CD58-pseudotyped viral particles and uses thereof

Abstract

Provided herein are recombinant targeting polypeptides. Some such polypeptides include an external region comprising a CD2 binding domain such as a CD58 extracellular domain, a transmembrane region, and an internal region comprising a cytoplasmic fragment of a henipavirus G protein. Some embodiments relate to a viral particle that includes a recombinant targeting polypeptide. Also disclosed are methods of using the recombinant targeting polypeptides or viral particles comprising them.

Description

Attorney Ref: 061479-515001WOCD58-PSEUDOTYPED VIRAL PARTICLES AND USES THEREOFCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 736.439, filed December 19, 2024, the entire contents of which are incorporated herein by reference.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 061479-515001WO. xml, created December 15, 2025, which is 67,433 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.BACKGROUND

[0003] There remains a need in the art for new compositions and methods that improve transduction of cells such as T cells by deliver}' vehicles such as lentiviruses. For example, use of some delivery vehicles may lead to off-target effects. Additionally, lentiviral particles pseudotyped with such glycoproteins may have reduced transduction efficiencies due to serum inhibition. Improved viral vectors are needed.SUMMARY

[0004] Disclosed herein, in some embodiments, arc particles such as viral particles that include a recombinant targeting polypeptide. Disclosed herein, in some embodiments, are viral particles, comprising: a polynucleotide cargo; a lipid envelope; and recombinant targeting polypeptide comprising: an external region comprising a CD58 extracellular domain, and a transmembrane region. In some embodiments, the recombinant targeting polypeptide comprises an internal region comprising a cytoplasmic tail of a henipavirus G protein. In some embodiments, the recombinant targeting polypeptide further comprises an internal region comprising a cytoplasmic fragment of a Nipah Virus (NiV) G protein. In some embodiments, the cytoplasmic fragment comprises a cytoplasmic tail of a NiV G protein. In some embodiments, the cytoplasmic fragment comprises a cytoplasmic tail comprising a GcA33 truncation variant or a GcA34 truncation variant. In some embodiments, the external region comprises a polypeptide sequence according to SEQ ID NO: 38, or at least 80%. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%. at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the transmembrane region comprises a transmembrane domain of the henipavirus G protein. In some embodiments, the transmembrane region comprises a transmembrane domain of CD58. In some embodiments, the targeting polypeptide comprises a stalk domain of the henipavirus G protein. In some embodiments, the stalk domain together with the transmembrane regionAttorney Ref: 061479-515001WO comprises the polypeptide sequence of SEQ ID NO: 34, or a polypeptide sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%. at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 34. In some embodiments, the stalk domain is connected through a linker to the CD58 extracellular domain. In some embodiments, the linker is 4 to 50 amino acids in length. In some embodiments, the linker comprises the polypeptide sequence of any of SEQ ID NOs: 35-37, or a polypeptide sequence at least 80%, at least 85%, at least 90%. at least 91%, at least 92%, at least 93%, at least 94%, at least 95%. at least 96%. at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 35-37. In some embodiments, the linker comprises the polypeptide sequence of any of SEQ ID NOs: 35. In some embodiments, the linker comprises the polypeptide sequence of any of SEQ ID NOs: 36. In some embodiments, the linker comprises the polypeptide sequence of any of SEQ ID NOs: 37. In some embodiments, the targeting polypeptide comprises a polypeptide sequence disclosed in Table 1, or at least 80%, at least 85%. at least 90%, at least 91%, at least 92%, at least 93%. at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the targeting polypeptide comprises the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence at least 80%. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% identical thereto.

[0005] Some embodiments include a multidomain fusion (MDF) protein comprising: a CD58 extracellular domain; a CD80 or CD86 extracellular domain; and an anti-CD3 antibody, an anti-CD3 antibody binding fragment, or an anti-CD3 single-chain variable fragment (scFv).

[0006] In embodiments, the CD58 extracellular domain comprises the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In embodiments, the CD80 extracellular domain comprises the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence at least 80%, at least 85%. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In embodiments, the CD86 extracellular domain comprises the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% identical thereto. In embodiments, anti-CD3 antibody or an anti- CD3 scFv comprises an complementarity detennining region (CDR) LI (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 26, an CDR-L2 comprising the amino acid sequence of SEQ ID NO: 27. and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 28; and an CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29, an CDR-H2 comprising the amino acid sequence of SEQ ID NO: 30, and CDR-H3 comprises the amino acid sequence of SEQ ID NO: 31. In embodiments, the multidomain fusion (MDF) comprises the amino acid sequence of any one of SEQ ID NOs: 13-16. or an amino acid sequence at least 80%, at least 85%, at least 90%. at leastAttorney Ref: 061479-515001WO91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0007] In some embodiments, the polynucleotide cargo comprises a promoter. In some embodiments, the polynucleotide cargo encodes a chimeric antigen receptor. In some embodiments, the polynucleotide cargo encodes a small molecule-activated cytokine receptor.

[0008] In some embodiments, the polynucleotide cargo encodes a rapamycin molecule-activated cytokine receptor (RACR). In embodiments, the viral particle comprises a henipavirus F protein. In embodiments, the viral particle comprises a Nipah virus F (NiV F) protein. In embodiments, the NiV F protein comprises the amino acid sequence of SEQ ID NOs: 10 or 40, or an amino acid sequence at least 80%, at least 85%. at least 90%, at least 91%, at least 92%, at least 93%. at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In embodiments, the NiV F protein is a truncation variant, wherein the 22 amino acid residues corresponding to the N- terminal of SEQ ID NO: 10 are deleted. In embodiments, the NiV F protein comprises the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0009] In some embodiments, the particle is a lentiviral particle. Disclosed herein, in some embodiments, are pharmaceutical compositions comprising the particle and one or more pharmaceutically acceptable solvents or diluents.

[0010] Disclosed herein, in some embodiments, are recombinant targeting polypeptides. In some embodiments, the recombinant targeting polypeptide is as comprised in a viral particle described herein. Disclosed herein, in some embodiments, are recombinant targeting polypeptides comprising: an extracellular domain that specifically binds CD2; a transmembrane domain; and a cytoplasmic domain comprising a cytoplasmic tail of a henipavirus G protein. In some embodiments, the extracellular domain comprises an antibody that specifically binds CD2. In some embodiments, the recombinant targeting polypeptide comprises a stalk domain of the henipavirus G protein. In some embodiments, the recombinant targeting polypeptide comprises a henipavirus F protein. In some embodiments, the recombinant targeting polypeptide comprises a Nipah Virus (NiV) F protein.

[0011] Disclosed herein, in some embodiments, are gene delivery vehicles comprising the recombinant polypeptide. Disclosed herein, in some embodiments, are polynucleotides encoding the polypeptide, operably linked to a promoter. Disclosed herein, in some embodiments, are host cells comprising the polynucleotide. Disclosed herein, in some embodiments, are methods of making viral particles. Disclosed herein, in some embodiments, are methods of making viral particles, comprising expressing a polynucleotide herein in a host cell configured to generate a viral particle comprising the expressed targeting polypeptide.

[0012] Disclosed herein, in some embodiments, are methods of making CAR T cells. The method may be in vitro. Disclosed herein, in some embodiments, are in vitro methods of making a CAR TAttorney Ref: 061479-515001WO cell, the method comprising contacting a T cell with a particle herein, thereby transducing the CAR T cell with a polynucleotide encoding a chimeric antigen receptor, operatively linked to a promoter. The method may be in vivo. Some embodiments relate to a method of treatment. Disclosed herein, in some embodiments, are in vivo methods of generating a CAR T cell or treating cancer or an autoimmune disease in a subject in need thereof, the method comprising administering to the subject, a particle herein, thereby transducing the CAR T cell with the a polynucleotide encoding a chimeric antigen receptor, operatively linked to a promoter, wherein the particle is administering in an amount effective to generate CAR T cells or treat cancer or an autoimmune disease in the subject. In some embodiments, the administration is intravenous, intratumoral, or intralymphatic. In some embodiments, the administration comprises an injection.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1A depicts an example of a lentiviral particle that includes Cocal and MDF proteins.

[0014] FIG. IB depicts an example lentiviral particle that includes NiV Env proteins and a polynucleotide payload.

[0015] FIG. 1C depicts an example lentiviral particle that includes NiV Env proteins and a polynucleotide payload, in addition to an MDF protein at the particle surface.

[0016] FIG. 2 includes an example of a NiV glycoprotein G.

[0017] FIG. 3 includes a depiction of a viral particle that includes NiV glycoproteins F and G, as well as binding to proteins of a target cell.

[0018] FIG. 4 includes a depiction of an example viral particle and engineered NiV glycoprotein G. The engineered NiV glycoprotein G is shown about to bind with a receptor of a target cell.

[0019] FIG. 5 includes a NiV G protein engineering and screening strategy.

[0020] FIG. 6 includes plots of flow cytometry data for transduction of cells with viral particles that included engineered NiV G proteins of various linker lengths.

[0021] FIG. 7 includes summary data and an example flow cytometry plot for transduction of cells with viral particles that included engineered NiV G proteins of various linker lengths.

[0022] FIG. 8 includes a plot of flow cytometry data generated from attempting to transduce some CD2- cells with NiV (here, a NiV G fusion protein) pseudotyped lentiviral particles (left plot), and a second plot of flow cytometry data generated from transducing some CD2+ cells with NiV pseudotyped lentiviral particles (right plot).

[0023] FIG. 9A includes a plot of flow cytometry data for transduction of cells with viral particles that included an engineered NiV G protein that included a binding domain of a CD80 protein.

[0024] FIG. 9B includes a plot of flow cytometry data for transduction of cells with viral particles that included an engineered NiV G protein that included a binding domain of a CD58 protein in a type I orientation.Attorney Ref: 061479-515001WO

[0025] FIG. 10 includes plots showing measurements relevant to PBMC activation in a study herein.

[0026] FIG. 11A includes plots showing measurements relevant to PBMC transduction at day 7 in a study herein.

[0027] FIG. 11B includes plots showing measurements relevant to PBMC transduction at day 11 in a study herein.

[0028] FIG. 12 includes a depiction of a study design that was used for proof of concept in vitro PBMC testing.

[0029] FIG. 13 includes plots showing day 3 activation of PBMCs through lentiviral particle use.

[0030] FIG. 14 includes plots showing day 7 transduction of PBMCs by lentiviral particle.

[0031] FIG. 15 depicts some aspects that may be used for NiV vector production.

[0032] FIG. 16A includes plots relating to a DoE titer improvement summary.

[0033] FIG. 16B includes plots relating to a DoE PPTU improvement summary.

[0034] FIG. 17 includes plots showing measurements of lentiviral particles produced by various cells.

[0035] FIG. 18 includes plots showing PPTU measurements.

[0036] FIG. 19 includes a depiction of a study design.

[0037] FIG. 20A includes data showing activation of NHP PBMCs by lentiviral particles that included NiV or Cocal proteins.

[0038] FIG. 20B includes data showing CAR+ cell production by lentiviral particles that included NiV or Cocal proteins.

[0039] FIG. 21 includes a plot of antibody measurements. Each separate shape in the figure represents data from a different animal. Data in die figure were generated from a MSD-based assay.

[0040] FIG. 22 is a plot showing NHP serum inhibition of transduction of matched-species PBMCs in vitro, where PtM serum inhibition was greatest

[0041] FIG. 23 outlines an evaluation of serum inhibition of Cocal- or NiV-pscudotypcd lentiviral particles.

[0042] FIG. 24 includes plots of flow cytometry' data related to lentiviral particle binding of PBMCs.

[0043] FIG. 25 includes plots of flow cytometry data related to activation of T cells by lentiviral particles.

[0044] FIG. 26 shows a dramatic increase in percent CAR observed with NiV pseudotyped lentiviral particles at study day 10, indicating that there was no inhibition of transduction by preincubation with NHP serum.

[0045] FIG. 27 shows that serum inhibition is still not observed with NiV pseudotyped vector at day 14, indicating that there was no inhibition of transduction by pre -incubation with NHP serum.Attorney Ref: 061479-515001WO

[0046] FIG. 28A shows results of a serum inhibition experiment in SUP-T1-CD2+ cells with pigtailed macaque serum.

