Lentiviral delivery of Anti-CD22 chimeric antigen receptors

Abstract

Provided herein are particles comprising polynucleotide constructs for generating cells expressing anti-CD22 chimeric antigen receptor, as well as vectors, such as lentiviral vectors, comprising same, cells comprising same, and methods of using same. Some embodiments include a surface-engineered lentiviral vector. In some embodiments, the lentiviral vector further encodes a synthetic receptor protein.

Description

LENTIVIRAL DELIVERY OF ANTI-CD22 CHIMERIC ANTIGEN RECEPTORSCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 574,150 filed Apr. 3, 2024, 63 / 696,081 filed Sep. 18, 2024, and 63 / 717,157 filed Nov. 6, 2024, 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 061479-516001WO_seqs.xml, created March 13, 2025, which is 256,810 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.BACKGROUND

[0003] Autoimmune disorders and cancers such as B-cell malignancies are devastating for many individuals. Improved treatments are needed.SUMMARY

[0004] Disclosed herein, in some embodiments, are lentiviral particles comprising: a polynucleotide encoding a chimeric antigen receptor (CAR) that specifically binds cluster of differentiation-22 (CD22). Some embodiments relate to a lentiviral particle, comprising: a polynucleotide encoding a chimeric antigen receptor (CAR) that specifically binds cluster of differentiation-22 (CD22), and (a) or (b): (a) a surface protein comprising: a heterologous viral glycoprotein (G protein), a CD58 extracellular domain sequence, a CD80 or CD86 extracellular domain sequence, an anti-CD3 antibody or antigen-binding fragment, or a combination thereof; or (b) a polynucleotide encoding a small molecule-activated cytokine receptor. Some embodiments relate to a lentiviral particle, comprising: a polynucleotide comprising a sequence that is at least 95% identical (e.g. 99% identical or 100% identical) to SEQ ID NO: 292 and encodes a chimeric antigen receptor (CAR) that specifically binds cluster of differentiation-22 (CD22); a fusion molecule comprising: a CD58 extracellular domain sequence, a CD80 or CD86 extracellular domain sequence, and an anti-CD3 antibody or antigen-binding fragment; and a cocal glycoprotein.

[0005] In some embodiments, the CAR comprises light chain complementarity-determining regions (CDRs) selected from any of the following groups: LCDR1 having the sequence of SEQ ID NO: 413, LCDR2 having the sequence set forth in SEQ ID NO: 414 and LCDR3 having the sequence set forth in SEQ ID NO: 415, LCDR1 having the sequence set forth in SEQ ID NO: 401, LCDR2 having the sequence set forth in SEQ ID NO: 402 and LCDR3 having the sequence set forth in SEQ ID NO: 403, LCDR1 having the sequence set forth in SEQ ID NO: 407, LCDR2 having the sequence set forth in SEQ ID NO: 408 and LCDR3 having the sequence set forth in SEQ ID NO: 409, or LCDR1 having the sequence set forth in SEQ ID NO: 441, LCDR2 having the sequence set forth in SEQ ID NO: 442 and LCDR3 having the sequence set forth in SEQ ID NO: 443 ; and wherein the CAR comprises heavy chainCDRs selected from any of the following groups: HCDR1 having the sequence set forth in SEQ ID NO: 416, HCDR2 having the sequence set forth in SEQ ID NO: 417 and HCDR3 having the sequence set forth in SEQ ID NO: 418, HCDR1 having the sequence set forth in SEQ ID NO: 404, HCDR2 having the sequence set forth in SEQ ID NO: 405 and HCDR3 having the sequence set forth in SEQ ID NO: 406, HCDR1 having the sequence set forth in SEQ ID NO: 410, HCDR2 having the sequence set forth in SEQ ID NO: 411 and HCDR3 having the sequence set forth in SEQ ID NO: 412, or HCDR1 having the sequence set forth in SEQ ID NO: 438, HCDR2 having the sequence set forth in SEQ ID NO: 439 and HCDR3 having the sequence set forth in SEQ ID NO: 440. In some embodiments, the CAR comprises a VL comprising a polypeptide 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: 299, 419, 421, or 423. In some embodiments, the VL is encoded by a polynucleotide 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: 298. In some embodiments, the CAR comprises a VH comprising a polypeptide 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: 295, 420, 422, or 424. In some embodiments, the VH is encoded by a polynucleotide 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: 294. In some embodiments, the CAR comprises a CD8 hinge and transmembrane domain 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: 299. In some embodiments, the CD8 hinge and transmembrane domain are encoded by a polynucleotide 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: 298. In some embodiments, the CAR comprises a 4-1BB 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: 84 or 201. In some embodiments, the 4-1BB is encoded by a polynucleotide 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: 200 or 302. In some embodiments, the CAR comprises a CD3zeta 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: 82 or 203. In some embodiments, the CD3zeta is encoded by a polynucleotide 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: 202 or 303. In some embodiments, the CARcomprises a polypeptide 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 any one of SEQ ID NOs: 292, 435-437 or 456. In some embodiments, the polynucleotide encoding the CAR comprises a 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: 454 or 455.

[0006] Some embodiments include a heterologous viral glycoprotein (G protein). In some embodiments, the viral G protein is or is derived from the cocal virus. In some embodiments, the viral G protein comprises a polypeptide 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: 74.

[0007] Some embodiments of the lentiviral particles disclosed herein include a surface displayed polypeptide selected from: an anti-CD3 antibody or antigen-binding fragment thereof, a CD28 ligand, a CD2 ligand, and / or a 4-1BB ligand. In some embodiments, the lentiviral particles disclosed herein include a surface displayed polypeptide selected from: an anti-CD3 antibody or antigen-binding fragment thereof, a CD58 protein or extracellular binding domain thereof, and / or a CD80 or CD86 protein or extracellular binding domain thereof. Some embodiments of the lentiviral particles disclosed herein include a surface-displayed fusion molecule comprising: a CD58 extracellular domain, or a functional fragment thereof, a CD80 or CD86 extracellular domain, or a functional fragment thereof, and an anti-CD3 antibody or antigen-binding fragment thereof. Some embodiments include a viral surface comprising a CD58 extracellular domain sequence. In some embodiments, the CD58 extracellular domain comprises a polypeptide 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: 10, 444 or 445. Some embodiments include a viral surface comprising a CD80 extracellular domain sequence. In some embodiments, the CD80 extracellular domain comprises a polypeptide 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: 12 or 250. Some embodiments include a viral surface comprising a CD86 extracellular domain sequence. In some embodiments, the CD86 extracellular domain comprises a polypeptide 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: 13. Some embodiments include a viral surface comprising an anti-CD3 antibody or antigen-binding fragment. In some embodiments, the anti-CD3 antibody or antigen-binding fragment comprises complementarity-determining regions (CDRs) selected from (i) or (ii): (i) HCDR1 having the sequence set forth in SEQ ID NO: 48, HCDR2 having the sequence set forth in SEQ ID NO: 49 and HCDR3 having the sequence set forth in SEQ ID NO: 50, LCDR1 having the sequence of SEQ ID NO: 51, LCDR2 having the sequence set forth in SEQ ID NO:52 and LCDR3 having the sequence set forth in SEQ ID NO: 53, or (ii) HCDR1 having the sequence set forth in SEQ ID NO: 54, HCDR2 having the sequence set forth in SEQ ID NO: 55 and HCDR3 having the sequence set forth in SEQ ID NO: 56, LCDR1 having the sequence set forth in SEQ ID NO: 57, LCDR2 having the sequence set forth in SEQ ID NO: 58 and LCDR3 having the sequence set forth in SEQ ID NO: 59. In some embodiments, the anti-CD3 antibody or antigen-binding fragment comprises the anti-CD3 antigen-binding fragment. In some embodiments, the anti-CD3 antigen-binding fragment comprises a single chain variable fragment (scFv). In some embodiments, the anti-CD3 antigen-binding fragment comprises a polypeptide 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: 31 or 249. In some embodiments, the fusion molecule comprises a polypeptide 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: 446 or 448-449.

