji (0.1%) and Jurkat (0.6%) cells using mutant VSV-G pseudotyped viruses was observed (see, e.g., FIG. IB), suggesting that the viral recognition of specific receptors on host cells is the first essential step for viral entry and integration.
  • VSV-G mutant is a good fusogen candidate to cooperate with user-defined ligands on the viral surface for viral infection in host cells expressing paired receptors
  • a well-established CD19-CAR chimeric antigen receptor
  • sc-Fv anti-CD19 single-chain antibody variable fragment
  • MA and NC are processed from Gag precursor protein which can be assembled in cis as 3000 copies of MA or NC per viral particle ((De Guzman et al., 1998; Kutluay et al., 2014)
  • VPR can be incorporated in trans into the viral particle via interaction with Gag protein as 500 copies of VPR per viral particle (Wu et al., 1995). It was observed that 80% of Raji cells bound by CD19-CAR presented viruses when GFP was fused with NC, which significantly outperforms MA-GFP and VPR-GFP (see, e.g., FIG. 1C).
  • NC-GFP viruses displaying CD19-CAR can recognize and bind to primary CD 19+ B cells from human blood
  • these viruses were incubated with naive or activated human primary B cells for 2 hour and detected the GFP signal on these B cells by flow cytometry. Similar to Raji B cell line, 80% of activated human primary B cells were bound by NC-GFP labeled CD19-CAR displayed viruses whereas 60% of naive B cells are GFP+ (see, e.g., FIG. ID).
  • the difference of CD 19 expression between naive and activated B cells might account for discrepancy of binding of CD19-CAR viruses.
  • the viral binding and fusion assay were performed (see, e.g., FIG. 1H).
  • the result showed that there were only 5% GFP+ cells after proteinase K treatment, indicating that very few cells have undergone viral fusion to prevent the proteinase K mediated degradation of surface bound GFP labeled viruses (see, e.g., FIG. II).
  • the reduction of surface CD19 is mostly resulted from the specific CD19 binding/masking from ENTER viruses rather than internalization, further highlighting that the ligand-displayed viruses are highly specific to the targeted receptors.
  • viruses were engineered to display either wild type CD40 ligand (CD40L) or mutant CD40L.
  • CD40L mutant contains two point-mutations (K142E, R202E), leading to decreased binding affinity towards CD40 (Pasqual et al., 2018)
  • K142E, R202E point-mutations
  • the flow cytometry results showed a significant decrease of GFP+ Raji cells when incubated with viruses displaying CD40L mutant compared to wild-type CD40L (FIGS. 1J-1L).
  • viruses displaying CD40L and fusogen were generated and then incubated them with anti-CD40L beads, anti-VSV-G beads, and IgG beads respectively. After incubation and extensive washing, the viral RNA was extracted for subsequent qRT-PCR analyses.
  • viral display platform e.g., ENTER
  • ENTER can capture a highly specific ligand-receptor interaction in a transient viral binding assay and is applicable to multiple categories of receptor- ligand interactions.
  • engineered lentiviral particles displaying specific ligands deliver fluorescent protein into target cells upon cognate receptor-ligand interaction, without genome integration or transgene transcription.
  • This Example describes the results of experiments performed to illustrate how ENTER with MHC-peptide (pMHC) displaying viruses maps TCR specificity, particularly how this viral display platform captures the interaction between pMHC and TCR.
  • B2M beta 2 microglobulin
  • covalently linked peptide see, e.g., FIG. 2A.
  • HLA human leukocyte antigen
  • HLA-A2 The surface expression of B2M is also abolished in HLA KO cells, suggesting all endogenous HLA alleles have been successfully deleted (see, e.g., FIG. 2G).
  • HLA-A2 The single chain of HLA-A*0201 fused with B2M and peptide was overexpressed in HLA KO cells and observed a high level of surface expression of HLA-A2 and B2M (see, e.g., FIG. 2G).
  • the viruses were collected and incubated with Jurkat T cells expressing a TCR that targets the cognate pMHC antigen.
  • virus displaying a well-established cancer-testis antigen NY-ESO-1 as a 9-mer peptide (SLLMWITQC) on HLA-A2 a most prevalent HLA allele in humans was successfully generated (Jager et al., 1998) (see, e.g., FIG. 2A).
  • Applicants further engineered viruses displaying diverse 9-mer antigen epitopes from cancer-testis antigen, CMV pp65 antigen (ny-eso-1157-165), CMV pp65 antigen (pp65495-503), and influenza matrix protein antigen (mlss-66), all of which are presented on HLA- A2 allele (Gotch et al., 1987; Wills et al., 1996).
  • the result showed over 87% of TCR-matching T cells were GFP+ after 2 hours of incubation with GFP fused viruses displaying cognate HLA- A2 -peptide whereas only 1% of these T cells were labeled by negative control antigen displayed GFP viruses (see, e.g., FIG. 2B).
  • the highly specific entry of pMHC displaying viruses into TCR matching T cells were observed with different antigen peptide lengths and distinct HLA alleles, highlighting the generality of the ENTER platform to present diverse pMHC antigens.
  • the CMV pp65495-503 antigen-specific TCR Jurkat T cells was first incubated with pp65495-503 displayed viruses and then stained with a widely used commercial pp65495-503 tetramer.
  • these T cells were incubated with the influenza mlss-eedisplayed viruses and commercial mlss-66 tetramer (see, e.g., FIG. 2H).