[0047] FIG. 28B shows results of a serum inhibition experiment in human PBMCs with pigtailed macaque serum.

[0048] FIG. 29 depicts an NHP study that was performed using lentiviral particles herein.

[0049] FIG. 30A is a plot showing circulating anti-CD20 CAR T cell kinetics in a NHP dosed with lentiviral particles herein.

[0050] FIG. 30B is a plot showing circulating CD20+ B cell kinetics in an NHP dosed with lentiviral particles herein.

[0051] FIG. 31 depicts an NHP study with three NHPs that was performed using lentiviral particles herein.

[0052] FIG. 32A is a plot showing circulating anti-CD20 CAR T cell kinetics in three NHPs dosed with lentiviral particles herein.

[0053] FIG. 32B is a plot showing circulating CD20+ B cell kinetics in three NHPs dosed with lentiviral particles herein.DETAILED DESCRIPTION

[0054] Nipah virus (NiV) envelope (Env) proteins were engineered to specifically transduce CD2- expressing immune cells. Viral particles including such engineered viral proteins in conjunction with other surface engineered proteins may be useful as a platform for targeting immune cells.

[0055] Engineered NiV Env proteins may be useful for transduction in CD2+ expressing cells, and may eliminate off-target effects when using other viral proteins. Additionally, kinetics for viral transduction may be faster when using viral particles engineered with a NiV Env protein compared to other viral proteins. As such, using an engineered NiV Env protein may improve in vivo gene deliver}’ outcomes.

[0056] Described herein, is a NiV Env protein engineered to specifically transduce CD2+ cells, improving cell specific entry and subsequent transduction, when expressed on a viral surface. The engineered envelope proteins herein may be useful to include in vectors such as viral vectors.

[0057] NiV envelope proteins may include 2 proteins: one for binding, called G protein, and another one for fusion, called F protein. The G protein or the F protein can be engineered. The G protein can be engineered to specifically bind target cells. G protein is an example of a targeting polypeptide. A recombinant G protein is an example of a recombinant targeting polypeptide.

[0058] Disclosed herein, in some embodiments, are recombinant targeting polypeptides. The recombinant targeting polypeptide may include a recombinant external region (e.g. comprising a CD58 extracellular domain), a transmembrane region, and an internal region comprising a cytoplasmic fragment of a henipavirus G protein. A recombinant targeting polypeptide may be a part of a particle such as a viral particle. The recombinant targeting polypeptide may be included in aAttorney Ref: 061479-515001WO viral particle shown in FIG. 1A-1C. The targeting polypeptide may include an external region comprising a CD58 extracellular domain, a transmembrane region, and an internal region comprising a cytoplasmic fragment of a henipavirus G protein. Some embodiments relate to methods of using the recombinant targeting polypeptide or particle.Gene delivery particles

[0059] Disclosed herein, in some embodiments, are particles. The particles may include gene delivery particles. A particle may be a gene delivery particle. Some examples of gene delivery particles include viral particles (e.g. a virion), lipid nanoparticles (LNPs), lipoplexes. liposomes, or nanocarriers. The particle may include 1, 2 or more a membrane proteins. The particle may include a pay load. The particle may include 1, 2 or more a membrane proteins and pay load. The particle may be included in a composition herein or a kit. The particle may be used in a method herein.

[0060] The membrane proteins may include a targeting polypeptide. The particle may include a targeting polypeptide. The particle may include a recombinant polypeptide. The particle may include a recombinant targeting polypeptide. Disclosed herein, in some embodiments, are particles that include a recombinant targeting polypeptide. Disclosed herein, in some embodiments, are gene delivery vehicles comprising the recombinant polypepride.

[0061] Disclosed herein, in some embodiments, are viral particles. In some embodiments, the particles include viral particles. A particle may be a viral particle. The particle may be or include a viral particle. The viral particle may be pseudotyped using a recombinant targeting polypeptide herein.

[0062] Examples of viral particles may include a lentiviral particle such as a retroviral particle, a henipavirus particle such as a Nipah virus (NiV) particle, or a paramyxoviral particle.

[0063] Disclosed herein, in some embodiments, are viral particles, comprising: a polynucleotide cargo; a lipid envelope; and recombinant targeting polypeptide comprising: an external region comprising a CD58 extracellular domain, and a transmembrane region. Disclosed herein, in some embodiments, are lentiviral particles, comprising: a polynucleotide cargo; a lipid envelope; and recombinant targeting polypeptide comprising: an external region comprising a CD58 extracellular domain, and a transmembrane region. In some embodiments, the recombinant targeting polypeptide further comprises an internal region comprising a cytoplasmic fragment of a henipavirus G protein. Disclosed herein, in some embodiments, are viral particles, comprising: a polynucleotide cargo; a lipid envelope; and recombinant targeting polypeptide comprising: an external region comprising a CD58 extracellular domain, a transmembrane region, and an internal region comprising a cytoplasmic fragment of a henipavirus G protein.

[0064] In some embodiments, the particle is a lentiviral particle. The lentiviral particle may include a gag gene. The lentiviral particle may include a pol gene. The lentiviral particle may include a env gene. The lentiviral particle may include a lentiviral structural protein such as gp!20, gp41, p24,Attorney Ref: 061479-515001WO p 17, or p7 / p9. The lentiviral particle may include a lentiviral enzyme such as a reverse transcriptase, integrase, protease, or dUTPase. The lentiviral particle may include a gene regulatory' protein such as tat or rev. The lentiviral particle may include an accessory protein such as Nef, Vpr, Vif, Vpu / Vpx, or p6.

[0065] The viral particle of any one of the previous claims, wherein the particle is a paramyxovirus comprising a paramyxovirus fusion (F) protein.

[0066] Disclosed herein, in some embodiments, are viral particles such as a viral particle comprising a recombinant targeting polypeptide. Some example viral particles are shown in FIG. 1A-1C. The example lentiviral particle in FIG. 1A includes a membrane surface that includes a membrane protein for pseudotyping. The membrane protein for pseudotyping in the figure is a Cocal glycoprotein, as shown in the close-up. The membrane surface in the figure further includes a multidomain (MDF) protein. The inside of the example virus in the figure includes a polynucleotide payload.

[0067] The example lentiviral particle in FIG. IB includes NiV Env proteins and a polynucleotide payload. The NiV Env proteins in the figure are NiV Env glycoproteins F and G, as indicated in the close-up. Binding of the NiV G protein may drive F protein conformational changes and fusion to a membrane of a target cell.

[0068] The example lentiviral particle in FIG. 1C includes NiV Env proteins (e.g. NiV Env glycoproteins F and G) and a polynucleotide payload, in addition to an MDF protein at the particle surface. The NiV Env proteins may include an engineered NiV G protein or other recombinant targeting polypeptide.

[0069] The viral particle may include a polynucleotide payload and a membrane protein. The viral particle may include multiple polynucleotide pay loads or multiple membrane proteins. Some examples of lentiviral particles, payloads, and membrane proteins are included in WO2024097992 and W02024098028, which are incorporated herein by reference in their entirety .Payloads

[0070] Disclosed herein, in some embodiments, are payloads. The payload may be included as part of a particle herein. The payload may be inside the particle. The payload may be encompassed by the particle, or by a membrane of the particle. The payload may be included in a composition or kit herein, or be used in a method herein.

[0071] An example of a payload includes a polynucleotide. The polynucleotide may include DNA. The polynucleotide may include RNA. The polynucleotide may encode a gene of interest, or may encode multiple genes of interest. The polynucleotide may include a promoter operably connected with the gene of interest. The gene of interest may include a chimeric antigen receptor (CAR). The gene of interest may include a small molecule-activated cytokine receptor such as a rapamycin molecule-activated cytokine receptor (RACR). The gene of interest may include the CAR and smallAttorney Ref: 061479-515001WO molecule-activated cytokine receptor. The gene of interest may include a resistance gene. The pay load may encode 1, 2, 3, 4, or more genes of interest.

[0072] In some embodiments, the polynucleotide cargo comprises a promoter. In some embodiments, the polynucleotide cargo encodes a CAR. In some embodiments, the polynucleotide cargo encodes a small molecule-activated cytokine receptor.

[0073] Disclosed herein, in some embodiments, are payloads that include a polynucleotide sequence encoding a CAR.

[0074] CARs are artificial membrane -bound proteins that direct a T lymphocyte to an antigen and stimulate the T lymphocyte to kill cells displaying the antigen. See, e.g, Eshliar, U.S. Pat. No. 7,741,465. Because T lymphocytes may require two signals, a primary activation signal and a costimulatory signal, in order to maximally activate. CARs can comprise a stimulatory and a costimulatory domain such that binding of the antigen to the extracellular domain results in transmission of both a primary activation signal and a costimulatory signal. Illustrative CARs may be designed in a modular fashion, e.g. as described in (see, e.g., Guedan S, Calderon H, Posey AD, Maus MV, Molecular Therapy- Methods & Clinical Development. 2019; 12: 145-156), incorporated by reference.

[0075] A CAR may include a genetically engineered receptor comprising an extracellular domain that binds to an antigen, e.g.. an antigen on a cell, an optional linker, a transmembrane domain, and an intracellular (cytoplasmic) domain comprising a costimulatory domain and / or a signaling domain that transmits an activation signal to an immune cell. With a CAR, a receptor can be programmed to both recognize a specific antigen and, when bound to that antigen, activate the immune cell to attack and destroy the cell bearing that antigen. When a CAR-recognized antigen exists on a tumor cell, an immune cell that expresses the CAR can target and kill the tumor cell.

[0076] CAR T therapies have been approved by the FDA and include Yescarta, Tecartus, Kymriah, Abecma, Carvykti, and Breyanzi. Exemplary' CARs are described, for example, in US Publication No. 2020 / 0246381; US Patent No. 10,918,665; US Publication No. 2019 / 0161553; US Publication No. 2022 / 0033509, US Publication No. 20160152723, US Patent No. 10,736,918, US Patent No. 10,357,514, and US Patent No. 7,446,190, each incorporated by reference.

[0077] In some embodiments, a particle disclosed herein comprises a polynucleotide that encodes a CAR comprising an extracellular domain, optionally a hinge domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a costimulatory domain and an activation domain. In some embodiments, the costimulatory and activation domains are a single domain, for example a single intracellular domain that provides both costimulation and activation signals to a cell. In other embodiments, the intracellular signaling domain comprises either a costimulatory domain or an activation domain.

[0078] Disclosed herein, in some embodiments, are payloads that include a polynucleotide sequence encoding a small molecule-activated cytokine receptor. The small molecule-activatedAttorney Ref: 061479-515001WO cytokine receptor may include RACR. The RACR may include two polypeptide chains such as a RACRg chain and a RACRb chain. A RACRg chain may include an FRB sequence and an IL2Rg sequence. Examples of RACRg chains may include SEQ ID NO: 1 or 2. A RACRb chain may include an FKBP sequence and an IL2RB sequence. An example of a RACRb chain may include SEQ ID NO: 3. Examples of amino acid sequences for RACR may include: a T2098L FRB sequence of SEQ ID NO: 4 linked with an IL2Rg sequence of SEQ ID NO: 5 or 6. and an FKBP sequence of SEQ ID NO: 7 linked w ith an IL2Rb sequence of SEQ ID NO: 8. The payload may encode one or more amino acid sequences at least 80%, at least 85%, at least 90%, at least 91%. at least 92%, at least 93%, at least 94%, at least 95%, at least 96%. at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 1-8. The payload may include a polynucleotide sequence encoding an amino acid sequence that is at least 80%, at least 85%, at least 90%. at least 91%, at least 92%, at least 93%, at least 94%. at least 95%, at least 96%, at least 97%. at least 98%, at least 99%. or 100% identical to SEQ ID NO: 1. The pay load may include a polynucleotide sequence encoding an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2. The payload may include a polynucleotide sequence encoding an amino acid sequence that is at least 80%, at least 85%, at least 90%. at least 91%, at least 92%. at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%. at least 99%, or 100% identical to SEQ ID NO: 3. The pay load may include a polynucleotide sequence encoding an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%. at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 4. The payload may include a polynucleotide sequence encoding an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%. at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 5. The pay load may include a polynucleotide sequence encoding an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 6. The payload may include a polynucleotide sequence encoding an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 7. The payload may include a polynucleotide sequence encoding an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%. at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 8.

