Recombinant viral particles and uses thereof

Abstract

Provided herein are recombinant viral particles presenting on their surface a peptide presented in the context of a major histocompatibility complex (MHC) molecule for recognition by a T cell. The recombinant viral particles may further present on their surface a co-stimulatory domain. Also provided are methods of using the recombinant viral particles, for example, for activating T cells and for the treatment of disease.

Description

PROV 188219.00209 RECOMBINANT VIRAL PARTICLES AND USES THEREOF CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 653,264, filed on May 30, 2024, which is hereby incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under R01AI172607 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention. REFERENCE TO A SEQUENCE LISTING

[0003] This application contains a Sequence Listing, which has been submitted electronically in xml format and is hereby incorporated by reference in its entirety. Said xml copy, created on May 19, 2025, is named SeqList-182219-00269.xml and is 82,583 bytes in size. FIELD

[0004] The present disclosure relates generally to the field of molecular biology and medicine. More particularly, the methods and compositions herein are useful for treating cancer. BACKGROUND

[0005] Many immunotherapies for the treatment of infection or cancer work by regulating T cell activity in vivo. At the core of the molecular events underlying these therapies is the engagement of the T cell receptor (TCR) with a small peptide antigen non-covalently presented by a major histocompatibility complex (MHC) molecule. Following epitope-specific cell targeting, the recruited T cells can be activated through general engagement of costimulatory molecules found on the antigen presenting cell as well as by cytokines produced by a range of immune cells. These signals (peptide presentation, co-stimulation and cytokine signaling) are utilized to drive T cell specificity, activation, differentiation and durability. During T cell development, a genomic editing process results in the expression of a unique TCR on every T cell, while a costimulatory molecule, such as CD28, is generally expressed on all T cells (or large T cell subsets) and cytokines, such as IL-2, are provided by a variety of immune cells. 172416522.1

[0006] Current immunotherapies often lack specificity since many approaches stimulate signaling through the TCR complex by binding the CD3 complex, which is identical in all T cells, and / or signaling through costimulatory molecules, etc., resulting in “global therapies”. While these global immunotherapies can be extremely potent, they indiscriminately target all T cells. As such, existing approaches can lead to undesirable side effects such as a hyperactive immune response and significant toxicity.

[0007] Accordingly, provided herein are therapies that take advantage of the uniquely specific interaction between a TCR and an MHC / peptide complex, allowing the specific regulation of T cells of interest in vivo. SUMMARY

[0008] Provided herein are viral particles and methods of using thereof.

[0009] Provided herein is a recombinant viral particle, the recombinant viral particle comprising a lipid envelope and: (1) an MHC / peptide complex comprising a first fusion polypeptide comprising (a) a class I MHC polypeptide or a fragment, mutant, or derivative thereof, (b) a β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof, (c) an epitope, and (d) a first lipid envelope anchor; and (2) a second fusion polypeptide comprising (a) a co-stimulatory domain and (b) a second lipid envelope anchor.

[0010] Provided herein is a recombinant viral particle, wherein the class I MHC polypeptide comprises a human class I MHC polypeptide derived from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G.

[0011] Provided herein is a recombinant viral particle, wherein the class I MHC polypeptide comprises a murine class I MHC polypeptide derived from H-2K, H-2D, H-2L, H-2Q, H-2M or H-2T.

[0012] Provided herein is a recombinant viral particle, wherein the class I MHC polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO:20.

[0013] Provided herein is a recombinant viral particle, wherein the class I MHC polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO:20.

[0014] Provided herein is a recombinant viral particle, wherein the class I MHC polypeptide comprises SEQ ID NO:20.

[0015] Provided herein is a recombinant viral particle, wherein the β2 microglobulin polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO:19. 2 172416522.1

[0016] Provided herein is a recombinant viral particle, wherein the β2 microglobulin polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO:19.

[0017] Provided herein is a recombinant viral particle, wherein the β2 microglobulin polypeptide comprises SEQ ID NO:19.

[0018] Provided herein is a recombinant viral particle, wherein the epitope is 5 to 20 amino acids long.

[0019] Provided herein is a recombinant viral particle, wherein the epitope is derived from a bacterial, viral, fungal, or protozoan polypeptide.

[0020] Provided herein is a recombinant viral particle, wherein the epitope is derived from a cytomegalovirus (CMV) polypeptide, a human immunodeficiency virus (HIV) polypeptide, a SARS-CoV-2 virus polypeptide, or a hepatitis virus polypeptide.

[0021] Provided herein is a recombinant viral particle, wherein the epitope comprises any one of SEQ ID NOs:1-3, SEQ ID NO:94, or SEQID NO:95.

[0022] Provided herein is a recombinant viral particle, wherein the epitope comprises a sequence derived from HIV polypeptide gp120, HIV-1 polypeptide p17, or melanoma antigen recognized by T cells 1 (MART-1).

[0023] Provided herein is a recombinant viral particle, wherein the epitope is a cancer antigen or fragment thereof.

[0024] Provided herein is a recombinant viral particle, wherein the first lipid envelope anchor and / or the second lipid envelope anchor is a transmembrane domain.

[0025] Provided herein is a recombinant viral particle, wherein the transmembrane domain is derived from human HLA-A*0201 MHC.

[0026] Provided herein is a recombinant viral particle, wherein the transmembrane domain comprises a sequence that is at least 80% identical to SEQ ID NO:22.

[0027] Provided herein is a recombinant viral particle, wherein the transmembrane domain comprises a sequence that is at least 90% identical to SEQ ID NO:22.

[0028] Provided herein is a recombinant viral particle, wherein the transmembrane domain comprises SEQ ID NO:22.

[0029] Provided herein is a recombinant viral particle, wherein the co-stimulatory domain is a 4-1BBL polypeptide, a B7-1 polypeptide, a B7-2 polypeptide, an ICOS-L polypeptide, an OX-40L polypeptide, a CD80 polypeptide, a CD70 peptide, a CD86 polypeptide, a CD40L polypeptide, a CD40 polypeptide, a PD-L1 polypeptide, or a PD-L2 polypeptide. 3 172416522.1

[0030] Provided herein is a recombinant viral particle, wherein the co-stimulatory domain is a CD28 agonist, an ICOS agonist, a 4-1BB agonist, a OX40 agonist, a CD27 agonist, a CD40 agonist, a CD40L agonist, a CTLA-4 agonist, or a PD-1 agonist.

[0031] Provided herein is a recombinant viral particle, wherein the co-stimulatory domain is a CD28 agonist.

[0032] Provided herein is a recombinant viral particle, wherein the co-stimulatory domain comprises a sequence that is at least 80% identical to SEQ ID NO:25.

[0033] Provided herein is a recombinant viral particle, wherein the co-stimulatory domain comprises a sequence that is at least 90% identical to SEQ ID NO:25.

[0034] Provided herein is a recombinant viral particle, wherein the co-stimulatory domain comprises SEQ ID NO:25.

[0035] Provided herein is a recombinant viral particle, wherein: (1) the first fusion polypeptide comprises from N-terminus to C-terminus: (a) the epitope; (b) the β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof; (c) the class I MHC polypeptide or a fragment, mutant, or derivative thereof; (d) the first transmembrane domain; and (e) optionally, a first cytoplasmic domain; and / or (2) the second fusion polypeptide comprises from N-terminus to C-terminus: (a) the co-stimulatory domain; (b) the second transmembrane domain; and (c) optionally, a second cytoplasmic domain.

[0036] Provided herein is a recombinant viral particle, wherein the first fusion polypeptide and / or the second fusion polypeptide further comprises one or more linkers.

[0037] Provided herein is a recombinant viral particle, wherein the one or more linkers are one or more polypeptide linkers or disulfide bond linkers.

[0038] Provided herein is a recombinant viral particle, wherein the one or more polypeptide linkers are one or more flexible linkers.

[0039] Provided herein is a recombinant viral particle, wherein the one or more flexible linkers predominantly consist of glycines, serines, and alanines.

[0040] Provided herein is a recombinant viral particle, wherein the one or more flexible linkers include one or more repetitions of any one of SEQ ID NOs:4-17.

[0041] Provided herein is a recombinant viral particle, wherein the one or more flexible linkers include SEQ ID NO:16.

[0042] Provided herein is a recombinant viral particle, wherein the one or more polypeptide linkers are one or more rigid linkers. 4 172416522.1

[0043] Provided herein is a recombinant viral particle, wherein the one or more rigid linkers include one or more repetitions of any one of SEQ ID NOs:27-55.

[0044] Provided herein is a recombinant viral particle, wherein the first fusion polypeptide and / or the second fusion polypeptide further include a signal peptide.

[0045] Provided herein is a recombinant viral particle, wherein the signal peptide comprises any one of SEQ ID NOs:18 or 56-93.

[0046] Provided herein is a recombinant viral particle, wherein the signal peptide is derived from the beta 2 microglobulin signal peptide.

[0047] Provided herein is a recombinant viral particle, wherein the signal peptide comprises a sequence that is at least 80% identical to SEQ ID NO:18.

[0048] Provided herein is a recombinant viral particle, wherein the signal peptide comprises a sequence that is at least 90% identical to SEQ ID NO:18.

[0049] Provided herein is a recombinant viral particle, wherein the signal peptide comprises SEQ ID NO:18.

[0050] Provided herein is a recombinant viral particle, wherein the first fusion polypeptide and the second fusion polypeptide are covalently linked.

[0051] Provided herein is a recombinant viral particle, wherein the recombinant viral particle is derived from a lentivirus.

[0052] Provided herein is a recombinant viral particle, wherein one or more of polypeptides present in the lipid envelope of a wild-type virus corresponding to the recombinant viral particle are absent or mutated so that said recombinant viral particle is not capable of binding to any cell targeted by the wild-type virus in the absence of the MHC / peptide complex.

[0053] Provided herein is a recombinant viral particle, wherein the epitope binds to a T cell receptor (TCR) of a CD8+T cell.

[0054] Provided herein is a nucleic acid or pair of nucleic acids encoding a MHC / peptide complex and / or a fusion polypeptide disclosed herein.

[0055] Provided herein is a method of activating a T cell comprising a TCR, the method comprising contacting the T cell with a recombinant viral particle disclosed herein, wherein the TCR of the T cell binds to the epitope.

[0056] Provided herein is a method of inducing expression of pro-inflammatory cytokines in a T cell comprising a TCR, the method comprising contacting the T cell with a recombinant viral particle disclosed herein, wherein the TCR of the T cell binds to the epitope. 5 172416522.1

[0057] Provided herein is a method of expanding a population of T cells each comprising a TCR, the method comprising contacting the population of T cells with a recombinant viral particle disclosed herein, wherein the TCR of the T cells binds to the epitope.

[0058] In some embodiments, the T cell has been genetically engineered to express the TCR that binds to the epitope.

[0059] Provided herein is a method of inducing killing of a cell infected by a pathogen, wherein the cell displays on its surface a polypeptide derived from the pathogen, the method comprising: (a) contacting a population of T cells with a recombinant viral disclosed herein, thereby generating a population of activated T cells, wherein each T cell comprises a TCR, wherein the TCR is capable of binding to the polypeptide derived from the pathogen and to the epitope; and (b) exposing the cell infected by the pathogen to the population of activated T cells.

[0060] In one embodiment, the population of T cells has been genetically engineered to express the TCR that is capable of binding to the polypeptide derived from the pathogen and to the epitope.

[0061] Provided herein is a method of inducing killing of a cancer cell, wherein the cancer cell displays on its surface a cancer antigen, the method comprising: (a) contacting a population of T cells with a recombinant viral particle disclosed herein, thereby generating a population of activated T cells, wherein each T cell comprises a TCR, wherein the TCR is capable of binding to the cancer antigen and to the epitope; and (b) exposing the cancer cell to the population of activated T cells.

[0062] In one embodiment, the population of T cells has been genetically engineered to express the TCR that is capable of binding to the cancer antigen and to the epitope.

[0063] In one embodiment, the population of activated T cells is further expanded before step (b).

[0064] The methods disclosed herein may be performed in vitro, in vivo, or ex vivo.

[0065] Provided herein is a method of treating an infection with a pathogen in a subject in need thereof, the method comprising administering to the subject a recombinant viral particle disclosed herein, wherein: the subject has a cell that displays on its surface a polypeptide derived from the pathogen; and the subject has a T cell that comprises a TCR, wherein the TCR is capable of binding to the polypeptide derived from the pathogen and to the epitope.

[0066] Provided herein is a method of treating an infection with a pathogen in a subject in need thereof, the method comprising administering to the subject a population of T cells, 6 172416522.1wherein: the population of T cells has previously been contacted with a recombinant viral particle disclosed herein; the subject has a cell that displays on its surface a polypeptide derived from the pathogen; and the population of T cells has been genetically engineered to express a TCR, wherein the TCR is capable of binding to the polypeptide derived from the pathogen and to the epitope presented by the recombinant viral particle.

[0067] In some embodiments, the pathogen is a bacterial, viral, or fungal pathogen. In one embodiment, the pathogen is a viral pathogen. In some embodiments, the viral pathogen is a CMV, an HIV, a SARS-CoV-2 virus, or a hepatitis virus.

[0068] Provided herein is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a recombinant viral particle disclosed herein, wherein: the subject has a cancer cell that displays on its surface a cancer antigen; and the subject has a T cell that comprises a TCR, wherein the TCR is capable of binding to the cancer antigen and to the epitope presented by the recombinant viral particle.

[0069] Provided herein is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a population of T cells, wherein: the population of T cells has previously been contacted with a recombinant viral particle disclosed herein; the subject has a cancer cell that displays on its surface a cancer antigen; and the population of T cells has been genetically engineered to express a TCR, wherein the TCR is capable of binding to the cancer antigen and to the epitope presented by the recombinant viral particle.

[0070] In some embodiments, the subject is a human.

[0071] Provided herein is a recombinant viral particle, the recombinant viral particle comprising a lipid envelope and: (1) a first MHC / peptide complex comprising a first fusion polypeptide comprising (a) a first class I MHC polypeptide or a fragment, mutant, or derivative thereof, (b) a first β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof, (c) a first epitope, and (d) a first lipid envelope anchor; (2) a second MHC / peptide complex comprising a second fusion polypeptide comprising (a) a second class I MHC polypeptide or a fragment, mutant, or derivative thereof, (b) a second β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof, (c) a second epitope, wherein the first epitope and the second epitope are different and (d) a second lipid envelope anchor; and (3) optionally, a third fusion polypeptide comprising (a) a co-stimulatory domain and (b) a third lipid envelope anchor. 7 172416522.1

[0072] In one embodiment, the first epitope and the second epitope are derived from the same polypeptide. In one embodiment, the first epitope and the second epitope are derived from different polypeptides from the same organism. BRIEF DESCRIPTION OF THE FIGURES

[0073] Fig. 1A and Fig.1B illustrate that an HLA-A derived transmembrane domain enables functional expression of an ^CD3 single chain antibody (sFv) in the lentivirus membrane. CD3+Jurkat E6.1 T cells were incubated with lentivirus (1:5 dilution) designed to mediate selective infection of CD3+T cells through expression of an ^CD3 sFv (TR66.opt clone) linked either to an IgM transmembrane domain (Fig. 1A) or human HLA-A transmembrane domain in the lentivirus membrane (Fig. 1B). Jurkat E6.1 cells were either untransduced or transduced with the indicated anti-CD3 / GFP construct and evaluated three days later for transduction by flow cytometric evaluation of expression of the GFP reporter gene.

[0074] Fig. 2A and Fig. 2B illustrate that lentiviral-like particles (LVPs) that do not express VSV-gmutare stable but unable to transduce cells. Fig. 2A. Lentiviral particles comprising a nucleic acid encoding GFP were generated to incorporate into their membranes (1) a VSV-gmutenvelope protein (possessing fusion capacity, but mutated to remove cell binding activity) and a membrane-anchored MHC / NLV (NLV wVSVG) (to direct selective MHC allele-specific binding) or (2) the membrane-anchored MHC / NLV complex alone (NLV modless), which possesses binding but not fusion activity. Fig. 2B. The indicated lentiviral particles were incubated with PBMCs containing CD8+T cells expressing an NLV-specific TCR. Transduction by the lentiviral particles was determined by quantification of GFP fluorescence by flow cytometry.

[0075] Fig. 3A and Fig. 3B illustrate the design of LVPs presenting both an MHC / peptide complex and a co-stimulatory domain and show TCR activation and T cell expansion in vitro. Fig 3A. Structural representation of the LVPs designed for NLV-specific T cell expansion. “NLV modless” = LVPs presenting on their surface the membrane-anchored MHC / NLV complex. “NLV αCD28” = LVPs presenting on their surface the membrane- anchored MHC / NLV complex and membrane-anchored agonist anti-CD28 scFv. Fig.3B. Luciferase assay to detect NLV TCR activation in Jurkat / Ma CD8+NFAT luciferase reporter gene T cell line expressing the NLV-specific TCR after stimulation with the indicated LVP constructs (from left to right: Unstimulated control (negative control); NLV modless; NLV- 8 172416522.1anti-CD28, soluble recombinant NLV Flag Immuno-STAT (positive control); soluble recombinant NLV Flag Immuno-STAT with anti-CD28 co-stimulatory domain (positive control)). Values on x axis refer to µl for NLV Modless and NLV aCD28 and refer to µM for NLV Flag IST and NLVaCD28 IST.

[0076] Fig. 4A, Fig. 4B, and Fig. 4C illustrate that LVP treatment results in selective activation and expansion of NLV-specific CD8+T cells in HLA-A*0201 donor PBMCs in vitro. PBMCs from an NLV-responsive donor were incubated with the indicated constructs. Two weeks later, the fraction of CD3+CD8+NLV-specific T cells was determined by flow cytometry (using an NLV tetramer to detect NLV-specific T cells). Plots were gated on live single cell CD8+T cells. The results of three different experiments using cells from the same donor are shown (Figs. 4A, 4B, 4C, respectively). “VirTac modless” or “NLV modless” = LVPs presenting on their surface the membrane-anchored MHC / NLV complex. “NLV-VirTac- αCD28” or “NVL αCD28” = LVPs presenting on their surface the membrane-anchored MHC / NLV complex and membrane-anchored agonist anti-CD28 scFv. “NLV wVSVG” = LVPs presenting a VSV-gmut envelope protein and membrane-anchored MHC / NLV. “NLV αCD28 IST” = soluble recombinant NLV Flag Immuno-STAT with anti-CD38 co-stimulatory domain (positive control). Negative control is no treatment.

[0077] Fig. 5 illustrates that NLV-specific CD8+T cells expanded by LVP treatment display robust production of pro-inflammatory cytokines after NLV-peptide presentation. Cytokine production by NLV-specific CD8+T cells stimulated with the different LVPs was measured by intracellular staining quantified by flow cytometric analysis 5 h after incubation with NLV-loaded T2 cells. “NLV wVSVG” = LVPs presenting a VSV-gmut envelope protein and membrane-anchored MHC / NLV. “NLV modless” = LVPs presenting the membrane-anchored MHC / NLV complex (no envelope protein). “NLV αCD28” = LVPs presenting the membrane-anchored MHC / NLV complex and membrane-anchored agonist anti- CD28 scFv (no envelope protein). Cytokines are presented from left to right as shown in legend (from top to bottom).

[0078] Fig. 6 illustrates that NLV-specific CD8+T cells expanded by treatment with LVP display potent cytotoxic activity against target cells presenting NLV-peptide. The cytotoxic activity of LVP-expanded, NLV-specific CD8+T cells was determined by coculturing the expanded T cells with T2 cells either loaded with NLV peptide or no peptide at 1:1 and 1:10 Effector:Target ratio for 24 h and then measuring the number of viable T2 cells. The data is presented as % cytotoxicity comparing killing of NLV-peptide-loaded T2 cells to 9 172416522.1T2 cells with no loaded peptide. “NLV wVSVG” = LVP presenting a VSV-gmut envelope protein and membrane-anchored MHC / NLV. “NLV modless” = LVPs presenting the membrane-anchored MHC / NLV complex (no envelope protein). “NLV αCD28” = LVPs presenting the membrane-anchored MHC / NLV complex and membrane-anchored agonist anti- CD28 scFv (no envelope protein). NLV modless IST = soluble recombinant Immuno-STAT presenting the NLV peptide with no costimulatory signal. The data shown from one donor (HG0055) is representative of similar results obtained using PBMCs from two other donors. Bars shown from left to right for constructs listed in figure (from top to bottom).

[0079] Fig. 7 illustrates that NLV-specific CD8+T cells expanded by LVP treatment are able to suppress CMV infection. The capacity of LVP-expanded, NLV-specific CD8+T cells to suppress CMV infection was determined in an infection assay. To that end, the expanded CD8+T cells were co-cultured with an MRC5 cell line, which was infected with CMV and expressed a luciferase reporter gene, at 1:1 ratio. After 48 h of co-culture, luciferase expression was assessed. Significance (p > 0.05) was calculated using a two-way ANOVA statistical test. “NLV modless” = LVPs presenting the membrane-anchored MHC / NLV complex (no envelope protein). “NLV ^-CD28” = LVPs presenting the membrane-anchored MHC / NLV complex and membrane-anchored agonist anti-CD28 scFv (no envelope protein). NLV modless IST = Immuno-STAT presenting the NLV peptide with no costimulatory signal. Anti-CMV drug = cidofovir.

[0080] Fig. 8 illustrates the results from a luciferase assay to detect antigen-specific TCR binding and activation of Jurkat cells expressing DMF5 (a MART1-TCR). 104Jurkat cells (wild-type or genetically modified to express DMF5) were incubated with the indicated volume of LVP construct. After 24 h, firefly luciferase activity was detected. WT Jurkat = wild-type Jurkat cells. DMF5 Jurkat = Jurkat cells genetically modified to express DMF5, a MART1- specific TCR. Anti-CD28 MART1 Virtac = LVPs presenting membrane-anchored MHC / MART-1 and membrane-anchored agonist anti-CD28 scFv. Modless Mart Virtac = LVPs presenting membrane-anchored MHC / MART-1.

[0081] Fig. 9 illustrates the expansion of MART-1-specific, CD8+T cells after LVP treatment. Donor PBMCs were either untreated or treated with the indicated LVP constructs. Expansion of the MART-1 T cell population was determined by flow cytometry (assessing binding to a MART-1 tetramer). Modless = LVPs presenting membrane-anchored MHC / MART-1. Anti-CD28 = LVPs presenting membrane-anchored MHC / MART-1 and membrane-anchored agonist anti-CD28 scFv. 10 172416522.1

[0082] Fig.10 illustrates stimulation of NLV-specific CD8+T cell expansion in mouse spleens with LVPs in vivo. PBMCs from HGK0055 donor were injected intrasplenically in NGS mice alone (unstimulated) or together with the indicated volume of LVP constructs. Nine days after stimulation, the mouse spleens were processed, and NLV-specific CD8+T cells were detected by staining with NLV tetramer and flow cytometry. “NLV αCD28” = LVPs presenting membrane-anchored MHC / NLV and membrane-anchored agonist anti-CD28 scFv (two mice). “NLV modless IST” = Immuno-STAT presenting the NLV peptide with no costimulatory signal (positive control, one mouse).

[0083] Fig. 11. illustrates stimulation of circulating NLV-specific CD8+T cell expansion in mouse peripheral blood with LVPs in vivo. PBMCs were injected intrasplenically, and the indicated LVP or Immuno-STAT were administered via retroorbital injection and combination of cytokines IL-7, IL-15, and IL-21 was injected intraperitoneally (3 mg / kg) on the same day. Ten days after the first injection, blood from the mice was extracted, processed and stained with NLV tetramer to quantify the fraction of circulating human CD8+T cells that were NLV-specific CD8+T cells (each plot represents a single mouse). “NLV αCD28” = LVPs presenting membrane-anchored MHC / NLV and membrane-anchored agonist anti-CD28 scFv. “NLV αCD28 IST” = Immuno-STAT presenting the NLV peptide with costimulatory signal (positive control).

