Ligand discovery and gene delivery via retroviral surface display

Abstract

To provide compositions of retroviruses and methods of using the same for gene delivery.SOLUTION: A composition for use in delivering a nucleic acid to a cell, wherein the composition is characterized in that (a) it comprises a retrovirus that comprises: a nucleic acid; a viral envelope protein comprising at least one mutation that reduces a native viral tropism of a mutant viral envelope protein compared to a non-mutant viral envelope protein, which is a VSV-G envelope protein, a measles virus envelope protein, a Nipah virus envelope protein, or a coccal virus G protein; and a non-viral membrane-bound protein comprising a membrane-binding domain and an extracellular targeting domain capable of binding to a homologous ligand of a cell, and that (b) the retrovirus is contacted with a cell, thereby delivering the nucleic acid to the cell.SELECTED DRAWING: None

Description

Technical Field

[0001] Related Applications This application claims the benefit of the filing date of U.S. Provisional Application No. 62 / 851,889, filed May 23, 2019, under 35 U.S.C. § 119(e), titled "LIGAND DISCOVERY AND GENE DELIVERY VIA RETROVIRAL SURFACE DISPLAY"; the entire content of which is incorporated herein by reference.

Background Art

[0002] Background It is well established that retroviruses (e.g., lentiviruses) can redirect their natural tropism to a desired target by pseudotyping the virus to express the envelope protein of another virus. This is most commonly achieved by pseudotyping with the VSV glycoprotein, which allows viral entry into a wide range of cells to target the LDL receptor. In recent years, groups have shown that C-terminal fusions to these viruses enable receptor-mediated entry into selected target cells when the lentivirus is pseudotyped with envelope proteins from paramyxoviruses such as measles virus or Nipah virus and mutations are created that abrogate the native tropism.

Summary of the Invention

[0003] Summary Herein, the inventors have surprisingly demonstrated that a combination of mutations that abrogate native function (e.g., tropism) and overexpression of a second membrane protein enables that second protein to function as the basis for viral entry. These discoveries described herein enable a new and innovative methodology for, for example, screening cells (e.g., T cells) that are notoriously difficult to screen for specific antigens and functions, and for the delivery of nucleic acids in a target-specific manner to target cells. Some aspects of the present disclosure provide retrovirus (e.g., lentivirus) compositions comprising: (i) a nucleic acid comprising a non-viral membrane-binding protein having the structure: S-ETD-MBD-IRES-R, where S encodes a signal sequence, ETD encodes an extracellular targeting domain; MBD encodes a membrane-binding domain, IRES encodes an internal ribosome entry site, and R encodes a reporter; and, (ii) a mutated viral envelope protein comprising at least one mutation that reduces its native function.

[0004] Some aspects of the present disclosure provide retrovirus (e.g., lentivirus) compositions comprising: (i) a CD80 protein domain; and (ii) a mutated viral envelope protein comprising at least one mutation that reduces its native function. The CD80 protein domain can be an extracellular domain. In some embodiments, the CD80 extracellular domain binds to a receptor on the target cell. Some aspects of the present disclosure provide a method of screening a cell population, the method comprising: (i) providing a retrovirus comprising a viral envelope protein comprising at least one mutation that reduces its native function, a non-viral membrane-binding protein comprising a membrane-binding domain and an extracellular targeting domain, and a nucleic acid encoding a reporter; (ii) combining the retrovirus with the cell population; and (iii) sorting the cell population based on the presence or absence of the reporter. In some embodiments, the retrovirus (e.g., lentivirus) comprises a nucleic acid comprising a non-viral membrane-binding protein having the structure: S-ETD-MBD-IRES-R, where S encodes a signal sequence, ETD encodes an extracellular targeting domain; MBD encodes a membrane-binding domain, IRES encodes an internal ribosome entry site, and R encodes a reporter.

[0005] In some embodiments, the cell is a somatic cell (e.g., an antigen-specific cell, e.g., a T cell or a B cell). In some embodiments, the cell is isolated from a subject (e.g., a human subject). In some embodiments, the cell is isolated from the blood or tumor of a subject. In some embodiments, the cell is maintained in liquid culture prior to combination with a retrovirus. In some embodiments, the viral envelope protein is a VSV-G envelope protein, a measles virus envelope protein, a Nipah virus envelope protein, or a coccus virus G protein. The VSV-G envelope protein can be mutated at any one or more of H8, K47, Y209, and / or R354. The measles virus envelope protein can be mutated at any one or more of Y481, R533, S548, and / or F549. The Nipah virus envelope protein can be mutated at any one or more of E501, W504, Q530, and / or E533. The coccus virus G protein can be mutated at K64 and / or R371.

[0006] In some embodiments, the non-viral membrane-binding protein includes a major histocompatibility complex (MHC) protein. In some embodiments, the extracellular targeting domain is a protein (e.g., interleukin-13), a peptide, or an antibody (e.g., an anti-CD19 antibody, an anti-TCR antibody, or an anti-CD3 antibody). In some embodiments, the reporter is a fluorescent protein (e.g., green fluorescent protein, yellow fluorescent protein, red fluorescent protein) or an antibiotic resistance marker. In some embodiments, the linker is disposed between the membrane-binding domain and the extracellular targeting domain. The linker can be a rigid linker (e.g., the PDGFR stalk or the CD8α stalk), a flexible linker (e.g., an amino acid sequence containing GAPGAS (SEQ ID NO: 5) or GGGGS (SEQ ID NO: 7)), or an oligomerization linker (e.g., the IgG4 hinge, or an amino acid sequence capable of forming a tetrameric coiled coil).

[0007] In some embodiments, the retrovirus is combined with a cell population at a temperature in the range of 1 minute to 72 hours and 4°C to 42°C in (ii). In some embodiments, the retrovirus and the cell population are combined in (ii) (a) in cell culture medium, optionally in the presence of RPMI or DMEM cell culture medium; (b) in buffered saline, optionally in the presence of phosphate buffered saline or HEPES buffered saline; and / or (c) in the presence of an enhancer of retroviral transduction, optionally heparin sulfate, polybrene, protamine sulfate, and / or dextran. In some embodiments, the extracellular targeting domain can bind to a cognate protein (e.g., a protein receptor) present on the cell surface of a subset of the cell population. In some embodiments, the cell population is washed between (ii) and (iii) (e.g., using phosphate buffered saline (PBS) to remove, for example, the retrovirus from the cell population). In some embodiments, the sorting of the cell population is performed using fluorescence-activated cell sorting, single cell next generation sequencing, or antibiotic selection.

[0008] In some embodiments, the method further comprises a second retrovirus, the second retrovirus comprising a different extracellular targeting domain and / or a different reporter as compared to the first retrovirus. Another aspect of the disclosure provides a method of delivering a nucleic acid (e.g., a gene of interest, e.g., one encoding a protein) to a cell, the method comprising: (i) providing a retrovirus comprising a nucleic acid, a viral envelope protein comprising at least one mutation that reduces its native function, and a non-viral membrane-binding protein comprising an extracellular targeting domain capable of binding to a cognate ligand of the cell; and (ii) contacting the retrovirus with the cell, thereby delivering the nucleic acid to the cell. In some embodiments, the retrovirus enters or infects the cell during (ii). Some aspects of the present disclosure provide methods for delivering nucleic acids to cells, the methods comprising: (i) providing a retrovirus comprising a nucleic acid, a viral envelope protein comprising at least one mutation that reduces its native function, and a CD80 protein domain; and (ii) contacting the retrovirus with a cell, thereby delivering the nucleic acid to the cell.

[0009] Still other aspects of the present disclosure provide methods for detecting an interaction between a retrovirus and a cell, the methods comprising: (i) contacting a sample comprising a retrovirus and a cell with an antibody, wherein the retrovirus comprises a viral envelope protein comprising at least one mutation that reduces its native function and a non-viral membrane-binding protein comprising an extracellular targeting domain, and wherein the antibody binds to the extracellular targeting domain of the retrovirus; (ii) optionally, removing unbound antibody from the sample; and (iii) imaging the sample to detect whether an antibody-retrovirus complex is bound to the cell. In some aspects, the antibody further comprises a fluorescent label, optionally wherein the antibody is covalently bound to the fluorescent label. In some aspects, the sample is imaged in (iii) using a confocal microscope or a fluorescence microscope. Some aspects of the present disclosure provide a library of retroviruses comprising a plurality of unique retroviruses, wherein each unique retrovirus comprises a viral envelope protein comprising at least one mutation that reduces its native function, a non-viral membrane-binding protein comprising a membrane-binding domain and an extracellular targeting domain (e.g., comprising at least 5, at least 10, at least 15, at least 20, or at least 50 amino acids), and a nucleic acid encoding a reporter, and wherein each unique retrovirus comprises a different unique extracellular targeting domain.

[0010] In some embodiments, the library can be screened against a population of antigen-specific cells, where optionally, the antigen-specific cells are B cells or T cells. The library can contain at least 10 2 , at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , or at least 10 10 unique retroviruses. In some embodiments, each different unique extracellular targeting domain is generated by site-directed mutagenesis. Some aspects of the disclosure provide a population of cells, where a subset of the population of cells contains a retrovirus comprising a viral envelope protein having at least one mutation that reduces its native function, a non-viral membrane-binding protein comprising a membrane-binding domain and an extracellular targeting domain, and a nucleic acid encoding a reporter. In some embodiments, a subset of a population of cells (e.g., antigen-specific cells, such as B cells or T cells) contains the retroviruses described herein. In some embodiments, a subset of the population of cells contains a retrovirus within each cell of the subset. A subset of the population containing the retrovirus can be isolated and / or selected from the cells of the population that do not contain the retrovirus. BRIEF DESCRIPTION OF THE DRAWINGS

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[0017] **Detailed Description of the Invention** In this specification, for example, new and innovative methods are provided for screening cells (e.g., T cells) that are notoriously difficult to screen for specific antigens and functions, and for delivering nucleic acids to target cells in a target-specific manner. In some aspects, described herein is, for example, a system that enables repertoire-scale analysis of T cell receptor (TCR)-peptide-major histocompatibility complex (pMHC) specificities, which has been a previously difficult bottleneck because determining what a previously described method could recognize (e.g., as in a typical immune response) for a single T cell clone required a significant amount of effort. In some aspects, described herein is a retrovirus-based system that re-uses virus tropism as a method of selection of molecular interactions, for example by encoding these protein variants on the corresponding transfer plasmid used to generate the virus, replacing the binding function of the wild-type virus surface protein with the binding function of the protein variant of interest, such that the resulting virus displays the protein variant on its surface and is guaranteed to package the corresponding gene sequence. Thus, when the virus infects a target cell (e.g., one having a receptor that binds to the extracellular targeting domain of the displayed protein variant), cell entry results in integration of the gene sequence of the displayed protein into the genome of the target cell.

[0018] Previous approaches for studying T cell specificity required a combination of generated T cell lines, recombinant expression of T cell receptors, and / or individual verification of T cell binding or activity by candidate antigen-based approaches. Each of these elements imposed limitations inherent to the throughput of the T cells or antigens screened. For example, yeast display-based methods for de-orphaning T cell receptors reduced the bottleneck of the number of antigens to be investigated (>10 8(by the ability to screen for ligands, but still severely limited by the need to recombinantly express the TCR. The current strategy of the invention described herein enables screening of more than 10 8 ligands and represents a major advance in T cell specificity research and T cell screening by eliminating the need for recombinant TCR expression.

[0019] retrovirus Described herein is a retrovirus comprising a viral envelope protein comprising at least one mutation that reduces its native function, a non-viral membrane-binding protein comprising a membrane-binding domain and an extracellular targeting domain, and a nucleic acid encoding a reporter. In some embodiments, the retrovirus comprises a viral envelope protein comprising at least one mutation that reduces its native function, and a non-viral membrane-binding protein comprising a membrane-binding domain and an extracellular targeting domain. The retroviruses disclosed herein contain one or more elements derived from a suitable species of retroviral genome (naturally occurring or modified). Retroviruses include seven families: alpharetrovirus (avian leukosis virus), betaretrovirus (mouse mammary tumor virus), gammaretrovirus (mouse leukemia virus), deltaretrovirus (bovine leukemia virus), epsilonretrovirus (walleye dermal sarcoma virus), lentivirus (human immunodeficiency virus 1), and spumavirus (human spumavirus). Six additional examples of retroviruses are provided in U.S. Patent No. 7,901,671.

[0020] In some embodiments, the retrovirus is a lentivirus. Lentiviruses are a genus of retroviruses that typically cause slow - progressing diseases because of their ability to integrate into the host genome. The modified lentiviral genome is useful as a viral vector for delivering nucleic acids to host cells. Host cells can be transfected with a lentiviral vector and, optionally, an additional vector for expressing lentiviral packaging proteins (e.g., VSV - G, Rev, and Gag / Pol) to produce lentiviral particles in culture medium. Retrovirus and lentivirus constructs are well - known in the art, and any suitable retrovirus can be used to construct the retroviruses (or a plurality of retroviruses or a library thereof) described herein. Non - limiting examples of retrovirus constructs include lentiviral vectors, human immunodeficiency virus (HIV) vectors, avian leukosis virus (ALV) vectors, murine leukemia virus (MLV) vectors, mouse mammary tumor virus (MMTV) vectors, mouse stem cell virus, and human T - cell leukemia virus (HTLV) vectors. These retrovirus constructs contain proviral sequences from the corresponding retroviruses.