[0079] Disclosed herein, in some embodiments, are payloads that include a polynucleotide sequence encoding a human protein domain (e.g. FRB) derived from the mammalian target of rapamycin (mTOR) complex that binds intracellular rapamycin-FKBP complexes to conferAttorney Ref: 061479-515001WO rapamycin resistance to transduced cells. An example of an amino acid sequence for a free FRB may include SEQ ID NO: 9. The payload may encode an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% identical to SEQ ID NO: 9.

[0080] In some embodiments, the pay load comprises a polynucleotide sequence disclosed in Table 1, or at least 80%, at least 85%, at least 90%. at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%. at least 97%, at least 98%, or at least 99% identical thereto.

[0081] In some embodiments, the pay load may include or encode one or more therapeutic cargos. Such cargos can comprise, without limitation, nucleic acids (e.g., DNA, RNA, mRNA, siRNA, miRNA, antisense oligonucleotides, small activating RNAs), gene-editing components (e.g., CRISPR / Cas systems, TALENs. zinc finger nucleases), immune-modulatory molecules (e.g, T-cell receptors (TCRs). therapeutic proteins or peptides, and other biologically active agents. These cargos may be designed to modulate gene expression, silence or activate target genes, correct genetic defects, or elicit desired immune responses. In some embodiments, the cargos are delivered via viral vectors, such as those disclosed herein.Membrane proteins

[0082] Disclosed herein, in some embodiments, are membrane proteins. The membrane protein may be part of a viral particle. The membrane protein may be included in a composition or kit herein, or be used in a method herein.

[0083] The membrane protein may interact with a target cell. The membrane protein may bind, target, or otherwise interact with an immune cell such as a T cell. A membrane protein may include a targeting polypeptide such as a recombinant targeting polypeptide.

[0084] NiV glycoprotein F and NiV glycoprotein G are examples of membrane proteins that may be included in a viral particle such as a lentiviral particle herein. In embodiments, “G protein” refers to a henipavirus envelope attachment glycoprotein G. In embodiments, “F protein” refers to a henipavirus fusion protein F. The F and G proteins may be a Nipah (NiV) virus, and may be a wildtype protein or may be a variant thereof that exhibits reduced binding for the native binding partner. The NiV F (fusion) and NiV G (attachment) glycoproteins mediate cellular entry of Nipah virus. The G protein initiates infection by binding to the cellular surface receptor ephrin-B2 (EphB2) or EphB3. The subsequent release of the viral genome into the cytoplasm is mediated by the action of the F protein, which induces the fusion of the viral envelope with cellular membranes.

[0085] An example of a membrane protein includes a recombinant targeting polypeptide. The recombinant targeting polypeptide may include an engineered NiV protein such as an engineered NiV glycoprotein G. The viral particle may include NiV glycoprotein F and an engineered NiV glycoprotein G. An engineered G may be referred to as an engineered NiV G protein or may include a recombinant NiV G protein.Attorney Ref: 061479-515001WO

[0086] NiV attachment glycoproteins (NiV G) contain a short N-terminal cytoplasmic tail, a transmembrane domain, and an extracellular domain containing an extracellular stalk and a globular head. The N-terminal cytoplasmic domain is within the inner lumen of the lipid bilayer and the C- tenninal portion is the extracellular domain that is exposed on the outside of the lipid bilayer. The receptor binding and antigenic sites reside on the extracellular domain. Regions of the stalk in the C- terminal region have been shown to be involved in interactions with the F protein and triggering of fusion with a target cell membrane (Liu et al. 2015 J of Virology 89: 1838). The F protein undergoes significant conformational change that facilitates the insertion of the fusion peptide into target membranes. The cytoplasmic tails play a role in particle formation, incorporation into packaged particles, and serves as a signal peptide to modulate protein maturation and surface transport (Sawatsky et al. 2016. J of Virology 97:1066-1076).

[0087] Disclosed herein, in some embodiments, are membrane proteins that include a NiV glycoprotein F (also referred to as a NiV F protein). An example NiV F protein may include the polypeptide sequence of SEQ ID NO: 10. The NiV F protein may have a polypeptide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%. at least 98%, at least 99%. or 100% identical to SEQ ID NO: 10. The NiV F protein may be engineered to include an exogenous signal peptide.

[0088] Disclosed herein, in some embodiments, are membrane proteins that include a NiV glycoprotein G (also referred to as a NiV G protein). An example NiV G protein may include the polypeptide sequence of SEQ ID NO: 11. The NiV G protein may have a polypeptide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%. at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 11.

[0089] Provided below is a reference NiV G protein sequence, SEQ ID NO: 11. The NiV G proteins are type II transmembrane glycoproteins containing an N-terminal cytoplasmic tail (e.g., corresponding to amino acid residues 1-48 of SEQ ID NO: 11), a transmembrane domain (e.g., corresponding to amino acid residues 49-69 of SEQ ID NO: 11), and an extracellular domain containing an extracellular stalk (e.g., corresponding to amino acid residues 70-168 of SEQ ID NO: 11), a neck domain (e.g. corresponding to amino acid residues 169-186 of SEQ ID NO: 11, often considered to be part of the stalk), and a globular head responsible for receptor binding (corresponding to amino acid residues 187-602 of SEQ ID NO: 11). The N-terminal cytoplasmic domain is within the inner lumen of the lipid bilayer (i.e. it is an “interior region” of the polypeptide) and the C-terminal portion is the extracellular domain that is exposed on the outside of the lipid bilayer (an “exterior region”). Regions of the stalk in the C-tenninal region (e.g. corresponding to amino acid residues 159-167 of SEQ ID NO: 11) have been shown to be involved in interactions with F protein and triggering of F protein fusion (Liu et al. 2015 J of Virology 89: 1838). In particular, the cysteine residues are considered to be relevant to this interaction process.Attorney Ref: 061479-515001WO1 MPAENKKVRF ENTTSDKGKI PSKVIKSYYG TMDIKKINEG LLDSKILSAF 51 NTVIALLGS I VIIVMNIMII QNYTRSTDNQ AVIKDALQGI QQQIKGLADK 101 IGTE IGPKVS LIDTSSTITI PANIGLLGSK ISQSTAS INE NVNEKCKFTL 151 PPLKIHECNI SCPNPLPFRE YRPQTEGVSN LVGLPNNICL QKTSNQILKP 201 KL ISYTLPW GQSGTCITDP LLAMDEGYFA YSHLERIGSC SRGVSKQRI I 251 GVGEVLDRGD EVPSLFMTNV WTPPNPNTVY HCSAVYNNEF YYVLCAVSTV 301 GDPILNSTYW SGSLMMTRLA VKPKSNGGGY NQHQLALRS I EKGRYDKVMP 351 YGPSGIKQGD TLYFPAVGFL VRTEFKYNDS NC PITKCQYS KPENCRLSMG 401 IRPNSHYILR SGLLKYNLSD GENPKWFIE ISDQRLS IGS PSKIYDSLGQ 451 PVFYQASFSW DTMIKFGDVL TVNPLWNWR NNTVISRPGQ SQC PRFNTC P 501 EICWEGVYND AFL IDRINWI SAGVFLDSNQ TAENPVFTVF KDNEILYRAQ 551 LASEDTNAQK TITNCFLLKN KIWC ISLVEI YDTGDNVIRP KLFAVKIPEQ 601 CT ( SEQ ID NO : 11 )

[0090] An example of a membrane protein may include a Cocal protein such as a Cocal glycoprotein (Cocal G). The Cocal glycoprotein may include a VSV-G glycoprotein. The VSV-G glycoprotein may include a mutation at position 47 such as K47Q, a mutation at position 354 such as R354A, or both. An example of a Cocal glycoprotein may include the polypeptide sequence of SEQ ID NO: 12. The Cocal glycoprotein may have a polypeptide sequence having at least 75%. at least 80%, at least 85%, at least 90%. at least 91%, at least 92%, at least 93%, at least 94%, at least 95%. at least 96%. at least 97%, at least 98%. at least 99%, or 100% sequence identity to SEQ ID NO: 12. Some embodiments do not include the Cocal protein or a portion thereof. Some embodiments do not include a VSV-G protein or a portion thereof. For example, some embodiments include a lentiviral particle that includes one or more NiV glycoproteins or an engineered NiV glycoprotein, and not a Cocal glycoprotein. Some embodiments include a lentiviral particle that includes one or more NiV glycoproteins or an engineered NiV glycoprotein, and not a VSV-G protein.

[0091] In some embodiments, the viral particle is pseudotyped with viral glycoproteins as described herein, such as a NiV F and / or Niv G protein. In embodiments, the viral particle is pseudotyped with a NiV G fusion, such as a NiV G-CD58 fusion. In some embodiments, the CD58 of the NiV G-CD58 fusion is a CD58 extracellular domain (ECD). In embodiments, the viral particle is pseudotyped with a NiV G fusion, such as a NiV G-CD58 extracellular domain fusion.

[0092] Disclosed herein, in some embodiments are membrane proteins that include a protein such as a multidomain protein (MDF protein). A key feature of said membrane proteins is that the binding or other active domain is presented on the outside of the lentiviral particle and does not function as a self-signaling protein if expressed on a cell. In some embodiments, a viral particle such as a lentiviral particle includes an MDF protein and one or more NiV proteins (e.g. aNiV F protein and a recombinant NiV G protein). A multidomain protein is an example of a multidomain molecule. The multidomain protein may in some instances be referred to as a multidomain molecule. The multidomain protein may include an adhesion protein. The multidomain protein may include a costimulatory protein. The multidomain protein may include an activation protein. The multidomain protein may include an adhesion protein and a costimulatory protein. The multidomain protein may include an adhesion protein, a costimulatory protein, and an activation protein. The multidomainAttorney Ref: 061479-515001WO molecule may include an adhesion molecule, a costimulatory molecule, an activation molecule, or a combination thereof. An MDF protein may include a CD58 sequence (such as a CD58 extracellular domain), a CD80 sequence (such as a CD80 extracellular domain), and an anti-CD3 antibody, binding fragment, or scFv. Some embodiments include a multidomain molecule comprising: a CD58 extracellular domain; a CD80 or CD86 extracellular domain; and an anti-CD3 antibody, an anti-CD3 antibody binding fragment, or an anti-CD3 single-chain variable fragment (scFv).

[0093] CD58, also known as LFA-3, is the natural ligand for CD2. CD58 / LFA-3 proteins are glycoproteins that are expressed on the surfaces of a variety of cell types (Dustin et al., 1991 , Annu. Rev. Immunol. 9:27) and play roles in mediating T-cell interactions with APCs in both antigendependent and antigen-independent manners (Wallner et ai., 1987, J. Exp. Med. 166:923). The sequence of human CD58 has the UniProt identifier P19256 (www.uniprot.org / uniprot / P19256).

[0094] The extracellular region of human CD58 comprises two 1g- like domains. The most N- terminal Ig-like domain, referred to as domain 1 , is of V-type, and the second Ig-like domain, named domain 2, is of C-type. Also within the scope of the disclosure are MDF proteins or NiV-G CD58 fusion proteins comprising CD58 variants, including those with substitutions, and / or deletions, for example, CD58 or CD58 extracellular domain (ECD) variants that do not alter binding to CD2.

[0095] In embodiments, the CD58 extracellular domain comprises the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%. at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In embodiments, the CD80 extracellular domain comprises the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence at least 80%, at least 85%. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In embodiments, the CD86 extracellular domain comprises the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In embodiments, anti-CD3 antibody comprises an CDR-L1 comprising the amino acid sequence of SEQ ID NO: 26, an CDR-L2 comprising die amino acid sequence of SEQ ID NO: 27, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 28; and an CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29, an CDR-H2 comprising the amino acid sequence of SEQ ID NO: 30, and CDR-H3 comprises the amino acid sequence of SEQ ID NO: 31.