[0084] Fig. 12A and Fig. 12B illustrate the ability of LVPs presenting MHC / SL9 to activate and expand SL9-specific TCR in vitro. Fig.12A. Jurkat / Ma CD8+NFAT-luciferase T cells expressing the SL9-specific TCR were incubated with the indicated LVP constructs and expanded. SL9-modless VirTac (left bars); SL9-^CD28 VirTac (right bars). Constructs shown include SL9 LVPs and SL9 ISTs. Fig.12B. Donor PBMCs (RC619) from a person with HIV were treated with the indicated LVPs. After 14 days of culture, the fraction of SL9-specific CD8+T cells was quantified by flow cytometry after gating on live single cell lymphocytes and CD8+cells (upper panel). SL9+populations from left to right: 3.54 %; 19.8 %; 22.8 %; 31.5 %. The middle panel shows SL9-specific CD8+T cells with different memory phenotypes (based on CD45Ro and CD62L expression). SL9+CD45Ro+CD62L- CD8+effector memory T cells populations from left to right: 87%; 95%; 94%; 94%. The lower panel shows CD8+T cells with different memory phenotypes that are non-SL9 reactive (SL9-). SL9- CD45Ro+CD62L+central memory populations from left to right: 58%; 59%; 58%; 52%. Plots were gated on live single cell lymphocytes CD8+SL9+or SL9- cells. “SL9-modless VirTac” = LVP presenting on its surface membrane-anchored MHC / SL9. “SL9 αCD28” = LVP presenting on its surface 11 172416522.1membrane-anchored MHC / SL9 and membrane-anchored agonist anti-CD28 scFv. “SL9 modless IST” = soluble recombinant Immuno-STAT presenting the NLV peptide with no costimulatory signal.

[0085] Fig. 13 illustrates that NLV-specific CD8+T cells expanded by treatment with LVP display potent cytotoxic activity against target cells presenting SL9-peptide. The cytotoxic activity of LVP-expanded, SL9-specific CD8+T cells was determined by coculturing the expanded T cells with T2 cells either loaded with SL9 peptide or no peptide at 1:1 and 1:10 Effector:Target ratio for 24 h and then measuring the number of viable T2 cells. The data is presented as % cytotoxicity comparing killing of SL9-peptide-loaded T2 cells to T2 cells with no loaded peptide. Constructs from left to right: “SL9 modless” = LVPs presenting the membrane-anchored MHC / SL9 complex (no envelope protein). “SL9 ^CD28” = LVPs presenting the membrane-anchored MHC / SL9 complex and membrane-anchored agonist anti- CD28 scFv (no envelope protein). “SL9 modless IST” = Immuno-STAT presenting the NLV peptide with no costimulatory signal. “SL9 ^CD28 IST” = Immuno-STAT presenting the NLV peptide with linked agonist anti-CD28 scFv costimulatory signal. The data shown are from one donor with HIV (RU169). Bars shown from left to right for constructs listed in figure (from top to bottom).

[0086] Fig. 14A and Fig. 14B illustrate the sustained in vitro stability of LVPs in the presence of human serum and incubation at 37ºC. Fig. 14A. To assess serum-mediated inactivation of lentiviral particles, donor PBMCs (HG0055) were incubated with NLV-specific LVPs for 14 days in the presence of the indicated concentrations of human serum from either an AB blood group donor (HG0055) or an O blood group donor (HG0123). The samples were evaluated for LVP-induced expansion of NLV-specific CD8+T cells. Significant (p > 0.05) differences between conditions were calculated using one-way or two-way ANOVA statistical tests. Left bars: Serum O. Right bars: Serum AB. Fig.14B. To evaluate the thermal stability of LVPs, modless NLV-LVPs or anti-CD28 NLV-LVPs, respectively, were preincubated at 37 °C for 0 to 7 days prior to treatment of donor PBMCs (HG0055), followed by a 14-day culture period. Then, quantification of LPV-induced expansion of NLV-specific CD8+T cells was performed. Expansion of donor NLV-specific CD8+T cells was assessed by tetramer staining and analyzed via flow cytometry. Left data points: Modless NLV-LVP. Right data points: anti- CD28 NLV-LVP.

[0087] Fig. 15A and Fig. 15B illustrate the expansion of antigen-specific CD8+T cells obtained from donors without HIV (n = 4 donors) and donors with HIV (n = 3 donors) 12 172416522.1by NLV or SL9 LVPs. PBMC from donors without HIV or with HIV (the latter marked with an asterisk), which were responsive to NLV peptide (n =5 ) or SL9 peptide (n = 2), respectively, were incubated with the indicated NLV-LPVs or SL9-LVPs, respectively, or no LVPs (unstimulated). CD8+T cells that were NLV-specific (Fig. 15A) or SL9-specific (Fig. 15B) were quantified by flow cytometry using tetramer staining. Significant (p > 0.05) differences between conditions were calculated using one-way or two-way ANOVA statistical tests. Bars from left to right: unstimulated; modless LVP; anti-CD28 LVP.

[0088] Fig. 16A and Fig. 16B illustrate the generation of LVPs expressing scMHC molecules displaying either a SARS-CoV-2 (YLQ) peptide or an HIV (IV9) peptide. Fig. 16A. LPVs presenting the YLQ peptide-scMHC complex on their surface (“YLQ VirTac”) or the IV9 peptide-scMHC complex (“IV9 VirTac”) were generated. PBMCs from YLQ-reactive donors, either without HIV (n = 3 donors) or with HIV (n = 1 donor indicated with an asterisk), were stimulated with modless YLQ-LVPs or anti-CD28 YLQ-LVPs, respectively, or were left unstimulated. CD8+YLQ-specific T cells were quantified by flow cytometry using tetramer staining. Bars from left to right: unstimulated; modless LVP; anti-CD28 LVP. Fig.16B. IV9- specific LVPs were tested for activation of an IV9-specific TCR expressed in a Jurkat / Ma cell line carrying a firefly luciferase reporter gene under the control of the NFAT promoter. Modless IV9-LVPs induced dose-dependent activation of the IV9-TCR in this reporter cell system. Significant (p > 0.05) differences between conditions were calculated using one-way or two-way ANOVA statistical tests.

[0089] Fig. 17A, Fig. 17B, and Fig. 17C illustrate that YLQ-specific or SL9-specific CD8+T cells, which have previously been expanded in vitro by treatment with YLQ- or SL9-LVPs, respectively, display functional activity. Fig. 17A. Production of interferon γ (IFNγ) and tumor necrosis factor α (TNFα) proinflammatory cytokines by donor YLQ-specific CD8+T cells (HG0055) stimulated with YLQ-LVPs was measured by intracellular staining and flow cytometric analysis 5 h after (a) being activated by incubation with YLQ-loaded T2 cells or (b) not being activated by incubation with T2 cells alone. Bars from left to right as in figure legend from top to bottom. Fig.17B. The cytotoxic activity of LVP-expanded YLQ- specific CD8 T+cells was determined by coculturing the T cells with T2 cells (either loaded with YLQ peptide or no peptide) at 1:1 and 1:10 Effector:Target ratios for 24 h. The number of viable T2 cells was measured by flow cytometry. The percentage of cytotoxicity is represented as the percentage of specific killing of peptide-loaded T2 cells compared with killing of T2 cells that do not present any peptide. Bars from left to right as in figure legend 13 172416522.1from top to bottom. Fig.17C. The capacity of SL9 LVP-treated CD8+T cells to suppress HIV replication was evaluated by measuring Renilla luciferase activity (RLU) after 48 h of co- culture with autologous CD4+T cells infected with BAL-HIV expressing Renilla luciferase reporter gene at 10:1 Effector:Target ratio. The anti-HIV drug Raltegravir (2 µM) was used as a positive control for HIV suppression. Not HIV infected cells served as a negative control. Bars from left to right as in figure legend from top to bottom.

[0090] Fig. 18 illustrates the in vivo half-life of LVPs in plasma after intravenous injection into mice. NSG mice were injected intravenously with NLV-specific LVPs (n = 2) or PBS as a vehicle control (n = 1). Peripheral blood was collected at multiple time points post- injection, and HIV-1 p24 antigen (pg / mL) levels in plasma were quantified as an indicator to measure VLP levels, were quantified by ELISA. Bottom trace: control.

[0091] Fig.19 illustrates the in vivo expansion of SL9-specific T cells in mouse spleens after intravenous injection of SL9-LVPs. NSG were humanized by intrasplenic injection with human PBMC donors with HIV and either remained unstimulated or were stimulated by intravenous injection with modless SL9-LVPs or anti-CD28 LVPs LVPs (VT). All mice were provided with human IL-7, IL-15, and IL-21 cytokines to support T cell proliferation by intraperitoneal injection with HCW9206 (3 mg / kg). Ten days later, the human T cell population in the blood was evaluated by flow cytometry. Summary data of percentage of SL9-specific CD8+T cells in the bloodstream of NSG mice (n = 2 mice / group) ten days after intravenous LVP treatment are shown. Significant (p > 0.05) differences between conditions were calculated using one-way or two-way ANOVA statistical tests. Bars from left to right as in figure legend from top to bottom.

[0092] Fig. 20A, Fig. 20B, and Fig. 20C illustrate LVPs that express two different scMHC complexes, each presenting a different peptide. These LVPs can be used to stimulate cognate CD8+T cells. Fig. 20A. To address potential immune escape by HIV-1 mutations, dual-specific LVPs (also referred to as duoVirTacs or duo-LVPs) capable of presenting two distinct peptide-scMHC complexes on their surface were engineered. In one example, SL9 and IV9 immunodominant peptides from HIV-1 are presented on the LVP. Fig. 20B. NLV-YLQ duo-LVP (presenting NLV and YLQ peptides). Fig. 20C. NLV-SL9 duo- LVP (presenting NLV and SL9 peptides). Fig.20D. Expansion of two different antigen- specific CD8+T cell populations from two distinct donors (HG0055 for NLV-YLQ duo-LVP; RC619 NLV-SL9 duo-LVP), was assessed 14 days post-treatment by dual-tetramer staining and flow cytometry. Fig. 20E. Cytotoxic activity was assessed by co-culturing duo-LVP- 14 172416522.1expanded CD8+T cells with T2 target cells loaded with NLV, YLQ, or SL9 peptides, respectively, for 24 hours. Target cell viability was measured by live-dead staining and analyzed by flow cytometry. Bars from left to right as in figure legend from top to bottom.

[0093] Fig.21A and Fig.21B illustrate the LVP-induced expansion of TCR-engineered antigen-specific CD8+T cells. CD8⁺ T cells were isolated from donor PBMCs. Genetic engineering was performed by transduction with a lentiviral vector encoding the SL9-specific TCR after activation with anti-CD3 and anti-CD28 antibodies. Following transduction, SL9- TCR-engineered CD8+T cells were stimulated with either modless SL9-LVPs or anti-CD28 SL9-LPVs for 14–21 days. Antigen-specific expansion was quantified by SL9-tetramer staining and flow cytometric analysis. SL9-specific CD8+T cells were expanded by both SL9- LVPs. DETAILED DESCRIPTION

[0094] Provided herein are recombinant viral particles that are, for example, useful for the selective ex vivo, in vitro or in vivo activation and expansion of T cells specific for an epitope of interest.

[0095] Viral particles

[0096] Provided herein is a recombinant viral particle capable of binding to a T cell receptor (TCR), the recombinant viral particle presenting on its surface (e.g., in a lipid envelope) a TCR- binding molecule. In one embodiment, the TCR-binding molecule comprises a peptide (also referred to as “epitope herein”) presented in the context of a major histocompatibility complex (MHC) molecule. The epitope may be bound to the MHC covalently or non-covalently. The recombinant viral particle may be capable of selective binding to the antigen-specific TCR on a surface of a cell, such as a T cell. The terms “antigen” and “epitope” may be used herein interchangeably. In one embodiment, the T cell is a CD8+T cell. In some embodiments, the recombinant viral particle is capable of binding the TCR-expressing cell only in the presence of the MHC / peptide complex presented on the particle’s surface.

[0097] As used herein, “recombinant viral particles” or “virus-like particles” are protein / lipid structures that mimic the organization and conformation of authentic native viruses but that do not contain the viral genome and are not capable of infecting cells.

[0098] In one embodiment, the recombinant viral particle is derived from an enveloped virus. Generally speaking, in enveloped viruses, the viral genome and core proteins are 15 172416522.1wrapped within one or more membranes. These membranes are acquired from the host cell during virus assembly and budding. The membrane may comprise a lipid bilayer.

[0099] Non-limiting examples of enveloped viruses from which the viral particles described herein can be derived include, but are not limited to, e.g., retroviruses (e.g., rous sarcoma virus, human and bovine T-cell leukemia virus (HTLV and BLV)), lentiviruses (e.g., human and simian immunodeficiency viruses (HIV and SIV), Mason-Pfizer monkey virus), foamy viruses (e.g., Human Foamy Virus (HFV)), herpes viruses (herpes simplex virus (HSV), varicella- zoster virus, VZVEBV, HCMV, HHV), hantaviruses, pox viruses (e.g., vertebrate and avian poxviruses, vaccinia viruses), orthomyxoviruses (e.g., influenza A, influenza B, influenza C viruses), paramyxoviruses (e.g., parainfluenza virus, respiratory syncytial virus, Sendai virus, mumps virus, measles and measles- like viruses), rhabdoviruses (e.g., vesicular stomatitis virus, rubella virus, rabies virus), coronaviruses (e.g., SARS, MERS), flaviviruses (e.g., Marburg virus, Reston virus, Ebola virus), alphaviruses (e.g., Sindbis virus), bunyaviruses, arenaviruses (e.g., LCMV, GTOV, JUNV, LASV, LUJV, MACV, SABV, WWAV), iridoviruses, and hepadnaviruses.

[0100] In one embodiment, the viral particle is derived from a lentivirus. As used herein, a recombinant virus that is derived from a lentivirus is also referred to herein as a lentivirus like particle (LVP). Compared to other gene transfer systems, lentiviral vectors offer a wide range of advantages, including their in vivo stability, ability to transduce a variety of cell types, to stably integrate transferred genetic material into the genome of the targeted host cell, and to express the transduced gene at significant levels.

[0101] In one aspect, one or more of the proteins present in the lipid envelope of a reference wild-type virus corresponding to the recombinant viral particle are absent or mutated so that said recombinant viral particle is not capable of binding to any of the target cells of the reference wild-type virus in the absence of the TCR-binding molecule. In one embodiment, all of the proteins which are normally present in the lipid envelope of a reference wild-type virus are absent in the viral particle.

[0102] In one embodiment, the recombinant particle does not contain vesicular stomatitis virus glycoprotein (VSV-g). In one embodiment, the recombinant viral particle comprises a mutated VSV-g. In one embodiment, the recombinant viral particle comprises a mutated VSV- g with significantly reduced or abolished ability to bind to the cellular receptor of VSV-g, e.g., the low-density lipoprotein receptor (LDRL). In one embodiment, the VSV-g mutant comprises a K47Q mutation. 16 172416522.1

[0103] In one embodiment, the viral particle comprises a viral core protein, such as p24.

[0104] In some embodiments, the viral particle comprises one or more proteins derived from the producer cells that produced the viral particle. Producer cells are discussed herein.

[0105] In one embodiment, the viral particle is substantially unable to infect a cell. In one embodiment, the viral particle is substantially unable to fuse with a cell. In one embodiment, the viral particle is replication deficient. In some embodiments, the viral particles described herein are replication deficient and only contain an incomplete genome of the virus from which they are derived. For example, in some embodiments, the viral particles do not comprise the genetic information for certain proteins involved in the assembly of the viral particle. In these cases, the minimal set of viral proteins needed to assemble the vector particle are provided in trans by means of a packaging cell line or a cell line transfected with vectors encoding the viral proteins.

[0106] In one embodiment, provided is a recombinant viral particle, the viral particle comprising a lipid envelope and an MHC / peptide complex comprising a fusion polypeptide comprising (a) a class I MHC polypeptide or a fragment, mutant, or derivative thereof, (b) a β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof, (c) an epitope, and (d) a lipid envelope anchor.

[0107] MHC Molecules

[0108] MHC molecules are generally classified into two categories: class I and class II MHCs.

[0109] An MHC class I molecule generally is an integral membrane protein comprising a glycoprotein heavy chain, also referred to herein as the α chain, which has three extracellular domains (e.g., α1, α2 and α3) and a transmembrane domain (TM) and a cytoplasmic (intracellular) domain. The heavy chain is noncovalently associated with a soluble subunit called β2-microglobulin (β2m or B2M). Generally, class I molecules bind peptides of about 8- 12 amino acids in length. Humans have between three and six different class I molecules, which can each bind many different types of peptides. For reference, an MHC class II protein generally is a heterodimeric integral membrane protein comprising one α chain and one β chain in noncovalent association. This α chain has two extracellular domains (α1 and α2), and a TM domain, and a cytoplasmic domain. The β chain contains two extracellular domains (β1 and β2), and two intracellular domains (a TM domain and CYT domain). CD4+T cells usually bind to the MHC class II molecules. CD8+T cells usually bind to MHC class I molecules. 17 172416522.1

[0110] Naturally-occurring MHC molecules are encoded by a cluster of genes on human chromosome 6 or mouse chromosome 17. Said MHCs are referred to as H-2 in mice and HLA (Human Leucocyte Antigen) in humans. MHC class I molecules specifically bind CD8 molecules expressed on cytotoxic T lymphocytes (CD8+T cells), whereas MHC class II molecules specifically bind CD4 molecules expressed on helper T lymphocytes (CD4+T cells). Examples of naturally occurring β2-microglobulins include β2-microglobulins encoded on human chromosome 15 and mouse chromosome 2.

[0111] The domain organization of MHCs forms the antigenic determinant binding site, or peptide binding groove. A “peptide binding groove” refers to a portion of an MHC protein that forms a cavity in which a peptide, e.g., antigenic determinant, can bind. The conformation of a peptide binding groove is capable of being altered upon binding of a peptide to enable proper alignment of amino acid residues important for TCR binding to the peptide-MHC (pMHC) complex. The pMHC complex is also referred to herein as MHC / peptide complex or MHC / epitope complex.

[0112] The MHCs described herein include fragments of MHC chains that are sufficient to form a peptide binding groove. For example, a peptide binding groove of a class I protein can comprise portions of the α1 and α2 domains of the heavy chain capable of forming two β- pleated sheets and two α helices. Inclusion of a portion of the β2-microglobulin chain stabilizes the complex.

[0113] Many human and other mammalian MHC molecules are well known in the art and may be used in the compositions and methods disclosed herein.

[0114] MHC molecules useful in the recombinant viral particles described herein include naturally occurring full-length MHC molecules as well as individual chains of MHC molecules (e.g., MHC class I α (heavy) chain, β2-microglobulin, individual subunits of such chains of MHCs (e.g., α1, α2 and / or α3 subunits of MHC class I α chain) as well as fragments, mutants, various derivatives thereof, or substantially engineered analogs, wherein such fragments, mutants, derivatives, and engineered analogs retain the ability to display an antigenic determinant for recognition by an antigen-specific TCR.

[0115] Also useful in the recombinant viral particles are engineered analogs of MHC molecules.

[0116] Also useful for the compositions and methods disclosed herein are engineered MHCs that preserve the peptide binding groove but comprise modifications that enhance stability and / or support additional functionality (e.g., additional receptor binding functions or 18 172416522.1enzymatic functions). Provided herein are derivatives of the MHC molecules disclosed herein that comprise modifications and / or amino substitutions that do not affect the functionality of the peptide binding groove.

[0117] In one embodiment, the MHC comprises a transmembrane domain embedded in the lipid envelope of the viral particle.

[0118] The MHCs useful in the viral particles described herein may be from, but are not limited to, any mammalian or avian species, for example, primates (e.g., humans), rodents, rabbits, equines, bovines, canines, felines, pigs, etc.

[0119] MHCs include, but are not limited to, HLA specificities such as A (e.g. A1-A74), B (e.g., B 1-B77), C (e.g., C1-C11), D (e.g., D1-D26), E, and G. More preferably, HLA specificities include A1, A2, A3, A11, A23, A24, A28, A30, A33, B7, B8, B35, B44, B53, B60, and B62. In one embodiment, the class I MHC polypeptide is a human class I MHC polypeptide selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In one embodiment, the class I MHC polypeptide is a human class I MHC polypeptide selected from the group consisting of HLA-A, HLA-B, and HLA-C. In another specific embodiment, the class I MHC polypeptide is a murine class I MHC polypeptide selected from the group consisting of H-2K, H-2D, H-2L, H2-IA, H2-IB, H2-IJ, H2- IE, and H2-IC.

[0120] In some embodiments, the viral particle comprises one or more MHC class I α heavy chains. In some embodiments, the MHC class I α heavy chain is fully human. In some embodiments, the MHC class I α heavy chain is humanized. Humanized MHC class I α heavy chains are described, e.g., in U.S. Pat. Pub. Nos. 2013 / 0111617, 2013 / 0185819 and 2014 / 0245467, which are incorporated herein by reference in their entireties. In some embodiments, the MHC class I α heavy chain comprises a human extracellular domain (human α1, α2, and / or α3 domains) and a cytoplasmic domain of another species. In some embodiments, the class I α heavy chain polypeptide is derived from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K, or HLA-L. In some embodiments, the HLA-A sequence can be an HLA-A*0201 sequence or be derived from a HLA-A*0201 sequence. In various aspects, the peptide-MHC can include all the domains of an MHC class I heavy chain.

[0121] In some embodiments, the MHC / peptide complex comprises an extracellular domain comprising SEQ ID NO:20 or a mutant or a derivative thereof. In some embodiments, the MHC / peptide complex comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID 19 172416522.1NO:20. In some embodiments, the MHC / peptide complex comprises a sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions as compared to SEQ ID NO:20. In one embodiment, the MHC / peptide complex comprises SEQ ID NO:20. Provided herein is MHC / peptide complex that at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:20 and that comprises one or more modifications and / or amino substitutions that do not affect the functionality of the peptide binding groove.

[0122] In some embodiments, the “amino acid substitutions” are “mutations” of a polypeptide sequence referenced herein are conservative amino acid substitutions.

[0123] As used herein, the terms “conservative amino acid substitutions” and “conservative modifications” refer to amino acid modifications that do not significantly affect or alter the function and / or activity of the presently disclosed proteins comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the proteins of this disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0124] Amino acids can be classified into groups according to their physicochemical properties such as charge and polarity. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid within the same group. For example, amino acids can be classified by charge: positively-charged amino acids include lysine, arginine, histidine, negatively-charged amino acids include aspartic acid, glutamic acid, neutral charge amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In addition, amino acids can be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; non-polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine.

[0125] In some embodiments, the MHC / peptide complex comprises a transmembrane domain comprising SEQ ID NO:21 or SEQ ID NO:22 or a mutant or a derivative thereof. In some embodiments, the MHC / peptide complex comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:21 or SEQ ID NO:22. In some embodiments, the MHC / peptide complex comprises a sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions 20 172416522.1as compared to SEQ ID NO:21 or SEQ ID NO:22. In some embodiments, the MHC / peptide complex comprises SEQ ID NO:21 or SEQ ID NO:22. In one embodiment, the MHC / peptide complex comprises SEQ ID NO:22.

[0126] In some embodiments, the MHC / peptide complex comprises an intracellular (cytoplasmic) domain comprising SEQ ID NO:23 or a mutant or a derivative thereof. In some embodiments, the MHC / peptide complex comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:23. In some embodiments, the MHC / peptide complex comprises a sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions as compared to SEQ ID NO:23. In one embodiment, the MHC / peptide complex comprises SEQ ID NO:23.