[0008] In some embodiments, the lentiviral particle comprises a polynucleotide encoding the small molecule-activated cytokine receptor. The small molecule may include rapamycin or a rapalog. Some embodiments include a polynucleotide sequence encoding a synthetic cytokine receptor comprising: a first polypeptide comprising a sequence that is at least 95% identical (e.g. 99% identical or 100% identical) to SEQ ID NO: 451, and a second polypeptide comprising a sequence that is at least 95% identical (e.g. 99% identical or 100% identical) to SEQ ID NO: 453. In some embodiments, the lentiviral particle includes a polynucleotide encoding a rapamycin activated cytokine receptor (RACR). In some embodiments, the lentiviral particle comprises a polynucleotide encoding a FKBP12- rapamycin binding (FRB) polypeptide or functional fragment thereof. In some embodiments, the FRB polypeptide comprises a polypeptide 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: 252. In some embodiments, the FRB polypeptide is encoded by a polynucleotide 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: 257. In some embodiments, the lentiviral particle comprises a polynucleotide encoding a cytokine gamma chain polypeptide. In some embodiments, the cytokine gamma chain polypeptide 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%, at least 99%, or 100% identical to SEQ ID NO: 264 or 265. In some embodiments, the cytokine gamma chain polypeptide is encoded by a polynucleotide 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: 261, 262, or 263. In some embodiments, the FRB polypeptide and the cytokine gamma chain polypeptide are fused together. In some embodiments, the lentiviral particle comprises a polynucleotide encoding a FKBP polypeptide.In some embodiments, the FKBP polypeptide comprises a polypeptide 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: 253. In some embodiments, the FKBP polypeptide is encoded by a polynucleotide 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: 268. In some embodiments, the lentiviral particle comprises a polynucleotide encoding a cytokine beta chain polypeptide. In some embodiments, the cytokine beta chain polypeptide 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%, at least 99%, or 100% identical to SEQ ID NO: 272. In some embodiments, the cytokine beta chain polypeptide is encoded by a polynucleotide 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: 270 or 271. In some embodiments, the FKBP polypeptide and the cytokine beta chain polypeptide are fused together. In some embodiments, the FRB polypeptide and cytokine beta chain polypeptide are fused together and the FKBP polypeptide and the cytokine gamma chain polypeptide are fused together. In other embodiments, the FRB polypeptide and cytokine gamma chain polypeptide are fused together and the FKBP polypeptide and the cytokine gamma chain polypeptide are fused together.

[0009] In some embodiments, the lentiviral particle comprises a polynucleotide encoding a free FKBP12-rapamycin binding (FRB) domain polypeptide. In some embodiments, the free FRB polypeptide comprises a 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: 251, 252, or 260. Some embodiments include a polynucleotide sequence encoding a free FRB polypeptide that comprises a sequence at least 95% identical SEQ ID NO: 251. In some embodiments, the free FRB polypeptide is encoded by a polynucleotide 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: 256, 257, or 258.

[0010] Disclosed herein, in some embodiments, are methods comprising administering the lentiviral particle to a subject. In some embodiments, the subject has cancer or is in need of cancer treatment. In some embodiments, the administration treats the cancer. Some embodiments treat cancer in the subject. In some embodiments, the cancer comprises CD22+ cancer cells. In some embodiments, the cancer is a B-cell malignancy. In some embodiments, the subject has an autoimmune disease or is in need of treatment for an autoimmune disorder. Some embodiments treat an autoimmune disorder in the subject. In some embodiments, the autoimmune disorder is caused or exacerbated by B cells. The autoimmune disorder may include lupus. In some embodiments, the autoimmune disorder includes systemic lupus erythematosus. In some embodiments, the autoimmune disorder includes lupus nephritis.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 depicts a viral particle modified with a fusion molecule and a glycoprotein on its surface. The particle in the figure includes an anti-CD22 chimeric antigen receptor (CAR) payload. Abbreviations: CAR= chimeric antigen receptor; MDF=multidomain fusion protein; scFv=single-chain fragment variable.

[0012] FIG. 2 depicts an example of an anti-CD22 CAR payload. The CAR shown includes (from N to C) an anti-CD22 single chain antibody fragment binding domain, a hinge domain, a transmembrane domain from CD8a, and 41BB and CD3z intracellular signaling domains.

[0013] FIG. 3A depicts a viral particle selectively binding, activating and transducing T cells in vivo to generate CAR T cells that express the anti-CD22 CAR. Abbreviations: CAR=chimeric antigen receptor; LDL-R=low-density lipoprotein receptor; MDF=multidomain fusion protein; RACR=rapamycin-activated cytokine receptor; RNA=ribonucleic acid; scFv=single-chain fragment variable.

[0014] FIG. 3B shows an example payload that encodes free FRB, a synthetic receptor, and a CAR.

[0015] FIG. 3C shows an example CAR T cell after transduction and transcription of a payload such as is included in FIG. 3B.

[0016] FIG. 4 depicts an example of infection, delivery of a payload and generation of T-cells expressing the anti-CD22 CAR. In the example in the figure: (1) the lentivirus particle binds and activates T cells via multidomain fusion and cocal glycoprotein and is internalized (2) the payload RNA genome is reverse transcribed and integrates into the host cell genome, which allows for expression of the CD22 CAR payload proteins and (3) expression of the anti-CD22 CAR by primary T-cells allows them to target and kill CD22+ cells.

[0017] FIG.5 shows diagrams of alentiviral particle, and demonstrates 2 examples of useful payloads.

[0018] FIG. 6 depicts a time-line for in vitro experiments described in Example 1.

[0019] FIG. 7 shows percentage of CD3+ T cells that are positive for the particle associated Cocal protein after 1 hour incubation with Product A and Product B particles.

[0020] FIG. 8 shows the percentage of CD3+ T cells that are CD25+ (3 days after infection) or anti- CD22 CAR+ (7 days after infection) after infection with Product A and Product B particles.

[0021] FIG. 9 shows the total number of tumor cells (Nalm6 or Raji) after being cocultured with CAR T cells that were transduced with Product A or Product B particles.

[0022] FIG. 10A shows total numbers of tumor cells (Raji wild-type or Raji CD22 Knockout) after being cocultured with CAR T cells that were transduced with Product A or Product B particles. FIG. 10A includes an overlay of the data from FIG. 10B + data obtained from using Product A.

[0023] FIG. 10B shows total numbers of tumor cells (Raji wild-type or Raji CD22 Knockout) after being cocultured with CAR T cells that were transduced with Product B particles.

[0024] FIG. 11 shows the quantification of cytokines produced by anti-CD22 expressing CAR-T cells cocultured with different tumor cell lines. CAR-T cells were generated by incubating PBMCs withProduct A and Product B particles. Shown within each plot from left to right are data from Nahn6 tumor cells, Raji tumor cells, K562.CD19 tumor cells, and Raji_CD22 knockout tumor cells.

[0025] BIG. 12 shows the percentage of CD3+, anti-CD22 CAR T positive T cells after exposure to indicated compounds.

[0026] FIG. 13 shows the number of anti-CD22 CAR T positive T cells after exposure to indicated compounds.

[0027] FIG. 14A is a schematic that shows an example fusion protein comprising a CD58 extracellular region and a-CD3 scFv fused to the N-terminus of a CD80 via a linker. The construct is termed “498.” The example is included with N to C from left to right.