[0096] Some example MDF proteins are provided as SEQ ID NOs: 13-16. A multidomain protein may include a polypeptide sequence at least 75%, at least 80%. at least 85%, at least 90%. at least 91%. at least 92%, at least 93%, at least 94%, at least 95%, at least 96%. at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 13. A multidomain protein may include a polypeptide sequence at least 75%, at least 80%. at least 85%, at least 90%. at least 91%, at least 92%. at least 93%, at least 94%. at least 95%, at least 96%, at least 97%. at least 98%, at least 99%, or 100%Attorney Ref: 061479-515001WO identical to SEQ ID NO: 14. A multidomain protein may include a polypeptide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 15. A multidomain protein may include a polypeptide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%. at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 16.

[0097] Some embodiments relate to an adhesion molecule, which may be included as part of a multidomain or membrane protein. An adhesion molecule may include a molecular component of a SMAC or other immune synapse, other than an activation molecule (e.g. TCR-binding agent) or a costimulatory molecule, which contributes to adhesion of a particle to target cells. An adhesion molecule may specifically bind a conjugate molecule with affinity sufficient to cause increased adhesion between the particle and the target cell compared to the adhesion of a reference particle lacking the adhesion molecule to the same or similar target cell. An adhesion molecule may include but is not limited to CD58, a CD58 extracellular domain (ECD), or a functional fragment of CD58. A functional fragment of a polypeptide such as CD58 may include a fragment of the polypeptide that retains a desired function (such as adhesion, costimulation, or activation). An adhesion protein may include a polypeptide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%. at least 95%, at least 96%, at least 97%. at least 98%, at least 99%, or 100% identical to SEQ ID NO: 17. An adhesion protein may include a polypeptide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 18. An adhesion protein may include a polypeptide sequence at least 75%, at least 80%, at least 85%, at least 90%. at least 91%, at least 92%, at least 93%, at least 94%. at least 95%, at least 96%, at least 97%. at least 98%, at least 99%, or 100% identical to SEQ ID NO: 19. An adhesion protein may include a polypeptide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 20.

[0098] Some embodiments relate to a costimulatory molecule, which may be included as part of a multidomain or membrane protein. A costimulatory molecule may include a molecule capable of providing a costimulatory signal to target cells. In T cell biology', the binding of the T cell receptor by an antigen can provide the primary stimulatory signal to the cell. Costimulatory' signals may be provided by accessory' molecules. An example costimulatory signal may include a signal provided by binding of CD28 on a T cell by a ligand. Some examples of ligands of CD28 include CD80 and CD86. Some examples of costimulatory molecules may include CD80, CD86, CD40L (also known as CD154). GITRL, OX40L, 41BBL, ICOSL, CD27. CD30L, LIGHT, LTalpha. MICA, or MICB. Each of the foregoing may be employed as a costimulatory molecules as a full-length protein, an extracellular domain, or functional fragment. A costimulatory protein may include a polypeptideAttorney Ref: 061479-515001WO sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 21. A costimulatory protein may include a polypeptide sequence at least 75%. at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%. at least 98%, at least 99%, or 100% identical to SEQ ID NO: 22. A costimulatory protein may include a polypeptide sequence at least 75%. at least 80%, at least 85%, at least 90%. at least 91%, at least 92%, at least 93%, at least 94%, at least 95%. at least 96%. at least 97%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 23. A costimulatory protein may include a polypeptide sequence at least 75%, at least 80%. at least 85%, at least 90%, at least 91%, at least 92%. at least 93%, at least 94%, at least 95%, at least 96%, at least 97%. at least 98%, at least 99%, or 100% identical to SEQ ID NO: 24. A costimulatory protein may include a polypeptide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%. at least 92%, at least 93%, at least 94%. at least 95%, at least 96%, at least 97%. at least 98%, at least 99%, or 100% identical to SEQ ID NO: 25.

[0099] Some embodiments relate to an activation molecule, which may be included as part of a multidomain or membrane protein. An activation molecule may include a protein or protein subunit that provides an activation signal to a target cell. An example of an acti \ ation molecule includes a TCR-binding molecule or TCR-binding protein. In some embodiments, a multidomain molecule includes an activation molecule. In some embodiments, a multidomain molecule does not include an activation molecule. A TCR-binding molecule may include a molecule capable of directly binding the extracellular portion of the T cell receptor (TCR) by contacting one or more components of the TCR or otherwise providing a primary or “signal 1” activation signal to a target cell (e.g. a T cell or NK cell). Some examples of TCR-binding molecules may include an antibody, or antigen binding fragment, that specifically binds CD3 (an anti-CD3 monoclonal antibody, or antigen binding fragment thereof). In some embodiments, tire activation molecule comprises an antibody, single domain antibody, antibody fragment, nanobody, or other binding protein specific for CD3. Illustrative example antibodies may include OKT3 (also known as Muromonab-CD3), otelixizumab, teplizumab, or visilizumab. The activation molecule may include anti-CD3 antibody, an antigenbinding fragment, or single-chain variable fragment (scFv). In some embodiments, the antigenbinding fragment of the anti-CD3 antibody, antigen -binding fragment, or scFv comprises a complementarity determining region (CDR) LI (CDR-L1) sequence comprising SASSSVSYMN (SEQ ID NO: 26), a CDR-L2 sequence comprising DTSKLAS (SEQ ID NO: 27). a CDR-L3 sequence comprising QQWSSNPFT (SEQ ID NO: 28), a CDR-H1 sequence comprising GYTFTRY (SEQ ID NO: 29), a CDR-H2 sequence comprising NPSRGY (SEQ ID NO: 30). and a CDR-H3 sequence comprising YYDDHYCLDY (SEQ ID NO: 31). In some embodiments, the anti-CD3 antibody, antigen-binding fragment, or scFv comprises an anti-CD3 scFv. The anti-CD3 antigenbinding fragment may include a polypeptide sequence that is at least 80%, at least 85%, at least 90%,Attorney Ref: 061479-515001WO at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 32.

[0100] In embodiments, the viral particle may include an MDF protein in addition to aNiV F protein and / or a NiV G-CD58 extracellular domain (ECD) fusion.

[0101] In some embodiments, the activation, costimulatory, and / or adhesion molecules are separately expressed on a particle surface. In some such embodiments, each of the activation, costimulatory, and / or adhesion molecules would comprise its own transmembrane domain or membrane anchor (such as the C-terminal signal sequence that directs the attachment of a GPI anchor). For example, a particle may comprise an activation protein on its surface, a costimulatory protein on its surface, an adhesion molecule on its surface or any combination thereof. In some embodiments, the particle comprises an activation protein and a costimulatory protein on its surface. In some embodiments, the particle comprises an activation protein and an adhesion protein on its surface. In some embodiments, the particle comprises an adhesion protein and a costimulatory protein on its surface.Recombinant targeting polypeptides

[0102] Disclosed herein, in some embodiments, are recombinant targeting polypeptides. The recombinant targeting polypeptide may be included as part of a particle herein such as a viral particle or a lentiviral particle. In some embodiments, the recombinant targeting polypeptide is as comprised in a viral particle described herein. The recombinant targeting polypeptide may be found at a surface (e.g. a membrane surface) of a particle. The recombinant targeting polypeptide may be included in a composition or kit herein, or be used in a method herein.

[0103] A recombinant targeting polypeptide may include aspects of a Nipah virus (NiV) protein or proteins. NiV typically includes two envelope glycoproteins, an attachment protein (G / RBP or G) and a fusion protein (F), which facilitate entry into host cells. Cellular receptors for the G / RBP protein may include ephrin-B2 and ephrin-B3. Lentiviral vectors can be pseudotyped with the NiV glycoproteins. Successful pseudotyping may include trimcation of the cytoplasmic tail of the fusion protein F. The cytoplasmic tail of the G protein may be truncated as well. In some embodiments, an ankyrin repeat protein (e.g. DARPin) can be fused an ectodomain of the G protein to target NiV- pseudotyped lentiviruses to desired cell populations.

[0104] Examples of NiV Env proteins include NiV glycoprotein F and NiV glycoprotein G. Depictions of these NiV Env proteins are shown in FIG. 2-3. A NiV glycoprotein may be engineered. An engineered NiV glycoprotein is an example of a recombinant targeting polypeptide. An engineered NiV glycoprotein G may include a target cell binding domain, a linker, a stalk, a transmembrane domain, or a combination thereof as shown in FIG. 2. The engineered NiV glycoprotein G may also include a transmembrane region, an internal region, or both, as shown in the figure.Attorney Ref: 061479-515001WO

[0105] NiV Env entry may be based on cell surface fusion. An example is shown in FIG. 3, which includes pre-fusion and fusion complexes. Such fusion may be employed by a lentivirus herein comprising a recombinant targeting polypeptide such as a recombinant NiV G protein and a NiV F protein. The cell specific receptor in the figure may be CD2, and the target cell binding domain in the figure may include a CD58 protein (such as a CD58 fragment that includes a CD58 extracellular domain).

[0106] A recombinant targeting polypeptide may include an extracellular region and a transmembrane region. A recombinant targeting polypeptide may include an extracellular region, a transmembrane region, and an internal region. In some embodiments, the recombinant targeting polypeptide includes an extracellular region without a transmembrane domain, and instead includes an anchor such as a lipid anchor.

[0107] Disclosed herein, in some embodiments, are recombinant targeting polypeptides, comprising: an external region comprising a CD58 extracellular domain, a transmembrane region, and an internal region comprising a cytoplasmic fragment of a henipavirus G protein. In some embodiments, the henipavirus is a Nipah virus (NiV).

[0108] Disclosed herein, in some embodiments, are recombinant targeting polypeptides comprising: an extracellular domain that specifically binds CD2; a transmembrane domain; and a cytoplasmic domain comprising a cytoplasmic tail of a henipavirus G protein.

[0109] In some embodiments, the targeting polypeptide comprises a polypeptide sequence disclosed in Table 1, or at least 80%, at least 85%, at least 90%. at least 91%, at least 92%, at least 93%, at least 94%, at least 95%. at least 96%, at least 97%. at least 98%, or at least 99% identical thereto.

[0110] A recombinant targeting polypeptides may include an engineered NiV glycoprotein. An engineered NiV glycoprotein may be designed to be ty pe I or type II, where type I denotes an orientation where the engineered protein has an external (e.g. extracellular or extraviral) N terminus and an internal (e.g. intracellular, cytoplasmic or intraviral) C terminus, and type II denotes an orientation where the engineered protein has an internal N terminus and an external C terminus. The type I or type II protein may be or include a single-pass transmembrane protein.[oni] A recombinant targeting polypeptide may be in a Type I orientation or a Type II orientation. In some embodiments, a recombinant targeting polypeptide is in a Type I orientation. In some embodiments, a recombinant targeting polypeptide is in a Type II orientation.

[0112] An engineered NiV glycoprotein may include an engineered NiV glycoprotein G (which may also be referred to as an engineered NiV G protein). The engineered NiV G protein may include a structure of FIG. 2. The engineered NiV G protein may include a target cell binding domain. The target cell binding domain may be a recombinant or engineered target cell binding domain. For example, the target cell binding domain may be or include a binding domain of another protein such as CD58. The engineered NiV G protein may include a stalk. The engineered NiV G protein mayAttorney Ref: 061479-515001WO include a linker linking the target cell binding domain to the stalk. The stalk may further be connected to the transmembrane region.

[0113] An example engineered NiV glycoprotein is shown in FIG. 4, which includes a NiV G protein that has a wild type target cell binding domain replace with a binding domain of a CD58 protein. The engineered Niv G protein in FIG. 4 may be referred to as NiV G fusion protein. CD58 may be used to bind CD2 of a target cell. The engineered NiV G protein of this example includes a linker length of 33 amino acids, which may optionally be up to, for example, 46 amino acids long, and is in a type II orientation.