[0127] In some embodiments, the viral particle comprises a β2-microglobulin. In some embodiments, the β2-microglobulin is fully human. In some embodiments, the β2- microglobulin is humanized. Humanized β2-microglobulin polypeptides are described, e.g., in U.S. Pat. Pub. Nos. 2013 / 0111617 and 2013 / 0185819, which are incorporated herein by reference in their entireties. In some embodiments, the MHC class I molecule comprises a mutation in a β2- microglobulin (β2m or Β2M) polypeptide and in the heavy chain sequence so as to affect a disulfide bond between the Β2M and the heavy chain. In some cases, the heavy chain is an HLA and wherein the disulfide bond links one of the following pairs of residues: Β2M residue 12, HLA residue 236; Β2M residue 12, HLA residue 237; Β2M residue 8, HLA residue 234; Β2M residue 10, HLA residue 235; Β2M residue 24, HLA residue 236; Β2M residue 28, HLA residue 232; Β2M residue 98, HLA residue 192; Β2M residue 99, HLA residue 234; Β2M residue 3, HLA residue 120; Β2M residue 31, HLA residue 96; Β2M residue 53, HLA residue 35; Β2M residue 60, HLA residue 96; Β2M residue 60, HLA residue 122; Β2M residue 63, HLA residue 27; Β2M residue Arg3, HLA residue Glyl20; Β2M residue His31, HLA residue Gln96; Β2M residue Asp53, HLA residue Arg35; Β2M residue Trp60, HLA residue Gln96; Β2M residue Trp60, HLA residue Aspl22; Β2M residue Tyr63, HLA residue Tyr27; Β2M residue Lys6, HLA residue Glu232; Β2M residue Gln8, HLA residue Arg234; Β2M residue TyrlO, HLA residue Pro235; Β2M residue Serl l, HLA residue Gln242; Β2M residue Asn24, HLA residue Ala236; Β2M residue Ser28, HLA residue Glu232; Β2M residue Asp98, HLA residue His 192; and Β2M residue Met99, HLA residue Arg234, first linker position Gly 2, Heavy Chain (HLA) position Tyr 84; Light Chain (Β2M) position Arg 12, HLA Ala236; and / or Β2M residue Argl2, HLA residue Gly237. See, e.g., Int. Pat. Appl. Pub. WO2015 / 195531, incorporated herein by reference in its entirety. In some embodiments, 21 172416522.1the β2-microglobulin sequence can comprise a full-length β2-microglobulin sequence. In certain embodiments, the β2-microglobulin sequence lacks the leader peptide sequence. As such, in some configurations, the β2-microglobulin sequence can comprise about 99 amino acids, and can be a mouse β2-microglobulin sequence (e.g., GenBank X01838). In some other configurations, the β2-microglobulin sequence can comprise about 99 amino acids, and can be a human β2-microglobulin sequence (e.g., GenBank AF072097.1).

[0128] In some embodiments, the β2-microglobulin comprises SEQ ID NO:19 or a mutant or a derivative thereof. In some embodiments, the β2-microglobulin comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:19. In some embodiments, the β2-microglobulin comprises a sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions as compared to SEQ ID NO:19. In one embodiment, the β2-microglobulin comprises SEQ ID NO:19.

[0129] Epitopes

[0130] Naturally occurring MHC class I molecules bind peptides derived from proteolytically degraded proteins, especially endogenously synthesized proteins, by a cell. Small peptides obtained accordingly are transported into the endoplasmic reticulum, where they associate with nascent MHC class I molecules before being routed through the Golgi apparatus and displayed on the cell surface for recognition by cytotoxic T lymphocytes.

[0131] As used herein, the peptide displayed on an MHC molecule may also be referred to as an “epitope” or an “antigenic determinant”. The terms “peptide,” “antigenic determinant” and “epitope” as used herein encompass not only those presented naturally by antigen- presenting cells (APCs) but may be any desired peptide so long as it is recognized by an immune cell, e.g., when presented appropriately to the cells of an immune system.

[0132] The epitope is usually between about 5 amino acids to about 35 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 6 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 7 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 8 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 9 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 10 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 11 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 12 amino acids in length. In some embodiments, the 22 172416522.1epitope (by itself or in combination with one or more linkers) is 13 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 14 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 15 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 16 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 17 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 18 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 19 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 20 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 21 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 22 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 23 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 24 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 25 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 26 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 27 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 28 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 29 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 30 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 31 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 32 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 33 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 34 amino acids in length. In some embodiments, the epitope (by itself or in combination with one or more linkers) is 35 amino acids in length.

[0133] In some embodiments, the antigenic determinant amino acid sequence can be that of a peptide that can be presented by an MHC class I molecule. In certain embodiments, the sequence can comprise from about 8 to about 15 contiguous amino acids. In certain embodiments, a peptide sequence can be that of a protein fragment, wherein the protein is a 23 172416522.1derived from, e.g., a portion of, an infectious agent or a cellular protein, such as, for example, a protein expressed by a cancer cell, and wherein the peptide can be bound to the MHC class I heavy chain.

[0134] In one embodiment, the epitope is derived from a bacterial pathogen. Non-limiting examples of bacterial proteins which may be a source of bacterial peptides, e.g., antigenic determinants, that can be used in the viral particles described herein include lipopolysaccharides isolated from gram-negative bacterial cell walls and staphylococcus- specific, streptococcus-specific, pneumococcus-specific (e.g., PspA; see PCT Publication No. WO 1992 / 14488, which is incorporated herein by reference in its entirety), Neisseria gonorrhea-specific, Borrelia-specific (e.g., OspA, OspB, OspC of Borrelia associated with Lyme disease such as Borrelia burgdorferi, Borrelia afzelli, and Borrelia garinii (see, e.g., U.S. Pat. No. 5,523,089; PCT Publication Nos. WO 90 / 04411, WO 91 / 09870, WO 93 / 04175, WO 96 / 06165, WO93 / 08306; PCT / US92 / 08697; Jonsson et al., Heterogeneity of outer membrane proteins in Borrelia burgdorferi: comparison of osp operons of three isolates of different geographic origins. Infect Immun. 1992 May;60(5):1845-53; Johnson et al., Incomplete protection of hamsters vaccinated with unlipidated OspA from Borrelia burgdorferi infection is associated with low levels of antibody to an epitope defined by mAb LA-2. Vaccine. 1995 Aug;13(12):1086-94; Edelman, The Sixth International Conference on Lyme Borreliosis: progress on the development of Lyme disease vaccines. 19-22 June 1994, Bologna, Italy. Vaccine. 1995 Jan;13(1):133-5, all of which are incorporated herein by reference in their entireties), and pseudomonas-specific proteins or peptides. Additional non-limiting examples of bacterial antigens include, e.g., antigens from Neisseria gonorrhea, Mycobacterium tuberculosis, Haemophilus vaginalis, Group B Streptococcus sp., Microplasma hominis, Hemophilus ducreyi, Granuloma inguinale, Lymphopathia venereum, Treponema pallidum, Brucella abortus. Brucella melitensis, Brucella suis, Brucella canis, Campylobacter fetus, Campylobacter fetus intestinalis, Leptospira pomona, Listeria monocytogenes, Brucella ovis, Chlamydia psittaci, Escherichia coli, Actinobacillus equuli, Salmonella abortus ovis, Salmonella abortus equi, Pseudomonas aeruginosa, Corynebacterium equi, Corynebacterium pyogenes, and Actinobaccilus seminis.

[0135] In one embodiment, the epitope is derived from a viral pathogen. In one embodiment, the epitope is derived from a viral protein, including, but not limited to, LCMV gp33, CMV pp65, HIV gag, EBV BMLF1. In some embodiments, the epitope is derived from influenza virus (e.g., surface glycoproteins hemagluttinin (HA) and neuramimidase (NA)); 24 172416522.1immunodeficiency virus (e.g., a human immunodeficiency virus antigens (HIV) such as gp120, gp160, p18 antigen Gag p17 / p24, Tat, Pol, Nef, and Env); herpesvirus (e.g., a glycoprotein from herpes simplex virus (HSV), Marek's Disease Virus, cytomegalovirus (CMV), or Epstein- Barr virus); hepatitis virus (e.g., Hepatitis B surface antigen (HBsAg)); papilloma virus; rous associated virus (e.g., RAV-1 env); infectious bronchitis virus (e.g., matrix and / or preplomer); flavivirus (e.g., a Japanese encephalitis virus (JEV) antigen, a Yellow Fever antigen, or a Dengue virus antigen); Morbillivirus (e.g., a canine distemper virus antigen, a measles antigen, or rinderpest antigen such as HA or F); rabies (e.g., rabies glycoprotein G); parvovirus (e.g., a canine parvovirus antigen); poxvirus (e.g., an ectromelia antigen, a canary poxvirus antigen, or a fowl poxvirus antigen); chicken pox virus (varicella zoster antigen); infectious bursal disease virus (e.g., VP2, VP3, or VP4); Hantaan virus, and mumps virus. In some embodiments, the epitope is derived from CMV, HIV, hepatitis virus, or SARS-CoV-2 virus.

[0136] In one embodiment, the epitope is derived from a pathogenic protozoa. In one embodiment, the epitope is derived from Plasmodium, such as P. falciparum, P. vivax, P. ovale or P. malariae.

[0137] In one embodiment, the epitope is derived from a malaria-specific protein, including, but not limited to, circumsporozoite (CS) protein, Thrombospondin Related Adhesion (Anonymous) protein (TRAP), also called Sporozoite Surface Protein 2 (SSP2), LSA I, hsp70, SALSA, STARP, Hep17, MSA, RAP-1, RAP-2.

[0138] In one embodiment, the epitope is derived from a fungal pathogen. Non-limiting examples of fungal proteins from which antigenic determinants may be isolated include those isolated from candida (e.g., MP65 from Candida albicans), trichophyton, and ptyrosporum.

[0139] In one embodiment, the epitope is a cancer antigen, also referred to herein as a tumor antigen. Non-limiting examples of tumor-associated proteins from which antigenic determinants may be isolated include, e.g., adipophilin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein (“AFP”), ARTC1, B-RAF, BAGE-1, BCLX (L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen (“CEA”), CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin D1, Cyclin-A1, dek-can fusion protein, DKK1, EFTUD2, Elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen (“ETA”), ETV6-AML1 fusion protein, EZH2, FGF5, FLT3-ITD, FN1, G250 / MN / CAIX, GAGE-1,2,8, GAGE-3,4,5,6,7, GAS7, glypican-3, GnTV, gp100 / Pme117, GPNMB, HAUS3, Hepsin, HER-2 / neu, HERV-K- MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDO1, 25 172416522.1IGF2B3, IL13Ralpha2, Intestinal carboxyl esterase, K-ras, Kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1 also known as CCDC110, LAGE-1, LDLR- fucosyltransferaseAS fusion protein, Lengsin, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE- A9, MAGE-C1, MAGE-C2, malic enzyme, mammaglobin-A, MART2, MATN, MC1R, MCSP, mdm-2, ME1, Melan-A / MART- 1, Meloe, Midkine, MMP-2, MMP-7, MUC1, MUC5AC, mucin, MUM-1, MUM-2, MUM-3, Myosin, Myosin class I, N-raw, NA88-A, neo-PAP, NFYC, NY-BR- 1, NY-ESO-1 / LAGE-2, OA1, OGT, OS-9, P polypeptide, p53, PAP, PAX5, PBF, pml-RARalpha fusion protein, polymorphic epithelial mucin (“PEM”), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, secernin 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1, survivin, SYT-SSX1 or - SSX2 fusion protein, TAG-1, TAG-2, Telomerase, TGF-betaRII, TPBG, TRAG- 3, Triosephosphate isomerase, TRP-1 / gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase (“TYR”), VEGF, WT1, XAGE-lb / GAGED2a, Kras, NY-ESO1, MAGE-A3, HPV E2, HPV E6, HPV E7, WT-1 antigen (in lymphoma and other solid tumors), ErbB receptors, Melan A [MART1], gp 100, tyrosinase, TRP-1 / gp 75, and TRP-2 (in melanoma); MAGE-1 and MAGE-3 (in bladder, head and neck, and non-small cell carcinoma); HPV EG and E7 proteins (in cervical cancer); Mucin [MUC- 1] (in breast, pancreas, colon, and prostate cancers); prostate-specific antigen [PSA] (in prostate cancer); carcinoembryonic antigen [CEA] (in colon, breast, and gastrointestinal cancers), and such shared tumor-specific antigens as MAGE-2, MAGE-4, MAGE-6, MAGE-10, MAGE-12, BAGE- 1, CAGE-1,2,8, CAGE-3 TO 7, LAGE-1, NY-ESO- 1 / LAGE-2, NA-88, GnTV, TRP2-INT2. In some embodiments, the protein is a neo-antigen. In some embodiments, the protein is a tumor specific antigen.

[0140] In some embodiments, the epitope is a helminth parasite peptide or an ectoparasite peptide.

[0141] In some embodiments, the peptide comprises any one of SEQ ID NOs:1-3, SEQ ID NO:94, or SEQID NO:95 or a mutant or derivative thereof. In some embodiments, the peptide comprises a sequence that is substantially identical to any one of SEQ ID NOs:1-3, SEQ ID NO:94, or SEQID NO:95. In some embodiments, the peptide comprises a sequence that has one or more amino substitutions as compared to any one of SEQ ID NOs:1-3, SEQ ID NO:94, or SEQID NO:95. 26 172416522.1

[0142] In some embodiments, the viral particle comprises more than one type of MHC / peptide complex. For example, provided is a recombinant viral particle, the recombinant viral particle comprising a lipid envelope and: (1) a first MHC / peptide complex comprising a first fusion polypeptide comprising (a) a first class I MHC polypeptide or a fragment, mutant, or derivative thereof, (b) a first β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof, (c) a first epitope, and (d) a first lipid envelope anchor; and (2) a second MHC / peptide complex comprising a second fusion polypeptide comprising (a) a second class I MHC polypeptide or a fragment, mutant, or derivative thereof, (b) a second β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof, (c) a second epitope, wherein the first and the second epitope are different and (d) a second lipid envelope anchor; and (3) optionally, a third fusion polypeptide comprising (a) a co-stimulatory domain and (b) a third lipid envelope anchor.

[0143] The viral particle can comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 types of MHCs that each display a different epitope. In some embodiments, some of the different epitopes are derived from the same polypeptide. In some embodiments, at least some of the different epitopes are derived from different polypeptides. In some embodiments, at least some of the different epitopes are derived from the same organism (e.g., the same pathogen). Such viral particles that display multiple peptides are particularly useful in the case of pathogens that have a high mutation rate.

[0144] Linkers

[0145] In some embodiments, the individual components of a fusion polypeptide are linked directly to each other without intervening sequences. In some embodiments, one or more of the individual components of a fusion polypeptide are linked to each by a linker.

[0146] Suitable linkers used herein can be of any of a number of suitable lengths, such as from 1 amino acid to 25 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0147] In some embodiment, the linker is a flexible linker. Non-limiting examples of linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n (where n is an integer of at least one) or any of the linkers in Table 1), glycine-alanine polymers, 27 172416522.1alanine-serine polymers, and other flexible linkers known in the art (see, e.g., Chichili et al., Linkers in the structural biology of protein-protein interactions. Protein Sci. 2013 Feb;22(2):153-67, which is incorporated herein in its entirety). Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Predicting Three-Dimensional Structures of Oligopeptides. In Reviews in Computational Chemistry, John Wiley & Sons, Ltd., 1992, Vol 3; pp 73−142).

[0148] In some embodiments, the linker is a rigid linker. Rigid polypeptide linkers comprise a sequence of amino acids that effectively separates protein domains by maintaining a substantially fixed distance / spatial separation between the domains, thereby reducing or substantially eliminating unfavorable interactions between such domains. Rigid polypeptide linkers thus may be employed where it is desired to minimize the interaction between the different domains of a fusion protein. Rigid peptide linkers include, but are not limited to, peptide linkers rich in proline, and peptide linkers having an inflexible helical structure, such as an a-helical structure.

[0149] Flexible or rigid linkers can be used, or a combination thereof.

[0150] In some embodiments, a linker polypeptide includes a cysteine residue that can form a disulfide bond with a cysteine residue present in a second polypeptide. Provided herein are also linkers that are combinations or that contain repeats of any of the linkers disclosed herein. In some embodiments, the linker comprises any one of SEQ ID NOs:4-17 or SEQ ID NOs:27- 55, or combinations thereof. Table 1. Selected amino acid sequences. Comment SEQ Amino acid sequence ID172416522.1Comment SEQ Amino acid sequence ID NO 1, 9, 8) 1,29 172416522.1Comment SEQ Amino acid sequence ID NO 8) 1, 8) 1, 0, 0, 9, 2,30 172416522.1Comment SEQ Amino acid sequence ID NO V T F H G R W L S R I N V E L S G F I N172416522.1Comment SEQ Amino acid sequence ID NO V E L S G F I N V E L S L G V A C Q K S32 172416522.1Comment SEQ Amino acid sequence ID NO172416522.1Comment SEQ Amino acid sequence ID NO172416522.1Comment SEQ Amino acid sequence ID NO

[0151] Lipid Envelope Anchors

[0152] Provided herein are recombinant viral particles comprising a lipid envelope and fusion polypeptides embedded in the envelope.

[0153] In one embodiment, provided is a fusion protein comprising a lipid envelope anchor. For example, provided is a recombinant viral particle, the viral particle comprising a lipid envelope and (1) an MHC / peptide complex comprising a first fusion polypeptide comprising 35 172416522.1(a) a class I MHC polypeptide or a fragment, mutant, or derivative thereof, (b) a β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof, (c) an epitope, and (d) a first lipid envelope anchor and (2) a second fusion polypeptide comprising (a) a co- stimulatory domain and (b) a second lipid envelope anchor. Provided is a recombinant viral particle, the viral particle comprising a lipid envelope and (1) an MHC / peptide complex comprising a first fusion polypeptide comprising (a) a class I MHC polypeptide or a fragment, mutant, or derivative thereof, (b) a β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof, (c) an epitope, (d) a first lipid envelope anchor, and (e) an optional first intracellular domain and (2) a second fusion polypeptide comprising (a) a co-stimulatory domain, (b) a second lipid envelope anchor, and (c) an optional second intracellular domain. As discussed herein, the fusion proteins may comprise additional linker sequences linking two or more of the individual components of the fusion proteins.

[0154] As used herein, a “lipid envelope anchor” or “membrane-anchoring polypeptide” allows conjugation of a fusion polypeptide to a viral particle disclosed herein. The membrane- anchoring polypeptide may comprise one or more transmembrane domains. In some embodiments, the membrane-anchoring polypeptide can comprise sequences derived from one or more naturally occurring or artificial proteins, including transmembrane proteins.

[0155] In one embodiment, the transmembrane domain comprises one or more alpha helices. In one embodiment, the transmembrane domain is derived from a multi-pass transmembrane protein. In some embodiments, the transmembrane domain is a multi-pass protein comprising alpha helices and / or one or more protein beta sheets.

[0156] In some embodiment, the transmembrane domain comprises SEQ ID NO:21 or SEQ ID NO:22 or a mutant or a derivative thereof. In some embodiment, the transmembrane domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:21 or SEQ ID NO:22. In some embodiment, the transmembrane domain comprises SEQ ID NO:21 or SEQ ID NO:22. In one embodiment, the transmembrane domain comprises SEQ ID NO:22. In some embodiments, the transmembrane domain comprises a sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions as compared to SEQ ID NO:21 or SEQ ID NO:22.

[0157] Co-Stimulatory Domains

[0158] In embodiments, provided are viral particles comprising both a membrane-anchored MHC / peptide complex and a membrane-anchored co-stimulatory domain. 36 172416522.1

[0159] As discussed herein, during an immune response, two signals provided by antigen- presenting cells (APCs) are required to activate antigen-specific naïve T cells. The first signal is an antigen-specific signal, resulting from the TCR’s recognition of its cognate peptide:MHC complex. The second signal is a co-stimulatory signal conveyed through one of several receptors on the T cell’s surface. For example, B7 located on the APC’s surface, stimulates CD28, on the T cell’s surface. Additional growth and differentiation signals are provided by cytokines, such as IL-2 and IL-15. Accordingly, as used herein, a “co-stimulatory domain” located on a viral particle is a polypeptide that binds to a receptor on the surface of a T cell and thereby contributing to T cell activation.

[0160] Co-stimulatory domains are known in the art. In some embodiments, the co- stimulatory domain binds to a molecule expressed on T cell as shown in Table 2. In some embodiments, the co-stimulatory domain is a ligand shown in Table 2, or a mutant or a derivative thereof.

[0161] In one embodiment, the co-stimulatory domain is a CD28 agonist. In some embodiments, the co-stimulatory domain is an agonist of any of the molecule expressed on T cell as shown in Table 2.

[0162] In one embodiment, the co-stimulatory domain comprises SEQ ID NO:25 or a mutant or a derivative thereof. In some embodiment, the co-stimulatory domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:21 or SEQ ID NO:25. In some embodiment, the co-stimulatory domain comprises SEQ ID NO:25. In some embodiments, the co-stimulatory domain comprises a sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions as compared to SEQ ID NO:25. The co-stimulatory domain may be expressed with or without a signal peptide. Table 2. Examples of co-stimulatory interactions. Molecule expressed on T cell Ligand D2 D D172416522.1

[0163] Signal Peptides

[0164] Provided herein are fusion proteins comprising a signal peptide, also referred to herein as a signal sequence. Sequences of suitable signal peptides are known in the art. A person skilled in the art may employ any signal sequence compatible with the compositions and methods disclosed herein. See, e.g., Owji et al., A comprehensive review of signal peptides: Structure, roles, and applications. Eur J Cell Biol.2018 Aug;97(6):422-441; O'Neill P, Mistry RK, Brown AJ, James DC. Protein-Specific Signal Peptides for Mammalian Vector Engineering. ACS Synth Biol. 2023 Aug 18;12(8):2339-2352, which are incorporated herein in their entirety.

[0165] In some embodiments, the signal peptide comprises any one of SEQ ID NOs:18 or 56-93 or a mutant or a derivative thereof. In some embodiments, the signal peptide comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:18 or 56-93. In some embodiments, the signal peptide comprises a sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions as compared to any one of SEQ ID NOs:18 or 56-93.

[0166] In some embodiments, the signal peptide comprises SEQ ID NO:18 or a mutant or a derivative thereof. In some embodiments, the signal peptide comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:18. In some embodiments, the signal peptide comprises SEQ ID NO:18. In some embodiments, the signal peptide comprises a sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions as compared to SEQ ID NO:18.

[0167] In one embodiments, the signal sequence is an beta 2 Microglobulin or IgG signal sequence or derivative thereof.

[0168] Fusion Proteins

[0169] Provided herein are membrane-anchored fusion proteins and viral particles comprising membrane anchored fusion proteins. In some embodiments, a fusion protein herein comprises an MHC / peptide complex or a co-stimulatory domain.

[0170] Provided is a recombinant viral particle comprising a lipid envelope and an MHC / peptide complex comprising a fusion polypeptide comprising (a) a class I MHC polypeptide or a fragment, mutant, or derivative thereof, (b) a β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof, (c) an epitope, (d) a lipid envelope anchor, and (e) optionally, a cytoplasmic domain. Provided is a recombinant viral particle comprising a lipid 38 172416522.1envelope and an MHC / peptide complex comprising a fusion polypeptide comprising (a) a class I MHC polypeptide or a fragment, mutant, or derivative thereof, (b) a β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof, (c) an epitope, (d) a transmembrane domain, and (e) optionally, a cytoplasmic domain.