[0028] FIG. 14B is a schematic that shows an illustrative fusion protein comprising a CD58 extracellular region fused to the N-terminus of a CD80 via a linker. The construct is termed “455.” a- CD3 scFv is expressed as a separate polypeptide in the producer cells. The example is included with N to C from left to right.

[0029] FIG. 15A is a diagram of an illustrative surface engineered viral particle displaying a CD58, CD80, dual-fusion polypeptide and an anti-CD3 scFv that binds NHP CD3 and a payload comprising a human-specific anti-CD20 CAR.

[0030] FIG. 15B is a diagram of an illustrative payload comprising a human-specific anti-CD20 CAR. The illustrative payload comprises:1. an optional MND promoter (not shown),2. an anti-CD20-based CAR which may include:1.Leul6 scFv - murine anti-human CD20 scFv (cross-reactive with NHP),2.CD8a hinge and transmembrane domain, and 3.4-1BB co-stimulatory domain and CD3 domain3. low-affinity nerve growth factor receptor (LNGFR) molecule as a marker of transduction.

[0031] FIG. 15C is a summary chart of the animals treated in an illustrative non-human primate study. Three animals were studied. * denotes at start of study.

[0032] FIG. 16 is a diagram of an illustrative study design and timeline for an NHP study. The engineered viral particles were injected into auxiliary lymph nodes (LN) of Animal #1 due to difficulty injecting inguinal lymph nodes (LN) due to the small size of the animal. The engineered viral particles were injected into inguinal lymph nodes in Animal #2 and Animal #3.

[0033] FIG. 17 is a graph showing the fraction of starting CD20+ cells in Animal #1, Animal #2, and Animal #3 over the course of the study to Day 56.

[0034] FIG. 18 is a panel of graphs showing serum levels of IL-6, ferritin, and C-reactive protein (CRP) in Animal #1, Animal #2, and Animal #3 over the course of the study to Day 56 and body temperature to Day 28.

[0035] BIG. 19A is a diagram of an illustrative surface engineered viral particle displaying a CD58, CD80, and anti-CD3 scFv tri-fusion polypeptide comprising an anti-CD3 scFv that binds NHP CD3 and a payload comprising a human-specific anti-CD20 CAR.

[0036] FIG. 19B is a diagram of an illustrative payload comprising a human-specific anti-CD20 CAR. The illustrative payload comprises:1. an MND promoter,2. an anti-CD20-based CAR comprising:1.Leul6 scFv - murine anti-human CD20 scFv (cross-reactive with NHP)2. FLAG tag3.CD8a hinge and transmembrane domain4.4-1BB co-stimulatory domain and CD3 domain.

[0037] FIG. 20 is a panel of flow cytometry staining graphs showing CAR expression in CD3+ T cells after 8 different donor PBMCs were transduced with viral particles displaying Human CD58-NHP- specific anti-CD3 scFv-Human CD80 multi-domain fusion (MDF) polypeptide at MOI=0.2.

[0038] FIG. 21A is a diagram of an illustrative study design and timeline for an NHP study.

[0039] FIG. 21B is a summary chart of the animals treated in an illustrative non-human primate study. Four animals were studied.

[0040] FIG. 22A-22B include a panel of flow cytometry staining graphs showing anti-CD20 CAR expression in CD3+ T cells through to Day 111 of the study in Animal #1.

[0041] FIG. 23 is a panel of flow cytometry staining graphs showing anti-CD20 CAR expression and CD25 expression in T cells through to Day 51 of the study in Animal #1.

[0042] FIG. 24A-24B include a panel of flow cytometry staining graphs showing CD20+ cells (B cells) and CD3+ T cells through to Day 111 of the study in Animal #1.

[0043] FIG. 25 is a graph showing CD20+ B cells and CD3+ CAR+ T cells through to Day 111 of the study in Animal #1.

[0044] FIG. 26A is a timeline of observed clinical symptoms over the course of the study.

[0045] FIG. 26B is a panel of graphs showing serum levels of IL-6, ferritin, and C-reactive protein (CRP) in Animal #1 over the course of the study to Day 56. The line depicting Anti-CD20 CAR T cells (CD3+ FLAG+) is identical in the 3 plots.

[0046] FIG. 27A-27B includes a panel of flow cytometry staining graphs showing CD20+ cells (B cells - top panels) and CAR+ (FLAG+) CD3+ T cells (bottom panels) through to Day 56 of the study in Animal #2.

[0047] FIG. 28A-28B includes a panel of flow cytometry staining graphs showing CD20+ cells (B cells - top panels) and CAR+ (FLAG+) CD3+ T cells (bottom panels) through to Day 37 of the study in Animal #3.

[0048] BIG. 29A-29B includes a panel of flow cytometry staining graphs showing CD20+ cells (B cells - top panels) and CAR+ (FLAG+) CD3+ T cells (bottom panels) through to day 21 of the study in Animal #4.

[0049] FIG. 30A includes a diagram of a pilot toxicology study in CD34-humanized NSG™ mice.

[0050] FIG. 30B is a table including treatment group details for a pilot toxicology study in CD34- humanized NSG™ mice.

[0051] FIG. 31 includes plots of B and T cell measurements prior to treatment with Product B.

[0052] FIG. 32 includes plots of B cell measurements before and after treatment with Product B.

[0053] FIG. 33 includes plots of CAR+ T cells.

[0054] FIG. 34 includes plots of circulating CAR+ T cells.

[0055] FIG. 35 depicts a study design.

[0056] FIG. 36A includes plots showing frequencies of cocal-positive cells of various cell types.

[0057] FIG. 36B includes plots showing frequencies and CD25 geometric mean fluorescence intensity (gMFT) in an example herein.

[0058] FIG. 36C is a plot showing frequencies of CAR+ T cells.

[0059] FIG. 37 includes plots of tumor count fold changes over time, separate for 3 individual donors.

[0060] FIG. 38 includes plots of CAR+ T cell concentrations over time.

[0061] FIG. 39A includes plots showing tumor burden in mice with cancer that were treated with vehicle or various indicated doses of lentiviral particles either alone or in combination with rapamycin.

[0062] FIG. 39B is a plot of survival over time in mice with cancer that were treated with vehicle or various indicated doses of lentiviral particles either alone or in combination with rapamycin.DETAILED DESCRIPTION

[0063] B-cell malignancies (BCMs) often originate from B lymphocytes or their progenitors. They may vary widely in clinical course and treatment, but can lead to significant morbidity and mortality over time. This group of malignancies may include large B-cell lymphomas (LBCLs), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), and small lymphocytic lymphoma (SLL), among others.

[0064] LBCLs are among the most aggressive of BCMs and can further be broken down into diffuse large B-cell lymphoma (DLBCL), either de novo or resulting from transformation of a more indolent lymphoma (FL, marginal zone lymphoma, CLL / SLL [e.g., Richter’s syndrome], etc.). DLBCL and FL are the most common non-Hodgkin’s lymphoma (NHL) subtypes in the USA and western Europe. DLBCL accounts for about 32% of NHL diagnoses worldwide. Some, but not all, are cured with frontline therapies. Follicular lymphoma Grade 3B (FL3B) has clinical characteristics akin to DLBCL and typically is treated with similar frontline treatment, warranting inclusion in this group of aggressive cancers.

[0065] Both healthy and malignant B cells may share a number of antigens throughout their development, including CD19, CD20, CD22, and CD79. A strategy of targeting these antigens in LBCLand other BCMs is useful in the treatment of these cancers. This has also led to side effects, such as B- cell aplasia, resulting from treatment effects on healthy B-cell populations. While efficacy has been shown with several agents in patients with BCMs, either alone or in combination therapy, most patients experience either relapsed or refractory disease and require further treatment. As such, there is an unmet need for therapies targeting other B-cell markers in patients who progress or relapse after being exposed to other therapies.