[0021] The retroviruses described herein can include the viral elements described herein from one or more suitable retroviruses that are RNA viruses having single-stranded positive-sense RNA molecules. Retroviruses include reverse transcriptase and integrase enzymes. Upon entry into a target cell, the retroviruses utilize reverse transcriptase to transcribe their RNA molecules into DNA molecules. Subsequently, the integrase enzyme is used to integrate the DNA molecules into the host cell genome. Once integrated into the host cell genome, the sequences from the retroviruses are called proviruses (e.g., proviral sequence or provirus sequence). The retroviral vectors described herein can further include additional functional elements known in the art to address safety concerns and / or to improve vector functions such as packaging efficiency and / or viral titer. Additional information can be found in US20150316511 and WO2015 / 117027, and the relevant disclosures of each are incorporated herein by reference for the purposes and subject matter referenced herein. Additional information regarding lentiviruses can be found, for example, in WO2019 / 056015, and the relevant disclosure thereof is incorporated herein by reference for this particular purpose. In some embodiments, the lentivirus can target target-specific cells via pMHC-TCR interactions or any other protein-protein, cell-cell interaction. In some embodiments, T cells with known relevant specificities can be enhanced (in the case of cancer or infection) or excised (in the case of autoimmunity) without affecting other T cells, dramatically limiting the risk of off-target effects. In some embodiments, the lentivirus can encode an extracellular domain and target any other surface-expressed molecule on the target cell.

[0022] Viral envelope protein The retroviruses described herein include viral envelope proteins that contain at least one mutation that reduces their native function (e.g., the wild-type function of a non-mutated viral envelope protein). In some embodiments, the viral envelope protein is any viral envelope protein of any retrovirus (e.g., lentivirus). The viral envelope protein can be a VSV-G envelope protein, a measles virus envelope protein, a Nipah virus envelope protein, or a mycoplasma virus G protein. In some embodiments, the native function reduced by the mutation in the viral envelope protein is the tropism of the virus (e.g., the ability to infect cells, the ability to bind to cells, etc.). In some embodiments, the viral envelope protein containing at least one mutation that reduces its native function is a mutated VSV-G envelope protein. In some embodiments, the viral envelope protein containing at least one mutation that reduces its native function is a mutated measles virus envelope protein. In some embodiments, the viral envelope protein containing at least one mutation that reduces its native function is a mutated Nipah virus envelope protein. In some embodiments, the viral envelope protein containing at least one mutation that reduces its native function is a mutated mycoplasma virus G protein.

[0023] In some embodiments, the mutated VSV-G envelope protein comprises a mutation at H8, K47, Y209, and / or R354. In some embodiments, the mutated VSV-G envelope protein comprises an H8A, K47A, K47Q, Y209A, R354A, and / or R354Q mutation. In some embodiments, the mutated VSV-G envelope protein is as described in Nikolic et al., “Structural basis for the recognition of LDL-receptor family members by VSV glycoprotein.” Nature Comm., 2018, 9:1029, the relevant disclosure of which is incorporated herein by reference for this particular purpose. In some embodiments, the mutated measles virus envelope protein comprises a mutation at Y481, R533, S548, and / or F549. In some embodiments, the mutated measles virus envelope protein comprises a Y481A, R533A, S548L, and / or F549S mutation. In some embodiments, the mutated Nipah virus envelope protein comprises a mutation at E501, W504, Q530, and / or E533. In some embodiments, the mutated measles virus envelope protein comprises an E501A, W504A, Q530A, and / or E533A mutation. In some embodiments, the mutated coccus virus G protein comprises a mutation at K64 and / or R371. In some embodiments, the mutated coccus virus G protein comprises a K64Q and / or R371A mutation.

[0024] In some embodiments, the mutated envelope protein is derived from any other enveloped virus, including but not limited to: baculovirus, herpes simplex virus (HSV), cytomegalovirus (CMV), lymphocytic choriomeningitis virus (LCMV), Epstein-Barr virus (EBV), vaccinia virus, hepatitis A, B, or C virus, vaccinia virus, alphavirus, dengue virus, yellow fever virus, Zika virus, influenza virus, hantavirus, Ebola virus, rabies virus, human immunodeficiency virus (HIV), coronavirus, and other members of the Rhabdoviridae family. In some embodiments, a viral envelope protein comprising at least one mutation comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, a viral envelope protein comprising at least one mutation comprises a nucleotide sequence and / or amino acid sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 97% identical to the wild-type viral envelope protein. In some embodiments, a viral envelope protein comprising at least one mutation that reduces its native function retains less than 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the function of the wild-type viral envelope protein. In some embodiments, a viral envelope protein comprising at least one mutation lacks all of its native function. In some embodiments, a retrovirus comprising a viral envelope protein comprising at least one mutation that reduces its native function has less than 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the cell infectivity of a retrovirus comprising the wild-type viral envelope protein.

[0025] Non-viral membrane-binding protein The retroviruses described herein include non-viral membrane-binding proteins. The non-viral membrane-binding protein can include a membrane-binding domain and an extracellular targeting domain. In some embodiments, the non-viral membrane-binding protein is a chimeric protein that includes sequences from at least two different proteins. In some embodiments, the non-viral membrane-binding protein is a full-length or truncated protein that includes sequences from a single protein. The membrane-binding domain is the protein or peptide having an amino acid sequence that enables the protein or peptide to be fully or partially embedded in or associated with the membrane of the retrovirus (e.g., envelope). In some embodiments, the membrane-binding domain enables the presentation and delivery of the extracellular targeting domain to the extracellular environment. In some embodiments, the membrane-binding domain includes an intracellular domain, a transmembrane domain, and / or an extracellular domain. In some embodiments, the membrane-binding domain includes an intracellular domain and a transmembrane domain. In some embodiments, the membrane-binding domain includes a major histocompatibility complex (MHC) protein or a fragment thereof. The MHC protein can be a class I or class II MHC protein.

[0026] In some embodiments, the membrane-binding domain includes a total of 10-50, 10-100, 25-100, 50-200, 50-150, 100-500, 100-250, 250-500, or any reasonable number of total amino acids. In some embodiments, the retroviruses present in the retrovirus library include the same membrane-binding domain as some or all of the other retroviruses in the library. In some embodiments, each retrovirus present in the retrovirus library includes a different membrane-binding domain compared to some or all of the other retroviruses in the library. In some embodiments, the extracellular targeting domain is any protein or peptide having an amino acid sequence and being a binding partner of a target molecule or ligand (e.g., cognate protein) on the cell surface. When present in the extracellular environment beyond the interior of the retrovirus, the extracellular targeting domain can bind to target cells. In some embodiments, the extracellular targeting domain binds to or targets a cognate protein or ligand (e.g., a protein receptor present on the target cell) present on the cell surface of a subset of cells or cell populations. In some embodiments, the extracellular targeting domain binds to a cognate protein or ligand present on the cell surface of a single T cell or a subset of a T cell population. In some embodiments, the binding interaction between the extracellular targeting domain of the retrovirus and the cognate protein or ligand of the cell enables the retrovirus to enter the cell (e.g., an antigen-specific cell, e.g., a T cell).

[0027] In some embodiments, the extracellular targeting domain comprises 10 - 50, 10 - 100, 25 - 100, 50 - 200, 50 - 150, 100 - 500, 100 - 250, 250 - 500, or any reasonable number of total amino acids. In some embodiments, the extracellular targeting domain comprises at least 5, at least 10, at least 15, at least 20, or at least 50 amino acids. In some embodiments, the extracellular targeting domain is a protein, antibody, or peptide. In some embodiments, the antibody is a full-length antibody, antibody fragment, nanobody, or single-chain antibody (scFv). In some embodiments, the extracellular targeting domain is an antibody that binds to a cognate protein of the target cell. In some embodiments, the extracellular targeting domain is an antibody that binds to a B cell or T cell antigen. In some embodiments, the extracellular targeting domain is an anti-CD19 antibody (e.g., an antibody that binds to CD19). In some embodiments, the extracellular targeting domain is an antibody that binds to any cell surface molecule. In some embodiments, the extracellular targeting domain is an antibody that binds to a lineage marker (e.g., CD3, CD20, integrin, or other receptor), a phenotypic marker (PD-1, CD25, CD45, or others). In some embodiments, the extracellular targeting domain is a protein or peptide that binds to a receptor (e.g., a receptor present on the surface of the target cell). In some embodiments, the extracellular targeting domain is a protein or peptide that binds to a cytokine receptor (e.g., the interleukin-13 (IL-13) receptor). In some embodiments, the extracellular targeting domain is a cytokine (e.g., IL-2, IL-6, IL-12, IL-13). In some embodiments, the extracellular targeting domain is a chemokine ligand (e.g., CXCL9, CXCL10, CXCL11, etc.). In some embodiments, the extracellular targeting domain is a cell receptor including a cytokine receptor (e.g., IL-13Rα1, IL-13Rα2, IL-2 receptor, common gamma chain), GPCR (including chemokine receptors such as CSCR3, CXCR4, etc.), and integrin. In some embodiments, the extracellular targeting domain is a peptide displayed by an MHC protein. In some embodiments, the non-viral membrane-binding protein includes a membrane-binding domain comprising an MHC protein or fragment, and an extracellular targeting domain comprising a peptide displayed by the MHC protein. In some embodiments, the extracellular domain binds to a T cell receptor and / or a B cell receptor.T cell receptors are normally expressed on the surface of T cells in nature as alpha / beta and gamma / delta heterodimeric integral membrane proteins, and each subunit contains a short intracellular segment, a single transmembrane alpha helix, and two globular extracellular Ig superfamily domains. B cell receptors are transmembrane receptor proteins on the outer surface of B cells.

[0028] In some embodiments, the extracellular targeting domain binds to a target cell or cell surface molecule with a binding affinity of 10 -9 ~10 -8 M, 10 -8 ~10 -7 M, 10 -7 ~10 -6 M, 10 -6 ~10 -5 M, 10 -5 ~10 -4 M, 10 -4 ~10 -3 M, or 10 -3 ~10 -2 M. In some embodiments, the extracellular targeting domain binds to a cognate protein or ligand of the target cell with a binding affinity of 10 -9 ~10 -8 M, 10 -8 ~10 -7 M, 10 -7 ~10 -6 M, 10 -6 ~10 -5 M, 10 -5 ~10 -4 M, 10 -4 ~10 -3 M, or 10 -3 ~10 -2 M. In some embodiments, the binding affinity between the extracellular targeting domain and the cognate protein or ligand is in the picomolar to nanomolar range (e.g., about 10 -12 ~ about 10 -9 M). In some embodiments, the binding affinity between the extracellular targeting domain and the cognate protein or ligand is in the nanomolar to micromolar range (e.g., about 10 -9 ~ about 10 -6is between M. In some embodiments, the binding affinity between the extracellular targeting domain and its cognate protein or ligand ranges from micromolar to millimolar (e.g., about 10 -6 to about 10 -3 M. In some embodiments, the binding affinity between the extracellular targeting domain and its cognate protein or ligand ranges from picomolar to micromolar (e.g., about 10 -12 to about 10 -6 M. In some embodiments, the binding affinity between the extracellular targeting domain and its cognate protein or ligand ranges from nanomolar to millimolar (e.g., about 10 -9 to about 10 -3 M).

[0029] As used herein, the term antibody generally refers to a protein comprising at least one immunoglobulin variable domain or immunoglobulin variable domain sequence. For example, an antibody can comprise a heavy (H) chain variable region (abbreviated herein as V H ), and / or a light (L) chain variable region (abbreviated herein as V L ). In another example, an antibody can comprise two heavy (H) chain variable regions and / or two light (L) chain variable regions. An antibody can have the structural characteristics of IgA, IgG, IgE, IgD, IgM (and its subtypes). The V H and V L regions can be further subdivided into hypervariable regions called "complementary determining regions" ("CDRs"), which are separated by more conserved regions called "framework regions" ("FRs"). Each V H and / or V L typically consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The V H or V LThe lock can further include a heavy or light chain constant region, thereby forming a heavy or light immunoglobulin chain, respectively. In some embodiments, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, where the heavy immunoglobulin chains and the light immunoglobulin chains are interconnected, for example, by disulfide bonds. In IgG, the heavy chain constant region includes three immunoglobulin domains, CH1, CH2, and CH3.

[0030] In some embodiments, the retroviruses present in the retrovirus library include the same extracellular targeting domain as some or all of the other retroviruses in the library. In some embodiments, each retrovirus present in the retrovirus library includes a different extracellular targeting domain compared to some or all of the other retroviruses in the library. In some embodiments, the non-viral membrane-binding protein further includes a signal sequence (also referred to as the signal peptide of the localization sequence). In some embodiments, the signal sequence is at the N-terminus or C-terminus of the non-viral membrane-binding protein. The signal sequence functions to move the non-viral membrane-binding protein to the membrane (or envelope) of the retrovirus. In some embodiments, the signal sequence is 5-10, 5-15, 10-20, 15-20, 15-30, 20-30, or 25-30 amino acids. In some embodiments, the signal sequence is an Ig kappa leader sequence (e.g., a mouse Ig kappa leader sequence including METDTLLLWVLLLWVPGSTG (SEQ ID NO: 1)) or a B2M signal peptide sequence (e.g., a B2M signal peptide sequence including MSRSVALAVLALLSLSGLEA (SEQ ID NO: 2)). In some embodiments, the retroviruses present in the retrovirus library include the same signal sequence as some or all of the other retroviruses in the library. In some embodiments, each retrovirus present in the retrovirus library includes a different signal sequence compared to some or all of the other retroviruses in the library.