[0171] Provided is a recombinant viral particle comprising a lipid envelope comprising a fusion polypeptide comprising (a) a co-stimulatory domain, (b) a lipid envelope anchor, and (c) optionally, a cytoplasmic domain. Provided is a recombinant viral particle comprising a lipid envelope comprising a fusion polypeptide comprising (a) a co-stimulatory domain, (b) a transmembrane domain, and (c) optionally, a cytoplasmic domain.

[0172] In one embodiment, provided is a recombinant viral particle, the viral particle comprising a lipid envelope and (1) an MHC / peptide complex comprising a first fusion polypeptide comprising (a) a class I MHC polypeptide or a fragment, mutant, or derivative thereof, (b) a β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof, (c) an epitope, (d) a first lipid envelope anchor, and (e) optionally, a first cytoplasmic domain, and (2) a second fusion polypeptide comprising (a) a co-stimulatory domain, (b) a second lipid envelope anchor, and (c) optionally, a second cytoplasmic domain. The first and / or the second lipid envelope anchor may be a transmembrane domain. The first and / or the second lipid envelope anchor may be the same. The first and / or the second lipid envelope anchor may be different. The first and the second cytoplasmic domain may be the same. The first and the second cytoplasmic domain may be different.

[0173] In some embodiments, the class I MHC polypeptide comprises any class I MHC polypeptide disclosed herein, or a fragment, mutant, or derivative thereof.

[0174] The class I MHC polypeptide or a fragment, mutant, or derivative thereof may be derived from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G. The class I MHC polypeptide or a fragment, mutant, or derivative thereof may be derived from H-2K, H-2D, H- 2L, H-2Q, H-2M or H-2T.

[0175] The class I MHC polypeptide or a fragment, mutant, or derivative thereof may comprise an extracellular domain derived from HLA-A, HLA-B, HLA-C, HLA- E, HLA-F, or HLA-G. The class I MHC polypeptide or a fragment, mutant, or derivative thereof may comprise an extracellular domain derived from H-2K, H-2D, H-2L, H-2Q, H-2M or H-2T.

[0176] In one embodiment, the class I MHC polypeptide or a fragment, mutant, or derivative thereof comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:20. In 39 172416522.1one embodiment, the class I MHC polypeptide or a fragment, mutant, or derivative thereof comprise SEQ ID NO:20.

[0177] In some embodiments, the β2 microglobulin comprises any β2 microglobulin polypeptide disclosed herein, or a fragment, mutant, or derivative thereof.

[0178] In some embodiments, the β2 microglobulin comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:19. In some embodiments, the β2 microglobulin comprises SEQ ID NO:19.

[0179] In some embodiments, the epitope comprises any epitope disclosed herein, or a fragment, mutant, or derivative thereof. The epitope may be derived from a bacterial, viral, fungal, or protozoan polypeptide. In some embodiments, the epitope comprises any one of SEQ ID NOs:1-3, SEQ ID NO:94, or SEQID NO:95, or a fragment, mutant, or derivative thereof. In embodiments, the epitope is between 5 to 20 amino acids long. In some embodiments, the epitope is derived from a cytomegalovirus (CMV) polypeptide, a human immunodeficiency virus (HIV) polypeptide, a SARS-CoV-2 virus polypeptide, or a hepatitis virus polypeptide. In one embodiment, the epitope is derived from HIV polypeptide gp120, HIV-1 polypeptide p17, or melanoma antigen recognized by T cells 1 (MART-1). In some embodiments, the epitope is a cancer antigen or fragment thereof.

[0180] In some embodiments, the lipid envelope anchor (e.g., the first and / or the second lipid envelope anchor) is a transmembrane domain. In embodiments, the transmembrane domain comprises a transmembrane domain disclosed herein, or a fragment, mutant, or derivative thereof. In one embodiment, the transmembrane domain is derived from human HLA-A*0201 MHC. In embodiments, the transmembrane domain comprises the transmembrane domain of any polypeptide disclosed herein. In some embodiments, the transmembrane domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:22. In some embodiments, the transmembrane domain comprises SEQ ID NO:22.

[0181] The cytoplasmic domain (e.g., the first and / or the second cytoplasmic domain) can be any cytoplasmic domain compatible with the embodiments of the instant disclosure. In embodiments, the cytoplasmic domain comprises a cytoplasmic domain disclosed herein, or a fragment, mutant, or derivative thereof. In embodiments, the cytoplasmic domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at 40 172416522.1least 99% identical to SEQ ID NO:23. In one embodiment, the cytoplasmic domain comprises SEQ ID NO:23.

[0182] In some embodiments, the co-stimulatory domain is a 4-1BBL polypeptide, a B7-1 polypeptide, a B7-2 polypeptide, an ICOS-L polypeptide, an OX-40L polypeptide, a CD80 polypeptide, a CD70 peptide, a CD86 polypeptide, a CD40L polypeptide, a CD40 polypeptide. a. In some embodiments, the co-stimulatory domain is a CD28 agonist, an ICOS agonist, a 4- 1BB agonist, a OX40 agonist, a CD27 agonist, a CD40 agonist, a CD40L agonist,. In one embodiment, the co-stimulatory domain is a CD28 agonist. The co-stimulatory domain may be a scFv. In some embodiments, the co-stimulatory domain is a CD28 agonist that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:25. In one embodiment, the co-stimulatory domain comprises SEQ ID NO:25.

[0183] In one embodiment, provided is a recombinant viral particle comprising a lipid envelope and a first and / or a second fusion protein, wherein: (1) the first fusion polypeptide comprises from N-terminus to C-terminus: (a) an epitope; (b) a β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof; (c) a class I MHC polypeptide or a fragment, mutant, or derivative thereof; (d) a transmembrane domain; and (e) optionally, a first cytoplasmic domain; and / or (2) the second fusion polypeptide comprises from N-terminus to C-terminus: (a) a co-stimulatory domain; (b) a transmembrane domain; and (c) optionally, a second cytoplasmic domain.

[0184] The first and the second fusion polypeptide may be covalently linked.

[0185] In one embodiment, provided is a recombinant viral particle comprising a lipid envelope and a first and / or a second fusion protein, wherein: (1) the first fusion polypeptide comprises from N-terminus to C-terminus: (a) an epitope; (b) optionally, a linker; (c) a β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof; (d) optionally, a linker; (e) a class I MHC polypeptide or a fragment, mutant, or derivative thereof; 41 172416522.1(f) optionally, a linker; (g) a transmembrane domain; (h) optionally, a linker; and (i) optionally, a first cytoplasmic domain; and / or (2) the second fusion polypeptide comprises from N-terminus to C-terminus: (a) a co-stimulatory domain; (b) optionally, a linker; (c) a second lipid envelope anchor; (d) optionally, a linker; and (e) optionally, a second cytoplasmic domain.

[0186] In some embodiments, any of the linkers in the fusion polypeptide is a polypeptide linker. In some embodiments, any of the linkers in the fusion polypeptide is a disulfide bond. In some embodiments, any of the linkers in the fusion polypeptide is a flexible linker. In some embodiments, the flexible linker predominantly consists of glycines, serines, and alanines. In some embodiments, the flexible linker comprises one or more repetitions of any one of SEQ ID NOs:4-17 or SEQ ID NOs:27-55. In one embodiment, the flexible linker comprises SEQ ID NO:16.

[0187] In one embodiment, provided is a recombinant viral particle comprising a lipid envelope and a first and / or a second fusion protein, wherein: (1) the first fusion polypeptide comprises from N-terminus to C-terminus: (a) a signal peptide; (b) an epitope; (c) a β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof; (d) a class I MHC polypeptide or a fragment, mutant, or derivative thereof; (e) a transmembrane domain; and (f) optionally, a first cytoplasmic domain; and / or (2) the second fusion polypeptide comprises from N-terminus to C-terminus: (a) a signal peptide; (b) a co-stimulatory domain; (c) a transmembrane domain; and (d) optionally, a second cytoplasmic domain.

[0188] In one embodiment: (1) the first fusion polypeptide comprises from N-terminus to C-terminus: 42 172416522.1(a) a signal peptide; (b) an epitope; (c) optionally, a linker; (d) a β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof; (e) optionally, a linker; (f) a class I MHC polypeptide or a fragment, mutant, or derivative thereof; (g) optionally, a linker; (h) a transmembrane domain; (i) optionally, a linker; and (j) optionally, a first cytoplasmic domain; and / or (2) the second fusion polypeptide comprises from N-terminus to C-terminus: (a) a signal peptide; (b) a co-stimulatory domain; (c) optionally, a linker; (d) a second lipid envelope anchor; (e) optionally, a linker; and (f) optionally, a second cytoplasmic domain.

[0189] The signal peptide can be any signal sequence compatible with the embodiments of the instant disclosure. In one embodiment, the signal peptide is derived from the beta 2 microglobulin signal peptide. In some embodiments, the signal peptide is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:18. In one embodiment, the signal peptide comprises SEQ ID NO:18.

[0190] Nucleic Acids

[0191] Also provided herein are nucleic acids (or pairs of nucleic acids) encoding any of the polypeptide disclosed herein, including the fusion polypeptides disclosed herein. Further provided are vectors comprising such nucleic acids and host cells comprising such nucleic acids or vectors.

[0192] The term “nucleic acid” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double- or multi- stranded DNA or RNA, genomic DNA, cDNA, DNA- RNA hybrids, or a polymer comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. 43 172416522.1

[0193] Provided herein is a nucleic acid that encodes a fusion protein disclosed herein. Provided herein is a set of nucleic acids encoding for two or more fusion proteins disclosed herein. The nucleic acids disclosed herein may be DNA, cDNA, RNA, synthetically produced DNA or RNA, or a recombinantly produced chimeric nucleic acid molecule comprising any of those polynucleotides either alone or in combination.

[0194] “Vector” means a recombinant plasmid, yeast artificial chromosome (YAC), mini chromosome, DNA mini-circle or virus (including virus derived sequences) that comprises a polynucleotide to be delivered into a host cell, either in vitro or in vivo. A “vector” includes, but is not limited to, a viral vector, a plasmid, an RNA vector or a linear or circular DNA or RNA molecule which may consist of a chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic acids. In some embodiments, the employed vectors are those capable of autonomous replication (episomal vector) and / or expression of nucleic acids to which they are linked (expression vectors). Large numbers of suitable vectors are known to those of skill in the art and commercially available.

[0195] Also provided herein are vectors comprising nucleic acids disclosed herein.

[0196] Methods of Producing Viral Particles

[0197] Other approaches that have been developed for the presentation of MHC / peptide complexes to T cells, such as Fc domains or artificial nanoparticles, often require the production and purification of proteins, which is time consuming and expensive. Moreover, these proteins are often difficult to synthesize and / or correctly fold at high yields. In contrast, the compositions and methods disclosed herein exploit the very efficient processes that viruses have developed of hijacking a host cell’s production machinery to induce the production of viral particles.

[0198] Provided herein is a method of producing a viral particle that is capable of binding to a TCR, the method comprising culturing a packaging cell (also referred to herein as a producer cell) in conditions sufficient for the production of a plurality of viral particles, wherein the packaging cell comprises one or more plasmids comprising (i) one or more viral elements involved in the assembly of the viral particle, (ii) a nucleotide sequence encoding a membrane- anchored MHC / peptide complex disclosed herein and (iii) optionally a sequence encoding a membrane-anchored co-stimulatory molecule. After transfection of the packaging cell, the cell produces viral particles that are released into the culture supernatant. In some embodiments, the method further comprises collecting the viral particles. In some embodiments, the 44 172416522.1collecting step comprises one or more of the following steps: clearing cell debris, treating the supernatant containing viral particles with DNase I and MgCl2, concentrating viral particles, and / or purifying the viral particles.

[0199] In some embodiments, the packaging cell is transfected with a plasmid comprising one or more of a Psi (ψ) packaging signal, a 5' Long Terminal Repeat (LTR), a 3' LTR, a Rev Response Element (RRE), a Central Polypurine Tract (cPPT), a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), a Unique 3' (U3), a Repeat (R) region, a Unique 5' (U5), a 3' LTR, a 3’LTR with the U3 element deleted (e.g., to make a lentivirus non- replicative), a Trans-activating response element (TAR), a derivative or mutant of any thereof, or any combination thereof. In some embodiments, the packaging cell is transfected with one or more plasmids comprising one of more or (a) GAG, (b) POL, and (c) TAT and / or REV lentiviral or retroviral elements, each of which may be considered involved with the assembly of the viral particle.

[0200] In some embodiments, the packaging cell is transfected with a plasmid comprising one or more viral elements encoding Human Immunodeficiency Virus (HIV) component(s), Bovine Immunodeficiency Virus (BIV) component(s), Feline Immunodeficiency Virus (FIV) component(s), Simian Immunodeficiency Virus (SIV) component(s), Equine Infectious Anemia Virus (EIAV) component(s), Murine Stem Cell Virus (MSCV) component(s), Murine Leukemia Virus (MLV) component(s), Avian leukosis virus (ALV) component(s), Feline leukemia virus (FLV) component(s), Bovine leukemia virus (BLV) component(s), Human T- lymphotropic virus (HTLV) component(s), feline sarcoma virus component(s), avian reticuloendotheliosis virus component(s), caprine arthritis encephalitis virus (CAEV) component(s), and / or Visna-Maedi virus (VMV) component(s).

[0201] The plasmids / vectors used for viral particle production can be introduced into the packaging cells using methods well known in the art such as, e.g., electroporation, Ca2+- mediated transfection or via liposomes, non- liposomal compounds, or nucleofection into cells.

[0202] Packaging cells useful for production of the viral particles described herein include, e.g., animal cells permissive for the virus, or cells modified to be permissive for the virus; or the packaging cell construct, for example, with the use of a transformation agent such as calcium phosphate. Non-limiting examples of packaging cell lines useful for producing viral particles described herein include, e.g., human embryonic kidney 293 (HEK-293) cells (e.g., American Type Culture Collection [ATCC] No. CRL-1573), HEK-293 cells that contain the SV40 Large T- antigen (HEK-293T or 293T), HEK293T / 17 cells, human sarcoma cell line 45 172416522.1HT-1080 (CCL-121), lymphoblast-like cell line Raj i (CCL-86), glioblastoma-astrocytoma epithelial-like cell line U87- MG (HTB-14), T-lymphoma cell line HuT78 (TIB-161), NIH / 3T3 cells, Chinese Hamster Ovary cells (CHO) (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), HeLa cells (e.g., ATCC No. CCL- 2), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), HLHepG2 cells, CAP cells, CAP-T cells, and the like. L929 cells, the FLY viral packaging cell system outlined in Cosset et al (1995) J Virol 69,7430-7436, NS0 (murine myeloma) cells, human amniocytic cells (e.g., CAP, CAP-T), yeast cells (including, but not limited to, S. cerevisiae, Pichia pastoris), plant cells (including, but not limited to, Tobacco NTl, BY-2), insect cells (including but not limited to SF9, S2, SF21, Tni (e.g. High 5)) or bacterial cells (including, but not limited to, E. coli).

[0203] For additional packaging cells and systems, see for example, Polo et al., Stable alphavirus packaging cell lines for Sindbis virus and Semliki Forest virus-derived vectors, Proc Natl Acad Sci USA. 1999 Apr 13;96(8):4598-603, incorporated herein by reference in its entirety. Methods of packaging include using packaging cells that permanently express the viral components, or by transiently transfecting cells with plasmids.

[0204] Any method known to one skilled in the art may be used for large scale production of viral particles, packaging cells, and vector constructs described herein. For example, master and working seed stocks may be prepared under GMP conditions in qualified primary CEFs or by other methods. Packaging cells may be plated on large surface area flasks, grown to near confluence and viral particles purified. Cells may be harvested, and viral particles released into the culture media isolated and purified, or intracellular viral particles released by mechanical disruption (cell debris can be removed by large-pore depth filtration and host cell DNA digested with endonuclease). Virus particles may be subsequently purified and concentrated by tangential-flow filtration, followed by diafiltration. The resulting concentrated bulk may be formulated by dilution with a buffer containing stabilizers, filled into vials, and lyophilized. Compositions and formulations may be stored for later use. For use, lyophilized viral particles may be reconstituted by addition of diluent.

[0205] Cells 46 172416522.1

[0206] Provided is a host cell comprising a nucleic acid molecule described herein, or a vector described herein. Provided herein is a cell that produces a viral particle disclosed herein. Cells include packaging cells disclosed herein.

[0207] The cell can be isolated. The cell may be a mammalian cell. The cell may be human.

[0208] Also provided herein are immune cells that have been contacted with a viral particle disclosed herein. Provided herein are immune cells that have been activated by a viral particle disclosed herein. The immune cell may be a T cell. The terms “T cell” and “T lymphocyte” are interchangeable and used synonymously herein. The T cell may be activated. The T cell may be genetically engineered to express a TCR or a chimeric antigen receptor.

[0209] Pharmaceutical Compositions

[0210] In one embodiment, provided herein is a pharmaceutical composition comprising a viral particle disclosed herein and a pharmaceutically acceptable carrier. Provided herein is a pharmaceutical composition comprising a therapeutically effective number of an immune cell, such as a T cell that has been activated by exposure to a viral particle disclosed herein, disclosed herein and a pharmaceutically acceptable carrier. Provided are pharmaceutical compositions that comprise substantially isolated / purified T cells and a pharmaceutically acceptable carrier in a form suitable for administration to a subject.

[0211] Pharmaceutically-acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. The pharmaceutical compositions may generally be formulated in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0212] The terms “pharmaceutically acceptable,” “physiologically tolerable,” as referred to compositions, carriers, diluents, and reagents, are used interchangeably and include materials are capable of administration to or upon a subject without the production of undesirable physiological effects to the degree that would prohibit administration of the composition. For example, “pharmaceutically-acceptable excipient” includes an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use.

[0213] Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. The use of such media 47 172416522.1and compounds for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or compound is incompatible with the compositions disclosed herein, use of the media or compound in the compositions disclosed herein is contemplated. In some embodiments, a second therapeutic agent, such as an anti-cancer or anti-tumor, can also be incorporated into pharmaceutical compositions.

[0214] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate-buffered saline (PBS). The composition may be sterile and fluid to the extent that easy syringeability exists. In embodiments, the compositions disclosed herein are stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, e.g., water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, e.g., by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

[0215] In some embodiments, the pharmaceutical composition further includes a cryo- protectant (e.g., glycerol, DMSO, PEG).

[0216] Methods

[0217] Provided herein are methods of using the compositions disclosed herein. The viral particles described herein can be used for various therapeutic applications (in vivo and ex vivo) and as research tools, e.g., in vitro.

[0218] As discussed herein, a T cell population of interest (i.e., the T cell population that the person skilled in the art wishes to expand) may specifically bind to the epitope displayed by a viral particle disclosed herein (wherein the epitope is displayed in the context of the particle’s MHC). Accordingly, provided is a method of activating a T cell comprising a TCR, the method comprising contacting the T cell with a viral particle disclosed herein, wherein the viral particle comprises a lipid envelope and (1) an MHC / peptide complex comprising a first fusion polypeptide comprising (a) a class I major histocompatibility (MHC) polypeptide or a fragment, mutant, or derivative thereof, (b) a β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof, (c) an epitope, wherein the TCR of the T cell binds to the epitope, 48 172416522.1(d) a first lipid envelope anchor; and (e) optionally a first cytoplasmic domain and, optionally (2) a second fusion polypeptide comprising (a) a co-stimulatory domain, (b) a second lipid envelope anchor, (c) optionally a second cytoplasmic domain. The T cell may be a T cell that has been isolated from a patient and genetically engineered to express the TCR that binds to the epitope. The engineered T cell may be reintroduced into the same or a different patient.

[0219] “Activation of a T cell” may refer to induction of signal transduction pathways in the T cell resulting in production of cellular products (e.g., interleukin-2) by that T cell. Methods of measuring T cell activation are known in the art. See, e.g., Phetsouphanh et al., Detecting Antigen-Specific T Cell Responses: From Bulk Populations to Single Cells. Int J Mol Sci. 2015 Aug 12;16(8):18878-93, which is incorporated herein in its entirety. T cell activation can be measured, for example, by detecting activation marker CD25 by flow cytometry. Activation can also be measured by, for example, measuring the amount of IL-2 produced by a T cell after a peptide / MHC complex has bound to the TCR. T cell activation may be determined, e.g., by measuring changes in the level of expression of cytokines and / or T cell activation markers, and / or the induction of antigen-specific proliferating cells. Techniques known to those of skill in the art, including, but not limited to, immunoprecipitation followed by Western blot analysis, ELISAs, flow cytometry, Northern blot analysis, and RT- PCR can be used to measure the expression cytokines and T cell activation markers. Cytokine release may be measured by measuring secretion of cytokines including, but not limited to, Interleukin-2 (IL-2), Interleukin- 4 (IL-4), Interleukin-6 (IL-6), Interleukin-12 (IL-12), Interleukin-16 (IL-16), PDGF, TGF-α, TGF-β, TNF-α, TNF-β, GCSF, GM-CSF, MCSF, IFN- α, IFN-β, IFN-γ, TFN-γ, IGF-I, and IGF-II.

[0220] In some embodiments, the activated T cells are further cultured. The term “culturing” or “expanding” refers to maintaining or cultivating cells under conditions in which they can proliferate and avoid senescence. For example, cells may be cultured in media optionally containing one or more growth factors, i.e., a growth factor cocktail. In some embodiments, the cell culture medium is a defined cell culture medium. The cell culture medium may include neoantigen peptides. Stable cell lines may be established to allow for the continued propagation of cells.

[0221] Provided is a method of inducing expression of pro-inflammatory cytokines in a T cell comprising a TCR, the method comprising contacting the T cell with a viral particle disclosed herein, wherein the viral particle comprises a lipid envelope and (1) an MHC / peptide complex comprising a first fusion polypeptide comprising (a) a class I MHC polypeptide or a 49 172416522.1fragment, mutant, or derivative thereof, (b) a β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof, (c) an epitope, wherein the TCR of the T cell binds to the epitope, (d) a first lipid envelope anchor; and (e) optionally a first cytoplasmic domain and, optionally (2) a second fusion polypeptide comprising (a) a co-stimulatory domain, (b) a second lipid envelope anchor, (c) optionally a second cytoplasmic domain. The T cell may be a T cell that has been isolated from a patient and genetically engineered to express the TCR that binds to the epitope. The engineered T cell may be reintroduced into the same or a different patient.

[0222] Methods of measuring pro-inflammatory cytokines are known in the art. For example, pro-inflammatory cytokines include, but are not limited, to IL-1, IL-2, IL-6, IL-8, IL- 12, IL-17, IL-18, IFN-γ, TNF-α, perforin, and granzyme B. Expression of these cytokines can be measured on the nucleic acid level (e.g., by RT-PCT) or on the protein level (e.g., by ELISA).

[0223] Provided is a method of expanding a population of T cells each comprising a TCR, the method comprising contacting the population of T cells with a viral particle disclosed herein, wherein the viral particle comprises a lipid envelope and (1) an MHC / peptide complex comprising a first fusion polypeptide comprising (a) a class I MHC polypeptide or a fragment, mutant, or derivative thereof, (b) a β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof, (c) an epitope, wherein the TCR of the T cell binds to the epitope, (d) a first lipid envelope anchor; and (e) optionally a first cytoplasmic domain and, optionally (2) a second fusion polypeptide comprising (a) a co-stimulatory domain, (b) a second lipid envelope anchor, (c) optionally a second cytoplasmic domain. The T cell may be a T cell that has been isolated from a patient and genetically engineered to express the TCR that binds to the epitope. The engineered T cell may be reintroduced into the same or a different patient.

[0224] The methods provided herein may be performed in vivo, in vitro, or ex vivo.

[0225] The T cells may further be exposed to one or more cytokines that enhance selective differentiation and / or expansion.