[0066] CD22 is a useful therapeutic target because it is often restrictively expressed by B cells and in particular, malignant B cells. CD22 may be considered a B cell-specific antigen that is expressed on most B-cell lymphomas and involved in regulation of B-cell function and profiferation. Thus, it may be a useful therapeutic target for BCMs.

[0067] Described herein are lentiviral vectors (LVV) designed to selectively bind, activate, and transduce T cells in vivo, resulting in in vivo anti-CD22 CAR T-cell generation. Some lentiviral particles herein may utilize an anti-CD22 binder with an identical CAR sequence to that is useful an ex vivo-generated CAR T-cell product. The unique characteristics of lentiviral envelope engineering described herein may be useful to generate potent CAR T cells in CAR T cell-naive subjects who may have T-cell dysregulation or low T-cell number / activity by virtue of their underlying disease pathology or previous therapies. Additionally, those who have received a previous CAR T-cell therapy may benefit from the activation signals of the lentiviral particles herein and / or rapamycin-induced signaling through the RACR system encoded in its payload.

[0068] Examples of lentiviral particles are shown in FIG. 1, BIG. 3A, and FIG. 5. The examples in the aforementioned figures include a fusion molecule, cocal glycoprotein, and payload including a transgene encoding an anti-CD22 CAR. Any aspect of these figures may be included in a lentiviral particle herein. The payload may further encode a RACR and free FRB. Examples of a payload and proteins encoded by the payload in a T cell are shown in FIG. 3B-3C and FIG. 4. Some embodiments relate to or include a surface engineered lentiviral vector with a fully human anti-CD22-CAR and RACR payload.

[0069] Some lentiviral particles herein may be given without the need for lymphodepletion, unlike more traditional ex vivo manufactured autologous CAR T-cell therapies. Additionally, as an “off-the- shelf” product, one manufacturing lot of some lentiviral particles herein may treat patients, overcoming a major challenge associated with ex vivo CAR T-cell manufacturing / supply.

[0070] The disclosure relates generally to a surface-engineered viral particle comprising a vector genome comprising a polynucleotide sequence encoding an anti-CD22 chimeric antigen receptor (CAR), wherein the viral particle transduces immune cells in vivo. The present disclosure includes particles comprising fusion molecules for use in transduction of target cells, such as immune cells, or specifically T cells. In one aspect, the disclosure provides, a particle for in vivo generation of CAR-T cells, comprising, displayed on the surface of the particle, a fusion molecule comprising an adhesion molecule linked to a costimulatory molecule, an activation molecule, or both.

[0071] Some embodiments relate to or include a lentiviral particle comprising: a polynucleotide encoding a chimeric antigen receptor (CAR) that specifically binds cluster of differentiation-22 (CD22). Some embodiments relate to or include an anti-CD22 CAR. Some embodiments relate to or include a particle such as a lentiviral particle for transducing an immune cell such as a T cell.

[0072] Some embodiments relate to or include a lentiviral particle, comprising: a polynucleotide encoding a chimeric antigen receptor (CAR) that specifically binds cluster of differentiation-22 (CD22), and (a) or (b): (a) a surface protein comprising: a heterologous viral glycoprotein (G protein), a CD58 extracellular domain sequence, a CD80 or CD86 extracellular domain sequence, an anti-CD3 antibody or antigen-binding fragment, or a combination thereof; or (b) a polynucleotide encoding a small molecule-activated cytokine receptor.

[0073] Some embodiments relate to or include a lentiviral particle, comprising: a polynucleotide comprising a sequence that is at least 95% identical (e.g. 99% identical or 100% identical) to SEQ ID NO: 292 and encodes a chimeric antigen receptor (CAR) that specifically binds cluster of differentiation-22 (CD22); a fusion molecule comprising: a CD58 extracellular domain sequence, a CD80 or CD86 extracellular domain sequence, and an anti-CD3 antibody or antigen-binding fragment; and a cocal glycoprotein.

[0074] The term “transduction” is used in its broadest sense to mean delivery of an agent to a cell, such as a therapeutic agent. The agent may be a small molecule, polynucleotide, or polypeptide. 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).

[0075] The fusion molecules of the disclosure combine an adhesion molecule with a costimulatory molecule, an activation molecule, or both. Without being bound by theory, it is believed that the inclusion of two or more of these types of molecules in a fusion molecule may cause such a particle, when it encounters a target cell, to form a macromolecular complex at the interface of the particle and cell that acts as artificial supramolecular activation cluster (SMAC).

[0076] T cells that encounter an antigen-presenting cell (APC) form an immune synapse known as a SMAC. In natural SMACs, the APC presents an antigen in complex with a major histocompatibility complex (MHC) molecule to the T cell receptor (TCR) on a T cell; CD80 or CD86 interact with CD28 to provide a costimulatory signal; and CD58 interacts with CD2 to adhere the APC to the T cell. The interactions between CD58 and CD2 may also provide an activatory or costimulatory signal. The adhesion molecule displayed on a particle may be CD58. SMACs may further present costimulatory molecules. Costimulatory molecules that may be displayed on a particle include CD80 and CD86.

[0077] As contemplated by the present disclosure, a particle may be engineered to display on its surface any of the foregoing adhesion molecules or costimulatory molecules; extracellular fragments thereof; or functional fragments thereof. Extracellular portions of these molecules may be identified in databases such as UniProt, which is available at www.uniprot.org, or may be predicted using methods, such as a method implemented by the TMHMM 2.0 program available at services.healthtech.dtu.dk.Furthermore, in some cases, functional fragments of each are identified in scientific literature or they may be identified using laboratory methods. For example, one may predict the identify fragments of a protein likely to form well-folded domains. Fragments may be tested in binding assays against a cognate molecule, or used in pull-down assays compared to the full molecule. Functional assays, such as expression of a fluorescence reporter under the control of a promoter activated by T-cell signaling (e.g. , the NKkB promoter) when a T cell is contacted with a cell or particle expressing a putative functional fragment. The sequence of the adhesion molecule, costimulatory molecule, or activation molecule may be varied to identify and use variants that retain function. For example, conservative mutations may be made to a molecule or a molecule may be randomly mutated with the function of the variant confirmed experimentally.

[0078] An adhesion molecule, costimulatory molecule, and activation molecule may be linked in any order with only the most N-terminal or C-terminal of the molecules connected to a transmembrane region or anchor. In a variant, the fusion molecule comprises or is associated with another membrane- associated molecule, thereby displaying the fusion molecule on the particle. The term “display” is used, in a broad sense, to mean the position on the surface of the particle such that the molecule may contact cognate molecules on the target cell.

[0079] In some embodiments, the lentiviral particle includes a heterologous viral glycoprotein (G protein). The viral G protein may include a Cocal virus G protein. An example of a Cocal G protein is provided as SEQ ID NO: 74, or a sequence at least 80%, at least 85%, 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. The viral G protein may include a Nipah virus G protein. The viral G protein may include a Vesicular stomatitis virus G protein.Anti-CD22 Chimeric Antigen Receptors (CARs)

[0080] In some embodiments, a particle such as a lentiviral particle described herein is used to transduce a nucleic acid sequence (polynucleotide) encoding one or more chimeric antigen receptor (CARs) into a cell (e.g., a T lymphocyte). In some embodiments, the transduction of the lentiviral particle results in expression of one or more CARs in the transduced cells.