[0031] In some embodiments, the nucleic acid encoding the non-viral membrane-binding protein further comprises an internal ribosome entry site (IRES) within the sequence. An IRES is an RNA sequence that enables the initiation of translation during protein synthesis. In some embodiments, the IRES is located at or near the C-terminus. In some embodiments, the IRES is located C-terminal to the membrane-binding domain and the extracellular targeting domain. In some embodiments, the IRES is a viral IRES. In some embodiments, the IRES is an IRES specific to retroviruses. In some embodiments, the IRES is a sequence derived from encephalomyocarditis virus (EMCV). In some embodiments, the retroviruses present in a library of retroviruses contain the same IRES as some or all of the other retroviruses in the library. In some embodiments, each retrovirus present in a library of retroviruses contains a different IRES as compared to some or all of the other retroviruses in the library.

[0032] In some embodiments, the non-viral membrane-binding protein further comprises a linker disposed between the membrane-binding domain and the extracellular targeting domain. The linker is an amino acid linker and can be a rigid linker, a flexible linker, or an oligomerized linker. A rigid linker is an amino acid sequence lacking flexibility (e.g., may contain at least one proline). In some embodiments, the rigid linker comprises a platelet-derived growth factor receptor (PDGFR) stalk or a CD8α stalk. In some embodiments, the PDGFR stalk comprises an amino acid sequence comprising AVGQDTQEVIVVPHSLPFK (SEQ ID NO: 3). In some embodiments, the PDGFR stalk comprises an amino acid sequence comprising ASAKPTTTPAPRPPTPAPTIASQPLSLRPEAARPAAGGAVHTRGLDFAK (SEQ ID NO: 4). A flexible linker is an amino acid sequence having many degrees of freedom (e.g., may contain a plurality of amino acids having small side chains, such as glycine or alanine). In some embodiments, the flexible linker comprises an amino acid sequence comprising GAPGAS (SEQ ID NO: 5). In some embodiments, the flexible linker comprises an amino acid sequence consisting of GAPGSGGGGSGGGGSAS (SEQ ID NO: 6). In some embodiments, the flexible linker comprises an amino acid sequence comprising GGGGS (SEQ ID NO: 7). In some embodiments, the flexible linker is (GAPGAS) N (SEQ ID NO: 29) or (G4S) NIt comprises an amino acid sequence containing (SEQ ID NO: 30), where N is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. The oligomerization linker is an amino acid capable of oligomerizing with another relevant amino acid. In some embodiments, the oligomerization linker is an amino acid sequence capable of forming a dimer, trimer, or tetramer. In some embodiments, the oligomerization linker comprises an IgG4 hinge domain (e.g., ASESKYGPPCPPCPAVGQDTQEVIVVPHSLPFK (SEQ ID NO: 8)). In some embodiments, the oligomerization linker comprises an amino acid sequence capable of forming a tetrameric coiled coil (e.g., ASGGGGSGELAAIKQELAAIKKELAAIKWELAAIKQGAG (SEQ ID NO: 9)). In some embodiments, the oligomerization linker comprises an amino acid sequence capable of forming a dimeric coiled coil (e.g., ASESKYGPPCPPCP (SEQ ID NO: 10)).

[0033] Reporter In some embodiments, the retroviruses described herein may contain a reporter (e.g., a reporter protein). In some embodiments, the retroviruses described herein contain a nucleic acid encoding a reporter (e.g., a reporter protein). As used herein, a reporter is generally a protein or gene that can be detected when expressed in a retrovirus and / or a target cell. In some embodiments, the presence or absence of a reporter in a target cell or a subset of target cells within a cell population enables the ability to sort the cells (e.g., using flow cytometry and / or fluorescence-activated cell sorting). In some embodiments, the reporter is a fluorescent protein. The fluorescent protein can be a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), or a red fluorescent protein (RFP). The fluorescent protein may be one described in U.S. Patent No. 7,060,869 entitled "Fluorescent protein sensors for detection of analytes". In some embodiments, the reporter is an antibiotic resistance marker. In some embodiments, the antibiotic resistance marker is a protein or gene that confers a competitive advantage to the target cells containing the marker. In some embodiments, the antibiotic resistance marker includes a hygromycin resistance protein or gene, a kanamycin resistance protein or gene, an ampicillin resistance protein or gene, a streptomycin resistance protein or gene, or a neomycin resistance protein or gene.

[0034] Cell The cells described herein can be any bacterial, mammalian, or yeast cells. In some embodiments, the cells are human, mouse, rat, or non-human primate cells. In some embodiments, the cells are somatic or germ cells. In some embodiments, the cells are epithelial cells, nerve cells, hormone-secreting cells, immune cells, secretory cells, blood cells, stromal cells, or embryonic cells. In some embodiments, the cells are antigen-specific cells (e.g., cells that bind to a specific antigen). In some embodiments, the antigen-specific cells are immune cells. In some embodiments, the antigen-specific cells are B cells or T cells. In some embodiments, the cells are target cells (e.g., those containing a cognate protein or ligand that can be targeted by the retroviruses described herein). The cell population described in this specification can be any bacterial, mammalian, or yeast cell population. In some embodiments, the cell population is a cell population of a human, mouse, rat, or non-human primate. In some embodiments, the cell population is a somatic cell population or a germ cell population. In some embodiments, the cell population includes epithelial cells, nerve cells, hormone-secreting cells, immune cells, secretory cells, blood cells, stromal cells, and / or embryonic cells. In some embodiments, the cell population includes antigen-specific cells (e.g., cells that bind to a specific antigen). In some embodiments, the antigen-specific cell population includes immune cells. In some embodiments, the antigen-specific cell population includes B cells and / or T cells. In some embodiments, the cell population includes a homogeneous cell population. In some embodiments, the cell population includes a heterogeneous cell population.

[0035] In some embodiments, the cell population is a cell population isolated from a subject. The subject can be a human subject (e.g., a human subject suffering from a disease), a mouse subject, a rat subject, or a non-human primate subject. In some embodiments, the cell population is isolated from the blood or tumor of the subject. In some embodiments, the cell population has been previously frozen and thawed (e.g., 1, 2, 3, 4, 5, or more freeze / thaw cycles). In some embodiments, the cell population is maintained in a liquid culture medium. In some embodiments, the cell population has been passaged 1, 2, 3, 4, 5, or more times using any known method. In some embodiments, the cell population is maintained in a liquid culture medium before being combined with a retrovirus or retroviruses. In some embodiments, the cell population is maintained in a liquid culture medium after being combined with a retrovirus or retroviruses. In some embodiments, the cell population is maintained in a liquid culture medium prior to being combined with a retrovirus or retroviruses.

[0036] In some embodiments, the cell population comprises any of the retroviruses described herein. In some embodiments, a subset of the cell population comprises any of the retroviruses described herein. In some embodiments, a subset of the cell population comprises a retrovirus within each cell of the subset (e.g., within the nucleus of each cell of the subset). In some embodiments, the cell population or a subset thereof expresses a reporter (e.g., a fluorescent protein or an antibiotic resistance marker). In some embodiments, the cell population or a subset thereof (e.g., containing a retrovirus) is isolated and / or sorted based on the presence or absence of the reporter. In some embodiments, a subset of the cell population containing a retrovirus described herein is isolated and / or sorted based on the presence or absence of the reporter away from cells of the population that do not contain the retrovirus. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, or 95% of the cell population prior to cell sorting contains a retrovirus. In some embodiments, at least 70%, 80%, 90%, 95%, or 100% of the cell population contains a retrovirus after isolation and / or sorting based on the presence or absence of the reporter.

[0037] Method of screening Described herein is a method of screening a cell population, comprising: (i) providing a retrovirus comprising a viral envelope protein comprising at least one mutation that reduces its native function, a non-viral membrane-binding protein comprising a membrane-binding domain and an extracellular targeting domain, and a nucleic acid encoding a reporter; (ii) combining the retrovirus with the cell population; and (iii) sorting the cell population based on the presence or absence of the reporter. In some embodiments, the retrovirus of (i) comprises a nucleic acid having the following structure: S-ETD-MBD-IRES-R, where S encodes a signal sequence, ETD encodes an extracellular targeting domain; MBD encodes a membrane-binding domain, IRES encodes an internal ribosome entry site, and R encodes a reporter; and further comprises a mutated viral envelope protein comprising at least one mutation that reduces its native function.

[0038] As used herein, the term "combining" (which is synonymous in some embodiments with the terms "providing" and "contacting") generally refers to the act of bringing the retrovirus into close physical contact with the cell population such that the extracellular targeting domain of the retrovirus can bind to cognate ligands present on a subset of the cells of the population. In some embodiments, combining the retrovirus with the cell population occurs when a solution containing the retrovirus is mixed with a solution containing the cell population. In some embodiments, combining the retrovirus with the cell population occurs when a lyophilized retrovirus is mixed with a solution containing the cell population. In some embodiments, combining the retrovirus with the cell population occurs when a lyophilized retrovirus is mixed with a lyophilized cell population and reconstituted with a solution. In some embodiments, the cells of the population are maintained in a monolayer of cells in cell culture medium and / or are attached to a tissue culture plate or Petri dish.

[0039] Generally, a retrovirus and a cell population are combined (e.g., physically combined or contacted) for a defined period. In some embodiments, the period is measured in seconds, minutes, hours, or days. In some embodiments, the period is 0 to 30 seconds, 15 to 45 seconds, 30 to 60 seconds, 45 to 90 seconds, 60 to 90 seconds, or 60 to 120 seconds. In some embodiments, the retrovirus and the cell population are combined and contacted for 0 to 30 seconds, 15 to 45 seconds, 30 to 60 seconds, 45 to 90 seconds, 60 to 90 seconds, or 60 to 120 seconds. In some embodiments, the period is 1 to 2 minutes, 1 to 5 minutes, 1 to 10 minutes, 2 to 10 minutes, 5 to 10 minutes, 5 to 20 minutes, 10 to 20 minutes, 25 to 30 minutes, 25 to 60 minutes, 30 to 45 minutes, 30 to 40 minutes, 40 to 60 minutes, 50 to 70 minutes, or 60 to 120 minutes. In some embodiments, the retrovirus and the cell population are combined and contacted for 1 to 2 minutes, 1 to 5 minutes, 1 to 10 minutes, 2 to 10 minutes, 5 to 10 minutes, 5 to 20 minutes, 10 to 20 minutes, 25 to 30 minutes, 25 to 60 minutes, 30 to 45 minutes, 30 to 40 minutes, 40 to 60 minutes, 50 to 70 minutes, or 60 to 120 minutes. In some embodiments, the period is 1 to 2 hours, 1 to 5 hours, 1 to 3 hours, 2 to 5 hours, 3 to 6 hours, 3 to 12 hours, 6 to 12 hours, 12 to 18 hours, 12 to 24 hours, 15 to 30 hours, 18 to 24 hours, 24 to 48 hours, 24 to 36 hours, or 36 to 50 hours. In some embodiments, the retrovirus and the cell population are combined and contacted for 1 to 2 hours, 1 to 5 hours, 1 to 3 hours, 2 to 5 hours, 3 to 6 hours, 3 to 12 hours, 6 to 12 hours, 12 to 18 hours, 12 to 24 hours, 15 to 30 hours, 18 to 24 hours, 24 to 48 hours, 24 to 36 hours, or 36 to 50 hours. In some embodiments, the period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 5 to 15 days. In some embodiments, the retrovirus and the cell population are combined and contacted for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 5 to 15 days.

[0040] In some embodiments, the cell population is sorted based on the presence or absence of a reporter. In some embodiments, a subset of the cell population that contains a reporter (e.g., expresses a reporter) is sorted from the remaining subset of the cell population that does not contain the reporter. In some embodiments, sorting of the cell population is performed using flow cytometry (e.g., fluorescence-activated cell sorting), next-generation genomic sequencing (e.g., single-cell next-generation sequencing), or antibiotic selection. In some embodiments, the condition of (ii), which enables the retrovirus to have an intercellular interaction with a subset of the cell population, includes combining the retrovirus and the cell population in a defined solution, composition, and at a specific temperature. In some embodiments, the retrovirus and the cell population are combined in the presence of a cell culture medium (e.g., RPMI or DMEM cell culture medium). In some embodiments, the retrovirus and the cell population are combined in the presence of buffered saline. In some embodiments, the buffered saline is phosphate-buffered saline or HEPES-buffered saline. In some embodiments, the buffered saline contains bovine serum albumin and / or EDTA. In some embodiments, the retrovirus and the cell population are combined in the presence of an enhancer of retroviral transduction (e.g., heparin sulfate, polybrene, protamine sulfate, or dextran). In some embodiments, the retrovirus and the cell population are combined at a temperature in the range of 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 in (ii).

[0041] In some embodiments, the screening method described herein further comprises washing the cell population with a washing solution between steps (ii) and (iii). In some embodiments, the washing solution is any liquid solution that allows for the maintenance of healthy cells (e.g., a solution having a neutral pH and a low to moderate level of ionic strength). In some embodiments, washing the cell population removes excess and / or residual retrovirus from the cell population. In some embodiments, the cell population is washed using a cell culture medium (e.g., RPMI or DMEM cell culture medium). In some embodiments, the cell population is washed using buffered saline. In some embodiments, the buffered saline is phosphate buffered saline or HEPES buffered saline. In some embodiments, the buffered saline contains bovine serum albumin and / or EDTA. In some embodiments, the cell population is washed at a temperature in the range of 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. In some embodiments, the cell population is maintained in liquid culture prior to being combined with the retrovirus. In some embodiments, the cell population is maintained in liquid culture after being combined with the retrovirus. In some embodiments, the cell population is maintained in liquid culture during the step of combining with the retrovirus. In some embodiments, the cell population is attached to a cell culture plate or Petri dish. In some embodiments, the cell population is maintained in a monolayer, embryoid body, or any cell aggregate.