[0226] Merely to illustrate, in one embodiment, the method of activating a T cell comprising a TCR is performed in vivo by administering to a patient the viral particle disclosed herein, wherein the viral particle activates the patient’s own T cells that bind to the epitope presented on the viral particle. In one embodiment, the method is performed ex vivo by removing T cells from a patient, activating the patient’s own T cells that bind to the epitope presented on the viral particle, optionally expanding the activated T cells, optionally washing the activated T cells, and reintroducing the activated T cells into the same patient or a different patient. In 50 172416522.1another embodiment, the T cells are activated in vitro by contacting the T cells with the viral particles.

[0227] In some embodiments, the viral particles disclosed herein are used in cell therapy with T cells engineered to express a T cell receptor (TCR), wherein the T cells have been modified to express specific TCRs that can recognize and target, for example, cancer cells or cells infected with viruses. This therapy is a form of adoptive cell therapy (ACT), where T cells are collected from a patient, engineered ex vivo to express TCRs with desired antigen specificity, and then infused back into the patient to target and kill diseased cells.

[0228] In some embodiments, T cells are autologous, allogeneic, syngeneic, or xenogeneic.

[0229] T cells to be used in the methods disclosed herein can be obtained from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, splenic tissue, and tumors. T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the person skilled in the art, such as the separation using a neutral, highly branched, high-mass, hydrophilic polysaccharide which dissolves readily in aqueous solutions. Circulating blood cells of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T lymphocytes, monocytes, granulocytes, B lymphocytes, other nucleated white blood cells, red blood cells, and platelets. The cells harvested by apheresis can be washed to remove the plasma fraction and place the cells in a suitable buffer or medium for the subsequent processing steps. The cells may be washed with phosphate-buffered saline (PBS). Alternatively, the wash solution may lack calcium and may lack magnesium or may lack many, if not all, divalent cations. As those of ordinary skill in the art would readily appreciate, a washing step can be achieved by methods known to those skilled in the art, such as using a semiautomatic continuous flow centrifuge. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as, for example, Ca2+free, PBS free Mg2+, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample can be removed, and the cells resuspended directly in a culture medium.

[0230] As described herein, T cells may be isolated from peripheral blood by lysis of red blood cells and depletion of monocytes, for example, by density gradient centrifugation or by countercurrent centrifugal elutriation. T cells may also be isolated from the spleen. T cells may be isolated by incubation with conjugated anti-CD3 beads for a sufficient period of time (i.e., 30 minutes to 24 hours) for positive selection of the desired T lymphocytes. For the isolation 51 172416522.1of T lymphocytes from patients with leukemia, the use of longer incubation times, such as 24 hours, can increase cellular performance. Longer incubation times can be used to isolate T lymphocytes in any situation where there are few T lymphocytes compared to other cell types, such as isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals. The person skilled in the art will recognize that multiple rounds of selection may also be used. It may be desirable to perform the selection procedure and use the “unselected” cells in the activation and expansion process. “Unselected” cells can also undergo new rounds of selection.

[0231] Enrichment of a population of T cells by negative selection can be performed with a combination of antibodies directed to unique surface markers for the negatively selected cells. One method is the sorting and / or selection of cells by positive or negative magnetic immune adherence or flow cytometry using a cocktail of monoclonal antibodies directed to cell surface markers present in the negatively selected cells. Alternatively, the regulatory T lymphocytes are depleted by anti-C25 conjugate beads or other similar selection method.

[0232] T cells can also be frozen after a washing step. Wishing not to be bound by theory, freezing and the following thawing step provide a more uniform product by eliminating granulocytes and, to some extent, monocytes in the cell population. After the washing step that removes the plasma and platelets, the cells can be suspended in a freezing solution. Although many solutions and freezing parameters are known in the art and will be useful in this context, one method involves the use of PBS containing 20% DMSO and 8% human serum albumin, or culture medium containing 10% dextran 40 and 5% dextrose human albumin and 7.5% DMSO or 31.25% Plasmalyte A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% of dextrose, 20% serum of human albumin and 7.5% of DMSO or other suitable cell freezing medium containing for example Hespan and PlasmaLyte A. The cells may then be frozen at - 80 °C at a rate of 1 °C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing can be used.

[0233] The cryopreserved cells may be thawed and washed as described herein and allowed to stand for one hour at room temperature before activation using the methods of the present disclosure. As described herein, T cells can be expanded, frozen, and used later. As described herein, samples may be collected from a patient shortly after the diagnosis of a particular disease as described herein, but before any treatment. The cells may be isolated from a blood sample or an apheresis of a subject before any number of relevant treatment modalities, including but not limited to, treatment with agents such as natalizumab, efalizumab, antiviral 52 172416522.1agents, chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunoablatories such as CAMPATH, anti-CD3 antibodies, cytoxane, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation.

[0234] Cells that are infected by a pathogen can present on their surface a polypeptide derived from the pathogen in the context of an MHC. This polypeptide / MHC can in turn be recognized by a T cell specific for the presented antigenic peptide. The T cell may then induce killing of the infected cell. Accordingly, provided herein are methods of expanding T cells targeting pathogen-infected cells using viral particles presenting on their surface a polypeptide derived from the pathogen. The T cells of interest are capable of both recognizing (1) the polypeptide derived from the pathogen, as presented on the infected cell (as part of an MHC) and (2) the epitope presented on the viral particle (as part of the particle’s MHC). In one embodiment, the polypeptide derived from the pathogen and the epitope are identical. In another embodiment, the polypeptide derived from the pathogen and the epitope differ from each other (but are still both recognized by the TCR on the T cells of interest). For example, the polypeptide derived from the pathogen could be longer or shorter than the epitope displayed by the MHC on the viral particle.

[0235] Similarly, cancer cells can present on their surface a cancer antigen in the context of an MHC. Any protein or polypeptide synthesized within the cancer cell can be the source of the antigenic peptide. Again, the cancer antigen / MHC can in turn be recognized by a T cell specific for the presented antigenic peptide. The T cell may then induce killing of the cancer cell. The T cells of interest are capable of both recognizing (1) the cancer antigen, as presented on the cancer cell (as part of an MHC) and (2) the epitope presented on the viral particle (as part of the particle’s MHC). In one embodiment, the cancer antigen and the epitope are identical. In another embodiment, the cancer antigen and the epitope differ from each other (but are still both recognized by the TCR on the T cells of interest). For example, the cancer antigen could be longer or shorter than the epitope displayed by the MHC on the viral particle.

[0236] Provided herein is a method of inducing killing of a cell infected by a pathogen, wherein the cell displays on its surface a polypeptide derived from the pathogen, the method comprising: (a) contacting a population of T cells with a viral particle disclosed herein, thereby generating a population of activated T cells, wherein the viral particle comprises a lipid envelope and (1) an MHC / peptide complex comprising a first fusion polypeptide comprising (a) a class I major histocompatibility (MHC) polypeptide or a fragment, mutant, or derivative 53 172416522.1thereof, (b) a β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof, (c) an epitope (d) a first lipid envelope anchor; and (e) optionally a first cytoplasmic domain and, optionally (2) a second fusion polypeptide comprising (a) a co-stimulatory domain, (b) a second lipid envelope anchor, (c) optionally a second cytoplasmic domain, wherein each T cell comprises a TCR, wherein the TCR is capable of binding to the polypeptide derived from the pathogen and to the epitope; and (b) exposing the cell infected by the pathogen to the population of activated T cells. In some embodiments, the pathogen is a bacterial, viral, or fungal pathogen. In one embodiment, the pathogen is a viral pathogen. In some embodiments, the viral pathogen is a CMV, HIV, or a hepatitis virus. In embodiments, the population of activated T cells is further expanded before step (b). In some embodiments, the population of T cells may be derived from a population of T cells that has been isolated from a patient and that has been genetically engineered to express the TCR is capable of binding to the polypeptide derived from the pathogen and to the epitope. The engineered T cell may be reintroduced into the same or a different patient.

[0237] Provided is a method of killing of a cancer cell, wherein the cell displays on its surface a cancer antigen, the method comprising: (a) contacting a population of T cells with the viral particle disclosed herein, thereby generating a population of activated T cells, wherein the viral particle comprises a lipid envelope and (1) an MHC / peptide complex comprising a first fusion polypeptide comprising (i) a class I major histocompatibility (MHC) polypeptide or a fragment, mutant, or derivative thereof, and (ii) a β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof, (iii) an epitope (iv) a first lipid envelope anchor; and (v) optionally a first cytoplasmic domain and, optionally (2) a second fusion polypeptide comprising (i) a co-stimulatory domain, (ii) a second lipid envelope anchor, (iii) optionally a second cytoplasmic domain, wherein each T cell comprises a TCR, wherein the TCR is capable of binding to the cancer antigen and to the epitope; and (b) exposing the cancer cell to the population of activated T cells. In embodiments, the population of activated T cells is further expanded before step (b). In some embodiments, the population of T cells may be derived from a population of T cells that has been isolated from a patient and that has been genetically engineered to express the TCR that is capable of binding to the cancer antigen and to the epitope. The engineered T cell may be reintroduced into the same or a different patient.

[0238] Anti-tumor responses of T cells contacted with viral particles described herein may be determined in xenograft tumor models. Tumors may be established using any human cancer cell line expressing the tumor associated antigen presented by the viral particles. The endpoint 54 172416522.1of the xenograft tumor models can be determined based on the size of the tumors, weight of animals, survival time and histochemical and histopathological examination of the cancer, using methods known to one skilled in the art.

[0239] Methods of Treatment

[0240] Provided herein are methods of treating disease in a subject in need thereof, the methods comprising administering to the subject a viral particle disclosed herein or a pharmaceutical composition comprising a viral particle disclosed herein.

[0241] As used herein, the terms “subject” and “patient” are used interchangeably irrespective of whether the subject has undergone treatment in the past or is currently undergoing any form of treatment. As used herein, the terms “subject” and “subjects” may refer to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgus monkey, chimpanzee, etc.) and a human). The subject may be a human or a non-human. In some embodiments, the subject is a human. In some embodiments, the subject is immune-depleted.

[0242] The terms “treat,” “treated,” “treating,” or “treatment” as used herein refer to therapeutic treatment, wherein the object is to slow down (lessen) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment. The terms “prevent”, “prevention”, and the like refer to acting prior to overt disease or disorder onset, to prevent the disease or disorder from developing or to minimize the extent of the disease or disorder or slow its course of development.

[0243] Provided is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a viral particle disclosed herein, wherein: 55 172416522.1the viral particle comprises a lipid envelope and (1) an MHC / peptide complex comprising a first fusion polypeptide comprising (a) a class I MHC polypeptide or a fragment, mutant, or derivative thereof, (b) a β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof, (c) an epitope (d) a first lipid envelope anchor; and (e) optionally a first cytoplasmic domain and, optionally (2) a second fusion polypeptide comprising (a) a co-stimulatory domain, (b) a second lipid envelope anchor, (c) optionally a second cytoplasmic domain; the subject has a cancer cell that displays on its surface a cancer antigen; and the subject has a T cell that comprises a TCR, wherein the TCR is capable of binding to the cancer antigen and to the epitope.

[0244] Provided herein is a method of treating cancer in a first subject in need thereof, the method comprising: isolating a population of T cells from a second subject; genetically engineering the population of T cells to express a TCR, thereby producing a genetically engineered population of T cells; contacting the genetically engineered population of T cells with a viral particle comprising a lipid envelope and (1) an MHC / peptide complex comprising a first fusion polypeptide comprising (a) a class I MHC polypeptide or a fragment, mutant, or derivative thereof, (b) a β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof, (c) an epitope (d) a first lipid envelope anchor; and (e) optionally a first cytoplasmic domain and, optionally (2) a second fusion polypeptide comprising (a) a co-stimulatory domain, (b) a second lipid envelope anchor, (c) optionally a second cytoplasmic domain; thereby producing an activated genetically engineered population of T cells; optionally, further expanding the activated genetically engineered population of T cells; and administering the activated genetically engineered population of T cells to the first subject, wherein: the first subject has a cancer cell that displays on its surface a cancer antigen; and the TCR is capable of binding to the cancer antigen and to the epitope.

[0245] The first and the second subject may be the same subject.

[0246] As discussed herein, the T cells of interest are capable of both recognizing (1) the cancer antigen, as displayed on the cancer cell (as part of an MHC) and (2) the epitope presented on the viral particle (as part of the particle’s MHC). 56 172416522.1

[0247] The epitope may be a cancer / tumor epitope disclosed herein.

[0248] The term “cancer” refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers, as well as dormant tumors or micrometastases. Accordingly, the term “cancer” as used herein refers to an uncontrolled growth of cells, which interferes with the normal functioning of the bodily organs and systems, including cancer stem cells and tumor vascular niches. A subject that has a cancer is a subject having objectively measurable cancer cells present in the subject's body. Included in this definition are benign and malignant cancers, as well as dormant tumors or micrometastases. Cancers that migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs. Hematopoietic cancers, such as leukemia, can out-compete the normal hematopoietic compartments in a subject, thereby leading to hematopoietic failure (in the form of anemia, thrombocytopenia and neutropenia) ultimately causing death.

[0249] The compositions (including pharmaceutical compositions) disclosed herein may be administered in therapeutically effective amounts. An “effective amount” or “therapeutically effective amount” refers to an amount of the compound or agent that is capable of producing a medically desirable result in a treated subject. The treatment method can be performed in vivo or ex vivo, alone or in conjunction with other drugs or therapy. A therapeutically effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.

[0250] Cancers that can be treated by the compositions and methods disclosed herein include tumors that are not vascularized or are not substantially vascularized, as well as vascularized tumors. Cancers may comprise non-solid tumors (such as hematologic tumors, e.g., leukemias and lymphomas) or may comprise solid tumors. The types of cancers to be treated with the compositions disclosed herein include, but are not limited to, carcinoma, blastoma and sarcoma, and certain leukemias or malignant lymphoid tumors, benign and malignant tumors and malignancies, e.g., sarcomas, carcinomas, and melanomas. Also included are adult tumors / cancers and pediatric tumors / cancers.

[0251] Hematologic cancers are cancers of the blood or bone marrow. Examples of hematologic (or haematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous myelogenous leukemia, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous 57 172416522.1leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin’s lymphoma (indolent and high-grade forms), myeloma Multiple, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0252] Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. The different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovium, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, carcinoma of the sweat gland, medullary thyroid carcinoma, papillary thyroid carcinoma, sebaceous gland carcinoma of pheochromocytomas, carcinoma papillary, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as glioma) (such as brainstem glioma and mixed gliomas), glioblastoma (also astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastasis).

[0253] Also provided herein are methods of reducing the growth of a tumor, reducing cancer stemness, reducing tumor-associated fibrosis, reducing tumor metastasis, increasing cytokine production in the tumor microenvironment, increasing anti-tumor immunity, increasing infiltration of a tumor with immune cells, or reducing T cell tolerance, the methods comprising administering to the subject a viral particle disclosed herein.

[0254] The compositions disclosed herein (e.g., the viral particles, cells and pharmaceutical compositions disclosed herein) can be administered in a manner appropriate to the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages can be determined by clinical trials. The precise amount of the compositions disclosed herein to be administered can be determined by a physician having 58 172416522.1accounted for individual differences in age, weight, tumor size, extent of infection or metastasis, and patient's condition (subject).

[0255] The compositions can also be administered several times at these dosages. The cells disclosed herein can be administered using infusion techniques that are commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dose and treatment regimen for a particular patient can be determined by one skilled in the art of medicine by monitoring the patient for signs of the disease and adjusting the treatment accordingly.

[0256] The administration of the present compositions can be carried out in any convenient way, including infusion or injection (e.g., intravenous, intrathecal, intramuscular, intraluminal, intratracheal, intraperitoneal, or subcutaneous), or other methods known in the art. Administration can be once every four weeks, once every three weeks, every two weeks, once a week, or more often, but the frequency may be decreased during a maintenance phase of the disease or disorder. In some embodiments, the composition is administered by intravenous infusion.

[0257] In certain cases, the compositions described herein are administered to a patient together with (e.g., before, simultaneously or consecutively) any number of relevant treatment modalities, including any of the LVPs as described herein. Also described herein, the compositions disclosed herein can be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablating agents such as CAMPATH, anti-cancer antibodies. CD3 or other antibody therapies, cytoxine, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation.

[0258] The viral particles described herein may be used for treating or preventing an infection. The infections include, without limitation, infections caused by viruses, bacteria, fungi, protozoa, parasites, helminths, and ectoparasites. The infectious agent can be, without limitation, a virus, a bacterium, a fungus, a protozoa, a parasite, a helminth, and an ectoparasite. In some embodiments, the infectious agent is a CMV, HIV, or a hepatitis virus.

[0259] Provided herein is a method of treating an infection with a pathogen in a subject in need thereof, the method comprising administering to the subject a viral particle disclosed herein, wherein: the viral particle comprises a lipid envelope and (1) an MHC / peptide complex comprising a first fusion polypeptide comprising (a) a class I MHC polypeptide or a fragment, mutant, or 59 172416522.1derivative thereof, (b) a β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof, (c) an epitope (d) a first lipid envelope anchor; and (e) optionally a first cytoplasmic domain and, optionally (2) a second fusion polypeptide comprising (a) a co-stimulatory domain, (b) a second lipid envelope anchor, (c) optionally a second cytoplasmic domain; the subject has a cell that displays on its surface a polypeptide derived from the pathogen; and the subject has a T cell that comprises a TCR, wherein the TCR is capable of binding to the polypeptide derived from the pathogen and to the epitope.

[0260] In some embodiments, the pathogen is a bacterial, viral, or fungal pathogen. In one embodiment, the pathogen is a viral pathogen.

[0261] Provided herein is a method of treating an infection with a pathogen in a first subject in need thereof, the method comprising: isolating a population of T cells from a second subject; genetically engineering the population of T cells to express a TCR, thereby producing a genetically engineered population of T cells; contacting the genetically engineered population of T cells with a viral particle comprising a lipid envelope and (1) an MHC / peptide complex comprising a first fusion polypeptide comprising (a) a class I MHC polypeptide or a fragment, mutant, or derivative thereof, (b) a β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof, (c) an epitope (d) a first lipid envelope anchor; and (e) optionally a first cytoplasmic domain and, optionally (2) a second fusion polypeptide comprising (a) a co-stimulatory domain, (b) a second lipid envelope anchor, (c) optionally a second cytoplasmic domain; thereby producing an activated genetically engineered population of T cells; optionally, further expanding the activated genetically engineered population of T cells; and administering the activated genetically engineered population of T cells to the first subject, wherein: the first subject has a cell that displays on its surface a polypeptide derived from the pathogen; and the TCR is capable of binding to the polypeptide derived from the pathogen and to the epitope.

[0262] The first and the second subject may be the same subject. 60 172416522.1

[0263] Combination Therapies

[0264] In some embodiments, the method may further include administering to the subject a second therapeutic agent. In some embodiments, the composition is administered to the subject before, after, or concurrently with the second therapeutic agent.

[0265] “Combination” therapy, as used herein, unless otherwise clear from the context, is meant to encompass administration of two or more therapeutic agents in a coordinated fashion, and includes, but is not limited to, concurrent dosing. Specifically, combination therapy encompasses both co-administration (e.g., administration of a co-formulation or simultaneous administration of separate therapeutic compositions) and serial or sequential administration, provided that administration of one therapeutic agent is conditioned in some way on administration of another therapeutic agent. For example, one therapeutic agent may be administered only after a different therapeutic agent has been administered and allowed to act for a prescribed period of time. See, e.g., Kohrt et al. (2011) Blood 117:2423.

[0266] In one embodiment, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different LVPs disclosed herein are administered to the patient, consecutively or concurrently.

[0267] In some embodiments, the second therapeutic agent is an anti-cancer or anti-tumor agent.

[0268] In some embodiments, the method further comprises administering a therapeutically effective amount of an immune checkpoint modulator. Checkpoint proteins interact with specific ligands that send a signal into the T cell and switch off or inhibit T cell function. By expressing high levels of checkpoint proteins on their surface, cancer cells can control the function of T cells that enter the tumor microenvironment, thus suppressing the anticancer immune response. Examples of immune checkpoint modulators include PD1, PDL1, CTLA4, TIM3, LAG3, and TRAIL. The immune checkpoint protein Programmed Death-1 (PD-1) is a key immune checkpoint receptor ex-pressed by activated T and B cells and mediates immunosuppression. PD-1 is a member of the CD28 family of receptors, which includes CD28, CTLA-4, ICOS, PD-1, and BTLA. Two cell surface glycoprotein ligands for PD-1 have been identified, Programmed Death Ligand-1 (PD-L1) and Programmed Death Ligand-2 (PD-L2), that are expressed on antigen-presenting cells as well as many human cancers and have been shown to downregulate T cell activation and cytokine secretion upon binding to PD-1 (Freeman et al., 2000; Latchman et al., 2001). Inhibition of the PD-1 / PD-L1 interaction can promote potent antitumor activity. Examples of PD-1 inhibitors include, but are not limited to, Pembrolizumab (MK-3475), Nivolumab (MDX-1106), Cemiplimab-rwlc (REGN2810), 61 172416522.1Pidilizumab (CT-011), Spartalizumab (PDR001), tislelizumab (BGB-A317), PF-06801591, AK105, BCD-100, BI 754091, JS001, LZM009, MEDI0680, MGA012, Sym021, TSR-042. Examples of PD-L1 inhibitors include, but are not limited to, Atezolizumab (MPDL3280A), Durvalumab (MEDI4736), Avelumab (MSB0010718C), BGB-A333, CK-301, CS1001, FAZ053, KN035, MDX-1105, MSB2311, SHR-1316. The checkpoint modulators may be administered simultaneously, separately, or concurrently with the compositions disclosed herein.

[0269] In some embodiments, the method further comprises administering a therapeutically effective amount of a “chemotherapeutic agent,” which is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXANTM); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, methyldopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotics chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, 62 172416522.1methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti- adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®.; razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2’’- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, xeloda, gemcitabine, KRAS mutation covalent inhibitors and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Additional examples include irinotecan, oxaliplatinum, and other standard colon cancer regimens.

[0270] For treatment of infections, combination therapy described herein can encompass co- administering compositions and methods described herein with an antibiotic, an anti-fungal drug, an anti-viral drug, an anti-parasitic drug, an anti-protozoal drug, or a combination thereof. 63 172416522.1

[0271] Non-limiting examples of useful antibiotics include lincosamides (clindomycin); chloramphenicols; tetracyclines (such as Tetracycline, Chlortetracycline, Demeclocycline, Methacycline, Doxycycline, Minocycline); aminoglycosides (such as Gentamicin, Tobramycin, Netilmicin, Amikacin, Kanamycin, Streptomycin, Neomycin); beta-lactams (such as penicillins, cephalosporins, Imipenem, Aztreonam); vancomycins; bacitracins; macrolides (erythromycins), amphotericins; sulfonamides (such as Sulfanilamide, Sulfamethoxazole, Sulfacetamide, Sulfadiazine, Sulfisoxazole, Sulfacytine, Sulfadoxine, Mafenide, p-Aminobenzoic Acid, Trimethoprim-Sulfamethoxazole); Methenamin; Nitrofurantoin; Phenazopyridine; trimethoprim; rifampicins; metronidazoles; cefazolins; Lincomycin; Spectinomycin; mupirocins; quinolones (such as Nalidixic Acid, Cinoxacin, Norfloxacin, Ciprofloxacin, Perfloxacin, Ofloxacin, Enoxacin, Fleroxacin, Levofloxacin); novobiocins; polymixins; gramicidins; and antipseudomonals (such as Carbenicillin, Carbenicillin Indanyl, Ticarcillin, Azlocillin, Mezlocillin, Piperacillin) or any salts or variants thereof. The antibiotic used will depend on the type of bacterial infection.