[0081] 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., Eshhar, U.S. Pat. No. 7,741,465. Generally, CARs are genetically engineered receptors 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 single 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 these antigens exist on tumor cells, an immune cell that expresses the CAR can target and kill the tumor cell. All other conditions being satisfied, when a CARis expressed on the surface of, e.g., a T lymphocyte, and the extracellular domain of the CAR binds to an antigen, the intracellular signaling domain transmits a signal to the T lymphocyte to activate and / or proliferate, and, if the antigen is present on a cell surface, to kill the cell expressing the antigen. 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. Some illustrative CARs are known in the art and may 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. In some embodiments, the antigen is CD22.

[0082] An example of an anti-CD22 CAR is shown in FIG. 2, which includes an scFv, hinge, transmembrane domain, 4-1BB protein sequence, and CD3zeta protein sequence.

[0083] In some embodiments, a lentiviral particle disclosed herein comprises a polynucleotide encoding a CAR comprising an extracellular domain that binds to CD22, 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. In some embodiments, the CAR comprises an extracellular domain, a CD8a hinge domain, a CD8a transmembrane domain, a 4-1BB costimulatory domain, and a CD3zeta signaling domain. In some embodiments, a lentiviral particle disclosed herein comprises a polynucleotide encoding a CAR comprising an extracellular domain, a CD8a hinge, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3zeta signaling domain.

[0084] In some embodiments, a CAR comprises a binding domain for CD22, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB costimulatory domain, and a CD3zeta signaling domain. In some embodiments, a CAR comprises a binding domain for CD22, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3zeta signaling domain. In some embodiments, a CAR comprises a binding domain for CD22, a CD28 hinge, a CD28 transmembrane domain, a CD28 costimulatory domain, and CD3zeta signaling domain.

[0085] In some embodiments, the lentiviral particle comprises a nucleic acid encoding an anti-CD22 CAR comprising a nucleic acid sequence that shares 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% identity to SEQ ID NO: 291.GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCC TGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGC TCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAG TGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGGCCGGACTCTTCCCTTACACTTTTGGCGGAGGGACCAAGGTTGAGATCAAATTCGT GCCCGTGTTCCTGCCCGCCAAACCTACCACCACCCCTGCCCCTAGACCTCCCACCCCAGCCCCAAC AATCGCCAGCCAGCCTCTGTCTCTGCGGCCCGAAGCCTGTAGACCTGCTGCCGGCGGAGCCGTGCA CACCAGAGGCCTGGACTTCGCCTGCGACATCTACATCTGGGCCCCTCTGGCCGGCACCTGTGGCGT GCTGCTGCTGAGCCTGGTGATCACCCTGTACTGCAACCACCGGAACAAACGGGGCAGAAAGAAAC TCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTA GCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGATCCGCC GACGCCCCTGCCTACCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGACGGGA AGAGTACGACGTGCTGGACAAGCGGAGAGGCCGGGACCCCGAGATGGGCGGAAAGCCCAGACGG AAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCG AGATCGGCATGAAGGGCGAGCGGAGGCGCGGCAAGGGCCACGATGGCCTGTACCAGGGCCTGAG CACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCCAGATGA (SEQ ID NO: 291)

[0086] Some examples of anti-CD22 CARs are included as SEQ ID NOs: 292, 435-437 and 456. In some embodiments, the anti-CD22 CAR comprises an amino add sequence that shares 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% identity to SEQ ID NO: 292. In some embodiments, the anti-CD22 CAR comprises an amino acid sequence that shares 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% identity to SEQ ID NO: NO: 456. In some embodiments, the anti-CD22 CAR comprises an amino acid sequence that shares 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% identity to any one of SEQ ID NO: 435-437.MALPVTALLLPLALLLHAARPQVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLE WIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARLPGYESAFDIWGQGTMVT VSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYG ASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQAGLFPYTFGGGTKVEIKFVPVFLPAKPTTTP APRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVn'LYCNHRNK RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLG RREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL STATKDTYDALHMQALPPR (SEQ ID NO: 292)QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVT ISVDKSKNQFSLKLSSVTAADTAVYYCARLPGYESAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSEIV LTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFT LTISRLEPEDFAVYYCQQAGLFPYTFGGGTKVEIKFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEAC RPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVn'LYCNHRNKRGRKKLLYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 456)MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLE WVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVGISSLHGMDVWGQG TTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTrrCRASQSISSWLAWYQQKPGKAPKL LIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYERFPWTFGGGTKVEIKFVPVFLPAKP TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVn'LYCN HRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNE LNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL YQGLSTATKDTYDALHMQALPPRGSGEGRGSLLTCGDVEENPG (SEQ ID NO: 435)MALPVTALLLPLALLLHAARPQVQLQESGPGLVKPSETLSLTCTVSGGSTSSYYWSWIRQPAGKGLEWI GRIYTSGSTNYNPSLKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARDLYRDGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLISDAS SLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQANTYSPTFGGGTKVEIKFVPVFLPAKPTTTPAP RPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNKRG RKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRR EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST ATKDTYDALHMQALPPRGSGEGRGSLLTCGDVEENPG (SEQ ID NO: 436)MALPVTALLLPLALLLHAARPQVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLE WIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARLPGYESAFDIWGQGTMVT VSSGGGGSGGGGSGGGGSErVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYG ASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQAGLFPYTFGGGTKVEIKFVPVFLPAKPTTTP APRPPTPAPnASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNK RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLG RREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL STATKDTYDALHMQALPPRGSGEGRGSLLTCGDVEENPG (SEQ ID NO: 437)

[0087] Some examples of a polynucleotide encoding an anti-CD22 CAR are included as SEQ ID NOs: 454 and 455. In some embodiments, a polynucleotide encoding the anti-CD22 CAR comprises a sequence that shares 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% identity to SEQ ID NO: 454. In some embodiments, a polynucleotide encoding the anti-CD22 CAR comprises a sequence that shares 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% identity to SEQ ID NO: 455.

[0088] Some CARs targeting CD22 and methods of using them are described, for example, in PCT Pub. No. WO2021197483 and U.S. Pub. No. 20230174654, the disclosures of which are incorporated herein by reference in their entirety.CAR Extracellular Domain

[0089] In some embodiments, the nucleic acid transduced into cells using the methods described herein comprises a sequence that encodes a polypeptide, wherein the extracellular domain of the polypeptide binds to an antigen of interest. In some embodiments, the extracellular domain comprises a receptor, or a portion of a receptor, that binds to said antigen. In some embodiments, the extracellular domain comprises, or is, an antibody or an antigen-binding portion thereof. In some embodiments, the extracellular domain comprises, or is, a single-chain Fv domain. The single-chain Fv domain can comprise, for example, a VL linked to VH by a flexible linker, wherein said VL and VH are from an antibody that binds said antigen.

[0090] In some embodiments, the extracellular domain of CAR may contain any polypeptide that binds the desired antigen (e.g. prostate neoantigen or antigen expressed on a tumor of interest). The extracellular domain may comprise a scFv, a portion of an antibody or an alternative scaffold. CARs may also be engineered to bind two or more desired antigens that may be arranged in tandem and separated by linker sequences. For example, one or more domain antibodies, scFvs, llama VHH antibodies or other VH only antibody fragments may be organized in tandem via a linker to provide bispecificity or multispecificity to the CAR.