  • Flow cytometry result showed that over 90% of tetramer positive cells were GFP+, indicating a strong concordance of pp65495-503 tetramer staining with the binding of pp65495-503 displayed GFP viruses.
  • the negative control influenza ml 58-66 tetramers and viruses did not label pp65495-503 TCR T cells (see, e.g., FIG. 2H). Additionally, it was observed that the pp65495-503 tetramer intensity and CD3 surface expression were significantly decreased after coincubation with viruses displayingpp65495-503 (see, e.g., FIG. 21), which is similar to the observation of decreased surface expression of CD 19 on Raji B cells after binding of CD19-CAR displayed viruses. This result indicated that pMHC displayed viruses specifically bind to and mask the TCR-CD3 complex, preventing later binding of pMHC tetramer and anti-CD3 antibody.
  • TCR-T cell line (lG4wt) that recognizes NY-ESO-1 antigen variants with different known TCR affinities (Kd range from 7-85 pM) (Zhang et al., 2021) was generated.
  • the NY-ESO-1 TCR T cell line utilized in previous experiments is very similar to lG4wt TCR T cell line except a few mutations on its TCR, resulting in very high binding affinity to ny-eso-1157-165 antigen (Robbins et al., 2008).
  • ENTER was then engineered to display different ny-eso-1157-165 antigen peptide variants such as wild-type peptide (SLLMWITQC), L3A mutant (SLAMWITQC), and T7A mutant (SLLMWIAQC). Then the percentage of GFP+ cells was measured after incubating different TCR-T cells with antigen variants displayed viruses after normalizing the titer of viruses (showed by viral p24 protein level). The result showed that ENTER is sensitive to detect TCR affinity as low as 10.8 uM when adding high titer of viruses (40ng p24).
  • the signal-to-noise ratio was over 150-fold even when the frequency of on-target T cells is as low as 1 in 1000, demonstrating a high specificity and sensitivity of the ENTER viral display platform (see, e.g., FIGS. 2F, 2J, and 2K).
  • This Example describes the results of experiments performed to illustrate that an exemplary decoding of B cell specificity by ENTER viruses that display B cell antigens.
  • B cells possess a high diversity of BCR that can specifically target foreign antigens from invading viruses and self-antigens.
  • Viral antigen- specific B cell can produce antibodies (secreted form of BCR), which is beneficial to prevent viral infection.
  • autoantigenspecific B cells can produce detrimental autoantibodies attacking our, contributing to autoimmune disorders (Burbelo et al., 2021; Tan, 1989).
  • Burbelo et al., 2021; Tan, 1989 can produce detrimental autoantibodies attacking our, contributing to autoimmune disorders.
  • it is important to decode B cell specificity which will facilitate the development of highly effective anti-viral antibodies, guide the rational design of vaccines, and provide a better understanding of the formation of autoreactive B cells (Ju et al., 2020).
  • this Example describes the results of experiments performed to explore the feasibility of capturing the interaction between BCR and antigens.
  • BCR can recognize antigen epitopes that are derived from not only cell surface proteins but also intracellular proteins, extracellular, and secreted proteins.
  • the main challenge of ENTER to decode B cell specificity is to display B cell antigens which do not contain their native transmembrane (TM) domains on the viral surface.
  • TM transmembrane
  • Nascent HIV-1 viruses can selectively incorporate certain host TM proteins while excluding other abundant host surface proteins during viral assembly and budding process (Bumie and Guzzo, 2019).
  • HLA-DRA MHC class I and II molecules
  • HLA-DRB HLA-DRB
  • HLA- A2 adhesion molecules
  • IAM1 CD43, CD162, CD62L
  • integrin family members CD49d, LFA-1 (see, e.g., FIG. 3A).
  • viruses were engineered to express a B cell antigen epitope derived from human papillomavirus (HPV) minor capsid antigen L2 (HPV16 L2 residuen-36) and fused with TM domains from the prioritized list.
  • HPV human papillomavirus
  • HPV16 L2 residuen-36 major capsid antigen L2
  • the percentage of GFP+ B cells was quantified to measure the efficiency and specificity (see, e.g., FIG. 3B).
  • the viruses were further engineered to fuse the B cell epitope with the TM domain from the fusogen VSV-G, a viral envelope protein that can be assembled in budding viruses.
  • the viruses were engineered to display Receptor Binding Domain (RBD) from SARS-CoV-2 spike protein (see, e.g., FIG. 3D).
  • RBD Receptor Binding Domain
  • the result showed that 88% of spike-RBD BCR+ B cells were labeled by RBD displayed viruses, indicating that ENTER with optimized TM domain can be applied to decode B cell specificity for both linear epitopes (HPV16 L217-36 antigens) and full antigen domains (SARS-CoV-2 spike RBD).
  • ENTER was engineered to display HER2 using its native TM domain.
  • HER2 is an epidermal growth factor receptor, which is overexpressed in breast cancer cells (Gutierrez and Schiff, 2011).
  • the experimental data showed that 76% of anti- HER2 BCR B cells were detected by the HER2 displayed viruses (see, e.g., FIGS. 3G-3H), highlighting the generalization of ENTER to display any B cell antigens from intracellular (HPV L2), extracellular (Spike RBD), and cell surface proteins (HER2).
  • on-target B cells with BCR recognizing SARS- CoV-2 Spike RBD antigen
  • off-target B cells with BCR recognizing HPV L2 antigen
  • the on-target B cells with cell trace violet dye. The signal-to-noise ratio was calculated based on the frequency of on- target GFP+ cells versus that of off-target GFP+ cells.