[0272] Non-limiting examples of useful anti-fungal agents include imidazoles (such as griseofulvin, miconazole, terbinafine, fluconazole, ketoconazole, voriconazole, and itraconizole); polyenes (such as amphotericin B and nystatin); Flucytosines; and candicidin or any salts or variants thereof.

[0273] Non-limiting examples of useful anti-viral drugs include interferon alpha, beta or gamma, didanosine, lamivudine, zanamavir, lopanivir, nelfinavir, efavirenz, indinavir, valacyclovir, zidovudine, amantadine, rimantidine, ribavirin, ganciclovir, foscarnet, and acyclovir or any salts or variants thereof.

[0274] Non-limiting examples of useful anti-parasitic agents include chloroquine, mefloquine, quinine, primaquine, atovaquone, sulfasoxine, and pyrimethamine or any salts or variants thereof.

[0275] Non-limiting examples of useful anti-protozoal drugs include metronidazole, diloxanide, iodoquinol, trimethoprim, sufamethoxazole, pentamidine, clindamycin, primaquine, pyrimethamine, and sulfadiazine or any salts or variants thereof.

[0276] In some embodiments, the viral particles disclosed herein are co-administered with one or more cytokines. This can serve to enhance the selective differentiation and / or expansion if desired T cells.

[0277] Kits 64 172416522.1

[0278] Further embodiments disclosed herein can concern kits for use with methods and compositions. Kits can also include a suitable container, for example, vials, tubes, mini- or microfuge tubes, test tube, flask, bottle, syringe or other container. Where an additional component or agent is provided, the kit can contain one or more additional containers into which this agent or component may be placed. Kits herein will also typically include a means for containing the viral particles and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. Optionally, one or more additional active agents such as, e.g., anti-inflammatory agents, anti-viral agents, anti-fungal or anti-bacterial agents or anti- tumor agents may be needed for compositions described.

[0279] Illustrative numbered embodiments

[0280] Non-limiting, illustrative embodiments are provide below.

[0281] Embodiment 1. A recombinant viral particle, the recombinant viral particle comprising a lipid envelope and: (1) a first MHC / peptide complex comprising a first fusion polypeptide comprising (a) a first class I MHC polypeptide or a fragment, mutant, or derivative thereof, (b) a first β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof, (c) a first epitope, and (d) a first lipid envelope anchor; (2) a second MHC / peptide complex comprising a second fusion polypeptide comprising (a) a second class I MHC polypeptide or a fragment, mutant, or derivative thereof, (b) a second β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof, (d) a second epitope, wherein the first epitope and the second epitope are different and (d) a second lipid envelope anchor; and (3) a third fusion polypeptide comprising (a) a co-stimulatory domain and (b) a third lipid envelope anchor.

[0282] Embodiment 2. The recombinant viral particle of embodiment 1, wherein the first and / or the second class I MHC polypeptide comprises a human class I MHC polypeptide derived from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G.

[0283] Embodiment 3. The recombinant viral particle of embodiment 1, wherein the first and / or the second class I MHC polypeptide comprises a murine class I MHC polypeptide derived from H-2K, H-2D, H-2L, H-2Q, H-2M or H-2T.

[0284] Embodiment 4. The recombinant viral particle of embodiment 1, wherein the first and / or the second class I MHC polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO:20. 65 172416522.1

[0285] Embodiment 5. The recombinant viral particle of embodiment 4, wherein the first and / or the second class I MHC polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO:20.

[0286] Embodiment 6. The recombinant viral particle of embodiment 5, wherein the first and / or the second class I MHC polypeptide comprises SEQ ID NO:20.

[0287] Embodiment 7. The recombinant viral particle of any one of embodiments 1-6, wherein the first and / or the second β2 microglobulin polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO:19.

[0288] Embodiment 8. The recombinant viral particle of embodiment 7, wherein the first and / or the second β2 microglobulin polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO:19.

[0289] Embodiment 9. The recombinant viral particle of embodiment 8, wherein the first and / or the second β2 microglobulin polypeptide comprises SEQ ID NO:19.

[0290] Embodiment 10. The recombinant viral particle of any one of embodiments 1-9, wherein the first and / or the second epitope is 5 to 20 amino acids long.

[0291] Embodiment 11. The recombinant viral particle of any one of embodiments 1-10, wherein the first and / or the second epitope is derived from a bacterial, viral, fungal, or protozoan polypeptide.

[0292] Embodiment 12. The recombinant viral particle of any one of embodiments 1-11, wherein the first and the second epitope are derived from the same polypeptide.

[0293] Embodiment 13. The recombinant viral particle of any one of embodiments 1-11, wherein the first and the second epitope are derived from two different polypeptides.

[0294] Embodiment 14. The recombinant viral particle of any one of embodiments 1-13, wherein the first and the second epitope are derived from different polypeptides from the same organism.

[0295] Embodiment 15. The recombinant viral particle of any one of embodiments 1-14, wherein the first and / or the second epitope is derived from a cytomegalovirus (CMV) polypeptide, a human immunodeficiency virus (HIV) polypeptide, a SARS-CoV-2 virus polypeptide, or a hepatitis virus polypeptide.

[0296] Embodiment 16. The recombinant viral particle of any one of embodiments 1-14, wherein the first and / or the second epitope comprises any one of SEQ ID NOs:1-3, SEQ ID NO:94, or SEQID NO:95. 66 172416522.1

[0297] Embodiment 17. The recombinant viral particle of any one of embodiments 1-14, wherein the first and / or the second epitope comprises a sequence derived from HIV polypeptide gp120, HIV-1 polypeptide p17, or melanoma antigen recognized by T cells 1 (MART-1).

[0298] Embodiment 18. The recombinant viral particle of any one of embodiments 1-10, wherein the first and / or the second epitope is a cancer antigen or fragment thereof.

[0299] Embodiment 19. The recombinant viral particle of any one of embodiments 1-18, wherein the first, the second, and / or the third lipid envelope anchor is a transmembrane domain.

[0300] Embodiment 20. The recombinant viral particle of any one of embodiments 1-18, wherein the first, the second, and / or the third lipid envelope anchor is a transmembrane domain derived from human HLA-A*0201 MHC.

[0301] Embodiment 21. The recombinant viral particle of embodiment 20, wherein the first, the second, and / or the third lipid envelope anchor is a transmembrane domain that comprises a sequence that is at least 80% identical to SEQ ID NO:22.

[0302] Embodiment 22. The recombinant viral particle of embodiment 21, wherein the first, the second, and / or the third lipid envelope anchor is a transmembrane domain that comprises a sequence that is at least 90% identical to SEQ ID NO:22.

[0303] Embodiment 23. The recombinant viral particle of embodiment 22, wherein the first, the second, and / or the third lipid envelope anchor is a transmembrane domain that comprises SEQ ID NO:22.

[0304] Embodiment 24. The recombinant viral particle of any one of embodiments 1-23, wherein the co-stimulatory domain is a 4-1BBL polypeptide, a B7-1 polypeptide, a B7-2 polypeptide, an ICOS-L polypeptide, an OX-40L polypeptide, a CD80 polypeptide, a CD70 peptide, a CD86 polypeptide, a CD40L polypeptide, a CD40 polypeptide, a PD-L1 polypeptide, or a PD-L2 polypeptide.

[0305] Embodiment 25. The recombinant viral particle of any one of embodiments 1-23, wherein the co-stimulatory domain is a CD28 agonist, an ICOS agonist, a 4-1BB agonist, a OX40 agonist, a CD27 agonist, a CD40 agonist, a CD40L agonist, a CTLA-4 agonist, or a PD- 1 agonist.

[0306] Embodiment 26. The recombinant viral particle of embodiment 25, wherein the co- stimulatory domain is a CD28 agonist.

[0307] Embodiment 27. The recombinant viral particle of embodiment 25, wherein the co- stimulatory domain comprises a sequence that is at least 80% identical to SEQ ID NO:25. 67 172416522.1

[0308] Embodiment 28. The recombinant viral particle of embodiment 27, wherein the co- stimulatory domain comprises a sequence that is at least 90% identical to SEQ ID NO:25.

[0309] Embodiment 29. The recombinant viral particle of embodiment 28, wherein the co- stimulatory domain comprises SEQ ID NO:25.

[0310] Embodiment 30. The recombinant viral particle of any one of embodiments 19-29, wherein: (1) the first fusion polypeptide comprises from N-terminus to C-terminus: (a) the first epitope; (b) the first β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof; (c) the first class I MHC polypeptide or a fragment, mutant, or derivative thereof; (d) the first transmembrane domain; and (e) optionally, a first cytoplasmic domain; (2) the second fusion polypeptide comprises from N-terminus to C-terminus: (a) the second epitope; (b) the second β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof; (c) the second class I MHC polypeptide or a fragment, mutant, or derivative thereof; (d) the second transmembrane domain; and (e) optionally, a second cytoplasmic domain; and / or (3) a third fusion polypeptide comprising (a) a co-stimulatory domain, (b) the third transmembrane domain, and (c) optionally, a third cytoplasmic domain.

[0311] Embodiment 31. The recombinant viral particle of any one of embodiments 1-30, wherein the first fusion polypeptide, the second fusion polypeptide, and / or the third fusion polypeptide further comprises one or more linkers.

[0312] Embodiment 32. The recombinant viral particle of embodiment 31, wherein the one or more linkers are polypeptide linkers or disulfide bond linkers.

[0313] Embodiment 33. The recombinant viral particle of embodiment 32, wherein the one or more polypeptide linkers are flexible linkers.

[0314] Embodiment 34. The recombinant viral particle of embodiment 33, wherein the one or more flexible linkers predominantly consist of glycines, serines, and alanines.

[0315] Embodiment 35. The recombinant viral particle of embodiment 33, wherein the one or more flexible linkers comprise one or more repetitions of any one of SEQ ID NOs:4-17.

[0316] Embodiment 36. The recombinant viral particle of embodiment 34, wherein the one or more flexible linkers comprise SEQ ID NO:16.

[0317] Embodiment 37. The recombinant viral particle of embodiment 32, wherein the one or more polypeptide linkers are rigid linkers.

[0318] Embodiment 38. The recombinant viral particle of embodiment 37, wherein the one or more rigid linkers comprise one or more repetitions of any one of SEQ ID NOs:27-55. 68 172416522.1

[0319] Embodiment 39. The recombinant viral particle of any one of embodiments 1-38, wherein the first fusion polypeptide, the second fusion polypeptide, and / or the third fusion polypeptide further comprise a signal peptide.

[0320] Embodiment 40. The recombinant viral particle of embodiment 39, wherein the signal peptide comprises any one of SEQ ID NOs:18 or 56-93.

[0321] Embodiment 41. The recombinant viral particle of embodiment 39, wherein the signal peptide is derived from the beta 2 microglobulin signal peptide.

[0322] Embodiment 42. The recombinant viral particle of embodiment 41, wherein the signal peptide comprises a sequence that is at least 80% identical to SEQ ID NO:18.

[0323] Embodiment 43. The recombinant viral particle of embodiment 42, wherein the signal peptide comprises a sequence that is at least 90% identical to SEQ ID NO:18.

[0324] Embodiment 44. The recombinant viral particle of embodiment 43, wherein the signal peptide comprises SEQ ID NO:18.

[0325] Embodiment 45. The recombinant viral particle of any one of embodiments 1-44, wherein the first and the third fusion polypeptide are covalently linked.

[0326] Embodiment 46. The recombinant viral particle of any one of embodiments 1-45, wherein the recombinant viral particle is derived from a lentivirus.

[0327] Embodiment 47. The recombinant viral particle of any one of embodiments 1-46, wherein one or more of polypeptides present in the lipid envelope of a wild-type virus corresponding to the recombinant viral particle are absent or mutated so that said recombinant viral particle is not capable of binding to any cell targeted by the wild-type virus in the absence of the first and the second MHC / peptide complex.

[0328] Embodiment 48. The recombinant viral particle of any one of embodiments 1-47, wherein the first and the second epitope bind to a T cell receptor (TCR) of a CD8+ T cell.

[0329] Embodiment 49. A nucleic acid or pair of nucleic acids encoding the first and / or second MHC / peptide complex and / or the third fusion protein of any one of embodiments 1-48.

[0330] Embodiment 50. A method of activating a first T cell comprising a first TCR and a second T cell comprising a second TCR, the method comprising contacting the first and the second T cell with the recombinant viral particle of any one of embodiments 1-48, wherein first TCR binds to the first epitope and wherein the second TCR binds to the second epitope.

[0331] Embodiment 51. A method of inducing expression of pro-inflammatory cytokines in a first T cell comprising a first TCR and a second T cell comprising a second TCR, the method comprising contacting the first and the second T cell with the recombinant viral particle 69 172416522.1of any one of embodiments 1-48, wherein the first TCR binds to the first epitope and wherein the second TCR binds to the second epitope.

[0332] Embodiment 52. A method of expanding a first population of T cells each comprising a first TCR and a second population of T cells comprising a second TCR, the method comprising contacting the first population of T cells and the second population of T cells with the recombinant viral particle of any one of embodiments 1-48, wherein the first TCR binds to the first epitope and wherein the second TCR binds to the second epitope.

[0333] Embodiment 53. The method of embodiment 52, wherein the first population of T cells has been genetically engineered to express the first TCR and wherein the second population of T cells has been genetically engineered to express the second TCR.

[0334] Embodiment 54. A method of inducing killing of a cell infected by a pathogen, wherein the cell displays on its surface a first polypeptide derived from the pathogen and a second polypeptide derived from the pathogen, the method comprising: (a) contacting a first population of T cells, wherein each T cell in the first population of T cells comprises a first TCR, and a second population of T cells, wherein each T cell in the second population of T cells comprises a second TCR, with the recombinant viral particle of any one of embodiments 1-17 or 19-48, thereby generating a first population of activated T cells and a second population of activated T cells, wherein the first TCR is capable of binding to the first polypeptide derived from the pathogen and to the first epitope and wherein the second TCR is capable of binding to the second polypeptide derived from the pathogen and to the second epitope; and (b) exposing the cell infected by the pathogen to the first population of activated T cells and the second population of activated T cells.

[0335] Embodiment 55. The method of embodiment 54, wherein the first population of T cells has been genetically engineered to express the first TCR and wherein the second population of T cells has been genetically engineered to express the second TCR.

[0336] Embodiment 56. A method of inducing killing of a cancer cell, wherein the cancer cell displays on its surface a first cancer antigen and a second cancer antigen, the method comprising: (a) contacting a first population of T cells, wherein each T cell in the first population of T cells comprises a first TCR, and a second population of T cells, wherein each T cell in the second population of T cells comprises a second TCR, with the recombinant viral particle of any one of embodiments 1-10 or 18-48, thereby generating a first population of activated T cells and a second population of activated T cells, wherein the first TCR is capable of binding to the first cancer antigen and to the first epitope and wherein the second TCR is 70 172416522.1capable of binding to the second cancer antigen and to the second epitope; and (b) exposing the cancer cell to the first population of activated T cells and to the second population of activated T cells.

[0337] Embodiment 57. The method of embodiment 56, wherein the first population of T cells has been genetically engineered to express the first TCR and wherein the second population of T cells has been genetically engineered to express the second TCR.

[0338] Embodiment 58. The method of any one of embodiments 54-57, wherein the first population of activated T cells and / or the second population of activated T cells is further expanded before step (b).

[0339] It is to be understood that this disclosure is not limited to the particular molecules, compositions, methodologies, or protocols described, as these may vary. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments disclosed herein. It is further to be understood that this disclosure includes all possible combinations of such particular features. For example, where a particular feature is disclosed in the context of a particular aspect or embodiment of the disclosure, or a particular claim, that feature can also be used, to the extent possible, in combination with and / or in the context of other particular aspects and embodiments disclosed herein.

[0340] Where reference is made herein to a method comprising two or more defined steps, the defined steps can be carried out in any order or simultaneously (except where the context excludes that possibility), and the method can include one or more other steps which are carried out before any of the defined steps, between two of the defined steps, or after all the defined steps (except where the context excludes those possibilities).

[0341] All other referenced patents and applications, scientific articles, book chapters, etc., are incorporated herein by reference in their entireties. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0342] To facilitate a better understanding of the present disclosure, the following examples of specific embodiments are given. The following examples should not be read to limit or define the entire scope of the disclosure. 71 172416522.1EXAMPLES

[0343] Example 1: Materials and Methods for Examples 1-20

[0344] Plasmid DNA

[0345] NLV, SL9, YLQ and IV9 peptide-scMHC and anti-CD28 expressing vectors were composed of a cytomegalovirus promoter, signal peptide, transmembrane coding sequence, and SV40 polyadenylation signal (Figs.2 and 3). Each peptide-scMHC plasmid encodes from N to C-terminus: the target peptide (NLV, SL9, YLQ or IV9, respectively), beta 2 microglobulin, an HLA-A*0102 heavy, an MHC-I transmembrane domain, and a cytoplasmic domain. The single change variable fragment (scFv) anti-CD28 plasmid comprises the anti- CD28 light and heavy single chains encoding sequences followed by a sequence encoding an IgG hinge.

[0346] Transfection / Lentivirus Production

[0347] HEK293T cells were seeded at a density of 10 × 106cells per plate in a 150 mm culture dish and incubated for 48 h. Culture media was replaced with DMEM supplemented with 15% FBS and 1% Glutamax 2 h before the transfection. Transfection was performed in >80% confluent HEK293T cells using JetPrime (Polyplus) reagents following the manufacturer’s instructions. VSVG plasmid (2.5 μg), psPAX plasmid (7.5 μg), peptide-scMHC plasmid (2.5 μg) and anti-CD28 plasmid (10 μg) were mixed with 40 μl of jetPrime reagent and 1200 μl of jetPrime buffer and added to each plate to generate modless VirTac models. Transfected cells were incubated for 48 h. Lentiviral particles were harvested by filtering supernatant from all the plates and ultracentrifuging at 20000 rpm for 2 h 15 min at 20 ºC. The pellet with the lentiviral particles was resuspended in a 500-600 μl PBS, aliquoted and frozen at -80 ºC.

[0348] TCR activation assay

[0349] Jurkat / MA cells, a TCR-β chain-deficient Jurkat-derivative engineered to express human CD8α and an NFAT-regulated luciferase reporter gene were obtained from Erik Hooijberg (VU University Medical Center, Amsterdam, The Netherlands) and cultured in I10 (Iscove culture media supplemented with 10% FBS, 1% glutamate, 1% penicillin / streptomycin, 1% HEPES). These cells were transfected for integrated expression of antigen-specific TCR (IV9). IV9-TCR Jurkat / MA cells were plated in a 96-well plate (1x105cells / well) and stimulated with IV9-VirTac or not stimulated (negative control) for 24h at 37 ˚C. Firefly luciferase activity was measured after adding the substrate from the Luciferase Assay System (Promega) in the a Victor luminometer (Perkin Elmer). 72 172416522.1

[0350] Expansion of Peptide-specific CD8+T cells using NLV-LVPs

[0351] Peripheral blood mononuclear cells (PBMCs) from donor HG55, HG86 and RC619 were cultured in R10 (RPMI supplemented with 10% FBS, 1% glutamate, 1% penicillin / streptomycin, 1% HEPES) and added IL-2 (100 units). In duplicate or triplicate wells for each condition, PBMCs (5 x 105 / well) were plated in a 48 well plate and stimulated with the indicated LVPs (also referred to as “VirTac” in the figures) or Immuno-STAT at the indicated concentration or unstimulated as a negative control. Antigen-specific T cells expansion was detected at day 7, day 14, and / or day 21 by flow cytometry in an Aurora full spectrum flow cytometer (Cytek Bioscience) using NLV-tetramer-BV421 (provided by the NIH) and 7-AAD dead dye (ThermoFisher). Cell activation, expression of co-stimulatory molecules and memory phenotype were determined by flow cytometry using the following antibodies from BioLegend: CD3-BUV395, CD8-PE, CD8-Pacific Blue, CD45ro-APC / Cy7, CD62L-BV650, CD28-FITC, 4-1BB-BV711, CD25-APC, LAG3-PE / Cy7.

[0352] Cytokine production of restimulated NLV-specific CD8+T cells / Intracellular Staining

[0353] Twenty days after LVP stimulation, cells were stimulated to produce cytokines by incubation with NLV-peptide loaded T2 cells or YLQ-loaded T2 cells, respectively, (5 x 105 / well) for 5 h at 37 ^C. Brefeldin and Monensin were added at 1:1000 dilution during the stimulation. Cells were stained with NLV tetramer-BV421 (provided by the NIH) or YLQ tetramer-BV421, respectively, Live Dead Blue (Invitrogen) and the following antibodies from BioLegend: CD3-FITC, CD8-Pacific Blue, CD4-PE / Cy7. For intracellular staining, the following antibodies were used (BioLegend): IFN^-BV605, sIL2-perCP / Cy5, Granzyme B- Alexa Fluor 700, Perforin-Alexa fluor 647, TNFa-BV711.

[0354] Cytotoxic activity of LVP-stimulated NLV-specific CD8+T cells

[0355] The cytotoxicity assay was performed using PBMCs (e.g., from donors HG55, RC619, HG86) 20 days after LVP stimulation. Effector cells (5 x 104 / well) were added to the 96 well plate to the 1:1 E:T ratio and (5 x 103 / well) to the 1:10 E:T ratio. Cells were cocultured with T2 (5 x 104 / well) (target cells), either loaded with NLV-peptide or YLQ peptide, respectively, at 1 ^M final concentration or no peptide, for 24 h at 37 ^C. All cells were collected and stained with 7-AAD dead dye (Invitrogen), CD3-BUV395, CD8-APC / Cy7, CD4-PE / Cy7 (BioLegend) and run in the Cytek Aurora flow cytometer. Live T2 cells were gated (i.e. identified) as CD8- CD3-, counted and normalized by cells / ^l using FlowJo software. Normalized cell counts were compared between conditions to assess specific cytotoxicity. 73 172416522.1Percentage of cytotoxicity was quantified by the ratio of the number of peptide-loaded T2 cells killed compared to the number of cells T2 cells with no loaded peptide to assess specific cytotoxicity.

[0356] Suppression of Cytomegalovirus infection using LVP-stimulated NLV-specific CD8+ T cells.

[0357] MRC5 cells were infected with cytomegalovirus (CMV)-Firefly luciferase reporter gene at 5 MOI in a 24-well plate for 24h. CMV-infected MRC5 cells (4 x 104cells / well) were cocultured with LVP-stimulated NLV-specific CD8+T cells from HG55 donor at a 1:1 ratio for 48 h at 37 ^C. CMV MRC5 cells alone, PBMCs alone, cidofovir (anti-CMV drug) at 50 nM and 500 nM were added as controls. CMV infection was measured after cell lysis by quantifying firefly luciferase units in the luminometer using the kit Luciferase Assay System (Promega).

[0358] In vivo expansion of NLV-specific CD8+ T cells

[0359] PBMCs from donor HG55 were intrasplenically injected in NSG mice (20 x 106cells / mouse) together with the indicated LVP at the indicated concentration or NLV-modless Immuno-STAT as a positive control. PBMCs alone were injected in the negative control mouse. Two weeks later, all mice were euthanized, and spleens were extracted and processed for flow cytometric analysis using the Cytek Aurora Spectrum Flow Cytometer (Cytek) after staining with the following antibodies: NLV tetramer-PE (provided by the NIH), Live Dead Blue (Invitrogen) and CD3-FITC, CD8-Pacific Blue, and CD45-APC / Cy7 (BioLegend).

[0360] Mass Spectrometry for LVP Characterization

[0361] Protein digestion

[0362] Proteins were reduced in a buffer containing 5 mM DTT and 50 mM ammonium bicarbonate (pH = 8) and incubated at room temperature for about 1 hour to allow disulfide bond reduction. Samples were then alkylated with 20 mM iodoacetamide in the dark for 30 minutes. Afterward, 500 ng of sequencing grade trypsin (Promega), diluted in 50 mM ammonium bicarbonate, was added into the sample and digested at 37 ˚C for 18 h.