[0114] Some embodiments relate to a recombinant targeting polypeptide such as an engineered NiV G protein. The engineered NiV G protein may include: a binding domain of a human CD58 protein (e.g. the extracellular domain of human CD58), the binding domain being connected through a linker to a NiV G protein stalk, the stalk being connected further to a NiV G protein transmembrane (TM) region, and the TM region being connected to a NiV G protein internal (e.g. cytoplasmic) region (e.g. as shown in FIG. 4). CD58 may specifically binds to CD2, which is expressed on T cells. Thus, inclusion of an engineered NiV G protein at a lentiviral particle surface (e.g. along with a NiV F protein) may be used to target the lentiviral particle to a T cell or other CD2 -expressing cell.

[0115] Some embodiments relate to or include a transmembrane region and a stalk domain. In some embodiments, the stalk domain together with the transmembrane region comprises the polypeptide sequence of SEQ ID NO: 34, or a polypeptide sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%. at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 34. In some embodiments, the stalk domain together with the transmembrane region comprises the polypeptide sequence of SEQ ID NO: 34. In some embodiments, the stalk domain together with the transmembrane region comprises a polypeptide sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 34.

[0116] An example of a recombinant targeting polypeptide sequence is included as SEQ ID NO: 33. The recombinant targeting polypeptide of SEQ ID NO: 33 is a NiV G fusion protein, and may be encoded by a polynucleotide having a sequence of SEQ ID NO: 39. Some embodiments relate to or include a recombinant targeting polypeptide having or including the amino acid sequence of SEQ ID NO: 33. Some embodiments relate to or include arecombinant targeting polypeptide that includes an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%. at least 93%. at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 33.

[0117] In embodiments, the NiV G fusion protein contains an N-terminal cytoplasmic tail corresponding to residues 1-15 of SEQ ID NO: 33, a transmembrane domain corresponding to residues 16-36 of SEQ ID NO: 33, a stalk domain corresponding to residues 37 to 135 of SEQ IDAttorney Ref: 061479-515001WONO: 33, a linker corresponding to residues 136-168 of SEQ ID NO: 33, and a CD58 extracellular domain (ECD) (i.e., CD2-binding domain) corresponding to residues 169-260 of SEQ ID NO: 33 (bold).1 MKKINEGLLD SKILSAFNTV IALLGSIVII VMNIMIIQNY TRSTDNQAVI 51 KDALQGIQQQ IKGLADKIGT EIGPKVSLID TSSTITIPAN IGLLGSKISQ 101 STASINENVN EKCKFTLPPL KIHECNISCP NPLPFGSTSG SGKPGSGEGS151 TKGGGSGGGG SGGGGSGGFS QQIYGWYGN VTFHVPSNVP LKEVLWKKQK 201 DKVAELENSE FRAFSSFKNR VYLDTVSGSL TIYNLTSSDE DEYEMESPNI 251 TDTMKFFLYV LESL ( SEQ ID NO : 33 )

[0118] Some embodiments relate to or include a nucleic acid sequence encoding a recombinant targeting polypeptide. In some embodiments, the nucleic acid sequence encoding the recombinant targeting polypeptide is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%. at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 39. In some embodiments, the nucleic acid sequence encoding the recombinant targeting polypeptide is 100% identical to SEQ ID NO: 39.

[0119] Some embodiments relate to a polynucleotide encoding a targeting polypeptide. The polynucleotide may include a promoter. Disclosed herein, in some embodiments, are polynucleotides encoding a targeting polypeptide, operably linked to a promoter. Disclosed herein, in some embodiments, are host cells comprising the polynucleotide.External regions

[0120] Disclosed herein, in some embodiments, is an external region of a polypeptide. Disclosed herein, in some embodiments, are recombinant targeting polypeptides that include an external region. The external region may be included in a composition or kit herein, or be used in a method herein.

[0121] A recombinant targeting polypeptide may include an external region. The external region may be or include an extracellular or extraviral protein domain. The external region may include a target cell binding domain. An example of a target cell binding domain includes a CD58 extracellular domain. The external region may include a CD58 extracellular domain. An example CD58 extracellular domain is provided as SEQ ID NO: 20. An external region may include a polypeptide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 20.

[0122] In some embodiments, the external region comprises a polypeptide sequence according to SEQ ID NO: 38, or at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.Attorney Ref: 061479-515001WO

[0123] The external region may include an external region of a NiV G protein, such as a stalk of a NiV G protein, a linker of a NiV G protein (e.g. a linker connecting a stalk to a target cell binding domain), or a linker and stalk of a NiV G protein.

[0124] The external region may include a linker connected to a target protein binding domain (e.g. a CD58 extracellular domain) and a stalk domain. The external region may include a linker connected to a target cell binding domain and a stalk domain.

[0125] In some embodiments, the external region includes a recombinant target cell binding domain. For example, a wild-type NiV glycoprotein G receptor binding domain may be switched out and replaced with a CD3 scFv (e.g. targeting CD3e). a CD80 protein (e.g. targeting CD28), a K12ecto protein (e.g. targeting CD7), a MDF protein (e.g. targeting CD3 / CD2 / CD28), or a CD58 protein (e.g. targeting CD2). In some embodiments, the extracellular domain comprises an antibody that specifically binds CD2.

[0126] A recombinant NiV G protein may include a fusion to a targeting moiety to target or bind CD2 of CD2-expressing cells. Such recombinant NiV G proteins are examples of a NiV G fusion protein. In some embodiments, a targeting moiety is fused to the Nipah virus F protein and / or a Nipah virus G protein. A targeting moiety may include an scFv, an antigen binding domain, a DARPIN. a VHH, or a FN3 domain. In some embodiments, the targeting moiety binds to protein such as a stem cell factor protein (e.g. SCF, KIT- ligand. KL, or steel factor). A targeting moiety may bind c-Kit (CD117), CD4, CD8, CD3, CD3D. CD3E, CD3G, CD3Z, CD5, CD6, CD7, CD2, TCR alpha, TCR beta. TCR gamma, TCR delta. CD 10, CD34, CD110, CD33, CD 14, CD68. CCR7, CD62L, CD25, CCR2, CCR3. CCR4, CCR5, CCR6. CCR7, and CXCR3. In some embodiments, the heterologous polypeptide of interest is a chimeric antigen receptor (CAR). Some such targeting moieties may be included as a target cell binding domain.

[0127] In some embodiments, the targeting polypeptide comprises a stalk domain of the henipavirus G protein. In some embodiments, the targeting polypeptide comprises a head domain of die henipavirus G protein. In some embodiments, the head domain comprises an amino acid substitution that decreases or prevents receptor binding. The henipavirus may be a NiV.

[0128] An external region of an engineered NiV glycoprotein G may include a target cell binding domain (e.g. a receptor binding domain), a linker, or a stalk. Some aspects that may be engineered include a length of the linker betw een the stalk and target cell binding domain, a size of the target cell binding domain, or a strength of binding of the target cell binding domain to a target cell or receptor.

[0129] Disclosed herein, in some embodiments, are engineered Niv G proteins that include a linker between a target cell binding domain and a stalk, or between a target cell binding domain and a transmembrane region. In some embodiments, a linker connects the target cell binding domain to the stalk. In some embodiments, a linker connects the target cell binding domain to the transmembrane region.Attorney Ref: 061479-515001WO

[0130] In some embodiments, the linker includes an amino acid length. The length may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37. 38, 39, 40, 41. 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids, or a range defined by any 2 of tire aforementioned numbers of amino acids. The length may be about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9. about 10, about 11, about 12. about 13, about14, about 15, about 16, about 17, about 18. about 19, about 20, about 21, about 22. about 23, about24, about 25, about 26, about 27, about 28. about 29, about 30, about 31, about 32. about 33, about34, about 35, about 36, about 37, about 38. about 39, about 40, about 41, about 42. about 43, about44, about 45. about 46, about 47, about 48, about 49, or about 50 amino acids, or a range thereof. The length may be at least 1, at least 2. at least 3, at least 4, at least 5, at least 6, at least 7. at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15. at least 16, at least 17, at least 18. at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25. at least 26, at least 1 , at least 28, at least 29. at least 30, at least 31. at least 32, at least 33. at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or at least 50 amino acids. In some embodiments, the length is less than 1, less than 2, less than 3. less than 4, less than 5. less than 6, less than 7. less than 8, less than 9, less than 1 , less than 1 1, less than 12, less than 13. less than 14. less than 15. less than 16, less than 17, less than 18, less than 19. less than 20, less than 21, less than 22, less than 23, less than 24, less than 25, less than 26. less than 27. less than 28, less than 29, less than 30, less than 31, less than 32, less than 33, less than 34, less than 35. less than 36, less than 37. less than 38, less than 39, less than 40, less than 41, less than 42. less than 43, less than 44, less than 45, less than 46, less than 47. less than 48, less than 49, or less than 50 amino acids. In some embodiments, the length includes 4-50 amino acids. In some embodiments, the length includes about 4-50 amino acids. In some embodiments, the length includes 4-46 amino acids. In some embodiments, the length includes about 33 amino acids. In some embodiments, the length includes about 15-40 amino acids. In some embodiments, the length includes about 15-37 amino acids.

[0131] In some embodiments, the stalk domain is comiected through a linker to the CD58 extracellular domain. In some embodiments, the linker is about 4-50 amino acids in length. In some embodiments, the linker comprises the polypeptide sequence of any of SEQ ID NOs: 35-37, or a polypeptide sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%. at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 35-37.

[0132] An example linker is provided as SEQ ID NO: 35. A linker connecting a target cell or target protein binding domain may include the amino acid sequence of SEQ ID NO: 35. A linker may include a sequence at least 75%. at least 80%, at least 85%, at least 90%. at least 91%, at least 92%, at least 93%. at least 94%, at least 95%. at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 35.Attorney Ref: 061479-515001WO

[0133] An example linker is provided as SEQ ID NO: 36. A linker connecting a target cell or target protein binding domain may include the amino acid sequence of SEQ ID NO: 36. A linker may include a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 36.

[0134] An example linker is provided as SEQ ID NO: 37. A linker connecting a target cell or target protein binding domain may include the amino acid sequence of SEQ ID NO: 37. A linker may include a sequence at least 75%. at least 80%, at least 85%, at least 90%. at least 91%, at least 92%, at least 93%, at least 94%, at least 95%. at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 37.Transmembrane regions

[0135] Disclosed herein, in some embodiments, is a transmembrane region of a polypeptide. Disclosed herein, in some embodiments, are recombinant targeting polypeptides that include a transmembrane region. The transmembrane region may be included in a composition or kit herein, or be used in a method herein.

[0136] A recombinant targeting polypeptide may include a transmembrane region. The transmembrane region may be or include a protein transmembrane domain. In some embodiments, the transmembrane region comprises a transmembrane domain of the henipavirus G protein. The transmembrane region may include a transmembrane region of a NiV G protein.

[0137] In some embodiments, the transmembrane region comprises a transmembrane portion of a polypeptide sequence herein (e.g. a transmembrane region of a NiV G sequence), or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical a transmembrane portion of a polypeptide sequence herein. In some embodiments, the transmembrane region comprises a transmembrane domain of CD58.

[0138] In some embodiments, the transmembrane region comprises a transmembrane domain of a NiV F protein. In some embodiments, the transmembrane region comprises a polypeptide sequence according to SEQ ID NO: 42, or at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.Internal regions

[0139] Disclosed herein, in some embodiments, is an internal region of a polypeptide. Disclosed herein, in some embodiments, are recombinant targeting polypeptides that include an internal region. The internal region may be included in a composition or kit herein, or be used in a method herein.Attorney Ref: 061479-515001WO

[0140] As used herein, the terms “internal region” and “cytoplasmic tail” are used interchangeably to refer to a portion of a membrane protein located on the interior of a cell membrane or a viral envelope.

[0141] A recombinant targeting polypeptide may include an internal region. The internal region may be or include an intracellular or intraviral protein domain. The internal region may be or include a cytoplasmic protein or a fragment of a cytoplasmic protein. The internal region may include a cytoplasmic fragment of a henipavirus G protein. The internal region may include an internal region of a NiV G protein.

[0142] In some embodiments, the recombinant targeting polypeptide comprises an internal region comprising a cytoplasmic tail of a henipavirus G protein. In some embodiments, the recombinant targeting polypeptide comprises an internal region comprising a cytoplasmic fragment of a Nipah Virus G protein.