[0042] In some embodiments, the screening method includes the use of multiple retroviruses. In one embodiment, the multiple retroviruses are at least 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 1010 and 10 11 or 10 12 contains a unique retrovirus. In some embodiments, among a plurality of retroviruses, at least 10 2 and 10 3 and 10 4 and 10 5 and 10 6 and 10 7 and 10 8 and 10 9 and 10 10 and 10 11 or 10 12 copies may be present. In some embodiments, the screening method includes screening a cell population using at least two different unique retroviruses. In some embodiments, the different unique retroviruses include different extracellular targeting domains and / or different reporters. In some embodiments, the screening method includes a first retrovirus and a second retrovirus, where the first and second retroviruses include different extracellular targeting domains and / or different reporters. In some embodiments, the screening method includes screening a cell population using 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100 or more different retroviruses. In some embodiments, the screening method includes screening a cell population using a library of retroviruses. In some embodiments, the library of retroviruses includes at least 10 2 at least 10 3 at least 10 4 at least 10 5 at least 10 6 at least 10 7 at least 10 8 at least 10 9 or at least 10 10 unique retroviruses.

[0043] Library of retroviruses Described herein is a library of retroviruses, where the library comprises a plurality of unique retroviruses, where each unique retrovirus comprises a viral envelope protein comprising at least one mutation that reduces its native function, a non-viral membrane-binding protein comprising a membrane-binding domain and an extracellular targeting domain, and a nucleic acid encoding a reporter, where each unique retrovirus comprises a different unique extracellular targeting domain. Also described herein is a library of cells comprising retroviruses, where the library comprises a plurality of unique cells, where each unique cell comprises a unique retrovirus. In some embodiments, the library comprises a pMHC-encoded (peptide / MHC-encoded) retrovirus (e.g., lentivirus) library for use in screening a T cell population. In such a library, the pMHC displayed on the virus surface will enable T cell infection in a TCR-specific manner. The infected T cells can be collected and sequenced, enabling the identification of pMHC ligands capable of infecting a subset of the T cell population of interest and the ability to simultaneously track TCR sequences and reactive pMHC ligands. In some embodiments, the pMHC retrovirus library minimally comprises a randomized transfer vector containing a randomized pMHC targeting element. In some embodiments, the randomly-derived library is generated using degenerate oligonucleotide primers. In some embodiments, a target library specific for a unique set of antigens (e.g., all possible viral or bacterial antigens for a particular target of interest - human immunodeficiency virus, tuberculosis TB, etc.; or all possible neoantigens for a particular subject) is generated.

[0044] In some embodiments, the library can be screened against a population of antigen-specific cells (e.g., B cells or T cells). In some embodiments, the library comprises at least 10 2 at least 10 3 at least 10 4 at least 10 5 at least 10 6 at least 10 7 at least 10 8 at least 10 9 or at least 10 10 unique retroviruses. In some embodiments, a library comprising unique retroviruses comprises an extracellular targeting domain that is at least 5, at least 10, at least 15, at least 20, or at least 50 amino acids in length. In some embodiments, each different unique extracellular targeting domain is generated by site-directed mutagenesis.

[0045] The size of the retrovirus or cell library can vary from hundreds to hundreds of thousands, millions, or more unique retroviruses or unique cells. In some embodiments, the libraries of the present disclosure contain at least 500,000 unique retroviruses or unique cells. The libraries of the invention include retrovirus libraries and cell libraries. A library is a collection of members having a common element and at least one different element that are synthesized (i.e., isolated, synthetically produced, with no components that are found together naturally in a cell, and purified before entering the library). The library contains 1,000 or more (e.g., at least 1,000; 2,000; 3,000; 4,000; 5,000; 10,000; 50,000; 100,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 2,000,000; 3,000,000; 4,000,000; or more) members. The upper limit of library size is defined by combinatorics of domains or modules and provides distinctness or diversity among the members. For example, the upper limit can be 4,000,000 members. Thus in some embodiments, the library is highly diverse and contains at least 500,000 distinct members. A highly diverse library can have a diversity of 10 6 or more. In some embodiments, the retrovirus library is generated using site-directed mutagenesis of the nucleic acids described herein. In some embodiments, site-directed mutagenesis includes the use of primers and low-fidelity RNA polymerase to allow for random mutagenesis of common nucleic acids as described herein.

[0046] Methods of delivering nucleic acids to cells Described herein is a method for delivering a nucleic acid to a cell, the method comprising: (i) providing a retrovirus comprising a nucleic acid, a viral envelope protein comprising at least one mutation that reduces its native function, and a non-viral membrane-binding protein comprising an extracellular targeting domain capable of binding to a cognate ligand of the cell; and (ii) contacting the retrovirus with the cell such that the retrovirus enters or infects the cell. In some embodiments, the nucleic acid encodes an mRNA molecule, where optionally the mRNA is a gene of interest. In some embodiments, the nucleic acid encodes double-stranded RNA, antisense RNA, microRNA, or any other RNA molecule. In some embodiments, the gene of interest encodes a protein. In some embodiments, the gene of interest encodes a therapeutic protein (e.g., a protein for compensating for a medical condition of a subject). In some embodiments, the nucleic acid is delivered to the cell when the retrovirus enters or infects the cell during step (ii). In some embodiments, the method for delivering a nucleic acid described herein does not require a transfection agent (e.g., a lipophilic transfection agent such as lipofectin).

[0047] Detection method Described herein is a method for detecting an interaction between a retrovirus and a cell, the method comprising: (i) contacting a sample comprising the retrovirus and the cell with an antibody, wherein the retrovirus comprises a viral envelope protein comprising at least one mutation that reduces its native function, a non-viral membrane-binding protein comprising an extracellular targeting domain, and wherein the antibody binds to the extracellular targeting domain of the retrovirus; (ii) optionally removing unbound antibody from the sample; and (iii) imaging the sample to detect whether the antibody-retrovirus complex is bound to the cell. In some embodiments, the antibody further comprises at least one fluorescent label. In some embodiments, the fluorescent label is a xanthene derivative (e.g., fluorescein, rhodamine, Oregon Green, eosin, and Texas Red), a cyanine derivative (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine), a naphthalene derivative (e.g., dansyl and prodan derivatives), a coumarin derivative, an oxadiazole derivative (e.g., pyridyloxazole, nitrobenzoxadiazole, and benzoxadiazole), a pyrene derivative (e.g., cascade blue), an oxazine derivative (e.g., Nile Red, Nile Blue, cresyl violet, and oxazine 170), an acridine derivative (e.g., proflavine, acridine orange, and acridine yellow), an arylmethine derivative (e.g., auramine, crystal violet, and malachite green), or a tetrapyrrole derivative (e.g., porphyrin, phthalocyanine, and bilirubin). The fluorescent label may be non-covalently or covalently bound to the antibody. In some embodiments, the sample is imaged in step (iii) using a confocal microscope or a fluorescence microscope. In some embodiments, the detection method can be performed using a standard microscope apparatus (e.g., a confocal microscope or a fluorescence microscope). In some embodiments, the sample is detected in a hyper-multiplexed format while being imaged using a standard confocal microscope or an epi-fluorescence microscope.

[0048] Nucleic acid As used herein, the term "nucleic acid" generally refers to a molecule comprising a plurality of linked nucleotides (i.e., a molecule comprising a sugar (e.g., ribose or deoxyribose) linked to an exchangeable organic base, wherein the exchangeable organic base is either a pyrimidine (e.g., cytosine (C), thymidine (T), or uracil (U)) or a purine (e.g., adenine (A) or guanine (G))). Nucleic acids include DNA and RNA, such as D-form DNA and L-form DNA, as well as various modifications. Modifications include base modifications, sugar modifications, and backbone modifications. It should be understood that the nucleic acids used in the retroviruses and methods of the present invention can be homogeneous or heterogeneous in nature. As an example, they can be entirely DNA in nature, or they can be composed of monomers or sequences of DNA and non-DNA (e.g., LNA). Thus, any combination of nucleic acid elements can be used. Modifications can make the nucleic acid more stable and / or less susceptible to degradation under certain conditions. For example, in some instances, the nucleic acid is nuclease resistant. Methods for synthesizing nucleic acids, including automated nucleic acid synthesis, are also known in the art.

[0049] Nucleic acids can contain modifications to their bases. Modified bases include modified cytosines (e.g., 5-substituted cytosines (e.g., 5-methyl-cytosine, 5-fluoro-cytosine, 5-chloro-cytosine, 5-bromo-cytosine, 5-iodo-cytosine, 5-hydroxy-cytosine, 5-hydroxymethyl-cytosine, 5-difluoromethyl-cytosine, and unsubstituted or substituted 5-alkynyl-cytosine), 6-substituted cytosines, N4-substituted cytosines (e.g., N4-ethyl-cytosine), 5-aza-cytosine, 2-mercapto-cytosine, isocytosine, pseudo-isocytosine, cytosine analogs having a condensed ring system (e.g., N,N'-propylene cytosine or phenoxazine), and uracil and its derivatives (e.g., 5-fluoro-uracil, 5-bromo-uracil, 5-bromovinyl-uracil, 4-thio-uracil, 5-hydroxy-uracil, 5-propynyl-uracil), modified guanines, e.g., 7-deazaguanine, 7-deaza-7-substituted guanines (7-deaza-7(C2C6)alkynyl guanine), 7-deaza-8-substituted guanines, hypoxanthine, N2-substituted guanines (e.g., N2-methyl-guanine), 5-amino-3-methyl-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione, 2,6-diaminopurine, 2-aminopurine, purine, indole, adenine, substituted adenines (e.g., N6-methyl-adenine, 8-oxo-adenine), 8-substituted guanines (e.g., 8-hydroxyguanine and 8-bromoguanine), and 6-thioguanine. Nucleic acids can contain universal bases (e.g., 3-nitropyrrole, P-base, 4-methyl-indole, 5-nitro-indole, and K-base) and / or aromatic ring systems (e.g., fluorobenzene, difluorobenzene, benzimidazole or dichloro-benzimidazole, 1-methyl-1H-[1,2,4]triazole-3-carboxamide). Specific base pairs that can be incorporated into the oligonucleotides of the present invention are the dZ and dP non-standard nucleic acid base pairs reported in Yang et al. NAR, 2006, 34(21):6095-6101.dZ, a pyrimidine analog, is 6-amino-5-nitro-3-(1’-β-D-2’-deoxyribofuranosyl)-2(1H)-pyridone, and its Watson-Crick complement dP, a purine analog, is 2-amino-8-(1’-β-D-1’-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one.

[0050] Amino acid substitution In some embodiments, the modification of the amino acid residue is a conservative amino acid residue substitution. As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequences known to those of skill in the art, for example, according to methods compiled in references such as Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0051] The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. The BLAST protein search can be performed using the XBLAST program, score = 50, word length = 3 to obtain an amino acid sequence homologous to the target protein molecule. When there are gaps between two sequences, gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When using the BLAST and gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used.

Example

[0052] Example 1. Generation of retroviruses targeting antigen-specific T cells The targeted lentivirus was generated by polyethyleneimine (PEI)-transfecting HEK293T cells with the following plasmids: an envelope plasmid encoding a mutated VSV-G envelope protein containing the K47Q and R354A mutations, at least one helper plasmid (pRRE, pRev, or psPAX2.1), and a transfer plasmid (Figure 1). The transfer plasmid used encoded a nucleic acid containing the structure: S-ETD-MBD-IRES-R, where S encoded the B2M signal sequence (provided by SEQ ID NO: 2), ETD encoded a variable extracellular targeting domain (e.g., cancer-testis antigen NYESO-1); MBD encoded a membrane-binding domain (e.g., MHC HLA-A2 domain), IRES encoded an internal ribosome entry site derived from encephalomyocarditis virus (EMCV), and R encoded a green fluorescent protein GFP reporter (Figure 2). The resulting virus was collected and purified by standard centrifugation techniques before being mixed (using pipette mixing) with T cell lines (such as Jurkat T cells) that express a T cell receptor (TCR) specific for a known pMHC in the presence of hexadimethrine bromide. When a virus displaying HLA-A2-NYESO-1 pMHC was mixed with Jurkat T cells expressing the IG4 T cell receptor (TCR) variant (SEQ ID NO: 27) (which efficiently recognizes the displayed NYESO-1 with a binding affinity of approximately 26 pM), efficient infection was observed, and 41.9% of the T cells expressed the GFP reporter after mixing (Figure 3A). This result indicates that 41.9% of the T cells in this cell population were infected with the virus. Conversely, when a virus displaying HLA-A2-NYESO-1 pMHC was mixed with cells that did not express the IG4 TCR, minimal infection was observed, and only 1.1% of the T cells expressed the GFP reporter after mixing (Figure 3B).

[0053] Mixing a virus displaying HLA-A2-NYESO-1 pMHC with T cells expressing different versions of the IG4 TCR (where different versions of the IG4 TCR show a decrease in binding to the NYESO-1 antigen compared to the IG4 TCR variant containing SEQ ID NO: 27) demonstrated that the virus could infect T cells even when the binding affinity between the NYESO-1 antigen and the IG4 TCR was reduced to 32 μM (Figure 4). When measuring GFP expression, a 32 μM binding affinity between the NYESO-1 antigen and the IG4 TCR variant (SEQ ID NO: 24) provided 3.6% transduction; an 84 nM binding affinity between the NYESO-1 antigen and the IG4 TCR variant (SEQ ID NO: 25) provided 17.6% transduction; a 5 nM binding affinity between the NYESO-1 antigen and the IG4 TCR variant (SEQ ID NO: 26) provided 18.1% transduction. 1G4-expressing T cells showed T cell activation when transduced / infected with a virus displaying HLA-A2-NYESO-1, as demonstrated by upregulation of CD69 (Figure 5A). Conversely, a virus displaying HLA-A2-SL9 neither transduced nor activated 1G4-expressing T cells (Figure 5B).