[0091] In some embodiments, the antigen is expressed on a B-cell malignancy cell, relapsed / refractory CD22-expressing malignancy cell, diffuse large B-cell lymphoma (DLBCL) cell, Burkitt’s type large B-cell lymphoma (B-LBL) cell, follicular lymphoma (FL) cell, chronic lymphocytic leukemia (CLL) cell, acute lymphocytic leukemia (ALL) cell, mantle cell lymphoma (MCL) cell, hematological malignancy cell, colon cancer cell, lung cancer cell, Ever cancer cell, breast cancer cell, renal cancer cell, prostate cancer cell, ovarian cancer cell, skin cancer cell, melanoma cell, bone cancer cell, brain cancer cell, squamous cell carcinoma cell, leukemia cell, myeloma cell, B cell lymphoma cell, kidney cancer cell, uterine cancer cell, adenocarcinoma cell, pancreatic cancer cell, chronic myelogenous leukemia cell, glioblastoma cell, neuroblastoma cell, medulloblastoma cell, or a sarcoma cell. In some embodiments, the antigen is expressed on a non-malignant B cell. In particular embodiments, there the disease to be treated is an autoimmune disease, the antigen may be expressed on non-malignant B cells.

[0092] In some embodiments, the lentiviral particle comprises a polypeptide comprising a hCSF2R (human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor a-chain) signal sequence that shares 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% identity to SEQ ID NO: 92.MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 92)

[0093] In some embodiments, the lentiviral particle comprises a polypeptide comprising a signal peptide sequence such as a CD8a signal peptide sequence. The CD8a signal peptide sequence may share 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% identity to SEQ ID NO: 79. The CD8a signal peptide sequence may be encoded by a nucleic acid sequence that shares 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% identity to SEQ ID NO: 293.MALPVTALLLPLALLLHAARP (SEQ ID NO: 79)ATGGCCCTGCCTGTGACAGCTCTGCTCCTCCCTCTGGCCCTGCTGCTCCATGCCGCCAGACCC (SEQ ID NO: 293)

[0094] In some embodiments, the lentiviral particle comprises a polynucleotide encoding a CAR whose extracellular domain comprises a aCD22 scFv (CD22 VL linked to a CD22 VH) that shares 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% identity to SEQ ID NO: 304. Some additional scFv examples are included as SEQ ID NOs: 425-428. In some embodiments, the lentiviral particle comprises a polynucleotide encoding a CAR whose extracellular domain comprises a aCD22 scFv (CD22 VL linked to a CD22 VH) that shares 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% identity to any one of SEQ ID NO: 425-428.QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVT ISVDKSKNQFSLKLSSVTAADTAVYYCARLPGYESAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSEIV LTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFT LTISRLEPEDFAVYYCQQAGLFPYTFGGGTKVEIK (SEQ ID NO: 304)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKVGISSLHGMDVWGQGTTVTVSSGGGGSGGGGSGGGGS DIQMTQSPSTLSASVGDRVTnCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEF TLTISSLQPDDFATYYCQQYERFPWTFGGGTKVEIK (SEQ ID NO: 425)QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPAGKGLEWIGRIYTSGSTNYNPSLKSRVTMS VDTSKNQFSLKLSSVTAADTAVYYCARDLYRDGMDVWGQGITVTVSSGGGGSGGGGSGGGGSDIQM TQSPSTLSASVGDRVTrrCRASQSISSWLAWYQQKPGKAPKLLISDASSLESGVPSRFSGSGSGTEFTLTI SSLQPDDFATYYCQQANTYSPTFGGGTKVEIK (SEQ ID NO: 426)QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVT ISVDKSKNQFSLKLSSVTAADTAVYYCARLPGYESAFDWGQGTMVTVSSGGGGSGGGGSGGGGSEIV LTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFT LUSRLEPEDFAVYYCQQAGLFPYTFGGGTKVEIK (SEQ ID NO: 427)QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSV KSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVSSGGGGSGGGGSG GGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGR GSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIK (SEQ ID NO: 428)

[0095] Example CDR sequences for the scFvs of SEQ ID NOs: 425-428 are included among SEQ ID NOs: 401-418. Example CDR sequences for the scFv of SEQ ID NO: 304 are included among SEQ ID NOs: 438-443. Any combination of these CDRs may be used.CDRH1 SSNWWS (SEQ ID NO: 438)CDRH2 EIYHSGSTNYNPSLKS (SEQ ID NO: 439)CDRH3 LPGYESAFDI (SEQ ID NO: 440)CDRL1 RASQSVSSSYLA (SEQ ID NO: 441)CDRL2 GASSRAT (SEQ ID NO: 442)CDRL3 QQAGLFPYT (SEQ ID NO: 443)

[0096] In some embodiments, the lentiviral particle comprises a nucleic acid encoding the extracellular domain of a CAR comprising a aCD22 scFv that shares 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% identity to SEQ ID NO: 305. SEQ ID NO: 305 encodes SEQ ID NO: 304.CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGGGACCCTGTCCCTCACCTG CGCTGTCTCTGGTGGCTCCATCAGCAGTAGTAACTGGTGGAGTTGGGTCCGCCAGCCCCCAGGGAA GGGGCTGGAGTGGATTGGGGAAATCTATCATAGTGGGAGCACCAACTACAACCCGTCCCTCAAGA GTCGAGTCACCATATCAGTAGACAAGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCG CCGCGGACACGGCGGTGTACTACTGCGCCAGACTTCCTGGATACGAGTCAGCTTTCGACATATGGG GTCAGGGTACAATGGTCACCGTCAGCTCAGGTGGCGGGGGCAGCGGCGGAGGCGGATCCGGAGG CGGAGGGAGTGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAG CCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAAC CTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGT TCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTG CAGTGTATTACTGTCAGCAGGCCGGACTCTTCCCTTACACTTTTGGCGGAGGGACCAAGGTTGAGA TCAAA (SEQ ID NO: 305)

[0097] In some embodiments, the CAR comprises light chain complementarity-determining regions (CDRs) selected from any of the following groups: LCDR1 having the sequence of SEQ ID NO: 413, LCDR2 having the sequence set forth in SEQ ID NO: 414 and LCDR3 having the sequence set forth in SEQ ID NO: 415, LCDR1 having the sequence set forth in SEQ ID NO: 401, LCDR2 having the sequence set forth in SEQ ID NO: 402 and LCDR3 having the sequence set forth in SEQ ID NO: 403, or LCDR1 having the sequence set forth in SEQ ID NO: 407, LCDR2 having the sequence set forth in SEQ ID NO: 408 and LCDR3 having the sequence set forth in SEQ ID NO: 409, or LCDR1 having the sequence set forth in SEQ ID NO: 441, LCDR2 having the sequence set forth in SEQ ID NO: 442 and LCDR3 having the sequence set forth in SEQ ID NO : 443 ; and wherein the CAR comprises heavy chain CDRs selected from any of the following groups: HCDR1 having the sequence set forth in SEQ ID NO: 416, HCDR2 having the sequence set forth in SEQ ID NO: 417 and HCDR3 having the sequence set forth in SEQ ID NO: 418; HCDR1 having the sequence set forth in SEQ ID NO: 404, HCDR2 having the sequence set forth in SEQ ID NO: 405 and HCDR3 having the sequence set forth in SEQ ID NO: 406; HCDR1 having the sequence set forth in SEQ ID NO: 410, HCDR2 having the sequence set forth in SEQ ID NO: 411 and HCDR3 having the sequence set forth in SEQ ID NO: 412, or HCDR1 having the sequence set forth in SEQ ID NO: 438, HCDR2 having the sequence set forth in SEQ ID NO: 439 and HCDR3 having the sequence set forth in SEQ ID NO: 440. In some embodiments, a CDR comprises 1 or 2 substitutions, deletions, or additions in relation to a CDR sequence herein. These CDR sequences are examples, and other CDR sequences may be predicted depending on the methodology used.