  • the signal-to-noise ratio was around 100- to 200-fold (see, e.g., FIG. 3F), indicating a profound specificity and sensitivity of the TM domain optimized viruses to display B cell antigen epitopes (see, e.g., FIGS. 3J-3K).
  • ENTER is a platform that can successfully capture the interaction of BCR and antigen in a highly specific and sensitive manner.
  • GFP transgene was used as a cargo to measure the delivery efficiency and specificity.
  • lentiviruses pseudotyped with wild-type VSV-G comparable transduction efficiency irrespective of TCR or BCR specificity was observed (see, e.g., FIG. 3L).
  • Additional experiments were then carried out to engineer viruses displaying pp65495-503 pMHC ligand and VSV-G-mutant fusogen, carrying GFP transgene on the viral RNA, and other viral components including wild-type integrase (see, e.g., General Methods in Example 11).
  • HSV-TK herpes simplex virus thymidine kinase
  • GCV drug ganciclovir
  • GCV drug was added to kill cells expressing HSV-TK and the cell survival was monitored for 4 days (see, e.g., FIG. 4E).
  • a specific cargo delivery in on-target T cells among a pool of mixed T cells post viral infection was observed (see, e.g., FIG. 3M).
  • a specific depletion of on-target T cells (CMV-pp65 TCR+) without impacting the off-target T cells was observed see, e.g., (see, e.g., FIG. 4F).
  • viruses with GFP gene as a negative control were generated.
  • HSV-TK gene delivery resulted in a significant ⁇ 8-fold depletion of on-target T cells compared to GFP gene delivery (see, e.g., FIG. 4G), further validating that selective depletion of one T cell clone is achieved by antigen- specific suicide gene delivery.
  • 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 addition of HER2 displayed viruses carrying suicide gene (see, e.g., FIGS. 4H and 3N).
  • pp65495-503 displayed carrying 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, e.g., FIG. 4K).
  • CMV-pp65 TCR+ on-target
  • NY-ESO-1 TCR+ off-target TCR+
  • the result showed a significant decrease of FAS protein surface expression in on-target T cells infected with shFAS #2 compared to shCtrl group or off-target uninfected T cells, indicating a targeted shRNA delivery (see, e.g., FIG. 4L).
  • the workflow of ENTER-seq comprises (1) generation of a pooled pMHC displayed GFP viruses, (2) incubation of these viruses with human T cells, (3) sorting virus-labeled GFP+ cells for droplet-based single cell profiling (e.g., 5 prime single-cell RNA-seq/V(D)J-seq by lOx Genomics), (4) generation and sequencing of three single-cell libraries including gene expression, V(D)J TCR repertoire, and antigen-peptide sequence (see, e.g., FIG. 5A).
  • droplet-based single cell profiling e.g., 5 prime single-cell RNA-seq/V(D)J-seq by lOx Genomics
  • generation and sequencing of three single-cell libraries including gene expression, V(D)J TCR repertoire, and antigen-peptide sequence see, e.g., FIG. 5A).
  • the single-chain PMHC information is stored in viral single-strand RNAs (ssRNAs) that are packaged into lentiviral particles.
  • the viral ssRNA is approximately 4.6 kb, making it difficult to reverse transcribe (RT) into full-length cDNA in droplets.
  • RT reverse transcribe
  • a capture tag was inserted in the linker region between B2M and MHC, and another PCR handle next to CMV promoter (see, e.g., FIG. 5B). This capture tag allows capture by commercially available 5’ GEM beads through hybridizing with the Template Switch Oligo (TSO) sequence conjugated on the beads.
  • TSO Template Switch Oligo
  • the PCR handle permits convenient amplification of targeted peptide sequence without spiking in additional primers during the cDNA amplification step (see, e.g., FIG. 5B). Further nested PCR and index PCR allow targeted enrichment of the antigen peptide sequence to generate the final antigen library for deep sequencing.
  • the insertion of the capture tag and PCR handle does not affect the display of pMHC on viruses and specific interaction with TCR-expressing T cells (see, e.g., FIGS. 4N-4O).
  • ENTER-seq was performed on a mixed TCR-expressing T cells with a pooled pMHC displaying viruses.
  • 10% of T cells was mixed with TCR recognizing ny-eso-1157-165 antigen and 90% of T cells with TCR recognizing CMV pp65495-503 antigen, and then incubated with pooled viruses displaying ny-eso-li57-i65antigen or pp65495-503 antigen (see, e.g., FIG. 5C).
  • the sensitivity of the ENTER-seq system was first validated using GFP viruses displaying CMV-pp65 antigen epitope presented on HLA-A2 allele, and primary T cells from HLA-A2+ patients with a history of CMV infection.
  • Primary T cells were incubated with pp65495-503 antigen displayed viruses and then stained with a widely used CMV pp65495-503 tetramer which serves as a positive control.
  • the tetramer staining analysis showed that 1% of the T cells were pp65495-503 antigen- specific (see, e.g., FIG. 4Q). 83% of the pp65495-503 tetramerpositive T cells were labeled by GFP viruses.
  • the GFP was replaced with mNeon, a monomeric green fluorescence protein that is substantially brighter than GFP (see, e.g., FIGS. 4P-4Q). Indeed, 98% of pp65495-503 tetramerpositive T cells were recovered by mNeon viruses displaying pp65495-503 epitope, but not negative control viruses, indicating a significantly higher efficiency than GFP viruses (see, e.g., FIGS. 4Q-4S).