[0054] Example 2. Generation of a retrovirus targeting the IL-13 receptor A lentivirus containing the interleukin-13 (IL-13) extracellular targeting domain was generated as described in Example 1. The IL-13 extracellular targeting domain consists of the full-length IL-13 protein connected to an IgG4 hinge linker protein linker and the PDGFR transmembrane domain (PDGFR transmembrane domain: VVVISAILALVVLTIISLIILIMLWQKKPR (SEQ ID NO: 28)) containing a truncated extracellular tail. When the resulting virus was mixed with Jurkat cells expressing the IL-13Rα1 receptor, efficient transduction was observed, and 85.2% of the cells expressed the GFP reporter after mixing (Figure 7A). Minimal infection was observed when the same virus was mixed with cells that did not express any IL-13 receptor (Figure 7B). Additional IL-13-expressing lentiviruses were generated using alternative protein linkers to connect the IL-13 extracellular targeting domain to the PDGFR transmembrane domain. Equal amounts of all the lentiviruses tested were mixed with Jurkat cells having the IL13Rα1 receptor, and the transduction efficiency was evaluated by a GFP reporter (Figure 12).

[0055] Short and long protein linkers were tested for their ability to enable cell-cell interactions between the extracellular targeting domain (IL-13) and cognate ligand (IL13Rα1 receptor). Lentiviruses containing short linkers were able to infect Jurkat cells slightly, with the PDGFR linker (AVGQDTQEVIVVPHSLPFK (SEQ ID NO: 3)) enabling infection of 6.1% of the cells; the GAPGAS linker (GAPGAS (SEQ ID NO: 5)) enabling infection of 1.0% of the cells; and the CAR ECD linker (ASESKYGPPCPPCP (SEQ ID NO: 10)) enabling infection of 1.6% of the cells. Lentiviruses containing long linkers were very successful in infecting Jurkat cells, with the CD8a stalk linker (ASAKPTTTPAPRPPTPAPTIASQPLSLRPEAARPAAGGAVHTRGLDFAK (SEQ ID NO: 4)) enabling infection of 88.5% of the cells; the IgG4 hinge linker (ASESKYGPPCPPCPAVGQDTQEVIVVPHSLPFK (SEQ ID NO: 8)) enabling infection of 85.2% of the cells; and the oligomerization linker containing an amino acid sequence capable of forming a tetrameric coiled coil (e.g., ASGGGGSGELAAIKQELAAIKKELAAIKWELAAIKQGAG (SEQ ID NO: 9)) enabling infection of 38.5% of the cells.

[0056] Example 3. Generation of Retroviruses Targeting CD19 Lentiviruses pseudotyped with Nipah virus F protein and Nipah virus G protein fused to an anti-CD19 scFv target CD19 +RajiB cells (cells expressing CD19) could be transduced, and 14.5% of the cells expressed the GFP reporter after mixing (Figure 9). Lentiviruses pseudotyped with VSV-mut (mutated VSV-G), anti-CD19 scFv fused to the IgG hinge domain, a truncated PDGFR stalk, and the PDGFR transmembrane domain could transduce CD19 + RajiB cells. When 1 μL of VSV-mut lentivirus (200-fold concentrated) was applied to CD19 + RajiB cells, 10.0% infected cells were obtained (based on GFP reporter expression). 10 μL of VSV-mut provided 10.7% infected cells (Figure 10). The same VSV-mut lentivirus construct did not transduce / infect CD19-Jurkat cells (cells not expressing CD19), indicating that the lentivirus is specific for cells expressing the cognate ligand of its extracellular targeting domain (anti-CD19 scFv). Lentiviruses pseudotyped with VSV-wt (wild-type VSV-G), anti-CD19 scFv fused to the IgG hinge domain, a truncated PDGFR stalk, and the PDGFR transmembrane domain could transduce CD19 + RajiB cells and CD19 - Jurkat cells. This comparison result demonstrates that lentiviruses containing mutated VSV-G (where the mutation reduces the native function of the VSV-G protein) are selective for the transduction / infection of cells expressing the cognate ligand targeted by the extracellular targeting domain of the lentivirus.

[0057] Example 4. Ability of targeted lentivirus to induce T cell signaling The virus was evaluated to determine whether infection via TCR targeting resulted in evidence of TCR signaling. The non-target VSV-wt virus efficiently infected T cells, but did not activate T cells as assessed by CD69 expression levels compared to the control (Figure 13, left panel). However, when Jurkat T cells expressing the high-affinity wild-type 1G4 TCR were targeted with a virus displaying HLA-A2-NYESO-1, upregulation of CD69 was observed in the transduced cells, indicating that TCR signaling occurred during infection (Figure 13 right panel). These results were repeated when the virus displaying HLA-A2-NYESO-1 was mixed with T cells expressing the low-affinity variant 1G4 TCR construct described in Example 1 (Figure 14).

[0058] Example 5. Lentiviral transduction with physiological affinity via TCR The Jurkat T cell population was purified based on expression of IG4 TCR before contacting with the virus displaying HLA-A2-NYESO-1. The virus efficiently transduced purified populations of Jurkat T cells expressing each of the affinity variants of 1G4 TCR based on expression of the GFP reporter (Figure 15). A binding affinity of 32 μM between the NYESO-1 antigen and the IG4 TCR variant provided 51.3% transduction; a binding affinity of 84 nM between the NYESO-1 antigen and the IG4 TCR variant provided 84.8% transduction; a binding affinity of 5 nM between the NYESO-1 antigen and the IG4 TCR variant provided 82.3% transduction; and a binding affinity of 26 nM between the NYESO-1 antigen and the IG4 TCR variant provided 73.2% transduction. Differences between different IG4 TCR affinity variants were further investigated by performing a dose response in which cells were incubated with viruses displaying various amounts (0.5 - 10 μL of virus) of HLA - A2 - NYESO - 1. Lentiviruses displaying NYESO - 1 transduced IG4 - expressing T cells even at high virus amounts (Figure 16A). In contrast, lentiviruses displaying SL - 1 (negative control) did not transduce IG4 - expressing T cells (SL - 1 and the IG4 TCR are not cognate binding partners).

[0059] The ability of constructs that display oligomerized NYESO - 1 by altering the protein structure of the membrane - bound NYESO - 1 - targeting protein to transduce T cells was tested. Lentiviruses expressing and displaying NYESO - 1 were generated using alternative protein linkers to connect the NYESO - 1 extracellular targeting domain to the transmembrane domain (a protein linker containing an IgG4 hinge (dimer); and an oligomerization linker containing an amino acid sequence capable of forming a tetrameric coiled - coil (tetramer)). As demonstrated in Figure 17, lentiviruses expressing these alternative structures were able to transduce T cells expressing 26 pM and 32 μM IG4 TCR variants.

[0060] Example 6. Virus Binding Assay Viruses were pseudotyped with any of the following: (1) wild - type VSV - G (VSV - G); (2) measles virus F protein and receptor - blind H protein fused to an anti - CD19 scFv and FLAG tag (MV - αCD19); (3) Nipah virus F protein and receptor - blind G protein fused to an anti - CD19 scFv and FLAG tag (NiV - αCD19); or (4) Nipah virus F and receptor - blind G proteins with a FLAG tag but no scFv (NiV - dead). These viruses were each directed against CD19 +It was mixed with Raji B cells and then exposed to a fluorescently labeled antibody against the FLAG tag. As demonstrated in FIGS. 18A-18B, the viruses expressing anti-CD19 scFv (MV-αCD19 and NiV-αCD19) bound to Raji B cells. The viruses not expressing anti-CD19 scFv (VSV-G, NiV-dead) did not bind to Raji B cells.

[0061] Example 7. Screening of a library of retroviruses against a T cell population A library of retroviruses (10 7 ~10 10 unique retroviruses) containing a mutated VSV-G envelope protein, a non-viral membrane-binding protein containing an MHC membrane-binding domain, a variable IL-13 extracellular targeting domain, and a GFP reporter. First, a library of nucleic acids encoding a unique non-viral membrane-binding protein containing an MHC membrane-binding domain and a variable IL-13 extracellular targeting domain is prepared using PCR primers that contain degenerate codons at positions known to interact with the IL-13 receptor and that induce random mutagenesis in the IL-13 extracellular targeting domain. The resulting nucleic acid constructs are assembled into viral transfer plasmids using standard molecular cloning techniques with a fluorescent reporter. These plasmids ("transfer plasmids") are transfected into retroviruses together with a helper plasmid and an envelope plasmid encoding a mutated VSV-G envelope protein (or equivalent pseudotype) to generate a library of retroviruses. The viruses are purified and mixed with a cell population expressing the IL-13 receptor of interest (such as a T cell line, HEK293 cell line, etc.). The viruses are incubated with the cell population at an appropriate temperature (e.g., 37°C) for an appropriate time (e.g., 1 - 48 hours). The mixing is carried out using hexadimethrine bromide in standard cell culture medium. After 24 - 48 hours, the cells are sorted based on the expression of the GFP reporter. The highly expressing cells are retained and the sorting process is repeated as necessary. After these rounds of selection by sorting, the retained cells are lysed and the RNA and DNA of the cells are isolated for analysis using next-generation sequencing methods to determine which IL-13 variants were able to mediate viral entry.

[0062] Example 8. Generation of additional retroviruses targeting antigen-specific T cells Additional target lentiviruses were generated using the protocol described in Example 1. As shown in Figure 19, the following: (i) a lentivirus displaying a mutated VSV-G envelope protein and an SL9 peptide, and (ii) 868 TCR-expressing T cells, were mixed, demonstrating that the virus was capable of transducing and infecting T cells even with a small amount of additional virus (about 70% transduction with 1 μL of virus). Conversely, when these viruses were mixed with off-target Jurkat cells, low levels of transduction were achieved (up to 10 μL of virus added, less than 5%). Similar experiments As also shown in Figure 19, the following: (i) a lentivirus containing a mutated VSV-G envelope protein and a pMHC displaying a cytomegalovirus (CMV) NLV peptide, and (ii) C7 TCR-expressing T cells, were mixed, demonstrating that the virus was capable of transducing and infecting T cells (~25% transduction with 10 μL of virus). Conversely, when these viruses were mixed with off-target Jurkat cells, low levels of transduction were achieved (up to 10 μL of virus added, less than 5%).

[0063] Example 9. Generation of Retroviruses Containing Targeted pMHC Stabilized by Disulfide Additional targeted lentiviruses were generated using the protocol described in Example 1. These additional lentiviruses further contained disulfides located within the pMHC to stabilize the structure of the pMHC. As shown in Figure 21, the following: (i) a lentivirus containing a disulfide-stabilized pMHC displaying a mutated VSV-G envelope protein and a targeting peptide, and (ii) target cells expressing the cognate receptor, were mixed, demonstrating that the targeted virus was capable of transducing and infecting the target cells. Conversely, when these targeted viruses were mixed with off-target cells, no or very limited transduction occurred (0.1%). Specifically, lentiviruses containing disulfide-stabilized CMV NLV pMHC transduced on-target C7 TCR-expressing T cells with a transduction efficiency of 21.8%; lentiviruses containing disulfide-stabilized EBV pMHC transduced on-target AS01 cells with a transduction efficiency of 4.8%; lentiviruses containing disulfide-stabilized SL9 pMHC transduced on-target 868 TCR-expressing T cells with a transduction efficiency of 22.1%; and lentiviruses containing disulfide-stabilized NYESO-1 pMHC transduced on-target IG4 TCR-expressing T cells with a transduction efficiency of 19.0%.

[0064] Example 10. Targeted viruses transduce primary cells Primary T cell lines specific for GL9 (presented by HLA-A2) were specifically and efficiently transduced by viruses having a mutated VSV-G envelope protein and displaying GL9 (Figures 22A - 22B). Approximately 50% of the primary T cells were transduced by these viruses targeting GL9. Conversely, these viruses targeting GL9 transduced only 0.3% of the T cells expressing IG4 TCR. T cells expressing IG4 TCR were specifically and efficiently transduced by viruses having a mutated VSV-G envelope protein and displaying NYESO-1 (55.9% transduction). Furthermore, the targeted viruses infect primary cells more efficiently than wild-type VSV-G virus. These results indicate that targeted viruses may be able to infect even unstimulated primary cells. Similarly, primary T cell lines specific for NYESO (presented by HLA-A2) were specifically and efficiently transduced by a virus having a mutated VSV-G envelope protein and displaying NYESO-1 (Figs. 23-24). Conversely, a virus targeting GL9 was unable to efficiently transduce primary T cell lines specific for NYESO. Collectively, these data demonstrate that primary NYESO-1-reactive cells, including expanded primary cells, may be specifically infected by viruses targeting NYESO.

[0065] Example 11. Generation of Retrovirus Containing CD80 Domain A virus pseudotyped with a mutated VSV-G envelope protein and containing the CD80 extracellular domain was generated using the protocol described in Example 1. This virus was able to specifically and efficiently infect Jurkat T cells (25.5% transduction) compared to B cells (0.0% transduction) (Fig. 25). Similarly, a virus pseudotyped with a mutated VSV-G envelope protein and containing NYESO-1 pMHC and the CD80 extracellular domain was generated using the protocol described in Example 1. This virus was able to specifically and efficiently infect Jurkat T cells (13.5% transduction) compared to B cells (0.2% transduction) (Fig. 25). The presence of the CD80 domain enabled transduction of this virus into Jurkat T cells; this is demonstrated by the inability of a virus containing only NYESO-1 pMHC pseudotyped with a mutated VSV-G envelope protein to infect these Jurkat T cells (0.9% transduction). Collectively, these data show that the presence of CD80 on the virus surface mediates specific infection of T cells, demonstrating that CD80 can be used to generally target viruses to T cells.