[0098] Antibody engineering methods may include precise identification of residues that have an impact on interaction or affinity of an antibody to a target antigen. Six CDRs in an antibody variable domain (e.g., three from the light chain and three from the heavy chain) may fold up together in 3- dimensional space to form an antibody binding site which docks onto a target antigen. The position and length of the CDRs can be defined by Kabat, E. et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1983, 1987. The position and length of the CDRs can be defined using the Chothia numbering scheme, Martin numbering scheme, Gelfnad number scheme, IMGT numbering scheme or Honnegar’s numbering scheme, e.g. as described in Dondelinger, M et al. Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface / Residue Definition, Front Immunol., 2018. A part of a variable region not contained in the CDRs may be referred to as a framework region, which may form a three-dimensional environment for the CDRs. A CDR sequence or framework region provided herein may be used in an embodiment that uses a CAR or binding fragment.

[0099] n some embodiments, the lentiviral particle disclosed herein comprises a nucleic acid encoding a CAR comprising a CD8 hinge domain comprising an amino acid sequence that shares 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% identity to SEQ ID NO: 192.

[0100] In some embodiments, the CAR comprises a VL comprising a polypeptide 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: 299, 419,421, or 423. In some embodiments, the CAR comprises a VL comprising a polypeptide 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: 299. In some embodiments, the CAR comprises the VL sequence of SEQ ID NO: 299.EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTD FTLTISRLEPEDFAVYYCQQAGLFPYTFGGGTKVEIK (SEQ ID NO: 299)DIQMTQSPSTLSASVGDRVTrrCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTE FrLTISSLQPDDFATYYCQQYERFPWTFGGGTKVElK (SEQ ID NO: 419)DIQMTQSPSTLSASVGDRVTrrCRASQSISSWLAWYQQKPGKAPKLLISDASSLESGVPSRFSGSGSGTEF TLTISSLQPDDFATYYCQQANTYSPTFGGGTKVEIK (SEQ ID NO: 421)EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTD FrLTISRLEPEDFAVYYCQQAGLFPYTFGGGTKVEIK (SEQ ID NO: 423)

[0101] In some embodiments, the VL is encoded by a polynucleotide 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: 298.GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCC TGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGC TCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAG TGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTA CTGTCAGCAGGCCGGACTCTTCCCTTACACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 298)

[0102] In some embodiments, the CAR comprises a VH comprising a polypeptide 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: 295, 420,422, or 424. In some embodiments, the CAR comprises a VH comprising a polypeptide 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: 295. In some embodiments, the CAR comprises the VH sequence of SEQ ID NO: 295.QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVT ISVDKSKNQFSLKLSSVTAADTAVYYCARLPGYESAFDIWGQGTMVTVSS (SEQ ID NO: 295)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRF T1SRDNSKNTLYLQMNSLRAEDIAVYYCAKVGISSLHGMDVWGQGTTVTVSS (SEQ ID NO: 420)QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPAGKGLEWIGRIYTSGSTNYNPSLKSRVTMS VDTSKNQFSLKLSSVTAADTAVYYCARDLYRDGMDVWGQGTTVTVSS (SEQ ID NO: 422)QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVT ISVDKSKNQFSLKLSSVTAADTAVYYCARLPGYESAFDIWGQGTMVTVSS (SEQ ID NO: 424)

[0103] In some embodiments, the VH is encoded by a polynucleotide 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: 294.CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGGGACCCTGTCCCTCACCTG CGCTGTCTCTGGTGGCTCCATCAGCAGTAGTAACTGGTGGAGTTGGGTCCGCCAGCCCCCAGGGAA GGGGCTGGAGTGGATTGGGGAAATCTATCATAGTGGGAGCACCAACTACAACCCGTCCCTCAAGA GTCGAGTCACCATATCAGTAGACAAGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCG CCGCGGACACGGCGGTGTACTACTGCGCCAGACTTCCTGGATACGAGTCAGCTTTCGACATATGGG GTCAGGGTACAATGGTCACCGTCAGCTCA (SEQ ID NO: 294)

[0104] In some embodiments, the VL is linked to the VH. The linker may include a G4S linker. The G4S linker may be encoded by a nucleic acid sequence that shares 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% identity to SEQ ID NO: 296. The G4S linker may include an amino acid sequence that shares 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% identity to SEQ ID NO: 297.GGTGGCGGGGGCAGCGGCGGAGGCGGATCCGGAGGCGGAGGGAGT (SEQ ID NO: 296) GGGGSGGGGSGGGGS (SEQ ID NO: 297)CAR Intracellular Domain

[0105] In some embodiments, the intracellular domain of the CAR is or comprises an intracellular domain or motif of a protein that is expressed on the surface of T lymphocytes and triggers activation and / or proliferation of said T lymphocytes. In some embodiments, such a domain or motif is able to transmit a signal for activation of a T lymphocyte in response to antigen binding to the CAR's extracellular portion. In some embodiments, this domain or motif comprises, or is, an ITAM (immunoreceptor tyrosine-based activation motif). ITAM-containing polypeptides suitable for CARs include, for example, the zeta CD3 chain (CD3Q or ITAM-containing portions thereof. In some embodiments, the intracellular domain is a CD3 intracellular signaling domain. In some embodiments, the intracellular domain is from a lymphocyte receptor chain, a TCR / CD3 complex protein, an Fc receptor subunit or an IL-2 receptor subunit. In some embodiments, the intracellular signaling domain of CAR may be the signaling domains of for example CD3 , CD3s, CD22, CD79a, CD66d or CD39. “Intracellular signaling domain” refers to the part of a CAR polypeptide that participates in transducing the message of effective CAR binding to a target antigen into the interior of the immune effector cell to elicit effector cell function, e.g., activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors to the CAR-bound target cell, or other cellular responses elicited following antigen binding to the extracellular CAR domain.

[0106] In some embodiments, the intracellular domain of the CAR is the zeta CD3 chain (CD3zeta).

[0107] In some embodiments, the CAR comprises a CD3zeta 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: 82 or 203. In some embodiments, the lentiviral particle comprises a polypeptide comprising a CAR whose intracellular domain comprises a CD3zeta domain that shares 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 least98%, at least 99%, or 100% identity to SEQ ID NO: 82. In some embodiments, the lentiviral particle disclosed herein comprises a polynucleotide that encodes a CAR comprising a CD3zeta signaling domain comprising a nucleic acid sequence that shares 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% identity to SEQ ID NO: 202. In some embodiments, the lentiviral particle disclosed herein encodes a CAR comprising a CD3zeta signaling domain comprising an amino acid sequence that shares 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% identity to SEQ ID NO: 203.RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 82)

[0108] In some embodiments, the lentiviral particle comprises a nucleic acid encoding the intracellular domain of a CAR comprising a CD3zeta domain that shares 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% identity to SEQ ID NO: 83.CGCGTGAAGTTCAGCCGGTCCGCCGATGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTATAA CGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGGGACCCA GAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTACAATGAGCTGCAGAAGG ACAAGATGGCCGAGGCCTATTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACA CGATGGCCTGTACCAGGGCCTGAGCACCGCCACAAAGGACACATATGATGCCCTGCACATGCAGG CCCTGCCACCTAGG (SEQ ID NO: 83)

[0109] In some embodiments, the CD3zeta is encoded by a polynucleotide 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: 202 or 303. In some embodiments, the lentiviral particle comprises a nucleic acid encoding a CD3zeta domain sequence that shares 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% identity to SEQ ID NO: 303.AGAGTGAAGTTCAGCAGATCCGCCGACGCCCCTGCCTACCAGCAGGGACAGAACCAGCTGTACAA CGAGCTGAACCTGGGCAGACGGGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCCGGGACCCC GAGATGGGCGGAAAGCCCAGACGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAG ACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGCGGCAAGGGCCA CGATGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAGG CCCTGCCCCCCAGA (SEQ ID NO: 303)

[0110] In some embodiments, the CAR additionally comprises one or more co-stimulatory domains or motifs, e.g., as part of the intracellular domain of the polypeptide.