[0143] In some embodiments, the cytoplasmic fragment comprises a cytoplasmic tail of a NiV G protein. In some embodiments, the cytoplasmic tail of the NiV G protein is truncated. Truncating the cytoplasmic domains NiV-G can improve pseudotyping efficiency and stability, enabling targeted gene delivery to endothelial cells. Witting S. R. et al., Gene Therapy. 2013;20:997-1005. In some embodiments, the cytoplasmic fragment comprises a GcA33 truncation variant, wherein the first 33 amino acid residues corresponding to the N-terminal portion of the cytoplasmic tail of SEQ ID NO: 11 are deleted. In some embodiments, the cytoplasmic fragment comprises a GcA34 truncation variant, wherein the first 34 amino acid residues corresponding to the N-terminal portion of the cytoplasmic tail of SEQ ID NO: 11 are deleted. An engineered NiV protein such as an engineered NiV F protein or an engineered NiV G protein may be truncated or exclude a cytoplasmic tail.Recombinant NiVF proteins

[0144] Disclosed herein, in some embodiments, are particles such as lentiviral particles diat include a NiV F protein. For example, a lentiviral particle herein may include a recombinant targeting polypeptide (e.g. an engineered NiV G protein) and a NiV F protein. Such proteins may be at a surface of the particle. The NiV F protein may be a wild-type NiV F protein. The NiV F protein may be engineered or recombinant. A NiV F protein, such as a recombinant NiV F protein, may be included with a recombinant targeting peptide such as a NiV G fusion protein or other engineered NiV G protein. The NiV F protein and recombinant targeting peptide may be together at a surface of a particle (e.g. lentiviral particle).

[0145] In embodiments, the variant NiV-F protein exhibits fusogenic activity. In embodiments, the F protein exhibits fusogenic activity with a target cell upon binding of the variant NiV-G protein to a target molecule on the target cell. In embodiments, the particles exhibit fusogenic activity mediatedAttorney Ref: 061479-515001WO by the NiV-G or a variant thereof along with any of the provided F proteins that facilitates merger or fusion of the two lumens of the viral envelope and the target cell membranes.

[0146] In some embodiments, the NiV F protein is truncated. In some embodiments, the NiV F protein is truncated at a N terminal end. A truncated NiV F protein is an example of an engineered or recombinant NiV F protein. Truncation may be at a N terminal end of the NiV F protein. The truncation may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14. 15, 16, 17, 18, 19, 20, 21, or 22 amino acids relative to a wild-type or endogenous NiV F protein, or a range defined by any 2 of the aforementioned numbers of amino acids. The truncation may include about 22 amino acids relative to a wild-type or endogenous NiV F protein. The truncation may include 22 amino acids relative to a wild-type or endogenous NiV F protein. In some embodiments, an engineered NiV F protein does not have a cytoplasmic tail.

[0147] In some embodiments, the NiV F protein is generated by a cell. The cell may include an expression construct that expresses the NiV F protein. The expression construct may encode an engineered NiV F protein.

[0148] In some embodiments, the NiV F protein is generated from a precursor NiV F protein. The precursor NiV F protein may include a signal peptide. The signal peptide may be a w ild-type or endogenous signal peptide. The precursor NiV F protein may be engineered by swapping the signal peptide for a different signal peptide. The different signal peptide may include a Gaussia luciferase signal peptide sequence. The different signal peptide may include an IFNa2 signal sequence. The signal peptide may be cleaved, for example by a cell generating the NiV F protein.

[0149] An example of a NiV F protein is included as SEQ ID NO: 40. Some embodiments relate to or include a NiV F protein having or including the amino acid sequence of SEQ ID NO: 40. Some embodiments relate to or include a NiV F protein that includes an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%. at least 92%, at least 93%, at least 94%, at least 95%. at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 40.

[0150] In embodiments, NiV F protein comprises the amino acid sequence of SEQ ID NO: 48, an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%. at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 48.

[0151] A NiV F protein may include an external portion or a transmembrane portion. A NiV F protein may include an external portion and a transmembrane portion. A NiV F protein may include an external portion, a transmembrane portion, or an internal portion. A NiV F protein may include an external portion, a transmembrane portion, and an internal portion.

[0152] A NiV F protein may include an external portion. An example amino acid sequence of an external portion of a NiV F protein is provided as SEQ ID NO: 41. Some embodiments relate to or include an external portion of a NiV F protein having or including the amino acid sequence of SEQ ID NO: 41. Some embodiments relate to or include an external portion of a NiV F protein thatAttorney Ref: 061479-515001WO includes an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 41.

[0153] A NiV F protein may include a transmembrane portion. An example amino acid sequence of a transmembrane portion of a NiV F protein is provided as SEQ ID NO: 42. Some embodiments relate to or include a transmembrane portion of a NiV F protein having or including the amino acid sequence of SEQ ID NO: 42. Some embodiments relate to or include a transmembrane portion of a NiV F protein that includes an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%. at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 42.

[0154] A NiV F protein may include an internal portion. An example amino acid sequence of an internal portion of a NiV F protein is provided as SEQ ID NO: 43. Some embodiments relate to or include an internal portion of a NiV F protein having or including the amino acid sequence of SEQ ID NO: 43. Some embodiments relate to or include an internal portion of a NiV F protein that includes an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%. at least 96%, at least 97%. at least 98%, or at least 99% identical to SEQ ID NO: 43.

[0155] Some embodiments relate to or include a nucleic acid sequence encoding a NiV F protein. In some embodiments, the nucleic acid sequence encoding the NiV F protein is at least 80%. at least 85%, at least 90%, at least 91%, at least 92%. at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 44. In some embodiments, the nucleic acid sequence encoding the NiV F protein is 100% identical to SEQ ID NO: 44. The nucleic acid sequence encoding the NiV F protein may further a signal sequence.

[0156] A NiV protein such as a NiV F protein may include a signal sequence. An example of a signal sequence is provided as SEQ ID NO: 45. Some embodiments include the signal sequence of SEQ ID NO: 45. Some embodiments include a signal sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 45.

[0157] The signal sequence may be encoded by a nucleic acid. An example nucleic acid sequence encoding a signal sequence of a NiV F protein is provided as SEQ ID NO: 46. Some embodiments include a nucleic acid sequence of SEQ ID NO: 46. Some embodiments include a nucleic acid sequence at least 80%, at least 85%. at least 90%, at least 91%. at least 92%, at least 93%. at least 94%. at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 46.Attorney Ref: 061479-515001WOCompositions

[0158] Disclosed herein, in some embodiments, are compositions. The composition may include a particle such as a viral particle or a lenti viral particle. The composition may be used in a method herein, or may be a part of a kit.

[0159] Disclosed herein, in some embodiments, are pharmaceutical compositions comprising the particle and one or more pharmaceutically acceptable solvents or diluents. In some embodiments, the disclosure provides a pharmacal composition comprising a particle according to the disclosure and a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier, excipient, or stabilizer may be included that is nontoxic to a recipient.Methods of use

[0160] Disclosed herein, in some embodiments, are methods such as methods of using a particle or composition herein. The method may include use of a particle such as a viral particle or a lentiviral particle. The method may include use of a composition herein.

[0161] Disclosed herein, in some embodiments, are methods of making a CAR T cells. The method may be in vitro. Disclosed herein, in some embodiments, are in vitro methods of making a CAR T cell, the method comprising contacting a T cell with a particle herein, thereby transducing the CAR T cell with the polynucleotide encoding a chimeric antigen receptor, operatively linked to a promoter.

[0162] The method may be in vivo. Some embodiments relate to a method of treatment. The method may include treating a disease. The disease may include a B cell disorder. The disease may include a B cell-related disorder. The disease may include cancer. The cancer may include a B cell malignancy. The disease may include an autoimmune disease. Disclosed herein, in some embodiments, are in vivo methods of generating a C AR-expressing cell or treating a disease such as cancer or an autoimmmie disease in a subject in need thereof, the method comprising administering to the subject, a particle herein, thereby transducing a cell with the a polynucleotide encoding a chimeric antigen receptor, operatively linked to a promoter, wherein the particle is administering in an amount effective to generate CAR-expressing cells or treat a disease such as cancer or an autoimmmie disease in the subject. In some embodiments, the method relates to treatment of a nonhuman primate. In some such methods, the non-human primate may not be in need of treatment, the method comprising administering to the non-human primate, a particle described herein, thereby transducing a cell in vivo and generating a transduced cell that expresses a payload. e.g. a CAR.

[0163] In some embodiments, the administration is intravenous, intratumoral, or intralymphatic. In some embodiments, the administration comprises an injection.

[0164] Disclosed herein, in some embodiments, are methods that include treating a subject or administering a composition such as a lentiviral particle described herein, a composition comprising the lentiviral particle, or a combination treatment (such as a lentiviral particle and rapamycin). InAttorney Ref: 061479-515001WO some embodiments, the lentiviral particle transduces T cells in the subject. In some embodiments, the lentiviral particle transduces T cells in the subject to generate CAR T cells. The method or administration may have an effect, such as improvement of a symptom of an disease. The treatment may result in an improvement of an aspect of the disease. The improvement may be reflected in a measurement. The improvement or measurement may be relative to a baseline, such as a baseline measurement.

[0165] In some embodiments, the administration treats or alleviates symptoms of the disease. In some embodiments, the administration treats the disease. In some embodiments, the administration alleviates symptoms of the disease. In some embodiments, the method or administration affects a biomarker measurement indicative of the disease.Manufacturing methods

[0166] Disclosed herein, in some embodiments, are manufacturing methods. A method may include a method of making a particle such as a viral particle or a lentiviral particle. A method may include a method of making a composition herein.

[0167] Disclosed herein, in some embodiments, are methods of making viral particles. Disclosed herein, in some embodiments, are methods of making viral particles, comprising expressing a polynucleotide herein in a host cell configured to generate a viral particle comprising the expressed targeting polypeptide.

[0168] In some embodiments, the disclosure provides a method of making a particle such as a lentiviral particle, or a composition herein. In some embodiments, the disclosure provides a method of making a particle, comprising introducing a polynucleotide encoding a vector genome into a host cell comprising a polynucleotide encoding a NiV G fusion protein. The NiV G fusion protein and the vector genome may be expressed by the host cell. The host cell may package the vector genome into a lentiviral particle comprising the NiV G fusion protein. Some embodiments include introducing to the host cell a polynucleotide encoding a multidomain protein. In some embodiments, the multidomain protein and the NiV G fusion protein are introduced to the host cell and expressed from separate polynucleotides. In some embodiments, the multidomain protein and the NiV G fusion protein are introduced to the host cell and expressed from the same polynucleotide.

[0169] Some embodiments include a method of making a recombinant targeting polypeptide. The method may include transcribing or translating a nucleic acid (such as a DNA or RNA) that encodes the recombinant targeting polypeptide. A NiV G fusion protein is an example of a recombinant targeting polypeptide. Some embodiments relate to or include a nucleic acid sequence encoding a recombinant targeting polypeptide. The nucleic acid may include a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 39.Attorney Ref: 061479-515001WO

[0170] Some embodiments include a method of making an adhesion molecule, a costimulatory molecule, an activation molecule, or a multidomain protein. The method may include transcribing or translating a nucleic acid (such as a DNA or RNA) that encodes a protein comprising the adhesion protein, costimulatory protein, activation protein, or a multidomain protein. The adhesion protein, costimulatory protein, activation protein, or multidomain protein may be produced, transcribed, or translated along with a recombinant targeting polypeptide.Kits

[0171] Disclosed herein, in some embodiments, are kits. In some embodiments, the kit includes a composition described herein. In some embodiments, the kit includes an engineered NiV Env protein. The kit may include instructions for use. The kit may be used in a method herein such as a method of making or a method of treatment.

[0172] In some embodiments, the kit includes an adhesion molecule. In some embodiments, the kit includes a costimulatory molecule. In some embodiments, the kit includes an activation molecule. In some embodiments, the kit includes a NiV G fusion protein. In some embodiments, the kit includes a NiV F protein. In some embodiments, the kit includes a multidomain protein. The kit may include a polynucleotide that encodes any one or more of such molecules.