[0066] Example 12. Mouse Anti-CD3 Antibody Mediates Infection of 58- / - Cells Transduced with TCR By displaying an antibody specific for the mouse TCR constant region (H57 antibody) or mouse CD3 (2C11 antibody) on the surface of the virus, the virus became capable of infecting mouse T cell lines. The virus containing the mutated VSV-G viral envelope protein and anti-TCR antibody was 58α - β - provided transduction of 11.0% of mouse T hybridoma cells; the virus containing the mutated VSV-G viral envelope protein and anti-CD3 antibody was 58α - β - provided transduction of 12.8% of mouse T hybridoma cells.

[0067] Example 13. Generation of virus containing inactivated cocal virus G protein Lentivirus was pseudotyped with cocal virus G protein (Cocal-dead; amino acids containing SEQ ID NO: 53) containing mutations to reduce its infectivity. These mutations at K64Q and R371A of the cocal virus G protein were similar to the mutations used in the VSV-dead variant described in Example 1. The Cocal-dead virus further contained a displayed anti-CD19 scFv antibody. This Cocal-dead virus was able to transduce CD19 + RajiB cells in a manner similar to the lentivirus containing the VSV-dead protein and displaying the anti-CD19 scFv antibody as shown in Figure 27 (8.6% transduction). These data demonstrate that the viral targeting strategy is very robust and that any viral envelope protein that can be mutated to reduce infectivity (e.g., mutated VSV-G, mutated Nipah envelope, mutated measles envelope, mutated cocal virus envelope) can be effectively used.

[0068] Other aspects All features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Thus, unless specifically stated otherwise, each feature disclosed is only an example of a general series of equivalent or similar features. From the above description, those skilled in the art can easily identify the essential characteristics of the present invention and make various changes and modifications to adapt the present invention to various usage methods and conditions without departing from its spirit and scope. Therefore, other aspects are also within the scope of the claims.

[0069] Equivalents Although some aspects of the present invention are described and illustrated herein, those skilled in the art can easily envision various other means and / or structures for performing the functions described herein and / or obtaining the results and / or one or more of the advantages described, and each such variation and / or modification is considered to be within the scope of the aspects of the present invention described herein. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials, and configurations described herein are exemplary, and that actual parameters, dimensions, materials, and / or configurations will depend on the particular application or applications for which the teachings of the present invention are used. Those skilled in the art will recognize, or be able to ascertain using only routine experimentation, many equivalents to the specific aspects of the present invention described herein. Accordingly, the foregoing aspects are presented by way of example only, and it is to be understood that within the scope of the appended claims and their equivalents, aspects of the present invention can be practiced otherwise than as specifically described and claimed. Aspects of the inventions disclosed herein are directed to each of the individual features, systems, articles, materials, kits, and / or methods described herein. Furthermore, where such features, systems, articles, materials, kits, and / or methods do not mutually conflict, any combination of two or more of such features, systems, articles, materials, kits, and / or methods is included within the scope of the inventions disclosed herein.

[0070] All definitions defined and used in this specification should be understood to take precedence over dictionary definitions, definitions in documents incorporated by reference, and / or the ordinary meaning of the defined terms. All references, patents, and patent applications disclosed in this specification are incorporated by reference for the subject matter to which each is cited, and these may, in some cases, include the entire document. The indefinite articles "a" and "an" used in this specification and the claims should be understood to mean "at least one" unless the contrary is explicitly stated. The phrase "and / or" used in this specification and the claims should be understood to mean "either or both" of the elements so joined, i.e., elements that exist conjunctively in some cases and disjunctively in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner; i.e., "one or more" of the elements so joined. Other elements may optionally exist, whether or not they are related to the specifically identified elements, other than those specifically identified by the "and / or" clause. Thus, by way of non-limiting example, a reference to "A and / or B", when used with open-ended language such as "comprising", can, in one aspect, refer to only A (optionally including elements other than B); in another aspect, it can refer to only B (optionally including elements other than A); in yet another aspect, it can refer to both A and B (optionally including other elements), and so on.

[0071] As used in this specification and the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be construed as inclusive; that is, including at least one of a number of elements or a list of elements, but also including more than one, and optionally also including additional unlisted items. Terms that clearly indicate the contrary, such as "only one" or "exactly one", or "consisting of" when used in the claims, refer to including exactly one element of a number of elements or a list of elements. In general, the term "or" as used in this specification should be construed as an exclusive alternative when preceded by an exclusive term such as "either", "one of", "only one", or "exactly one" (i.e., "one or the other but not both"). "Consisting essentially of" shall have the ordinary meaning as used in the field of patent law when used in the claims.

[0072] As used in this specification and the claims, the phrase "at least one" in connection with a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, provided that it is not necessary to include at least one of each of the specifically recited elements in the list of elements, and that it does not exclude any combination of elements in the list of elements. This definition also allows for the possibility that elements other than those specifically identified in the list of elements referred to by the phrase "at least one" may optionally be present, whether or not they are related to the specifically identified elements. Thus, by way of non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently "at least one of A and / or B") can refer to the following: In one aspect, at least one, optionally two or more of A, and no B (and optionally including elements other than B); in another aspect, at least one, optionally two or more of B, and no A (and optionally including elements other than A); in yet another aspect, at least one, optionally two or more of A, and at least one, optionally two or more of B (and optionally including other elements), and so on. It should also be understood that, unless explicitly stated to the contrary, in a method described herein that includes two or more steps or acts, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are described.

[0073] Array >Kappa leader sequence, amino acid (SEQ ID NO: 1): METDTLLLWVLLLWVPGSTG >B2M signal peptide sequence, amino acid (SEQ ID NO: 2): MSRSVALAVLALLSLSGLEA >PDGFR short stalk, amino acid (SEQ ID NO: 3): MSRSVALAVLALLSLSGLEA >PDGFR long stalk, amino acid (SEQ ID NO: 4): ASAKPTTTPAPRPPTPAPTIASQPLSLRPEAARPAAGGAVHTRGLDFAK >Short flexible linker, amino acids (SEQ ID NO: 5): GAPGAS >Long flexible linker, amino acids (SEQ ID NO: 6): GAPGSGGGGSGGGGSAS >Short flexible linker, amino acids (SEQ ID NO: 7): GGGGS >IgG4 hinge domain, amino acids (SEQ ID NO: 8): ASESKYGPPCPPCPAVGQDTQEVIVVPHSLPFK >Tetrameric coiled coil, amino acids (SEQ ID NO: 9): ASGGGGSGELAAIKQELAAIKKELAAIKWELAAIKQGAG >Dimeric coiled coil, amino acids (SEQ ID NO: 10): ASESKYGPPCPPCP >Wild-type VSV-G envelope protein (with leader sequence), DNA sequence (SEQ ID NO: 11):

[0074] >Wild-type VSV-G envelope protein (with leader sequence), amino acid sequence (SEQ ID NO: 12): MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTAIQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITQSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0075] >Wild-type VSV-G envelope protein, amino acid sequence (SEQ ID NO: 13): KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTAIQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITQSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0076] >VSV-G envelope protein (with leader sequence), DNA sequence (SEQ ID NO: 14):

[0077] >VSV-G envelope protein (with leader sequence), amino acid sequence (SEQ ID NO: 15): MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPQSHKAIQADGWMCHASKWVTTCDFRWYGPKYITQSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTEAELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0078] >I41L / K47Q / R354A VSV-G envelope protein, amino acid sequence (SEQ ID NO: 16): KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPQSHKAIQADGWMCHASKWVTTCDFRWYGPKYITQSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTEAELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0079] >K47Q / R354A VSV-G envelope protein, amino acid sequence (SEQ ID NO: 17): KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTAIQVKMPQSHKAIQADGWMCHASKWVTTCDFRWYGPKYITQSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTEAELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0080] >Exemplary wild-type measles envelope protein (with leader sequence), DNA sequence (SEQ ID NO: 18):

[0081] >Exemplary wild-type measles envelope protein, amino acid sequence (SEQ ID NO: 19): MGSRIVINREHLMIDRPYVLLAVLFVMFLSLIGLLAIAGIRLHRAAIYTAEIHKSLSTNLDVTNSIEHQVKDVLTPLFKIIGDEVGLRTPQRFTDLVKFISDKIKFLNPDREYDFRDLTWCINPPERIKLDYDQYCADVAAEELMNALVNSTLLETRTTNQFLAVSKGNCSGPTTIRGQFSNMSLSLLDLYLGRGYNVSSIVTMTSQGMYGGTYLVEKPNLSSKRSELSQLSMYRVFEVGVIRNPGLGAPVFHMTNYLEQPVSNDLSNCMVALGELKLAALCHGEDSITIPYQGSGKGVSFQLVKLGVWKSPTDMQSWVPLSTDDPVIDRLYLSSHRGVIADNQAKWAVPTTRTDDKLRMETCFQQACKGKIQALCENPEWAPLKDNRIPSYGVLSVDLSLTVELKIKIASGFGPLITHGSGMDLYKSNHNNVYWLTIPPMKNLALGVINTLEWIPRFKVSPYLFTVPIKEAGGDCHAPTYLPAEVDGDVKLSSNLVILPGQDLQYVLATYDTSRVEHAVVYYVYSPSRSFSYFYPFRLPIKGVPIELQVECFTWDQKLWCRHFCVLADSESGGHITHSGMVGMGVSCTVTREDGTNDYKDDDDK

[0082] >Exemplary mutant measles envelope protein, DNA sequence (SEQ ID NO: 20):

[0083] >Exemplary mutant measles envelope protein, amino acid sequence (SEQ ID NO: 21): MGSRIVINREHLMIDRPYVLLAVLFVMFLSLIGLLAIAGIRLHRAAIYTAEIHKSLSTNLDVTNSIEHQVKDVLTPLFKIIGDEVGLRTPQRFTDLVKFISDKIKFLNPDREYDFRDLTWCINPPERIKLDYDQYCADVAAEELMNALVNSTLLETRTTNQFLAVSKGNCSGPTTIRGQFSNMSLSLLDLYLGRGYNVSSIVTMTSQGMYGGTYLVEKPNLSSKRSELSQLSMYRVFEVGVIRNPGLGAPVFHMTNYLEQPVSNDLSNCMVALGELKLAALCHGEDSITIPYQGSGKGVSFQLVKLGVWKSPTDMQSWVPLSTDDPVIDRLYLSSHRGVIADNQAKWAVPTTRTDDKLRMETCFQQACKGKIQALCENPEWAPLKDNRIPSYGVLSVDLSLTVELKIKIASGFGPLITHGSGMDLYKSNHNNVYWLTIPPMKNLALGVINTLEWIPRFKVSPALFNVPIKEAGGDCHAPTYLPAEVDGDVKLSSNLVILPGQDLQYVLATYDTSAVEHAVVYYVYSPSRSFSYFYPFRLPIKGVPIELQVECFTWDQKLWCRHFCVLADSESGGHITHSGMVGMGVSCTVTREDGTNDYKDDDDK

[0084] >Exemplary mutant Nipah envelope protein, DNA sequence (SEQ ID NO: 22):

[0085] >Exemplary mutant Nipah envelope protein, amino acid sequence (SEQ ID NO: 23): MKKINEGLLDSKILSAFNTVIALLGSIVIIVMNIMIIQNYTRSTDNQAVIKDALQGIQQQIKGLADKIGTEIGPKVSLIDTSSTITIPANIGLLGSKISQSTASINENVNEKCKFTLPPLKIHECNISCPNPLPFREYRPQTEGVSNLVGLPNNICLQKTSNQILKPKLISYTLPVVGQSGTCITDPLLAMDEGYFAYSHLERIGSCSRGVSKQRIIGVGEVLDRGDEVPSLFMTNVWTPPNPNTVYHCSAVYNNEFYYVLCAVSTVGDPILNSTYWSGSLMMTRLAVKPKSNGGGYNQHQLALRSIEKGRYDKVMPYGPSGIKQGDTLYFPAVGFLVRTEFKYNDSNCPITKCQYSKPENCRLSMGIRPNSHYILRSGLLKYNLSDGENPKVVFIEISDQRLSIGSPSKIYDSLGQPVFYQASFSWDTMIKFGDVLTVNPLVVNWRNNTVISRPGQSQCPRFNTCPAICAEGVYNDAFLIDRINWISAGVFLDSNATAANPVFTVFKDNEILYRAQLASEDTNAQKTITNCFLLKNKIWCISLVEIYDTGDNVIRPKLFAVKIPEQCTGGGGSGGGGSGGGGSASDYKDDDDK

[0086] >IG4 TCR (variant that binds to the NYESO-1 antigen with a binding affinity of 32 μM), amino acid sequence (SEQ ID NO: 24): MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSATNFSLLKQAGDVEENPGPMETLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGSSTLYIAASQPGDSATYLCAVRPTSGGSYIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSAAA

[0087] >IG4 TCR (variant that binds to NYESO-1 antigen with a binding affinity of 84 nM), amino acid sequence (SEQ ID NO: 25): MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAQTTDQGEVPNGYNVSRSTIEDFPLRLLSAAPSQTSVYFCASSYLGNTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSATNFSLLKQAGDVEENPGPMETLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGSSTLYIAASQPGDSATYLCAVRPMIGGTYIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSAAA

[0088] >IG4 TCR (variant that binds to the NYESO-1 antigen with a binding affinity of 5 nM), amino acid sequence (SEQ ID NO: 26): MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGTTDRGEVPNGYNVSRSTIEDFPLRLLSAAPSQTSVYFCASSYVGDTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSATNFSLLKQAGDVEENPGPMETLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGSSTLYIAASQPGDSATYLCAVRPLLDGTYIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSAAA

[0089] >IG4 TCR (variant that binds to NYESO-1 antigen with a binding affinity of 26 pM), Amino acid sequence (SEQ ID NO: 27): MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVAIQTTDQGEVPNGYNVSRSTIEDFPLRLLSAAPSQTSVYFCASSYLGNTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSATNFSLLKQAGDVEENPGPMETLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLITPWQREQTSGRLNASLDKSSGSSTLYIAASQPGDSATYLCAVRPLLDGTYIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSAAA

[0090] >868 TCR (beta chain - P2A - alpha chain), Amino acid sequence (SEQ ID NO: 31): MSIGLLCCAALSLLWAGPVNADAGVTQSPTHLIKTRGQQVTLRCSPKQGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSDTVSYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSATNFSLLKQAGDVEENPGPMETLLGLLILWLQLQWVSSKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYISLLIRDSKLSDSATYLCAVRTNSGYALNFGKGTSLLVTPHIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0091] >CMV C7 TCR, Amino acid sequence (SEQ ID NO: 33): MGTRLLFWVAFCLLGADHTGAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQRLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGESVSTLTIQRTQQEDSAVYLCASSQTQLWETQYFGPGTRLLVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSATNFSLLKQAGDVEENPGPMEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCAFITGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSAAA

[0092] >SL9 pMHC (The SL9 peptide part is in bold), Amino acid sequence (SEQ ID NO: 35):

Table 1

[0093] >CMV pMHC (The CMV NLV peptide part is in bold), Amino acid sequence (SEQ ID NO: 37):

Table 2

[0094] >Disulfide trap MHC containing a Y84C mutation (bold) and having a C (bold) at the 2nd position of the linker; shown with the HGH signal peptide (underlined) and the GL9 peptide (italic), amino acid sequence (SEQ ID NO: 39):

Table 3

[0095] >GL9 pMHC (the GL9 peptide part is in bold), amino acid sequence (SEQ ID NO: 41):

Table 4

[0096] >CD80, amino acid sequence (SEQ ID NO: 43): MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRE

[0097] >CD86, amino acid sequence (SEQ ID NO: 45): MDPQCTMGLSNILFVMAFLLSGAAPLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGVMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIPWITAVLPTVIICVMVFCLILWKWKKKKR

[0098] > Mouse anti-TCR beta clone H57-597 Fab antibody (light chain - P2A - heavy chain, PDGFR transmembrane domain), Amino acid sequence: (SEQ ID NO: 47): METDTLLLWVLLLWVPGSTGADYKDDDDKDIQMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADGVPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLEIKRADAKPTVSIFPPSSEQLGTGSATLVCFVNNFYPKDINVKWKVDGSEKRDGVLQSVTDQDSKDSTYSLSSTLSLTKADYERHNLYTCEVTHKTSTAAIVKTLNRNECGSGATNFSLLKQAGDVEENPGPMVPCTLLLLLAAALAPTQTRAEVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVAYITSSSINIKYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMVTVSSAKTTAPSVYPLAPACDSTTSTTNTVTLGCLVKGYFPEPVTVIWNSGALTSGVHTFPSVLHSGLYSLSSSVTVPSSTWPSQTVTCNVAHPASSTTVDLKIEAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR

[0099] >Mouse anti-CD3 epsilon clone 145-2C11 Fab antibody (light chain-P2A-heavy chain, PDGFR transmembrane domain), amino acid sequence (SEQ ID NO: 49): METDTLLLWVLLLWVPGSTGADYKDDDDKYELIQPSSASVTVGETVKITCSGDQLPKNFAYWFQQKSDKNILLLIYMDNKRPSGIPERFSGSTSGTTATLTISGAQPEDEAAYYCLSSYGDNNDLVFGSGTQLTVLRGPKSSPKVTVFPPSPEELRTNKATLVCLVNDFYPGSATVTWKANGATINDGVKTTKPSKQGQNYMTSSYLSLTADQWKSHNRVSCQVTHEGETVEKSLSPAECLGSGATNFSLLKQAGDVEENPGPMVPCTLLLLLAAALAPTQTRAEVYLVESGGDLVQPGSSLKVSCAASGFTFSDFWMYWVRQAPGKGLEWVGRIKNIPNNYATEYADSVRGRFTISRDDSRNSIYLQMNRLRVDDTAIYYCTRAGRFDHFDYWGQGTMVTVSSATTTAPSVYPLAPACDSTTSTTDTVTLGCLVKGYFPEPVTVSWNSGALTSGVHTFPSVLHSGLYSLSSSVTVPSSTWPKQPITCNVAHPASSTKVDKKIEPRAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR*

[0100] >Coccus virus glycoprotein, amino acid sequence: (SEQ ID NO: 51): MNFLLLTFIVLPLCSHAKFSIVFPQSQKGNWKNVPSSYHYCPSSSDQNWHNDLLGITMKVKMPKTHKAIQADGWMCHAAKWITTCDFRWYGPKYITHSIHSIQPTSEQCKESIKQTKQGTWMSPGFPPQNCGYATVTDSVAVVVQATPHHVLVDEYTGEWIDSQFPNGKCETEECETVHNSTVWYSDYKVTGLCDATLVDTEITFFSEDGKKESIGKPNTGYRSNYFAYEKGDKVCKMNYCKHAGVRLPSGVWFEFVDQDVYAAAKLPECPVGATISAPTQTSVDVSLILDVERILDYSLCQETWSKIRSKQPVSPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRIDIDNPIISKMVGKISGSQTERELWTEWFPYEGVEIGPNGILKTPTGYKFPLFMIGHGMLDSDLHKTSQAEVFEHPHLAEAPKQLPEEETLFFGDTGISKNPVELIEGWFSSWKSTVVTFFFAIGVFILLYVVARIVIAVRYRYQGSNNKRIYNDIEMSRFRK*

[0101] >Coccus virus glycoprotein, DNA sequence: (SEQ ID NO: 52):

[0102] >Cocal-dead (mutations for removing native orientation are shown in bold in the protein sequence; these are K64Q and R371A counted from the start codon), amino acid sequence: (SEQ ID NO: 53):

Table 5

[0103] >Cocal-dead (mutation for removing native orientation), DNA sequence: (SEQ ID NO: 54):

Claims

**Claim 1** A method for screening a cell population, comprising: (i) providing a retrovirus comprising a viral envelope protein comprising at least one mutation that reduces its native function, a non-viral membrane-binding protein comprising a membrane-binding domain and an extracellular targeting domain, and a nucleic acid encoding a reporter; (ii) combining the retrovirus with the cell population; and (iii) selecting the cell population based on the presence or absence of the reporter. **Claim 2** A method for screening a cell population, comprising: (i) a retrovirus comprising: a nucleic acid comprising a non-viral membrane-binding protein having the structure S-ETD-MBD-IRES-R, where S encodes a signal sequence, ETD encodes an extracellular targeting domain; MBD encodes a membrane-binding domain, IRES encodes an internal ribosome entry site, and R encodes a reporter; and a mutated viral envelope protein comprising at least one mutation that reduces its native function; providing the retrovirus; (ii) combining the retrovirus with the cell population; and (iii) selecting the cell population based on the presence or absence of the reporter. **Claim 3** The method according to any one of claims 1 or 2, wherein the cell is a somatic cell, optionally an antigen-specific cell. **Claim 4** The method according to claim 3, wherein the antigen-specific cell is a T cell or a B cell. **Claim 5** The method according to any one of claims 1 to 3, wherein the retrovirus is a lentivirus. **Claim 6** The method according to any one of claims 1 to 5, wherein the viral envelope protein is a VSV-G envelope protein, a measles virus envelope protein, a Nipah virus envelope protein, or a mycoplasma virus G protein. **Claim 7** The method according to claim 6, wherein at least one mutation of the VSV-G envelope protein is a mutation selected from the group consisting of H8, K47, Y209, and R354. **Claim 8** The method according to claim 6, wherein at least one mutation of the measles virus envelope protein is a mutation selected from the group consisting of Y481, R533, S548, and F549. **Claim 9** The method according to claim 6, wherein at least one mutation of the Nipah virus envelope protein is a mutation selected from the group consisting of E501, W504, Q530, and E533.

10. The method according to claim 6, wherein at least one mutation of the coccus virus G protein is a mutation selected from the group consisting of K64 and R371.

11. The method according to any one of claims 1 to 10, wherein the non-viral membrane-binding protein comprises a major histocompatibility complex (MHC) protein.

12. The method according to any one of claims 1 to 11, wherein the extracellular targeting domain is a protein, a peptide, or an antibody.

13. The method according to claim 12, wherein the protein is an interleukin-13 or a CD80 protein domain.

14. The method according to claim 12, wherein the antibody is an anti-CD19 antibody, an anti-TCR antibody, or an anti-CD3 antibody.

15. The method according to any one of claims 1 to 14, wherein the reporter is a fluorescent protein (e.g., green fluorescent protein, yellow fluorescent protein, red fluorescent protein) or an antibiotic resistance marker.

16. The method according to any one of claims 1 to 15, wherein the linker is disposed between the membrane-binding domain and the extracellular targeting domain.

17. The method according to claim 16, wherein the linker is a rigid linker and optionally comprises a PDGFR stalk or a CD8α stalk.

18. The method according to claim 16, wherein the linker is a flexible linker and optionally comprises an amino acid sequence containing GAPGAS (SEQ ID NO: 5) or GGGGS (SEQ ID NO: 7).

19. The method according to claim 16, wherein the linker is an oligomerizing linker and optionally comprises an IgG4 hinge or an amino acid sequence capable of forming a tetrameric coiled coil.

20. The method according to any one of claims 1 to 19, wherein the retrovirus is combined with the cell population for 1 minute to 72 hours in (ii).

21. The method according to any one of claims 1 to 20, wherein the retrovirus and the cell population are combined in (ii) in the presence of: a) a cell culture medium, optionally an RPMI or DMEM cell culture medium; b) a buffered saline, optionally phosphate-buffered saline or HEPES-buffered saline; and / or c) Enhancers for retroviral transduction, optionally heparin sulfate, polybrene, protamine sulfate, and / or dextran.

22. The method according to any one of claims 1 to 21, wherein the retrovirus and the cell population are combined at a temperature in the range of 4°C to 42°C in (ii).

23. The method according to any one of claims 1 to 22, wherein the extracellular targeting domain can bind to a cognate protein present on the cell surface of a subset of the cell population.

24. The method according to claim 23, wherein the cognate protein is a protein receptor.

25. The method according to any one of claims 23 or 24, wherein the extracellular targeting domain of a single retrovirus binds to a cognate protein present on the cell surface of a single T cell during (ii), resulting in the entry of the retrovirus into the T cell.

26. The method according to any one of claims 1 to 25, further comprising washing the cell population between (ii) and (iii), optionally using phosphate-buffered saline (PBS), and optionally removing the retrovirus from the cell population.

27. The method according to any one of claims 1 to 26, wherein the sorting of the cell population is carried out using fluorescence-activated cell sorting, single-cell next-generation sequencing, or antibiotic selection.

28. The method according to any one of claims 1 to 27, wherein the cell population is a cell population isolated from a subject, optionally wherein the subject is human.

29. The method according to claim 28, wherein the cell population is isolated from the blood or tumor of a subject.

30. The method according to any one of claims 1 to 29, wherein the cell population is maintained in liquid culture before being combined with the retrovirus.

31. The method according to any one of claims 1 to 30, further comprising a second retrovirus, wherein the second retrovirus comprises a different extracellular targeting domain and / or a different reporter as compared to the first retrovirus.

32. The method according to any one of claims 2 to 31, wherein: a) the signal sequence comprises SEQ ID NO: 1 (METDTLLLLWVLLLWVP GSTG) or SEQ ID NO: 2 (MSRSVALAVLALLSLSGL EA); and / or b) the IRES encodes a sequence derived from encephalomyocarditis virus (EMCV).

33. Retroviruses comprising: (i) a nucleic acid comprising a non-viral membrane-binding protein having the structure: S-ETD-MBD-IRES-R, where S encodes a signal sequence, ETD encodes an extracellular targeting domain; MBD encodes a membrane-binding domain, IRES encodes an internal ribosome entry site, and R encodes a reporter; and (ii) a mutated viral envelope protein comprising at least one mutation that reduces its native function.

34. The retrovirus according to claim 33, wherein the retrovirus is a lentivirus.

35. The retrovirus according to any one of claims 33 or 34, wherein the viral envelope protein is a VSV-G envelope protein, a measles virus envelope protein, a Nipah virus envelope protein, or a coccus virus G protein.

36. The retrovirus according to claim 35, wherein at least one mutation of the VSV-G envelope protein is a mutation selected from the group consisting of H8, K47, Y209, and R354.

37. The retrovirus according to claim 36, wherein at least one mutation of the measles virus envelope protein is a mutation selected from the group consisting of Y481, R533, S548, and F549.

38. The retrovirus according to claim 36, wherein at least one mutation of the Nipah virus envelope protein is a mutation selected from the group consisting of E501, W504, Q530, and E533.