  • Anti-viral T cells are essential to control viral replication and dissemination. Adoptive transfer of in-vitro expanded CMV-specific T cells has shown great efficacy to control CMV infection in patients receiving transplantation. However, it is largely unexplored how CMV peptide-induced antigen- specific expansion in vitro impacts the molecular phenotype, clonal expansion and potential function of CMV-specific T cells.
  • ENTER-seq was used to characterize the transcriptional program, antigen specificity and TCR clonality of CMV-specific T cells expanded via CMV antigen peptide stimulation.
  • human peripheral mononuclear cells (PBMCs) from CMV seropositive donors were first cultured with a pool 12 CMV antigen peptides for 10 days (Lehmann et al., 2020; Liibke et al., 2020; Solache et al., 1999) (see, e.g., FIG. 4T).
  • the peptides were processed and presented by autologous antigen- presenting cells which then stimulated CMV antigen- specific T cells for later expansion.
  • T cells were expanded using peptide pp65495-503 and then incubated with pp65495-503 antigen presented mNeon viruses, followed by staining with pp65495-503 tetramer.
  • Flow cytometry analysis showed that -99% of tetramer+ T cells were labeled by viruses (see, e.g., FIG. 4U), demonstrating a high specificity and sensitivity of ENTER to detect peptide enriched antigenspecific T cells.
  • a pool of ENTER viruses displaying these 12 CMV antigen epitopes were prepared and incubated with expanded T cells from 4 different CMV seropositive HLA-A2 positive donors (see, e.g., Figure S4G). Dramatic expansion of CMV antigens- specific T cells was observed in 2 out of 4 donors (19.4% in donor#l and 8.78% in donor #2) (see, e.g., FIG. 4V). Next, ENTER-seq was performed on expanded T cells from these two donors, and labeled each donor sample with a unique hashtag antibody.
  • ENTER-seq was combined with CfTE-seq through staining cells with DNA barcoded antibodies targeting cell surface proteins CD45RA, CD45RO, and IL7R (see, e.g., FIG. 6A).
  • RNA-seq data of CMV pp65 TCR-T cells was compared with incubation of pp65495-503 displayed ENTER viruses or pp65495-503 tetramers.
  • 28 genes were identified as being differentially expressed between ENTER group and tetramer group (2-fold change and adjusted P value ⁇ 0.01) (see, e.g., FIG. 4W).
  • peptide-enriched CMV-specific T cells were mainly effector memory T (TEM) cells (CD45RO+CD45RA-) while ENTER- cells were a mixture of naive and central memory T (TCM) cells (see, e.g., FIGS. 6C and 5H-5I).
  • ENTER+ cells were potentially protective T cells based on high expression of effector molecules such as IFNG, TNF, and cytotoxic molecules including granzymes and perforin (see, e.g., FIG. 5J).
  • HSPA1A HSPA1A+; (10): proliferating TEM: CD45RO+KI67+ (see, e.g., FIGS. 6D and 5H-5L).
  • Comparison of subset frequency between donors showed that ENTER- bystander T cells were relatively similar between two donors while ENTER+ CMV antigen-specific T cells were phenotypically different between two donors, suggesting that two donors may have different immune responses to CMV antigens (see, e.g., FIGS. 5M-5N).
  • ULIOO2OO-2O8- specific T cells have high expression of FOXP3, IL2RA(CD25 and CTLA4, which are characteristics of regulatory T (Treg) cells (Billerbeck et al., 2007; Churlaud et al., 2015; Fontenot et al., 2003; Wing et al., 2008), indicating that ULIOO2OO-2O8- specific T cells resemble CD8+ Treg cells (see, e.g., FIG. 6G) (Vieyra-Lobato et al., 2018).
  • ENTER-seq not only uncovers donor- specific viral epitopes but also reveals distinct molecular blueprints of antigen- specific T cells upon recognizing different antigen epitopes from the same virus.
  • This Example describes the results of experiments performed to illustrate inter-clonal phenotypic diversity underlying the same antigen specificity.
  • TCR clonotypes were defined by the identity of CDR3 nucleotide sequences (Yassai et al., 2009).
  • Peptide enriched CMV-specific T cells (ENTER+) exhibited high clonal expansion (maximum 3856 cells per TCR clone) compared to bystander T cells (ENTER-, maximum 174 cells per TCR clone) (see, e.g., FIG. 6J).
  • TCR beta chain sequences (CASSFQGYTEAFF; SEQ ID NO: 54 and CASSYQTGASYGYTF; SEQ ID NO: 55) are identical with published pp65495-503-specific TCRs in the IEDB database, further validating the specificity of the ENTER platform disclosed herein (see, e.g., FIG. 60).
  • ENTER-seq can functionally characterize both the TCR binding specificity and the TCR-associated cell states.
  • ENTER viruses were engineered for displaying top 3 CMV antigen epitopes identified previously and performed ENTER-seq on primary T cells isolated directly from patient blood without in vitro expansion (see, e.g., FIG. 7A). Integrative analysis of CITE-seq and gene expression profiles showed that CMV-specific T cells (ENTER+) in patients were mainly terminally differentiated effector memory T cells (TEMRA, CD45RO-CD45RA+CCR7-) (see, e.g., FIGS. 7B-7C). This observation is consistent with previous studies showing the accumulation of TEMRA CMV- specific T cells in CMV seropositive patients (Appay et al., 2002; Derhovanessian et al., 2011).