[0173] In some embodiments, the kit includes a viral particle. The viral particle may include a lentiviral particle. The viral particle may include an engineered NiV Env protein. The viral particle may include an adhesion protein, a costimulatory protein, an activation protein, or a multidomain protein. The adhesion protein, costimulatory protein, activation protein, or multidomain protein may be included with a NiV G fusion protein. The viral particle may include a polynucleotide. The polynucleotide of the viral particle may encode a CAR, RACR, or a combination thereof.

[0174] In some embodiments, the disclosure provides a kit comprising the particle and instructions for use in transduction of target cells or treatment of a subject. The kit may include a pharmaceutically acceptable carrier. The kit may include an injection device. The kit may include suitable tubing for administering particles.Definitions

[0175] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Specifically, features described in one section may be combined with features in any other section of the description.

[0176] Unless otherwise defined, all terms (including technical and scientific tenns) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0177] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range fonnat is merely for convenience and brevity andAttorney Ref: 061479-515001WO should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3. from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of die breadth of the range.

[0178] The singular forms “a”, “an”, and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0179] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0180] A percent sequence identity may be detennined by comparing two optimally aligned sequences over a comparison w indow7, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (z.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window7of comparison and multiplying the result by 100 to yield the % sequence identity. A sequence identity may include a sequence identity to a reverse complement.

[0181] In determining a sequence identity’, thymine (T) and uracil (U) may be interchangeable. T and U may be interchangeable when describing an oligonucleotide. In some embodiments, Ts and Us are interchangeable depending on whether the oligonucleotide is an RNA or DNA, where RNA includes U and DNA includes T.

[0182] Any discrepancies between the written description and a sequence listing submitted herein may ty pically be resolved in favor of the written description.

[0183] “Subject,” “individual,” or “patient” are often used interchangeably herein. A “subject” can be an animal. A subject can be a mammal. A mammal can be a primate. The primate can be a human. The primate can be a pigtailed macaque. A subject may be diagnosed or suspected of being at risk for a disease such as cancer or an autoimmune disease.

[0184] “Transduction” may include delivery of an agent such as a polynucleotide to a cell, such as a therapeutic agent. A combination of agents may be delivered, such as several polynucleotides or a protein-nucleic acid complex (e.g, a gene-editing nuclease in complex with guide nucleic acid).

[0185] “Treatment” and “treating” may be used in reference to a pharmacal or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results may include a therapeutic benefit. A therapeutic benefit may include eradication or amelioration of symptoms or of an underlying disorder being treated. A therapeutic benefit may beAttorney Ref: 061479-515001WO achieved with eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder.

[0186] A ‘’transduction unit” or “TU” as it relates to a viral particle may indicate a functional unit capable of transducing a cell. For example, 1 TU of viral particles may mean 1 functional viral particle. TUs may be measured by flow cytometry.EXAMPLESExample 1. Viral particles pseudotyped with NiV G-CD58-ECD fusion protein + NiV F protein transduced CD2+ target cells

[0187] Experiments were conducted to generate engineered NiV G proteins, which are examples of recombinant targeting polypeptides. A NiV G protein engineering strategy was devised as shown in FIG. 5. Constructs were screened using SUP-T1 cells overexpressing CD2. Over 40 variations of engineered NiV G protein were screened, including: CD3 scFv (e.g. targeting CD3e), CD80 (e.g. targeting CD28), K12ecto (e.g. targeting CD7), MDF1 and MDF2 (e.g. targeting CD3 / CD2 / CD28), and CD58 (e.g. targeting CD2), as well as type I and II protein conversions.

[0188] An engineered NiV G protein was produced where a wild ty pe target cell binding domain was removed and replaced with a binding domain of a CD58 protein. Lentiviral particles were produced that included the engineered NiV G protein in a ty pe II orientation along with a NiV F protein within the lentiviral particle’s membrane. Transduction efficiency data for lentiviral particles that included the engineered NiV G protein were generated, and are shown next to transduction data from lentiviral particles pseudotyped with a Cocal protein, which transduced both cell types agnostically in FIG. 6. Modifications were made to the engineered NiV G protein to extend the length of its linker between a NiV stalk and the binding domain of a CD58 protein from 4 amino acids to 10 or 15 amino acids, and surprisingly increasing the linker length improved target cell transduction efficiency (FIG. 6). Additional experiments were performed to find an optimal linker length or range of linker lengths, and summary data from different experiments used to provide a general understanding of the linker length are provided in FIG. 7. Transduction with the engineered NiV G protein was specific for CD2 expressing cells (FIG. 8). FIG. 8 demonstrates that FPLC methods may be used to purify and concentrate NiV vector. The concentrated vector’s transduction was also cell type specific.

[0189] Some modified NiV G proteins worked less well than others. For example, a NiV G protein with its target cell binding domain was replaced with a binding domain of a CD80 protein, or a NiV G protein with its target cell binding domain was replaced with a binding domain of a CD58 protein and switched to a ty pe I orientation, had a lower target cell transduction efficiencies (FIG. 9A-9B).

[0190] The experiments herein demonstrated, among other things, production of a recombinant NiV-pseudotyped lentiviral particles that transduced CD2+ cells including CD2+ cell lines andAttorney Ref: 061479-515001WO peripheral blood mononuclear cells (PBMCs). Atempts were made at producing engineered NiV proteins with many different binding domains, including a CD58 protein binding domain. NiV protein linker lengths were tested and optimized to increase lentiviral transduction efficiency.

[0191] In addition to modifying NiV G protein, NiV F proteins were also engineered and produced. Different signal peptide sequences were tested for NiV F protein, including an endogenous signal peptide sequence, a Gaussia luciferease signal peptide sequence, and an IFNa2 signal peptide sequence. This was done because, for example, the Gaussia luciferase signal peptide sequence had a higher cleavage prediction (close to 100% versus closer to 50% for the endogenous signal peptide sequence).Example 2. NiV G + F pseudotyped viral particles transduce PBMCs

[0192] The transductions in Example 1 of NiV pseudotyped lentiviral vectors were performed in cultured cell lines. A question w as whether the lentiviral particles that included engineered NiV G proteins would transduce peripheral blood mononuclear cells (PBMCs). An experimental design was followed, PBMCs from multiple healthy donors were thawed and lentiviral vectors were transduced at different multiplicities of infection (MOI). As shown in FIG. 10, lentiviral particles that included MDF surface engineering activated cells. As shown in FIG. 11A-11B, NiV vectors were able to transduce PBMCs. In fact, the NiV vector transduction continued to increase w ith later time points, demonstrating a potential slower kinetics compared to Cocal G pseudotyped vectors in vitro. The NiV vectors that transduced the PBMCs included an MDF protein.

[0193] Additional experiments were performed to confirm these findings with other pay loads, low er multiplicities of infection (MOIs), and additional donors, as outlined in FIG. 12. Activation of PBMCs was similar across payloads and vector envelopes (FIG. 13). NiV vectors with different pay loads transduced PBMCs (FIG. 14).Example 3. Vector production and manufacturing for NiV F + G

[0194] One aspect provided herein is optimizing NiV vector production. Viral particles were produced as shown in FIG. 15. A DoE was performed to optimize plasmid ratios. A goal w as to create a vector that could be used to test a serum inhibition hypothesis with workable titer and PPTU. PPTU is a ratio of physical particles (from p24 ELISA quantification) over transducing particles, and is useful for determining how clean a vector is and how efficient cells are at producing a vector. Because of the different entry mechanism, many ratios that varied the following were tested: payload, GagPol, NiV F protein, NiV G protein, and MDF.

[0195] Some optimization data are shown in FIG. 16A-16B. Having a 2:1 ratio of F:G protein transduced cells best. Decreasing MDF helped increase the vector titer. Also, lowering GagPol increased titer and decreased the PPTU.Attorney Ref: 061479-515001WO

[0196] A stable cell line was created for consistent and increased yields. First, stably expression of NiV F protein in L2F9 cells was performed to make consistent vector production. Single cell clones were tested. NiV stable F cells produced 10X higher titer vector compared to a transient control (FIG. 17). NiV stable F cells produced much lower PPTU compared to control (FIG. 18).Example 4. Transduction of primate PBMCs

[0197] A question was whether the NiV pseudotyped vector could be used to test routes of administration in non-human primates (NHPs). A study was devised to test NiV vectors for activation and transduction on NHP peripheral blood mononuclear cells (PBMCs) as compared to Cocal pseudotyped vectors. A study design that was followed is shown in FIG. 19. All vectors contained a CD20-FLAG CAR payload and included NHP MDF 1 surface engineering.

[0198] As shown in FIG. 20A. NiV G protein + NiV F protein pseudotyped (without Cocal G and without MDF) (“NiV”) and Cocal G pseudotyped NHP vectors (with MDF construct CD58- antiCD3-CD80) both activated NHP PBMCs (3 donors). Cocal pseudotyped and NiV pseudotyped vectors both exhibited good activation, which may have been at least partly due to their MDF surface engineering comprising construct CD58-antiCD3-CD80. The NiV pseudotyped vector initially resulted in lower percentages of CAR+ cells compared to a Cocal-pseudotyped vector, but dramatically increased between Day 7 and Day 11 (FIG. 20B).Example 5. Use of NiV to reduce serum inhibition in NHPs

[0199] Experiments were performed in which it was found that pigtailed macaque (PtM) serum inhibited viral transduction when Cocal G pseudotyped lentiviral particles were incubated with macaque serum prior to ex vivo transduction of macaque PBMCs with the lentiviral particles. The lentiviral particles included a multi-domain (MDF) protein at their surface and carried a nucleic acid pay load that encoded a chimeric antigen receptor (CAR). The experiments indicated that Cocal G pseudoty ped lentiviral particles may face serum inhibition when administered to a non-human primate (NHP) such as a PtM (FIG. 21-22). The serum inhibition was specific to transduction with the lentiviral particles, and did not affect T cell activation. The data suggest the NHP system was improved in its capacity for in vivo modeling by replacing Cocal pseudotyped vectors with NiV pseudotyping to help overcome species-specific differences in serum inhibition.

[0200] In accordance with the finding that PtM serum inhibited ex vivo transduction by Cocal pseudotyped lentiviral particles, it was found in vivo that serum anti-Cocal antibodies were detected soon after administration of the Cocal pseudotyped lentiviral particles to PtMs.

[0201] Because Cocal G pseudotyped lentiviral particles could face serum inhibition in vivo in some NHPs, a further experiment was set up to compare serum inhibition of lentiviral particles pseudotyped with Cocal or Nipah virus (NiV) (here, NiV G fusion protein + NiV F protein). As such, PtM serum inhibition was evaluated in an extracorporeal (EC) delivery model (with 2 donors),Attorney Ref: 061479-515001WO as shown in FIG. 23. As shown in FIG. 24, NiV pseudotyped and Cocal pseudo typed vectors had similar binding to human PBMCs after 1 hour. PtM serum did not inhibit activation of T cells (FIG. 25), consistent with observations that PtM serum did not seem to inhibit activation of PBMCs, only transduction. The results showed a dramatic increase in percent CAR generation with NiV pseudotyped lentiviral particles. Flow cytometry results at study day 10, shown in FIG. 26, demonstrated no serum inhibition with the NiV pseudotyped vector, for each of 4 serum samples, whereas serum inhibition with the Cocal pseudotyped vector remained consistent. The 4 serum samples came from 4 different PtMs. There was still no observed serum inhibition with the NiV pseudotyped lentiviral particles at study day 14 (FIG. 27). at which point the serum inhibition effect of Cocal pseudotyped vector still remained consistent. As shown in FIG. 28A-28B, naive PtM serum did not inhibit in vitro transduction of SUP-T 1 -CD2+ cells or human PBMCs in the context of a NiV G fusion protein-pseudotyped vector.