[0363] Sample Desalting

[0364] Prior to mass spectrometry analysis, samples were desalted using a 96-well plate filter (Orochem) packed with 1 mg of Oasis HLB C-18 resin (Waters). Briefly, the samples were resuspended in 100 µl of 0.1% TFA and loaded onto the HLB resin, which was previously equilibrated using 100 µl of the same buffer. After washing with 100 µl of 0.1% TFA, the 74 172416522.1samples were eluted with a buffer containing 70 µl of 60% acetonitrile and 0.1% TFA and then dried in a vacuum centrifuge.

[0365] LC-MS / MS Acquisition and Analysis

[0366] Samples were resuspended in 10 µl of 0.1% TFA and loaded onto a Dionex RSLC Ultimate 300 (Thermo Scientific), coupled online with an Orbitrap Fusion Lumos (Thermo Scientific). Chromatographic separation was performed with a two-column system, consisting of a C-18 trap cartridge (300 µm ID, 5 mm length) and a picofrit analytical column (75 µm ID, 25 cm length) packed in-house with reversed-phase Repro-Sil Pur C18-AQ 3 µm resin. Peptides were separated using a 90 min gradient from 4-30% buffer B (buffer A: 0.1% formic acid, buffer B: 80% acetonitrile + 0.1% formic acid) at a flow rate of 300 nl / min. The mass spectrometer was set to acquire spectra in a data-dependent acquisition (DDA) mode. Briefly, the full MS scan was set to 300-1200 m / z in the orbitrap with a resolution of 120,000 (at 200 m / z) and an AGC target of 5x10e5. MS / MS was performed in the ion trap using the top speed mode (2 secs), an AGC target of 1x10e4 and an HCD collision energy of 35.

[0367] Proteome raw files were searched using Proteome Discoverer software (v2.5, Thermo Scientific) using SEQUEST search engine and the SwissProt and custom database. The search for total proteome included variable modification of N-terminal acetylation, and fixed modification of carbamidomethyl cysteine. Trypsin was specified as the digestive enzyme with up to 2 missed cleavages allowed. Mass tolerance was set to 10 pm for precursor ions and 0.2Da for product ions. Peptide and protein false discovery rate was set to 1%. Following the search, data was processed as described in Rosen et al., Parallel genome-scale CRISPR-Cas9 screens uncouple human pluripotent stem cell identity versus fitness, Nat Commun.2024 Oct 17;15(1):8966.

[0368] HIV Suppression Assay

[0369] An HIV clade B (BAL) [AN: AY426110] infectious molecular clone, which encodes the Renilla luciferase reporter (BAL-HIV-1-Renilla), was used as a surrogate marker to quantify productive HIV infection.

[0370] Autologous CD4+ T cells were isolated using MACS magnetic beads (Miltenyi Biotec) and activated with phytohemagglutinin A (PHA-L, 4 μg / mL) and 100 IU / ml of IL-2 for 72h. Activated CD4+target cells were infected with BAL-HIV-1-Renilla at 1 x 106IU / ml per 5 x 104cells for three days. Target cells were co-cultured with isolated CD8+ 14-days stimulated with either SL9 VirTac or nothing as a negative control for 48h at 1:1 effector: target ratio. CD4+T cells alone (No HIV), target BAL-HIV-1-Renilla-infected CD4+ (only HIV) and 75 172416522.1BAL-HIV-1-Renilla-infected CD4+ treated with Raltegravir at 2µM were added as assay controls. HIV infection was measured after cell lysis by quantifying relative light units in the luminometer using the kit Renilla Luciferase Assay System (Promega).

[0371] In Vivo Antigen-Specific CD8+T cells Stimulation by LVPs

[0372] NSG (NOD-scid IL2Rγnull) mice were purchased from the Jackson Laboratory. Donor PBMCs were intrasplenically injected into NSG mice (2 x 107cells / mouse). Simultaneously, LVPs were injected retroorbitally and HCW9206 (HCW Biologics Inc.), a scaffold linking human IL-7, IL-15 and IL-21, was injected intraperitoneally to support human T cell growth in the mouse system. No LVP was injected into negative control mice. Ten days later, blood was collected and analyzed by flow cytometric analysis using the Aurora Spectrum Flow Cytometer (Cytek) after staining with the following antibodies: tetramer-BV421 (provided by the NIH), Live Dead Blue (Invitrogen) and CD3-BUV395, CD8-PE, and CD45- APC / Cy7 (BioLegend).

[0373] Statistics

[0374] Statistical analyses were performed using GraphPad Prism version 8.2. To compare differences in various groups, a 2-way ANOVA followed by Sidak’s multiple comparisons test was used. Statistical significance was considered when P values were less than 0.05.

[0375] Example 2: Design of stable lentiviral-like particles (LVP) presenting an MHC / NLV complex

[0376] Lentiviral vectors designed for gene therapy require expression of envelope proteins such as vesicular stomatitis virus glycoprotein (VSV-G). These envelope proteins enable the lentiviral vectors to bind to target cells and initiate the process of infection including fusion of the virion membrane with the cellular membrane, entry of the viral capsid, reverse transcription of the viral genomic RNA encoding the therapeutic gene(s) into cDNA, and integration of the cDNA into cellular chromosomes.

[0377] In the experiments disclosed herein, LVPs were designed that are capable of extracellularly engaging the TCR of a T cell. Since a primary purpose of the LVPs was to activate antigen-specific T cells through TCR:MHC-peptide engagement, rather than to infect T cells, the LVPs were designed to be unable to infect cells. Specifically, LVPs disclosed herein lack the viral envelope proteins that contain the binding and fusion domains required for infection. As such, the LVPs are unable to mediate membrane fusion and infection. The LVPs 76 172416522.1also lack any genomic RNA or DNA, thus exogenous genetic material cannot be introduced into target cells if the LVPs do bind and fuse with a cell.

[0378] The LVPs present on their surface a single chain MHC complex fused to a peptide, herein also referred to as an epitope, which in turn is capable of binding to the peptide’s cognate TCR expressed by T cells. The structure of the single chain MHC complex was as follows: signal peptide – epitope (e.g., NLV) – linker – beta 2 microglobulin – linker – extracellular domain of a major histocompatibility complex (MHC) class I molecule – transmembrane domain – cytoplasmic domain of MHC class I molecule.

[0379] To start, a suitable transmembrane protein was identified to anchor the MHC / peptide complex into the viral envelope. To that end, the ability of an HLA-A*0201 transmembrane domain and an IgM transmembrane domain, respectively, to express an agonistic anti-CD3 scFv (αCD3-TR66.opt), capable of activating T cells, was compared. Plasmids encoding the following fusion proteins were designed: (1) green fluorescent protein (GFP), (2) the CD3- specific TR66.opt scFV (comprising a (G4S)3(SEQ ID NO:16) linker between the VLand VHchains), and (3) the transmembrane (TM) domain of membrane IgM (mIgM) or of the human HLA-A*0201 MHC molecule. LVPs presenting the membrane-anchored anti-CD3 scFv were generated by transducing 293T cells with the anti-CD3 scFv / GFP / TM plasmid, accessory packaging plasmids and a plasmid encoding a vesicular stomatitis virus glycoprotein (VSV-g) envelope protein. This protein was mutated (at position K47Q) to abolish binding of VSV-g to its cellular receptor, the low-density lipoprotein receptor (LDRL, VSV-gmut). Yet, the mutations did not affect the ability of VSG-gmutto mediate viral fusion after cellular binding. The resulting LVP bound to CD3 presented on the surface of the T cell and fused with the bound T cell through the VSV-gmutin the LVP membrane. Functional expression of the anti- CD3 scFV on the LVP envelope was evaluated by determining the capacity of the LVPs to selectively transduce a CD3+Jurkat E6.1 cell line. Specifically, transduction was assessed by determining the fraction of cells expressing the GFP reporter gene three days post-transduction with the LVP presenting GFP / anti-CD3 fusions anchored in the LVP envelope by either the HLA-A*0201 MHC TM domain or the mIgM-derived TM domain.

[0380] Flow cytometric analysis of the CD3+Jurkat E6.1 cells for GFP expression demonstrated that more T cells (CD3+GFP+) were transduced with the LVP presenting the anti- CD3 scFv linked to the MHC TM, as compared to the LVP presenting the anti-CD3 scFv linked to the mIgM-derived transmembrane domain (Fig.1A and Fig.1B). 77 172416522.1

[0381] Because these results showed that use of the MHC TM domain as compared to the mIgM-derived transmembrane domain resulted efficient anchoring of the anti-CD3 scFv in the LVP envelope, this TM domain was used in subsequent experiments.

[0382] For the MHC / peptide complex, as proof of concept, an LVP was generated that presented a single chain HLA-A*0201 complex loaded with the well-described human cytomegalovirus (CMV) peptide NLV (amino acids495NLVPMVATV503) (SEQ ID NO:1), herein referred to as “NLV”). The resulting LVP is referred to herein as scpMHC-NLV. The single chain MHC complex presenting the NLV peptide was expressed from plasmid pCL- CMVp-NLV-scMHC under the control of a CMV promoter. The specific structure of the single chain MHC complex was as follows: signal peptide – NLV peptide – (G4S)3linker – human β2M-HLA-A2 MHC domain – (G4S)3 linker– extracellular domain of HLA-A2*0201 MHC-I protein – transmembrane domain of HLA-A2*0201 MHC-I protein – cytoplasmic domain of HLA-A2*0201 MHC-I protein.

[0383] Example 3: LVPs presenting an MHC / NLV complex, which lack viral envelope proteins, are stable and have lost the ability to infect cells

[0384] First, it was demonstrated that LVPs expressing a membrane-anchored MHC / peptide complex are structurally stable even in the absence of viral envelope proteins and are unable to infect T cells unless co-expressed with a mutant VSVG envelope protein. The protein was mutated to eliminate binding to the envelope protein’s cellular receptor, VSV- gmut(“NLV wVSVG”). Yet, the mutated protein was still able to mediate virus-cell fusion and entry in combination with another molecule capable of binding T cells, such as the membrane- anchored MHC / NLV complex.

[0385] To that end, an LVP was generated comprising a nucleic acid encoding a GFP reporter protein and incorporating into its envelope (1) the membrane-anchored MHC / NLV complex and VSV-gmut (“NLV wVSVG”) or (2) the membrane-anchored MHC / NLV complex alone (“NLV modless”) (Fig.2A). The LVPs were incubated with PBMCs containing CD8+T cells that expressed an NLV-specific TCR. The LVPs’ capacity to transduce NLV-specific T cells was assessed by quantification of GFP fluorescence by flow cytometry.

[0386] While the LVPs incorporating both membrane-anchored MHC / NLV complex (cell binding) and VSV-gmut(virus-cell fusion) into their membranes were able to selectively transduce NLV-specific T cells, the LVPs comprising the membrane-anchored MHC / NLV complex alone, and unable to mediate virus-cell fusion, did not transduce T cells (Fig.2B). 78 172416522.1

[0387] These results demonstrated that despite the ability of the MHC / NLV complex to bind the NLV-specific TCR (also see Example 4 below), the LVPs were not capable of transducing T cells in the absence of the VSV-G fusion domain. This shows that the LVPs are safe for in vivo use. Safety is further enhanced by the complete lack of genetic material (i.e., RNA or DNA) inside the LVP.

[0388] Example 4: LVPs presenting an MHC / NLV complex selectively bind to NLV- specific TCRs and activate NLV-specific CD8+T cells

[0389] During an immune response, two signals provided by antigen-presenting cells (APCs) are required to activate antigen-specific naïve T cells. The first signal is an antigen- specific signal, resulting from the TCR’s recognition of its cognate peptide:MHC complex. The second signal is a co-stimulatory signal conveyed through one of several receptors on the T cell’s surface. For example, B7 located on the APC’s surface, stimulates CD28, on the T cell’s surface. The co-stimulatory signal is important for the activation of naïve T cells, since an antigen-specific signal delivered to a naive T cell without a co-stimulatory signal does not stimulate proliferation but instead induces clonal anergy, which restricts subsequent T-cell proliferation. As such, LVPs were generated that presented on their surface a membrane- anchored MHC / NLV complex in presence or absence of membrane-anchored agonist anti- CD28 scFv (Fig. 3A). The agonist anti-CD28 scFv mimics the B7 molecule located on the APC’s surface. The capacity of the two constructs to selectively activate and expand NLV- specific CD8+T cells was determined.

[0390] In an initial experiment, LVPs were incubated with Jurkat / Ma CD8+NFAT-Firefly luciferase reporter cells. These cells had been transduced with a lentiviral vector encoding an NLV-specific TCR. Activation of the TCR can be assessed by quantifying luciferase fluorescence.

[0391] Luciferase activity was markedly increased in Jurkat / MA cells expressing the NLV- TCR that had been treated with LVPs presenting on their surface the membrane-anchored MHC / NLV complex for both LVPs presenting on their surface the membrane-anchored MHC / NLV complex as well as LVPs presenting on their surface the membrane-anchored MHC / NLV complex and membrane-anchored agonist anti-CD28 scFv (Fig.3B). Increases in luciferase fluorescence (representing TCR activation) occurred in a dose responsive manner. Note that Jurkat cells do not express CD28. Protein-based Immuno-STAT proteins (IST) expressing the MHC / NLV complex alone or linked to the agonist anti-CD28 scFv were used 79 172416522.1as positive controls. Immuno-STAT proteins comprise a covalent fusion of peptide epitope, MHC class I allele, co-modulator, and Fc. See, e.g., Seidel et al., Peptide-HLA-based immunotherapeutics platforms for direct modulation of antigen-specific T cells, Sci Rep.2021 Sep 28;11(1):19220 (incorporated herein by reference in its entirety).

[0392] These results indicated that LVPs successfully presented the NLV peptide to its cognate TCR on NVP-specific T cells.

[0393] Example 5: LVPs presenting an MHC / NLV complex induce expansion of NLV- specific CD8+T cells in HLA-A*0201 donor PBMCs in vitro

[0394] To determine if the LVPs were able to activate and expand donor NLV-specific- CD8+T cells, PBMCs from an HLA-A*0201- and CMV-seropositive donor were treated with the following constructs: (1) LVPs presenting on their surface membrane-anchored MHC / NLV complex and VSV-gmut lacking a cellular binding domain but with an intact fusion domain (“NLV wVSVGmut”), (2) LVPs presenting on their surface membrane-anchored MHC / NLV complex alone (“NLV modless” = “NLV-VirTac modless”), (3) LVPs presenting on their surface the membrane-anchored MHC / NLV complex and membrane-anchored agonist anti- CD28 scFv (“NLV-VirTac-αCD28” or “NVL αCD28”), or (4) Immuno-STAT protein (IST) expressing the MHC / NLV complex linked to the agonist anti-CD28 scFv (“NLV-^CD28 IST”, positive control). Two weeks later, the expansion of the NLV-specific CD8+T cell population was assessed by flow cytometric quantification.

[0395] As shown in Figs.4A, 4B, and 4C, incubation with LVPs presenting the membrane- anchored MHC / NLV complex alone resulted in an expansion of NLV-specific CD8+T cell that was significantly higher than what was observed for treatment with PBS (control). Similarly, incubation with LVPs presenting the membrane-anchored MHC / NLV complex and membrane-anchored agonist anti-CD28 scFv resulted in an expansion of NLV-specific CD8+T cell that was significantly higher than what was observed for treatment with PBS (Figs.4A, 4B, and 4C). Of note, as compared to naïve T cells, CD8+T cells that have already transitioned from naïve to memory cells after TCR and CD28 signaling isolated from human donors are less dependent on activation by CD28 when they encounter their cognate antigen again, given the latter cells tend to exhibit a T memory phenotype which is less dependent on the need for both TCR and costimulatory signaling. Yet, in two of the three experiments shown, inclusion of the anchored agonist anti-CD28 scFv lead to increased expansion of NLV-specific CD8+T cell. 80 172416522.1

[0396] These results demonstrated that LVP treatment results in selective activation and expansion of NLV-specific CD8+T cells, similar to that induced by the protein-based NLV- ^CD28 Immuno-STAT, which was used as a positive control.

[0397] Example 6: LVPs presenting an MHC / NLV complex induce robust production of pro-inflammatory cytokines after NLV-peptide presentation.

[0398] To evaluate the functional activity of NLV-specific CD8+T cells expanded by LVP treatment (see Example 5), the T cells were incubated with NLV-peptide loaded T2 cells, and the production of proinflammatory cytokines and mediators by the expanded NLV-specific CD8+T cells was quantified by flow cytometric analysis.

[0399] NLV-specific CD8+T cells expanded by treatment with the indicated LVP constructs displayed increased levels of IFN^, TNF^, IL-2, perforin, and granzyme B as compared to unstimulated cells (Fig.5). The highest increase in immune modulator production was observed in PBMCs stimulated with LVPs presenting the membrane-anchored MHC / NLV complex and membrane-anchored agonist anti-CD28 scFv (no envelope protein), indicating an additional functional benefit of T cell activation induced by the NLV-specific TCR in presence of the additional CD28 co-stimulatory molecule.

[0400] Example 7: NLV-specific CD8+T cells, which have been expanded by treatment with LVPs presenting an MHC / NLV complex, display potent cytotoxic activity against target cells presenting the NLV-peptide

[0401] Next, the cytotoxic activity of expanded, NLV-specific CD8+T cells was determined. To that end, 20 days after treatment with different LVP constructs, the T cells were incubated at effector:target ratios of 1:1 and 1:10 with T2 cells. The T2 cells were loaded either with the NLV peptide or no peptide.

[0402] NLV-specific T cells treated with the indicated LVP constructs displayed potent and significant specific cytotoxic activity indicated by lysis of NLV-peptide loaded T2 cells, as compared to T2 cells that had not been loaded with peptide (Fig.6).

[0403] Example 8: NLV-specific CD8+T cells, which have been expanded by treatment with LVPs presenting an MHC / NLV complex, are able to suppress CMV infection 81 172416522.1

[0404] To further evaluate the functional capacity of LVP-expanded NLV-specific CD8+T cells, the capacity of the expanded T cells to suppress infection of CMV-susceptible MRC5 fibroblast cells with CMV was assessed in vitro.

[0405] PBMCs comprising NLV-specific CD8+T cells were expanded in presence of the indicated LVP constructs. After 20 days, the T cells were incubated with the CMV-infected MRC5 fibroblast cells, which expressed a firefly luciferase reporter. Two days later, the level of CMV infection in MRC5 cells was quantified by harvesting the cells and measuring the luciferase activity in the cell lysates.

[0406] LVP-expanded, NLV-specific CD8+T cells suppressed CMV infection significantly more than untreated T cells. The highest level of CMV infection suppression was mediated by T cells stimulated with LVPs presenting the membrane-anchored MHC / NLV complex and membrane-anchored agonist anti-CD28 scFv, indicating an additional functional benefit to T cell activation induced by the NLV-specific TCR in the presence of the CD28 co-stimulatory molecule. As a positive control, cells were treated with an anti-HIV drug, Cidofovir, which completely suppressed infection (Fig.7).

[0407] Example 9: LVPs presenting an MHC / MART-1 complex and a membrane- anchored co-stimulatory signal activate naïve MART-1-specific CD8+T cells

[0408] To demonstrate that the LVP platform described herein works with a variety of peptides, an LVP presenting a different peptide was constructed. Specifically, a fusion protein was designed comprising a single chain construct comprising MHC class I HLA-A*0201, presenting the well-characterized melanoma-associated antigen (MART-1) peptide (26-35, ELAGIGILTV) (SEQ ID NO:2). The MART-1 epitope is targeted by a relatively high frequency of CD8+T cells within the naïve T cell population of HLA-A*0201 individuals, providing sufficient naïve MART-1-reactive cells for evaluation by ex vivo stimulation assays.

[0409] LVP constructs were generated presenting on their surface (1) membrane-anchored MHC / MART-1 (“MART-1-Modless VirTac”) or (2) membrane-anchored MHC / MART-1 and membrane-anchored agonist anti-CD28 scFv (“MART-1-αCD28 VirTac”) The membrane- anchored MHC / MART-1 and the membrane-anchored agonist anti-CD28 scFv were not covalently linked to each other. The ability of the LVP constructs to bind to and activate MART-1-specific TCR was assessed using Jurkat T cells, which expressed a MART-1-specific TCR and an NFAT-regulated luciferase reporter gene. The reporter gene was activated by TCR engagement. 82 172416522.1

[0410] Both MART-1-^CD28 VirTac and MART-1-modless VirTac constructs induced expression of the luciferase reporter in the Jurkat cells as compared to transduced cells, indicating successful binding of the MART-1 to the MART-1-specific TCR and T cell activation by the LVP constructs (Fig.8).

[0411] These results indicated that the LVPs are inducing antigen-specific activation of these cells and the LVPs are not activating the T cells through another pathway.

[0412] Example 10: LVPs presenting an MHC / MART-1 complex and a membrane- anchored co-stimulatory signal expand naïve MART-1-specific CD8+T cells

[0413] Naïve CD8+T cells were isolated from the PBMCs of a healthy donor by immunomagnetic sorting. The T cells were stimulated with LVP constructs presenting on their surface (1) membrane-anchored MHC / MART-1 (“MART-1-Modless VirTac”) or (2) membrane-anchored MHC / MART-1 and membrane-anchored agonist anti-CD28 scFv (“MART-1-αCD28 VirTac”), respectively. The membrane-anchored MHC / MART-1 and the membrane-anchored agonist anti-CD28 scFv were not covalently linked to each other. On day 7 and 14 after LVP treatment, the naïve CD8+T cells were assessed using flow cytometry for selective MART-1-specific CD8+T cell expansion.

[0414] Selective expansion of MART-1-specific CD8+T cells was observed by day 14 after MART-1-αCD28 VirTac treatment for all concentrations tested, but not after MART-1- modless treatment (Fig.9).

[0415] CD28 co-stimulation, in addition to TCR signaling, is important for the effective priming, proliferation, and differentiation of naïve MART-1-specific CD8+T cells. As such, these data demonstrated the capacity of LVPs to deliver both a TCR and CD28 signal to cognate MART-1-specific T cells.

[0416] Example 11: LVPs presenting an MHC / NLV complex induce expansion of NLV-specific CD8+T cells in vivo

[0417] In a proof-of-concept experiment, the capacity of LVPs to selectively expand NLV- specific CD8+T cells in vivo was examined using a humanized mouse model. Donor PBMCs (donor HGK0055) were co-injected intrasplenically into NSG mice either alone or mixed with different LVP constructs. Nine days later, the mouse spleens were harvested and the fraction of CD8+T cells that were NLV-specific were quantified by flow cytometric analysis. 83 172416522.1

[0418] In vivo treatment with the indicated LVP constructs induced in vivo expansion of the NLV-specific CD8+T cell population as compared to the unstimulated population in a dose dependent manner (~25-fold expansion for lower dose; ~32-fold expansion for higher dose) (Fig.10).

[0419] To determine the capacity of systemically administrated LVPs to stimulate expansion of peripheral circulating NLV-specific CD8+T cells, donor PBMCs (donor HGK0055) were injected intrasplenically into NSG mice and either unstimulated (n = 1 mouse) or treated by intravenous injection of the indicated volume of “NLV ^CD28 LVP” (n = 2 mice) or “NLV ^CD28 Immuno-STAT” (n = 1 mouse). The mice were given one dose intraperitoneally of human IL-7 / IL-15 / IL-21 on day 1 because murine cytokines do not adequately support human T cell expansion. The mice were bled ten days after the first injection processed and stained with NLV tetramer to determine circulating NLV-specific CD8+T cells (each plot represents a single mouse).