  • TEMRA #1 cluster contains high expression of cytotoxic genes like IFNG, TNF, and PRF1 but not GZMK whereas TEMRA #4 cluster is IFNG- TNF-PRF1+G7M + (see, e.g., FIGS. 7C and 7L).
  • TEMRA #2 cluster in CMV- specific T cells contains low expression of all cytotoxic genes but high expression of type-I IFN stimulated genes (ISG) such as ISG15, ISG20, IFIT1, and OASL etc. (see, e.g., FIGS. 7C and 7L).
  • ISG type-I IFN stimulated genes
  • FIGS. 7C and 7L Such upregulation of ISG genes reflected a specific induction of type-I IFN response in a small subset of CMV-specific T cells, which might be stimulated by local production of type-I IFN responding to CMV viruses or bystander production of type-I IFN from other pathogens in patients.
  • CMV-specific T cells were found to predominantly pp65495-503-specific T cells associated with a wide range of clonal expansion, confirming that pp65495-503 is a highly immunogenic CMV epitope (see, e.g., FIGS. 7E-7F). Further observed was a rare population of T cells (43 cells) that were labeled by US874-82PMHC ENTER viruses (see, e.g., FIG. 7N).
  • TCR sequencing revealed that up to 60% of the US874-82 - specific cells at rest share TCRs with peptide-expanded US874-82 -specific T cells, confirming their clonal identity (see, e.g., FIG. 70).
  • donor #2 contain the most expanded TCR clone, whereas the TCR clone in donor #1 rarely expand (see, e.g., FIGS. 7P-7Q).
  • the number of pMHC bound per cell was also quantified to measure the binding strength of pMHC displayed ENTER viruses.
  • the result showed significantly higher binding of pMHC in highly expanded T cell clones (clone size >50) than lowly expanded T cells (see, e.g., FIG. 7G).
  • ENTER pMHC binding positively correlated with TCR affinity see, e.g., FIG. 2F
  • the experimental data described herein suggested that high TCR affinity is associated with and likely drives greater T cell clonal expansion.
  • TCR clone #2 is composed of type-I IFN ISG+ TEMRA, stressed IFNG hi FASLG 10 TEMRA, and IFNG lo FASLG hi TEMRA (see, e.g., FIGS. 7R-7S).
  • This data revealed an intra-clonal phenotypic diversity underlying the same TCR clones, suggesting that the T cell state is impacted by both TCR binding specificity and local microenvironment.
  • This Example describes the results of experiments performed to demonstrate phenotypic transition and clonal divergence of CM V- specific T cells upon ex vivo antigen peptide-induced expansion.
  • TEMRA T cells After antigen-induced expansion, 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., FIG. 7H). This observation was validated in both donors by flow cytometry (see, e.g., FIG. 71).
  • antigenic activation can also evoke inter-clonal phenotypic diversity.
  • T helper 2 Th2 cytokine gene IL13 but a spectrum of expression of the memory T cell transcription factor EOMES at rest (see, e.g., FIG. 7J).
  • clone 1 now produced cells that expressed either IL13, EOMES, or both;
  • clone 3 produced cells that expressed IL13 or EOMES in a mutually exclusive manner; clone 2 increased the frequency of expression of EOMES but never IL13 (see, e.g., FIG. 7J).
  • each TCR may recognize the same antigen differently to drive diverse transcriptional programs and cell states.
  • ENTER-seq enabled a systematic dissection of T cell specificities, resting cell states, and antigen-evoked cell fate potentials after a viral infection in patients.
  • Anti-CMV T cells transition in cytotoxicity and type-I IFN response from TEMRA T cells to TEM T cells, accompanied by upregulation of Th2 cytokine genes in specific T cell clones after peptide induced antigen- specific expansion (see, e.g., FIG. 7K).
  • psPAX2-D64V-NC-MS2 (addgene# 122944) was digested with SphI and BspEI sequentially. Then part of gag and eGFP or mNeon were assembled together with backbone. GFP-VPR is obtained from Addgene (#83374).
  • DNA fragments encoding the light and heavy chain of a RBD antibody (Protein Data Bank under accession number 7BWJ, (Ju et al., 2020)) were codon optimized and synthesized (Twist Bio). Afterward, a signal peptide was added to each chain and heavy chain was further extended to full length with a human IgGl Fc and a PDGFR TM sequence. The BCR was then inserted into a lentiviral vector driven by a SFFV promoter with hygromycin resistance.
  • CD19-CAR vector was generated by inserting a scFv CD 19 (kindly provided by Mackall lab) with a CD8 stalking linker and TM into the lentiviral plasmid followed by 2A-puromycin and 2A-eGFP.
  • scFv CD19 and TMs were replaced to generate other antigen candidates including HPV-L2 antigen, CD40L (addgene#125795) and CD40L mutant (addgene#125796).
  • TM domain screening the TM was swapped with 10 alternatives in the HPV-L2 antigen viral vector (Table 4).
  • DNA fragment of SAR2 spike RBD domain was synthesized and inserted the lentiviral expression vector followed by CD8 stalking linker and TM domain similar to the above description.
  • truncated Her2 (al- a700) fragment including its native TM and additional 55-aa cytoplasmic tail was amplified from WT HER2 (addgene# 16257), and inserted into the above vector.
  • VSV-G in pMD vector was first replaced with different envelope proteins such as RBD, HER2, pp65-HLA-A2 in the same approach as VSV-g mutant.
  • cargo delivery vectors were constructed where cargos such as HSV-TK-2A-egfp (HSV-TK from addgene#333O8), and eGFP only were driven by Efla promoter in a lentiviral vector.