39. The retrovirus according to claim 36, wherein at least one mutation of the coccus virus G protein is a mutation selected from the group consisting of K64 and R371.

40. The retrovirus according to any one of claims 33 to 39, wherein the non-viral membrane-binding protein comprises a major histocompatibility complex (MHC) protein.

41. The retrovirus according to any one of claims 33 to 40, wherein the extracellular targeting domain is a protein, a peptide, or an antibody.

42. The retrovirus according to claim 41, wherein the protein is an interleukin-13 or a CD80 protein domain.

43. The retrovirus according to claim 41, wherein the antibody is an anti-CD19 antibody, an anti-TCR antibody, or an anti-CD3 antibody.

44. The retrovirus according to any one of claims 33 to 43, wherein the reporter is a fluorescent protein (e.g., green fluorescent protein, yellow fluorescent protein, red fluorescent protein) or an antibiotic resistance marker.

45. The retrovirus according to any one of claims 33 to 44, wherein the linker is disposed between the membrane-binding domain and the extracellular targeting domain.

46. The retrovirus according to claim 45, wherein the linker is a rigid linker and optionally comprises a PDGFR stalk or a CD8α stalk.

47. The retrovirus according to claim 45, wherein the linker is a flexible linker and optionally comprises an amino acid sequence containing GAPGAS (SEQ ID NO: 5) or GGGGS (SEQ ID NO: 7).

48. The retrovirus according to claim 45, wherein the linker is an oligomerizing linker and optionally comprises an IgG4 hinge or an amino acid sequence capable of forming a tetrameric coiled coil.

49. The retrovirus according to any one of claims 33 to 48, wherein the extracellular targeting domain can bind to a cognate protein present on the cell surface of the cell, and optionally wherein the cell is an antigen-specific cell (e.g., a T cell or a B cell).

50. The retrovirus according to any one of claims 33 to 49, wherein the signal sequence comprises SEQ ID NO: 1 (METDTLLLLWVLLLWVP GSTG) or SEQ ID NO: 2 (MSRSVALAVLALLSLSGL EA).

51. The retrovirus according to any one of claims 33 to 50, wherein the IRES encodes a sequence derived from encephalomyocarditis virus (EMCV).

52. A method for delivering a nucleic acid to a cell, the method comprising: (i) providing a retrovirus comprising a nucleic acid, a viral envelope protein comprising at least one mutation that reduces its native function, and a non-viral membrane-binding protein comprising an extracellular targeting domain capable of binding to a cognate ligand of the cell; and (ii) contacting the retrovirus with the cell, thereby delivering the nucleic acid to the cell.

53. The method according to claim 51, wherein the nucleic acid encodes a gene of interest, and optionally wherein the gene of interest encodes a protein.

54. The method according to any one of claims 52 or 53, wherein the cognate ligand is a protein, optionally a protein receptor.

55. The method according to any one of claims 52 to 54, wherein the retrovirus penetrates or infects the cell during (ii).

56. The method according to any one of claims 52 to 55, wherein the cell is a somatic cell, optionally an antigen-specific cell.

57. The method according to claim 56, wherein the antigen-specific cell is a T cell or a B cell.

58. The method according to any one of claims 52 to 57, wherein the retrovirus is a lentivirus.

59. The method according to any one of claims 52 to 58, wherein the viral envelope protein is a VSV-G envelope protein, a measles virus envelope protein, a Nipah virus envelope protein, or a mycoplasma virus G protein.

60. The method according to claim 59, wherein at least one mutation of the VSV-G envelope protein is a mutation selected from the group consisting of H8, K47, Y209, and R354.

61. The method according to claim 59, wherein at least one mutation of the measles virus envelope protein is a mutation selected from the group consisting of Y481, R533, S548, and F549.

62. The method according to claim 59, wherein at least one mutation of the Nipah virus envelope protein is a mutation selected from the group consisting of E501, W504, Q530, and E533.

63. The method according to claim 59, wherein at least one mutation of the mycoplasma virus G protein is a mutation selected from the group consisting of K64 and R371.

64. The method according to any one of claims 52 to 63, wherein the non-viral membrane-binding protein comprises a major histocompatibility complex (MHC) protein.

65. The method according to any one of claims 52 to 64, wherein the extracellular targeting domain is a protein, a peptide, or an antibody.

66. The method according to claim 65, wherein the protein is an interleukin-13 or a CD80 protein domain.

67. The method according to claim 65, wherein the antibody is an anti-CD19 antibody, an anti-TCR antibody, or an anti-CD3 antibody.

68. The method according to any one of claims 52 to 67, wherein the linker is disposed between the membrane-binding domain and the extracellular targeting domain.

69. The method according to claim 68, wherein the linker is a rigid linker and optionally comprises a PDGFR stalk or a CD8α stalk.

70. The method according to claim 68, wherein the linker is a flexible linker and optionally comprises an amino acid sequence comprising GAPGAS (SEQ ID NO: 5) or GGGGS (SEQ ID NO: 7).

71. The method according to claim 68, wherein the linker is an oligomerization linker and optionally comprises an IgG4 hinge or an amino acid sequence capable of forming a tetrameric coiled coil.

72. The method according to any one of claims 52 to 71, wherein the extracellular targeting domain is capable of binding to a cognate protein present on the cell surface of a T cell.

73. A method for detecting an interaction between a retrovirus and a cell, comprising: (i) contacting a sample containing a retrovirus and a cell with an antibody, wherein the retrovirus comprises a viral envelope protein containing at least one mutation that reduces its native function and a non-viral membrane-binding protein containing an extracellular targeting domain, and wherein the antibody binds to the extracellular targeting domain of the retrovirus); (ii) optionally removing unbound antibody from the sample; and (iii) imaging the sample to detect whether the antibody-retrovirus complex is bound to the cell.

74. The method according to claim 73, wherein the antibody further comprises a fluorescent label, and optionally wherein the antibody is covalently bound to the fluorescent label.

75. The method according to any one of claims 73 or 74, wherein the sample is imaged in (iii) using a confocal microscope or a fluorescence microscope.

76. The method according to any one of claims 73 to 75, wherein the cell is a somatic cell, optionally an antigen-specific cell.

77. The method according to claim 76, wherein the antigen-specific cell is a T cell or a B cell.

78. The method according to any one of claims 73 to 77, wherein the retrovirus is a lentivirus.

79. The method according to any one of claims 73 to 78, wherein the viral envelope protein is a VSV-G envelope protein, a measles virus envelope protein, a Nipah virus envelope protein, or a streptococcal virus G protein.

80. The method according to claim 79, wherein at least one mutation of the VSV-G envelope protein is a mutation selected from the group consisting of H8, K47, Y209, and R354.

81. The method according to claim 79, wherein at least one mutation of the measles virus envelope protein is a mutation selected from the group consisting of Y481, R533, S548, and F549.

82. The method according to claim 79, wherein at least one mutation of the Nipah virus envelope protein is a mutation selected from the group consisting of E501, W504, Q530, and E533.

83. The method according to claim 79, wherein at least one mutation of the coccus virus G protein is a mutation selected from the group consisting of K64 and R371.

84. The method according to any one of claims 73 to 83, wherein the non-viral membrane-binding protein comprises a major histocompatibility complex (MHC) protein.

85. The method according to any one of claims 73 to 84, wherein the extracellular targeting domain is a protein or a peptide.

86. The method according to claim 85, wherein the protein is an interleukin-13 or a CD80 protein domain.

87. The method according to any one of claims 73 to 86, wherein the linker is disposed between the membrane-binding domain and the extracellular targeting domain.

88. The method according to claim 87, wherein the linker is a rigid linker and optionally comprises a PDGFR stalk or a CD8α stalk.

89. The method according to claim 87, wherein the linker is a flexible linker and optionally comprises an amino acid sequence comprising GAPGAS (SEQ ID NO: 5) or GGGGS (SEQ ID NO: 7).

90. The method according to claim 87, wherein the linker is an oligomerization linker and optionally comprises an IgG4 hinge or an amino acid sequence capable of forming a tetrameric coiled coil.

91. A library of retroviruses comprising a plurality of unique retroviruses, wherein each unique retrovirus comprises a viral envelope protein comprising at least one mutation that reduces its native function, a non-viral membrane-binding protein comprising a membrane-binding domain and an extracellular targeting domain, and a nucleic acid encoding a reporter, and Here, each unique retrovirus is said library, which contains different unique extracellular targeting domains.

92. The library according to claim 91, wherein the library can be screened against a population of antigen-specific cells, and optionally here, the antigen-specific cells are B cells or T cells.

93. The library has at least 10 ２ , at least 10 ３ , at least 10 ４ , at least 10 ５ , at least 10 ６ , at least 10 ７ , at least 10 ８ , at least 10 ９ , or at least 10 １０ unique retroviruses, the library according to claim 91 or 92.

94. The library according to any one of claims 91 to 93, wherein each extracellular targeting domain contains at least 5, at least 10, at least 15, at least 20, or at least 50 amino acids.

95. The library according to any one of claims 91 to 94, wherein the non-viral membrane-binding protein contains a major histocompatibility complex (MHC) protein.

96. The library according to any one of claims 91 to 95, wherein each different unique extracellular targeting domain is generated by site-directed mutagenesis.

97. A cell population containing a retrovirus, wherein a subset of the cell population contains a viral envelope protein with at least one mutation that reduces its native function, a non-viral membrane-binding protein containing a membrane-binding domain and an extracellular targeting domain, and a nucleic acid encoding a reporter.

98. The cell population according to claim 13, wherein a subset of the cell population contains the retrovirus according to any one of claims 33 to 51.

99. The population according to claim 97 or 98, wherein the cells are antigen-specific cells, optionally B cells or T cells.

100. The population according to any one of claims 97 or 98, wherein a subset of the cell population contains a retrovirus within each cell of the subset, optionally within the nucleus of each cell of the subset.

101. The population according to any one of claims 97 to 100, wherein a subset of the population expresses a reporter, and optionally here, the reporter is a fluorescent protein.

102. The population according to any one of claims 97 to 101, wherein a subset of the population containing the retrovirus is isolated and / or sorted from cells of a population not containing the retrovirus.

103. A retrovirus comprising: i) a CD80 protein domain; and ii) a mutated viral envelope protein containing at least one mutation that reduces its native function.

104. The retrovirus according to claim 103, wherein the retrovirus is a lentivirus.

105. The retrovirus according to any one of claims 103 or 104, wherein the viral envelope protein is a VSV-G envelope protein, a measles virus envelope protein, a Nipah virus envelope protein, or a coccus virus G protein.

106. The retrovirus according to claim 105, wherein at least one mutation of the VSV-G envelope protein is a mutation selected from the group consisting of H8, K47, Y209, and R354.

107. The retrovirus according to claim 105, wherein at least one mutation of the measles virus envelope protein is a mutation selected from the group consisting of Y481, R533, S548, and F549.

108. The retrovirus according to claim 105, wherein at least one mutation of the Nipah virus envelope protein is a mutation selected from the group consisting of E501, W504, Q530, and E533.

109. The retrovirus according to claim 105, wherein at least one mutation of the coccus virus G protein is a mutation selected from the group consisting of K64 and R371.

110. The retrovirus according to any one of claims 103 to 109, wherein the CD80 protein domain is an extracellular domain, and optionally here, the extracellular domain binds to a receptor on the target cell.

111. The retrovirus according to any one of claims 103 to 110, further comprising a non-viral membrane-binding protein comprising an extracellular targeting domain capable of binding to a cognate ligand of the cell.

112. The retrovirus according to claim 111, wherein the non-viral membrane-binding protein comprises a major histocompatibility complex (MHC) protein.

113. The retrovirus according to claim 111 or 112, wherein the extracellular targeting domain is a protein, a peptide, or an antibody.

114. A method for delivering a nucleic acid to a cell, the method comprising: (i) providing a retrovirus comprising a nucleic acid, a viral envelope protein comprising at least one mutation that reduces its native function, and a CD80 protein domain; and (ii) contacting a retrovirus with a cell, thereby delivering a nucleic acid to the cell. **Claim 115** The method according to claim 114, wherein the nucleic acid encodes a gene of interest, and optionally here, the gene of interest encodes a protein. **Claim 116** The method according to any one of claims 114 or 115, wherein the retrovirus penetrates or infects the cell during (ii). **Claim 117** The method according to any one of claims 114 to 116, wherein the cell is a somatic cell, optionally an antigen-specific cell. **Claim 118** The method according to claim 117, wherein the antigen-specific cell is a T cell or a B cell. **Claim 119** The method according to any one of claims 114 to 118, wherein the retrovirus is a lentivirus. **Claim 120** The method according to any one of claims 114 to 119, wherein the viral envelope protein is a VSV-G envelope protein, a measles virus envelope protein, a Nipah virus envelope protein, or a coccus virus G protein. **Claim 121** The method according to claim 120, wherein at least one mutation of the VSV-G envelope protein is a mutation selected from the group consisting of H8, K47, Y209, and R354. **Claim 122** The method according to claim 120, wherein at least one mutation of the measles virus envelope protein is a mutation selected from the group consisting of Y481, R533, S548, and F549. **Claim 123** The method according to claim 120, wherein at least one mutation of the Nipah virus envelope protein is a mutation selected from the group consisting of E501, W504, Q530, and E533. **Claim 124** The method according to claim 120, wherein at least one mutation of the coccus virus G protein is a mutation selected from the group consisting of K64 and R371. **Claim 125** The method according to any one of claims 114 to 124, wherein the CD80 protein domain is an extracellular domain, and optionally here, the extracellular domain binds to a receptor on the cell.

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