[0420] Remarkably, in vivo treatment with the indicated LVP constructs into the spleen induced in vivo expansion of the NLV-specific CD8+T cell population in the peripheral blood as compared to the unstimulated population (~5-fold expansion in the first mouse and; ~10- fold expansion in the second mouse), with both mice displaying greater expansion than a mouse treated with the protein-based NLV ^CD28 Immuno-STAT (positive control) (Fig.11).

[0421] Example 12: LVPs presenting an MHC / SL9 complex activate T cells targeting SL9 and induce an effector memory phenotype in these T cells with cytotoxic activity

[0422] Expansion of HIV-specific CD8+T cells may facilitate the ability of people with HIV to control HIV infection without the need to continue to take antiretroviral drugs. Accordingly, LVPs were generated that present on their surface (1) an HLA-A*0201 MHC complex presenting the well-conserved immunodominant peptide of the HIV gp120 glycoprotein, SL9 (SEQ ID NO:3), or (2) an MHC / SL9 complex and the agonist anti-CD28 scFv. The ability of the LVPs to activate SL9-specific TCRs was assessed using Jurkat / Ma CD8+NFAT-Firefly luciferase reporter cells. These cells had been genetically modified to express a TCR that specifically recognizes SL9 peptide. The Jurkat cells were incubated with different LVP constructs and luciferase activity was determined.

[0423] The indicated LVP constructs were able to bind to and activate the SL9-TCR in a dose responsive manner (Fig. 12A). These Jurkat cells do not express CD28 and thus would not be expected to display increase activation in the presence of ^CD28 signaling. 84 172416522.1

[0424] Next, PBMCs from a person with HIV whose peripheral blood contains a population of CD8+T cells expressing the SL9-specific TCR (donor RC619) were treated with different LVP constructs. The indicated LVPs induced expansion of SL9-specific CD8+T cells, as assessed by flow cytometric quantification of the population of SL9-tetramer binding CD8+T cells (Fig. 12B, upper panel). As compared to the untreated cells (3.54 %), the SL9-specific CD8+T cell population was expanded by treatment with an LVP presenting membrane- anchored MHC / SL9 (to 19.8%) or an LVP presenting membrane-anchored MHC / SL9 and membrane-anchored agonist anti-CD28 scFv (to 22.8%), comparable to expansion by a protein-based MHC / SL9 Immuno-STAT (31.5%).

[0425] Additionally, the impact of LVP treatment on the memory phenotype of T cells was assessed by characterizing CD45Ro and CD62L expression for T cells that were either SL9- tetramer positive or negative (Fig. 12B). After treatment with LVPs, >94% of the expanded SL9-specific CD8+T cell population (SL9+, Fig. 12B, middle panel) displayed an effector memory phenotype as compared to ~ 60% of the CD8+T cell population that did not recognize SL9 peptide (SL9-, Fig.12B, lower panel).

[0426] Next, the cytotoxic activity of expanded, SL9-specific CD8+T cells was determined. To that end, about 3 weeks days after treatment with different LVP constructs, the T cells were incubated at effector:target ratios of 1:1 and 1:10 with T2 cells. The T2 cells were loaded either with the SL9 peptide or no peptide.

[0427] SL9-specific T cells treated with the indicated LVP constructs displayed potent and significant specific cytotoxic activity indicated by lysis of SL9-peptide loaded T2 cells, as compared to T2 cells that had not been loaded with peptide (Fig.13).

[0428] Example 13: Characterization of LVPs presenting an MHC / NLV complex (NLV-LVPs) by mass spectrometry

[0429] Mass spectrometry was performed to confirm incorporation of peptide-loaded MHC complexes and co-stimulatory agonists (like the agonist anti-CD28 scFv) into the LVPs.

[0430] Both modless (Table 3) and anti-CD28-containing (Table 4) NLV-LVPs, respectively, incorporated the NLV peptide-MHC complex. Notably, only the anti-CD28 LPVs incorporated the scFv anti-CD28 agonist, confirming the specific presence of the CD28 co- stimulatory binder in the engineered anti-CD28 LVP construct. Table 3. Selected mass spectrometry results for modless NLV-LVPs 85 172416522.1Description Coverage # # # MW Mod T % raw [%] Peptides PSMs Unique [kDa] abundance Pe tidesDescription Coverage [%] # Peptides # PSMs # Unique PeptidesDescription MW [kDa] CD28 by Trypsin % raw Abundance NLV MHC 41.6 1.91E+07 0.84ged incubation at 37ºC

[0432] Because serum can inactivate lentiviral particles expressing the VSVg envelope, which may limit the particles’ in vivo function, it was examined whether serum inactivates LVPs.

[0433] NLV-specific LVPs were incubated with human serum from group AB and O donors for 14 days.

[0434] LVPs did not display any reduction in functional activity after incubation with human serum, as indicated by the LVPs’ continued ability to expand NLV-specific CD8+T cells at the same or higher level than when no serum was added (Fig.14A).

[0435] Thermal stability assays demonstrated that incubation at 37 °C for up to 7 days did not impair LVP-induced antigen-specific T cell expansion (Fig. 14B), indicating robust stability of the LVPs in the presence of human serum factors and 37 ºC temperature.

[0436] Example 15: NLV-LVPs expand NLV-specific CD8⁺ T cells, and SL9-LVPs expand SL9-specific CD8⁺ T cells, respectively, in both HIV⁻ and HIV⁺ donors

[0437] PBMCs from both donors without HIV (n = 4) and donors with HIV (n = 3) were treated with NLV- or SL9-specific LVPs, respectively.

[0438] A highly significant expansion of antigen-specific CD8⁺ T cells was observed in NLV-responsive donors (up to 80%) and SL9-responsive donors (up to 20%) regardless of HIV 86 172416522.1status, confirming the broad applicability of LVP stimulation across a wide range of donors (Fig.15A and Fig.15B).

[0439] Example 16: Generation of LPVs presenting immunodominant peptides YLQ and IV9 on their surface

[0440] LVP platforms were engineered to present other immunodominant peptides, such as YLQ (YLQPRTFLL (SEQ ID NO:94), derived from SARS-CoV-2) and IV9 (ILKEPVHGV (SEQ ID NO:95), derived from HIV), in the scMHC complex on the LVPs’ surface.

[0441] PBMCs from YLQ-reactive donors (n = 4) responded to the YLQ-LVPs with up to 50% YLQ-specific T cell expansion after YLQ-LVP treatment (Fig.16A).

[0442] Modless IV9-LVP function was demonstrated by verifying its ability to bind and activate IV9-specific TCR in a Jurkat cell line, which had been transduced to express only IV9- TCR and contained a luciferase reporter gene under the NFAT promoter. Luciferase activity was measured after IV9-LPV treatment.

[0443] TCR activation was observed in a dose-responsive manner, confirming effective IV9 peptide-MHC presentation, TCR engagement, and downstream signaling activation (Fig.16B).

[0444] Example 17: YLQ- and SL9-LVPs, respectively, successfully promote the in vitro expansion of functional YLQ-specific CD8+T cells or SL9-specific CD8+T cells, respectively

[0445] Donor YLQ-specific CD8+T cells were expanded in vitro by treating the cells with YLQ-LVPs. 80-100% of the resulting T cells demonstrated the ability to produce IFNγ and TNFα after exposure to cognate peptide-pulsed targets T2 cells (Fig.17A). No IFNγ and TNFα production was induced when exposed to non-peptide loaded T2 cells.

[0446] The YLQ-LPV-expanded, YLQ-specific CD8+T cells also exhibited 80-100% specific cytotoxicity calculated by YLQ-loaded T2 cells killing compared to non-peptide loaded T2 cells. (Fig.17B).

[0447] Finally, SL9-LVP-expanded CD8+T cells suppressed HIV replication in autologous CD4⁺ T cells infected with HIV, as measured by reduced luciferase reporter gene activity compared to unstimulated cells (Fig.17C).

[0448] These results confirm that LVP-expanded T cells are functionally competent and exhibit antiviral activity. 87 172416522.1

[0449] Example 18: In vivo half-life of NLV-LVPs

[0450] The half-life of NLV-LVPs was examined after intravenous injection into NSG mice.

[0451] Viral particle levels in plasma were measured using an HIV p24 antigen ELISA.

[0452] Detectable levels of NLV-LVPs in the first 3 hours after the injection indicated sufficient in vivo stability for the LVPs to circulate in the bloodstream, migrate into lymphoid tissues, and encounter the target cells (Fig. 18). The decrease in LVPs observed over time indicated a reduced likelihood that the LVP would generate an inflammatory or exacerbated immune response.

[0453] Example 19: Ability of SL9-LVPs to expand SL9-specific CD8+T cells after intravenous injection into NSG

[0454] The ability of SL9-LVPs to expand SL9-specific CD8+T cells was examined after intravenous injection into NSG mice was examined.

[0455] Humanized NSG mice were intrasplenically injected with PBMCs from an HIV- positive donor. SL9-LVPs were administered intravenously (Fig. 19). After 10 days, circulating human SL9-specific CD8+T cells were analyzed.

[0456] A significant population (30-38%) of expanded SL9-specific CD8+T cells was observed in LVP-treated groups compared to the 2% SL9-specific T cell starting population (unstimulated). This confirms the capacity of LVPs to migrate to lymphoid tissues and induce in vivo expansion of antigen-specific human T cells.

[0457] Example 20: Generation of LVPs presenting two different peptides (duo-LVPs)

[0458] LVPs were designed to express more than one type of scMHC complex, thus presenting more than one peptide. For example, a Duo-LVP can expand two different antigen- specific CD8+T cell populations.

[0459] To address potential immune escape by HIV-1 mutations, LVPs capable of presenting two distinct peptides (IV9 and SL9)-scMHC complexes, respectively, on the same viral particle were generated (Fig.20A). Combinations such as NLV-YLQ and NLV-SL9 were also successfully produced, establishing proof of concept for multi-specific LVPs (Fig. 20B and Fig.20C). 88 172416522.1

[0460] Donor PBMCs were stimulated with NLV-YLQ- or NLV-SL9-duo-LVPs, respectively.

[0461] Dual tetramer staining revealed simultaneous expansion of NLV (59 %) and YLQ (14.3 %)-specific CD8⁺ T cell populations when treated with the NLV-YLQ-duo-LVPs, and NLV (46.2 %) and SL9 (6.55 %) expansion of the respective antigen-specific CD8⁺ T cell when stimulated with NLV-SL9-duo-LVPs (Fig.20D).

[0462] NLV-YLQ duoVirTac expanded cells demonstrated ≈100% specific cytotoxicity against NLV loaded T2 cells and 50-60% against YLQ loaded T2 cells (compared to T2 cells loaded with a non-specific (SL9) peptide). This data shows that the duo-LVPS exhibit a functional dual specificity and cytotoxic capacity (Fig.20E).

[0463] Example 21: LVP expansion of TCR-engineered antigen-specific CD8+T cells

[0464] LVP can further be used to expand T cells that have been genetically engineered to express a TCR recognizing a specific antigen. To that end, donor-isolated CD8+T cells were engineered to express an SL9-specific TCR. The T cells were then stimulated with modless SL9-LVPs or anti-CD28 SL9-LVPs, respectively (Fig.21A). Antigen-specific expansion of up to 3- and 4-fold was observed after treatment with modless SL9-LVPs or anti-CD28 SL9- LVPs, respectively, compared to unstimulated SL9-TCR transduced CD8+T cells (Fig.21B).

[0465] This data shows that LVPs can be used to enhance the numbers of T cells used in TCR T cell therapy.

[0466] In sum, the experiments herein demonstrate the flexibility of the LVP platform to activate, expand, and differentiate functional CD8+T cells targeting diverse epitopes both in vitro and in vivo after intravenous injection. 89 172416522.1

Claims

CLAIMS We claim:

1. A recombinant viral particle, the recombinant viral particle comprising a lipid envelope and: (1) an MHC / peptide complex comprising a first fusion polypeptide comprising (a) a class I major histocompatibility (MHC) polypeptide or a fragment, mutant, or derivative thereof, (b) a β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof, (c) an epitope, and (d) a first lipid envelope anchor; and (2) a second fusion polypeptide comprising (a) a co-stimulatory domain and (b) a second lipid envelope anchor.

2. The recombinant viral particle of claim 1, wherein the class I MHC polypeptide comprises a human class I MHC polypeptide derived from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G.

3. The recombinant viral particle of claim 1, wherein the class I MHC polypeptide comprises a murine class I MHC polypeptide derived from H-2K, H-2D, H-2L, H-2Q, H-2M or H-2T.

4. The recombinant viral particle of claim 1, wherein the class I MHC polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO:

20.

5. The recombinant viral particle of claim 4, wherein the class I MHC polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO:

20.

6. The recombinant viral particle of claim 5, wherein the class I MHC polypeptide comprises SEQ ID NO:

20.

7. The recombinant viral particle of any one of claims 1-6, wherein the β2 microglobulin polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO:

19.

8. The recombinant viral particle of claim 7, wherein the β2 microglobulin polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO:

19.

9. The recombinant viral particle of claim 8, wherein the β2 microglobulin polypeptide comprises SEQ ID NO:

19.

10. The recombinant viral particle of any one of claims 1-9, wherein the epitope is 5 to 20 amino acids long.

11. The recombinant viral particle of any one of claims 1-10, wherein the epitope is derived from a bacterial, viral, fungal, or protozoan polypeptide. 90 172416522.

112. The recombinant viral particle of claim 11, wherein the epitope is derived from a cytomegalovirus (CMV) polypeptide, a human immunodeficiency virus (HIV) polypeptide, a SARS-CoV-2 virus polypeptide, or a hepatitis virus polypeptide.

13. The recombinant viral particle of claim 11, wherein the epitope comprises any one of SEQ ID NOs:1-3, SEQ ID NO:94, or SEQID NO:

95.

14. The recombinant viral particle of claim 11, wherein the epitope comprises a sequence derived from HIV polypeptide gp120, HIV-1 polypeptide p17, or melanoma antigen recognized by T cells 1 (MART-1).

15. The recombinant viral particle of any one of claims 1-10, wherein the epitope is a cancer antigen or fragment thereof.

16. The recombinant viral particle of any one of claims 1-15, wherein the first lipid envelope anchor and / or the second lipid envelope anchor is a transmembrane domain.

17. The recombinant viral particle of claim 16, wherein the transmembrane domain is derived from human HLA-A*0201 MHC.

18. The recombinant viral particle of claim 17, wherein the transmembrane domain comprises a sequence that is at least 80% identical to SEQ ID NO:

22.

19. The recombinant viral particle of claim 18, wherein the transmembrane domain comprises a sequence that is at least 90% identical to SEQ ID NO:

22.

20. The recombinant viral particle of claim 19, wherein the transmembrane domain comprises SEQ ID NO:

22.

21. The recombinant viral particle of any one of claims 1-20, wherein the co-stimulatory domain is a 4-1BBL polypeptide, a B7-1 polypeptide, a B7-2 polypeptide, an ICOS-L polypeptide, an OX-40L polypeptide, a CD80 polypeptide, a CD70 peptide, a CD86 polypeptide, a CD40L polypeptide, a CD40 polypeptide, a PD-L1 polypeptide, or a PD-L2 polypeptide.

22. The recombinant viral particle of any one of claims 1-20, wherein the co-stimulatory domain is a CD28 agonist, an ICOS agonist, a 4-1BB agonist, a OX40 agonist, a CD27 agonist, a CD40 agonist, a CD40L agonist, a CTLA-4 agonist, or a PD-1 agonist.

23. The recombinant viral particle of claim 22, wherein the co-stimulatory domain is a CD28 agonist.

24. The recombinant viral particle of claim 22, wherein the co-stimulatory domain comprises a sequence that is at least 80% identical to SEQ ID NO:

25. 91 172416522.

125. The recombinant viral particle of claim 24, wherein the co-stimulatory domain comprises a sequence that is at least 90% identical to SEQ ID NO:

25.

26. The recombinant viral particle of claim 25, wherein the co-stimulatory domain comprises SEQ ID NO:

25.

27. The recombinant viral particle of any one of claims 16-26, wherein: (1) the first fusion polypeptide comprises from N-terminus to C-terminus: (a) the epitope; (b) the β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof; (c) the class I MHC polypeptide or a fragment, mutant, or derivative thereof; (d) the first transmembrane domain; and (e) optionally, a first cytoplasmic domain; and / or (2) the second fusion polypeptide comprises from N-terminus to C-terminus: (a) the co-stimulatory domain; (b) the second transmembrane domain; and (c) optionally, a second cytoplasmic domain.

28. The recombinant viral particle of any one of claims 1-27, wherein the first fusion polypeptide and / or the second fusion polypeptide further comprises one or more linkers.

29. The recombinant viral particle of claim 28, wherein the one or more linkers are one or more polypeptide linkers or disulfide bond linkers.

30. The recombinant viral particle of claim 29, wherein the one or more polypeptide linkers are one or more flexible linkers.

31. The recombinant viral particle of claim 30, wherein the one or more flexible linkers predominantly consist of glycines, serines, and alanines.

32. The recombinant viral particle of claim 30, wherein the one or more flexible linkers comprise one or more repetitions of any one of SEQ ID NOs:4-17.

33. The recombinant viral particle of claim 31, wherein the one or more flexible linkers comprise SEQ ID NO:

16.

34. The recombinant viral particle of claim 29, wherein the one or more polypeptide linkers are one or more rigid linkers.

35. The recombinant viral particle of claim 34, wherein the one or more rigid linkers comprise one or more repetitions of any one of SEQ ID NOs:27-55. 92 172416522.

136. The recombinant viral particle of any one of claims 1-35, wherein the first fusion polypeptide and / or the second fusion polypeptide further comprise a signal peptide.

37. The recombinant viral particle of claim 36, wherein the signal peptide comprises any one of SEQ ID NOs:18 or 56-93.

38. The recombinant viral particle of claim 36, wherein the signal peptide is derived from the beta 2 microglobulin signal peptide.

39. The recombinant viral particle of claim 38, wherein the signal peptide comprises a sequence that is at least 80% identical to SEQ ID NO:

18.

40. The recombinant viral particle of claim 39, wherein the signal peptide comprises a sequence that is at least 90% identical to SEQ ID NO:

18.

41. The recombinant viral particle of claim 40, wherein the signal peptide comprises SEQ ID NO:

18.

42. The recombinant viral particle of any one of claims 1-41, wherein the first fusion polypeptide and the second fusion polypeptide are covalently linked.

43. The recombinant viral particle of any one of claims 1-42, wherein the recombinant viral particle is derived from a lentivirus.

44. The recombinant viral particle of any one of claims 1-43, wherein one or more of polypeptides present in the lipid envelope of a wild-type virus corresponding to the recombinant viral particle are absent or mutated so that said recombinant viral particle is not capable of binding to any cell targeted by the wild-type virus in the absence of the MHC / peptide complex.

45. The recombinant viral particle of any one of claims 1-44, wherein the epitope binds to a T cell receptor (TCR) of a CD8+T cell.

46. A nucleic acid or pair of nucleic acids encoding the MHC / peptide complex and / or the second fusion polypeptide of any one of claims 1-45.

47. A method of activating a T cell comprising a TCR, the method comprising contacting the T cell with the recombinant viral particle of any one of claims 1-45, wherein the TCR of the T cell binds to the epitope.

48. A method of inducing expression of pro-inflammatory cytokines in a T cell comprising a TCR, the method comprising contacting the T cell with the recombinant viral particle of any one of claims 1-45, wherein the TCR of the T cell binds to the epitope. 93 172416522.

149. A method of expanding a population of T cells each comprising a TCR, the method comprising contacting the population of T cells with the recombinant viral particle of any one of claims 1-45, wherein the TCR of the T cells binds to the epitope.

50. The method of any one of claims 47-49, wherein the T cell has been genetically engineered to comprise the TCR that binds to the epitope.

51. A method of inducing killing of a cell infected by a pathogen, wherein the cell displays on its surface a polypeptide derived from the pathogen, the method comprising: (a) contacting a population of T cells with the recombinant viral particle of any one of claims 1-14 or 16-45, thereby generating a population of activated T cells, wherein each T cell comprises a TCR, wherein the TCR is capable of binding to the polypeptide derived from the pathogen and to the epitope; and (b) exposing the cell infected by the pathogen to the population of activated T cells.

52. The method of claim 51, wherein the population of T cells has been genetically engineered to express the TCR that is capable of binding to the polypeptide derived from the pathogen and to the epitope.

53. A method of inducing killing of a cancer cell, wherein the cancer cell displays on its surface a cancer antigen, the method comprising: (a) contacting a population of T cells with the recombinant viral particle of any one of claims 1-10 or 15-45, thereby generating a population of activated T cells, wherein each T cell comprises a TCR, wherein the TCR is capable of binding to the cancer antigen and to the epitope; and (b) exposing the cancer cell to the population of activated T cells.

54. The method of claim 53, wherein the population of T cells has been genetically engineered to express the TCR that is capable of binding to the cancer antigen and to the epitope.

55. The method of any one of claims 51-54, wherein the population of activated T cells is further expanded before step (b).

56. A method of treating an infection with a pathogen in a subject in need thereof, the method comprising administering to the subject the recombinant viral particle of any one of claims 1-14 or 16-45, wherein: the subject has a cell that displays on its surface a polypeptide derived from the pathogen; and 94 172416522.1the subject has a T cell that comprises a TCR, wherein the TCR is capable of binding to the polypeptide derived from the pathogen and to the epitope.

57. A method of treating an infection with a pathogen in a subject in need thereof, the method comprising administering to the subject a population of T cells, wherein: the population of T cells has previously been contacted with the recombinant viral particle of any one of claims 14 or 16-45; the subject has a cell that displays on its surface a polypeptide derived from the pathogen; and the population of T cells has been genetically engineered to express a TCR, wherein the TCR is capable of binding to the polypeptide derived from the pathogen and to the epitope.

58. The method of any one of claims 51, 52, or 55-57 wherein the pathogen is a bacterial, viral, or fungal pathogen.

59. The method of claim 58, wherein the pathogen is a viral pathogen.

60. The method of claim 59, wherein the viral pathogen is a CMV, an HIV, a SARS-CoV- 2 virus, or a hepatitis virus.

61. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject the recombinant viral particle of any one of claims 1-10 or 15-45, wherein: the subject has a cancer cell that displays on its surface a cancer antigen; and the subject has a T cell that comprises a TCR, wherein the TCR is capable of binding to the cancer antigen and to the epitope.

62. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a population of T cells, wherein: the population of T cells has previously been contacted with the recombinant viral particle of any one of claims 1-10 or 15-45; the subject has a cancer cell that displays on its surface a cancer antigen; and the population of T cells has been genetically engineered to express a TCR, wherein the TCR is capable of binding to the cancer antigen and to the epitope.

63. The method of any one of claims 56-62, wherein the subject is a human.

64. A recombinant viral particle, the recombinant viral particle comprising a lipid envelope and: 95 172416522.1(1) a first MHC / peptide complex comprising a first fusion polypeptide comprising (a) a first class I MHC polypeptide or a fragment, mutant, or derivative thereof, (b) a first β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof, (c) a first epitope, and (d) a first lipid envelope anchor; (2) a second MHC / peptide complex comprising a second fusion polypeptide comprising (a) a second class I MHC polypeptide or a fragment, mutant, or derivative thereof, (b) a second β2 microglobulin polypeptide or a fragment, mutant, or derivative thereof, (c) a second epitope, wherein the first epitope and the second epitope are different and (d) a second lipid envelope anchor; and (3) a third fusion polypeptide comprising (a) a co-stimulatory domain and (b) a third lipid envelope anchor.

65. The recombinant viral particle of claim 64, wherein the first epitope and the second epitope are derived from the same polypeptide.

66. The recombinant viral particle of claim 64, wherein the first epitope and the second epitope are derived from different polypeptides from the same organism. 96 172416522.1

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