  • HSV-TK-2A-egfp HSV-TK-2A-egfp
  • eGFP only were driven by Efla promoter in a lentiviral vector.
  • shRNA delivery different shRNA were placed under human U6 promoter in a lentiviral vector containing eGFP and puromycin as fluorescent and selection markers.
  • mScarlet transgene was inserted after EFl short promoter in a lentiviral vector with puromycin resistance for labeling cells with a red fluorescent protein.
  • HEK 293T were transfected with a viral expression vector (2ug), pMD2.G (VSV-G wild type) (lug), and psPax2 (2ug) with lipofectamine 3000.
  • the media was changed one next day, and viral supernatant were collected twice at 48 hours and 72 hours respectively.
  • the virus was concentrated with 4x Lenti-X according to manufacturer’ s protocol, and stored at 20x concentrated in -80°C.
  • VSV-G mutant was used instead.
  • VSV-G mutant and fluorescent protein fused version (NC- eGFP or NC-mNeon) of psPAX2-D64V (D64V mutation on integrase) vectors were mixed with antigen expressing vector according to above ratio to transfect the HEK 293T cells.
  • N- eGFP or NC-mNeon fluorescent protein fused version of psPAX2-D64V (D64V mutation on integrase) vectors were mixed with antigen expressing vector according to above ratio to transfect the HEK 293T cells.
  • HLA-KO HEK 293T cells were used for transfection. Viruses were collected, concentrated to 40x, and stored in -80°C.
  • HEK 293T were transfected with a cargo expression vector (1.6 pg), pMD2.G VSV-g mut (0.8 pg), psPax2 (1.6 pg), and envelope plasmid (1 pg) with lipofectamine 3000.
  • Virus was collected as described above, concentrated to 40x, and stored in -80°C before use.
  • Lentiviral titer was determined by Lenti-X GoStix Plus kit (Takarabio) according to the manufacture’s protocol.
  • HEK 293T related cells were cultured in RPMI supplemented with 10% FBS (Invitrogen) and IX pen/strep.
  • HEK 293T related cells were maintained in DMEM supplemented with 10% FBS and IX pen/strep.
  • HLA-KO HEK 293T cells were generated by electroporation of Cas9 RNP targeting HLA-A, HLA-B, and HLA-C alleles and further sorted HLA-KO cells based on surface expression of HLA-A/B/C.
  • Ramos cells were obtained.
  • Jurkat TCR negative -76 cells and Jurkat expressing CMV pp65 TCR, and flu-ml TCR were obtained.
  • Ramos or Jurkat cells were infected with viruses, and selected by sorting or using drug after 4-5 days.
  • NYESO-TCR Jurkat and RBD-BCR Ramos cells were infected with red mScarlet virus and selected with puromycin to generate mScarlet red fluorescent labeled cell lines.
  • FIG. IB 30uL concentrated lentiviruses were added into 250K Raji or Jurkat cells in 12 well plate. 3 days later, GFP signal was measured by flow cytometry.
  • 200K target cells were collected in tubes and the supernatant was removed after centrifugation. The cell pellet was resuspended in 30uL concentrated GFP fused lentiviruses and incubated at 37°C. After 2hr incubation, cells were stained with flow cytometry antibodies for 10 min at 4°C (if needed), washed by RPMI medium for twice, and finally subjected to flow cytometry.
  • the viral titer was normalized and incubated 100K NY-ESO-1 TCR T cells (1G4 wild-type or a95:LY mutant) or off-target CMV-pp65 TCR+T cells with a titration (4ng, 20ng, 40ng p24 level) of ENTER viruses displaying antigen variants. Upon 2-hour incubation, cells were washed and subjected to flow cytometry to quantify GFP+ cells.
  • lOpL Protein G Dynabeads were incubated in ImL blocking buffer (PBS with 0.1%BSA) for 20 min at room temperature.
  • 2pg anti-CD40L antibody Cat#157009, Biolegend
  • anti-VSV-G antibody clone 8G5F11, Millipore sigma
  • IgG antibody were added into beads with lOOpL blocking buffer and rotated for 30 min at 4°C.
  • the antibody conjugated beads were washed three times and the supernatant was removed.
  • 30pL CD40L displayed viruses were added into beads with 30pL blocking buffer and rotated for 1 hour at room temperature. 5pL CD40L displayed virus from the same batch was prepared as input samples.
  • RNA extraction was performed using Stratagene Brilliant II SYBR Green QRT-PCR Master Mix (Agilent).
  • ganciclovir (GCV, Invivogen) was added to a final concentration of O.lpg/ml, which was counted as day 0.
  • Cell media and drug were refreshed every 3 days.
  • 300pL of cell culture were taken every day, stained with IgG or CD3 before analyzed by flow cytometry.
  • the ratio of live on-target cells over off-target cells were calculated and plotted over the days (normalized to Day 0).
  • raw count of live cells for targeted or NT population at day 4 of treatment were also compared between TK and eGFP only delivery.
  • Flu-TCR expressing Jurkat T cells were labeled by CellTrace Violet dye (#C34571, Thermo Fisher) according to manufacturer’s protocol. Violet labeled Flu-ml TCR+ T cells were mixed with NY-ESO-1-TCR+ T cells at diverse ratios including 1:1, 1:10, 1:100, 1:1000. The mixed T cells were incubated with 40pL concentrated HLA-A2-Flu antigen displayed GFP viruses for 2 hours at 37°C. T cells were stained with CD3- APC (clone HIT3a, BioLegend) antibody, washed twice, and subjected to flow cytometry.