[0202] This example tested NiV pseudotyped vectors for activation and transduction on non-human primate (NHP) PBMCs as compared to Cocal pseudotyped vectors. This study further evaluated the feasibility of NiV pseudotyped vectors for extracorporeal delivery and for reduced inhibition of transduction with PtM serum as compared to Cocal pseudotyped vectors. Here, a simple serum inhibition assay was developed, and used for screening and selection of NHPs, as well as analysis of post lentiviral particle administration serum.Example 6. Administration of NiV G-CD58 ECD fusion protein + NiV F protein pseudotyped lentiviral particles to primates

[0203] An in vivo study shown in FIG. 29 was performed, in which lentiviral particles with NiV G-CD58 extracellular domain (ECD) fusion protein and NiV glycoprotein F were intravenously (IV) delivered to a pigtailed macaque (PtM). The lentiviral particles also included a multi-domain (MDF) protein and a payload expressing a CD20-targeting chimeric antigen receptor (CAR) linked to a hemagglutinin (HA) tag.

[0204] NiV G fusion protein-pseudotyped vector drove generation of HA+ and anti-CD20 CAR T cells when administered IV. and robust function of these CAR T cells as measured by depletion of circulating CD20+ B cells (FIG. 30A-30B).

[0205] Here, a NiV G fusion protein-pseudotyped vector administered via an IV route of administration (ROA) drove generation and activity of CAR T cells in a PtM primate model, with similar kinetics and comparable or better efficacy as Cocal pseudotyped vectors (data not shown). Thus. NiV G fusion protein-pseudotyped vectors are useful, and can be used for delivering payloads and generating CAR T cells, or for modeling IV ROA efficacy, potency, and safety in PtM.Attorney Ref: 061479-515001WOExample 7. NiV G-CD58 ECD fusion protein + NiV F protein viral particles transduce T cells in primates and kill the target cells

[0206] This example expands on the study described in Example 6 above, which evaluated the effects of intravenous administration of NiV G-CD58 ECD fusion protein + NiV F protein- pseudotyped lentiviral vectors in pigtailed macaques to successfully generate functional CD20- targeting CAR T cells.

[0207] In this study, an in vivo study shown in FIG. 31 was performed in three NHPs, in which lentiviral particles with NiV G-CD58 ECD fusion protein and NiV glycoprotein F were intravenously (IV) delivered to pigtailed macaques (PtM) at the indicated dose levels. The lentiviral particles also included a multi-domain (MDF) protein and a payload either expressing a CD20- targeting chimeric antigen receptor (CAR) (comprising SEQ ID NO: 209 of WO / 2024 / 238153), or a RACR-CD20-targeting CAR, as described in WO / 2024 / 238153 (comprising SEQ ID NOs: 209 and 254 of WO / 2024 / 238153). The CAR payloads were linked to either a hemagglutinin (HA) or FLAG tag for detection of generated CAR T cells by flow cytometry.

[0208] The function of these anti-CD20 CAR T cells was measured as depletion of circulating CD20+ B cells. Consistent w ith the findings in Example 6, NiV G-CD58 ECD fusion protein- pseudotyped vector drove generation of anti-CD20 CAR T cells when administered IV. (FIG. 32A- 32B). Specifically, this study demonstrated evidence of RACR activity', as indicated by elevated activation markers in CAR+T cells and suppressed activation in CAR T cells. Importantly, sustained B cell depletion was observed in the animal while on rapamycin, followed by rapid B cell repopulation upon withdrawing rapamycin.In sum, NiV G fusion protein-pseudotyped vectors administered intravenously drove generation and activity of CAR T cells in a PtM primate model across a dose range, with similar kinetics and comparable or better efficacy as Cocal pseudotyped vectors. As such, the results from this study demonstrates that NiV G fusion protein-pseudotyped vectors are useful, and can be used for delivering payloads and generating CAR T cells in vivo.SEQUENCES

[0209] Some embodiments relate to or include a sequence provided in Table 1. Some embodiments include a sequence at least 80%. at least 85%, at least 90%, at least 91%. at least 92%, at least 93%, at least 94%. at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% identical to a sequence in Table 1.Table 1. SequencesAttorney Ref: 061479-515001WOAttorney Ref: 061479-515001WOAttorney Ref: 061479-515001WOAttorney Ref: 061479-515001WOAttorney Ref: 061479-515001WOAttorney Ref: 061479-515001WOAttorney Ref: 061479-515001WOAttorney Ref: 061479-515001WO***

[0210] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, controls. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment, or any form of suggestion, that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0211] While illustrative embodiments have been described and depicted, it will be appreciated that various changes can be made to these illustrative embodiments without departing from the spirit and scope of the invention.

Claims

Attorney Ref: 061479-515001WOCLAIMS1. A viral particle, comprising: a polynucleotide cargo; a lipid envelope; and a recombinant targeting polypeptide comprising: an external region comprising a CD58 extracellular domain, and a transmembrane region.

2. The viral particle of claim 1, wherein the recombinant targeting polypeptide further comprises an internal region comprising a cytoplasmic tail of a henipavirus G protein.

3. The viral particle of claim 1, wherein the recombinant targeting polypeptide comprises an internal region comprising a cytoplasmic tail of a Nipah Virus (NiV) G protein.

4. The viral particle of any one of the claims 1-3, wherein the cytoplasmic tail comprises a fragment comprising a GcA33 truncation variant or a GcA34 truncation variant.

5. The viral particle of any one of the claims 1-4, wherein the external region comprises a polypeptide sequence according to SEQ ID NO: 38, or at least 80%. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%. at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% identical thereto.

6. The viral particle of any one of the claims 1-5, wherein the transmembrane region comprises a transmembrane domain of the henipavirus G protein.

7. The viral particle of any one of the claims 1-6, wherein the transmembrane region comprises a transmembrane domain of CD58.

8. The viral particle of any one of the claims 1-7, wherein the recombinant targeting polypeptide comprises a stalk domain of the henipavirus G protein.

9. The viral particle of any one of the claims 1-8, wherein the stalk domain together with the transmembrane region comprises the polypeptide sequence of SEQ ID NO:

34. or a polypeptide sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%. at least 95%, at least 96%. at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 34.

10. The viral particle of any one of the claims 1-9, wherein the stalk domain is connected through a polypeptide linker to the CD58 extracellular domain.Attorney Ref: 061479-515001WO11. The viral particle of any one of the claims 1-10, wherein the polypeptide linker is 4 to 50 amino acids in length.

12. The viral particle of any one of the claims 1-11, wherein the polypeptide linker comprises the polypeptide sequence of any of SEQ ID NOs: 35-37, or a polypeptide sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%. at least 94%, at least 95%, at least 96%, at least 97%, at least 98%. or at least 99% identical to any of SEQ ID NOs: 35-37.

13. The viral particle of any one of the claims 1-12, wherein the recombinant targeting polypeptide comprises a polypeptide sequence disclosed in Table 1, or at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

14. The viral particle of any one of the claims 1-13, wherein the recombinant targeting polypeptide comprises the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence at least 80%, at least 85%. at least 90%, at least 91%, at least 92%. at least 93%, at least 94%, at least95%, at least 96%, at least 97%. at least 98%, or at least 99% identical thereto.

15. The viral particle of any one of the claims 1-14, wherein the external region comprises a multidomain fusion (MDF) protein comprising: a CD58 extracellular domain; a CD80 or CD86 extracellular domain; and an anti-CD3 antibody, an anti-CD3 antibody binding fragment, or an anti-CD3 single-chain variable fragment (scFv).

16. The viral particle of any one of claims 1-15, wherein the CD58 extracellular domain comprises the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence at least 80%. at least 85%, at least 90%. at least 91%, at least 92%, at least 93%. at least 94%, at least 95%, at least 96%, at least 97%, at least 98%. or at least 99% identical thereto.

17. The viral particle of any one of claims 1-16. wherein the CD80 extracellular domain comprises the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

18. The viral particle of any one of claims 1-17, wherein the CD86 extracellular domain comprises the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%. at least 94%, at least 95%, at least 96%. at least 97%, at least 98%, or at least 99% identical thereto.Attorney Ref: 061479-515001WO19. The viral particle of any one of claims 1-18, wherein the anti-CD3 antibody an anti-CD3 scFv comprises an CDR-L1 comprising the amino acid sequence of SEQ ID NO: 26, an CDR-L2 comprising the amino acid sequence of SEQ ID NO: 27 and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 28; and an CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29, an CDR-H2 comprising the amino acid sequence of SEQ ID NO:

30. and CDR-H3 comprises the amino acid sequence of SEQ ID NO: 31.

20. The viral particle of any one of claims 1-19, wherein the multidomain fusion (MDF) comprises the amino acid sequence of any one of SEQ ID NOs: 13-16, or an amino acid sequence at least 80%, at least 85%, at least 90%. at least 91%, at least 92%. at least 93%, at least 94%, at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% identical thereto.

21. The viral particle of any one of claims 1-20, wherein the polynucleotide cargo comprises a promoter.

22. The viral particle of any one of claims 1-21, wherein the polynucleotide cargo encodes a chimeric antigen receptor.

23. The viral particle of any one of claims 1-22. wherein the polynucleotide cargo encodes a small molecule-activated cytokine receptor, optionally a rapamycin molecule-activated cytokine receptor (RACR).

24. The viral particle of any one of claims 1-23, wherein the viral particle comprises a henipavirus F protein.

25. The viral particle of claim 24, wherein the henipavirus F protein is a Nipah Virus (NiV) F protein.

26. The viral particle of any one claims 1-25, wherein the NiV F protein comprises the amino acid sequence of SEQ ID NOs: 10 or 40, or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. l. The viral particle of any one of claims 1-25, wherein the NiV F protein lacks residues 1-22 of SEQ ID NO: 10.

28. The viral particle of any one of claims 1-25. wherein NiV F protein comprises the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence at least 80%, at least 85%. at least 90%, at least 91%, at least 92%, at least 93%. at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.Attorney Ref: 061479-515001WO29. The viral particle of any one of claims 1-28, wherein the particle is a lentiviral particle.

30. A recombinant targeting polypeptide as comprised in the particle of any one of claims 1- 29.

31. A recombinant targeting polypeptide comprising: an extracellular domain that specifically binds CD2; a transmembrane domain; and a cytoplasmic domain comprising a cytoplasmic tail of a henipavirus G protein.

32. The polypeptide of claim 31, wherein the extracellular domain comprises an antibody that specifically binds CD2.

33. The polypeptide of claim 31 or claim 32, wherein the recombinant targeting polypeptide comprises a stalk domain of the henipavirus G protein.

34. The polypeptide of any one of claims 31-33, wherein the viral particle comprises a henipavirus F protein.

35. The polypeptide of claim 34, wherein the henipavirus F protein is a Nipah Virus (NiV) F protein.

36. A polynucleotide encoding the polypeptide of any one of claims 1 -35, operably linked to a promoter.

37. A gene delivery vehicle comprising the polynucleotide of claim 36.

38. A host cell comprising the polynucleotide of claim 36.

39. A method of making a viral particle, comprising expressing the polynucleotide of claim 36 in a host cell configured to generate a viral particle comprising the expressed targeting polypeptide.

40. An in vitro method of making a CAR T cell, the method comprising contacting a T cell with the particle of any one of claims 1-35, thereby transducing the CAR T cell with a polynucleotide encoding a chimeric antigen receptor, operatively linked to a promoter.

41. An in vivo method of generating a CAR T cell in a subject in need thereof, the method comprising administering to the subject the viral particle of any one of claims 1-35, thereby transducing T cells with a polynucleotide cargo comprising a polynucleotide sequence encoding a chimeric antigen receptor, operatively linked to a promoter.Attorney Ref: 061479-515001WO42. A method of killing cancer cells in a subject in need thereof, tire method comprising administering to the subject the viral particle of any one of claims 1-35, wherein the polynucleotide cargo comprises a polynucleotide sequence encoding a therapeutic polypeptide or a chimeric antigen receptor.

43. A method of treating cancer or autoimmune disease in a subject in need thereof, the method comprising administering to the subject tire viral particle of any one of claims 1-35. wherein the polynucleotide cargo comprises a polynucleotide sequence encoding a therapeutic polypeptide or a chimeric antigen receptor.

44. The method of any one of claims 41-43, wherein the administration is intravenous, intratumoral, or intralymphatic.

45. The method of any one of claims 41-44, wherein the administration comprises an injection.

46. A pharmaceutical composition comprising the particle of any one of claims 1-35 and one or more pharmaceutically acceptable solvents or diluents.

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