  • CD3- APC clone HIT3a, BioLegend
  • HPV-BCR expressing Ramos B cells were labeled by CellTrace Violet dye and mixed with HPV-L2 BCR expressing Ramos B cells at diverse ratios including 1:1, 1:10, 1:100, 1:1000.
  • the mixed cells were incubated with 40uL concentrated RBD-antigen displayed GFP viruses for 2 hours at 37°C.
  • Signal-to-noise ratio (percentage of GFP+ on-target cells among total on-target cells)/(percentage of GFP+ off-target cells among total off-target cells).
  • PBMC Peripheral blood mononuclear cells
  • Lymphoprep Cat# 07811, STEMCELL Technologies
  • B cells were purified from thawed PBMCs by negative selection using EasySep Human B Cell Enrichment Kit (Cat#19844, STEMCELL Technologies) according to the manufacturer’s protocol.
  • Isolated B cells were cultured in IMDM medium supplemented with 10% FBS and 55 mM beta-mercaptoethanol at 1X10 6 cell/mL and activated by CellXVivo Human B cell expander (1:250 dilution, R&D system) and 50 ng/mL IL2 (Cat#200-02-10ug, PeproTech) for two days.
  • LRS chambers from HLA-A2+ donors with CMV infections (CMV seropositive) were obtained from Stanford Blood Center with consent forms.
  • PBMCs were isolated and stored as above.
  • CD8+ T cells were purified from thawed PBMCs by negative selection using EasySep Human CD8+ T Cell Enrichment Kit (Cat#19053, STEMCELL Technologies) according to the manufacturer’s protocol.
  • Short 9-mer peptides encoding CMV epitopes were synthesized by Elimbio in lyophilized powders. The peptides were dissolved in DMSO in lOmg/mL.
  • PBMC were isolated from donor blood described as above. PBMC were cultured in T cell medium (RPMI medium supplemented with 10% FBS, IX penstrep, lOOmM HEPES, 55 mM beta-mercaptoethanol). Individual peptide (lOug/mL) or pooled peptides (lug/mL for each peptide) were added into PBMC for culturing 10 days in T cell medium. 50ng/mL IL-2 were added every two days. After peptide enrichment, PBMCs were incubated with viruses and/or PE- tetramer and then analyzed by flow cytometry.
  • FIG. ID B cells were incubated with viruses for 2 hours and then stained with Human TruStain FcXTM (Fc Block, BioLegend), CD19-APC (clone HIB19) and CD20-V450 (clone L27) antibody in cell staining buffer (BioLegend) for 10 min at 4C.
  • FIG.2 Jurkat T cells were incubated with viruses for 2 hours and then stained with CD3-APC (clone HIT3a) and PE labeled tetramer loaded with peptides (NIH tetramer core) for 30 min at 4C.
  • FIG. ID B cells were incubated with viruses for 2 hours and then stained with Human TruStain FcXTM (Fc Block, BioLegend), CD19-APC (clone HIB19) and CD20-V450 (clone L27) antibody in cell staining buffer (BioLegend) for 10 min at 4C.
  • FIG.2 Jurkat T cells were incubated
  • cells were incubated with viruses for 2 hours and then stained with human Fc Block, CD3-APC, CD8- BV711 (clone SKI), tetramer-PE if needed, and viability dye for 30 min at 4°C. After staining, cells were washed twice by cell straining buffer and analyzed by flow cytometry (Attune, Thermo Fisher). All antibodies are from BioLegend if not specified. Tetramers were from NIH tetramer core.
  • RNA-seq libraries were incubated with 30uL pp65495-503 displayed ENTER viruses or Ipg pp65495-503 tetramer for 2 hours. Cells were washed twice and subject to RNA extraction. RNA was extracted using Quick-RNA Miniprep Kit with on-column Dnase digestion (Zymo Research). At least lOOng RNA was used to prepare the RNA-seq library using TruSeq® Stranded mRNA Library Prep Kit (Cat# 20020594, Illumina) for each sample following the manufacturer's instruction. The library was sequenced on an Illumina Nextseq to generate 2X150 paired-end reads.
  • DEG differentially expressed genes
  • the cDNA was amplified and cleaned up to generate the transcriptome according to manufacturer’s protocol. [316] During cDNA cleanup, the supernatant that contains shorter fragment of HLA peptide and TCR information was further mixed with SPRISelect beads to 0.9x, and cleaned up. The library that encodes HLA peptide were generated through 2 round of nested PCRs and a final round of indexing PCR. First, the HLA peptide cDNA was enriched by 8 cycles of PCR (98c for 45 min, then 8x of 20sec at 98c 20 sec, 20 sec at 59c and 30sec at 72c) with 0.5uM 10x_5pRNA_Fw, and HLA_nested_fw.
  • TCR identity was generated.
  • the TCR DNA were enriched by nested PCR (specifically, 98c for 45 min, then 8x of 20sec at 98c 20 sec, 20 sec at 59c and 30sec at 72c) with 0.5pM 10x_5pRNA_Fw and 0.5uM mix of nyeso_TRAc_rev and hs_TRAc_rev targeting two different TCR.
  • Peptide stimulated donor PBMC were collected and stain with a mixture of 12 viruses displaying CMV antigen epitopes including IElsi-89, IEI316-324, US150Ai52-i6i, US874-82, ULIOO2OO-2O8, UL46IOO-IO8, pp654i7-425, pp65325-333, pp65188-196, pp65i20-i28, pp65495-503, and pp6514- 22.
  • the peptide sequences for CMV antigens were listed in Table 5.
  • the scRNA-seq reads were aligned to GRCh38 genome and quantified using CellRanger count (lOx Genomics).
  • the CITE-seq reads were processed using CellRanger count with antibody oligo barcode as feature reference.
  • the TCR-seq reads were mapped to VDJ compatible reference (refdata- CellRanger -vdj-GRCh38-alts-ensembl-5.0.0) using CellRanger vdj (lOx Genomics).
  • HLA peptide reads were processed using CellRanger count with peptide sequence as feature reference.
  • RNA-seq and CITE-seq were performed using SCANPY (Wolf et al., 2018). Cells with less than 200 genes detected or greater than 10% mitochondrial RNA reads were excluded from analysis. Doublet cells were removed using CITE-seq analysis of barcoded hashtag antibody labeling donor origins. For cell clustering, raw UMI counts were first normalized by total counts to correct library size and then log-normalized. Variable genes were called using scanpy.pp.highly_variable_genes() with default parameters. Variable TCR genes were removed before principal component analysis (PC A) to prevent clustering bias from variable TCR transcripts.
  • PC A principal component analysis
  • Initial clusters were annotated using expression of known markers including 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-. Terminal effector cells re-expressing CD45RA (TEMRA) were CD45RA+CCR7-CD45RO-, MAIT cells were KLRB1+CXCR6+TRAV1-2+.
  • the gene set of cytotoxic genes were curated from well-established cytotoxic molecules.
  • the T cell exhaustion genes, T cell activation genes, and type-I IFN response genes were selected from previous literature (Yost et al., 2019).
  • TCR relevant analyses were performed using Scirpy (Sturm et al., 2020).
  • the contig annotation files generated by CellRanger vdj were used as input for TCR analysis.
  • TCR qualities were analyzed using scirpy. tl.chain_qc().
  • the TCR clonotypes were defined using scirpy.pp.ir_dist() and scirpy.tl.define_clonotypes() with default parameters based on CDR3 nucleotide sequence similarity.
  • the CDR3 amino acid compositions were generated using weblogo (Crooks et al., 2004). Cells with more than 5 raw count of HLA peptides for any individual antigens were labeled as antigen-specific T cells. The antigen peptide count per cell was quantified using log(count+l) transformation. The pp65-specific T cells were divided into diverse clonally expanded cells with clone size >50, or > 10, or >1. The distribution of antigen peptide count bound per cell in different clonally expanded T cells were showed in a violin plot.
  • 2D density plots were generated using kdeplotQ function to show cytotoxic gene score and type-I IFN gene score of T cells with same TCR sequence before and after antigen-induced expansion. All plots (e.g. violin plots, scatter plots, density plots and Bar plots) were generated by Python matplotlib and seaborn.
  • 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.
  • 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. 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. 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. Chandran, S.S., and Klebanoff, C.A. (2019). T cell receptor-based cancer immunotherapy: Emerging efficacy and pathways of resistance.
  • Cytotoxic T lymphocytes recognize a fragment of influenza virus matrix protein in association with HLA-A2. Nature 326, 881-882. doi.org/10.1038/32688 laO. 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. 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.
  • T-Scan A Genome-wide Method for the Systematic Discovery of T Cell Epitopes. Cell 178, 1016-1028. el3. doi.org/10.1016/j.cell.2019.07.009. 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. Lee, M.N., and Meyerson, M. (2021).
  • Lentiviral vector-based insertional mutagenesis identifies genes associated with liver cancer. Nat Methods 10, 155— 161. doi.org/10.1038/nmeth.2331. Rasmussen, A.-M., Borelli, G., Hoel, H.J., Lislerud, K., Gaudemack, G., Kvalheim, G., and Aarvak, T. (2010).
  • 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).

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WO2025212851A2 (en) 2024-04-03 2025-10-09 Orbital Therapeutics, Inc. mRNA COMPOSITIONS AND USES THEREOF IN VARICELLA ZOSTER VIRUS VACCINES
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EP4281569A4 (en) * 2021-01-25 2025-04-02 The Regents Of The University Of California Methods and compositions for labeling and selecting antigen-specific T cells
WO2024081820A1 (en) * 2022-10-13 2024-04-18 Sana Biotechnology, Inc. Viral particles targeting hematopoietic stem cells
WO2025036498A1 (zh) * 2023-08-16 2025-02-20 深圳市济因生物科技有限公司 一种病毒载体及其在感染b细胞中的应用
WO2025072615A1 (en) 2023-09-29 2025-04-03 Orbital Therapeutics, Inc. Improved methods of making rna by splinted ligation and compositions thereof
WO2025212851A2 (en) 2024-04-03 2025-10-09 Orbital Therapeutics, Inc. mRNA COMPOSITIONS AND USES THEREOF IN VARICELLA ZOSTER VIRUS VACCINES
WO2025259931A1 (en) 2024-06-14 2025-12-18 Orbital Therapeutics, Inc. Compositions and methods for rna circularization
WO2026006203A2 (en) 2024-06-24 2026-01-02 Orbital Therapeutics, Inc. Compositions and methods for making circular rna
WO2026006687A1 (en) 2024-06-28 2026-01-02 Orbital Therapeutics, Inc. Circular rna compositions and uses thereof for diseases caused by varicella-zoster virus

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