[0111] Co-stimulatory molecules may include cell surface molecules other than antigen receptors or Fc receptors that provide a second signal useful for efficient activation and function of T lymphocytes upon binding to antigen. The one or more co-stimulatory domains or motifs can, for example, be, or comprise, one or more of a co-stimulatory CD27 polypeptide sequence, a co-stimulatory CD28 polypeptide sequence, a co-stimulatory 0X40 (CD134) polypeptide sequence, a co-stimulatory 4-1BB(CD137) polypeptide sequence, or a co-stimulatory inducible T-cell costimulatory (ICOS) polypeptide sequence, or other costimulatory domain or motif, or any combination thereof. In some embodiments, the one or more co-stimulatory domains are selected from the group consisting of intracellular domains of 4-1BB, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70.

[0112] In some embodiments, the co-stimulatory domain is an intracellular domain of 4-1BB, CD28, or 0X40. Illustrative CAR constructs comprising a CD28 signaling domain are disclosed in US Patent No. 7,446,190, incorporated by reference. Illustrative CAR constructs comprising a 4-1BB signaling domain are disclosed in US Patent No. 9,856,322 and US Patent No. 8,399,964, each incorporated by reference.

[0113] In some embodiments, the lentiviral particle comprises a polynucleotide or a polypeptide that encodes a CAR comprising a CD8a linker operatively linked to a CD8a transmembrane domain operatively linked to a 4-1BB co-stimulatory domain operatively linked to a CD3zeta signaling domain.

[0114] In some embodiments, the lentiviral particle comprises a polypeptide comprising a CAR whose intracellular domain comprises a co-stimulatory 4-1BB polypeptide sequence that shares 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% identity to SEQ ID NO: 84. KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 84)

[0115] In some embodiments, the CAR comprises a 4-1BB 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: 84 or 201. In some embodiments, the lentiviral particle comprises a nucleic acid encoding the intracellular domain of a CAR comprising a co-stimulatory 4-1BB sequence that shares 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% identity to SEQ ID NO: 85. In some embodiments, the lentiviral particle disclosed herein comprises a polynucleotide that encodes a CAR comprising a 4-1BB co-stimulatory domain comprising a nucleic acid sequence that shares 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% identity to SEQ ID NO: 200. In some embodiments, the lentiviral particle disclosed herein encodes a CAR comprising a 4-1BB co-stimulatory domain comprising an amino acid sequence that shares 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% identity to SEQ ID NO: 201.CGCGTGAAGTTCAGCCGGTCCGCCGATGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTATAA CGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGGGACCCA GAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTACAATGAGCTGCAGAAGG ACAAGATGGCCGAGGCCTATTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTGAGCACCGCCACAAAGGACACATATGATGCCCTGCACATGCAGG CCCTGCCACCTAGG (SEQ ID NO: 85)

[0116] In some embodiments, the 4-1BB is encoded by a polynucleotide 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: 200 or 302. In some embodiments, the lentiviral particle comprises a nucleic add encoding a 4-1BB sequence that shares 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% identity with SEQ ID NO: 302.AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTAC TCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO: 302)

[0117] In some embodiments, the lentiviral particle comprises a polypeptide comprising a CAR whose intracellular domain comprises a CD8 linker operatively linked to a CD8 transmembrane domain operatively linked to a co-stimulatory 4-1BB polypeptide operatively linked to a CD3zeta domain.

[0118] In some embodiments, the intracellular domain can be further modified to encode a detectable, for example, a fluorescent, protein (e.g. , green fluorescent protein) or any variants thereof.CAR Transmembrane Region

[0119] The transmembrane region can be any transmembrane region that can be incorporated into a functional CAR, e.g., a transmembrane region from a CD28, CD4, or a CD8 molecule. As used herein, a transmembrane domain of a protein (e.g. a CAR, a synthetic cytokine receptor, and / or a surface protein on a particle) may be referred to as a TM domain, a TM region, or simply TM.

[0120] In some embodiments, the transmembrane domain of CAR may be the transmembrane domain of CD8, an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDlla, CD18), ICOS (CD278), 4-1 BB (CD137), 4-1 BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFT), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB- A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, NKG2D “in reverse orientation”, and / or NKG2C. In some embodiments, the transmembrane domain of the CAR may be the transmembrane domain of CD28. In some embodiments, the transmembrane domain of a CAR may be the transmembrane domain of CD8, for example, CD8a.CAR Linker Region

[0121] The optional linker or hinge of CAR positioned between the extracellular domain and the transmembrane domain may be a polypeptide of about 2 to over 100 amino acids in length. The linker can include or be composed of flexible residues such as glycine and serine so that the adjacent protein domains are free to move relative to one another. Longer tinkers may be used, e.g., when it is desirable to ensure that two adjacent domains do not sterically interfere with one another. Longer tinkers may also be advantageous when the target antigen is closer to the cell surface.

[0122] In some embodiments, the tinker is from a hinge region or portion of the hinge region of any immunoglobulin or other transmembrane protein. For example, the hinge region may be from IgGl, IgG2, IgG3, IgG4, PD1, CD8, or CD28, or a portion thereof. In some embodiments, the tinker is from a portion of an immunoglobulin, for example IgG4. In some embodiments, the tinker is a portion of an immunoglobulin, for example IgGl. In some embodiments, the tinker is a portion of the extracellular domain of CD28. In other embodiments, the tinker is a portion of the extracellular domain of CD8. In other embodiments, the tinker is a portion of the extracellular domain of PD1.

[0123] In some embodiments, the CAR comprises a CD8 hinge or transmembrane domain sequence. In some embodiments, the CAR comprises a CD8 hinge and transmembrane domain sequence. In some embodiments, the CAR comprises a CD8 hinge and transmembrane domain 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: 299. In some embodiments, the CAR comprises a CD8 hinge 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 a hinge sequence of SEQ ID NO: 299. In some embodiments, the CAR comprises a CD8 transmembrane domain 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 a transmembrane domain sequence of SEQ ID NO: 299.EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTD FTLTISRLEPEDFAVYYCQQAGLFPYTFGGGTKVEIK (SEQ ID NO:299)

[0124] In some embodiments, the CD8 hinge and transmembrane domain are encoded by a polynucleotide 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: 298. In some embodiments, the CD8 hinge sequence is encoded by a polynucleotide 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 a hinge sequence of SEQ ID NO: 298. In some embodiments, the CD8 transmembrane domain sequence is encoded by a polynucleotide 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 a transmembrane domain sequence of SEQ ID NO: 298.GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCC TGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGC TCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAG TGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTA CTGTCAGCAGGCCGGACTCTTCCCTTACACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO:298)

[0125] In some embodiments, the lentiviral particle disclosed herein comprises a nucleic acid encoding a CAR comprising a CD8 hinge domain comprising a nucleic add sequence that shares 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% identity to SEQ ID NO: 196. In some embodiments, the lentiviral particle disclosed herein comprises a nucleic acid encoding a CAR comprising a CD8 hinge domain comprising an amino acid sequence that shares 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% identity to SEQ ID NO: 197.Adhesion Molecules

[0126] Disclosed herein, in some embodiments, are particles comprising adhesion molecules displayed on their surface. The adhesion molecule may be included as part of a fusion molecule. The adhesion molecule may be included as part of a particle (e.g. on a particle surface).

[0127] As used herein, the term “adhesion molecule” refers, in a broad sense, to 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 in turn contributes to adhesion of a particle to target cells. Adhesion molecules from natural sources may be molecules expressed, natively, on antigen-presenting cells and adapted for use here on particles. Both naturally occurring adhesion molecules, and their variants, and artificial adhesion molecules, such as antibodies, or fragments thereof, are contemplated. An adhesion molecule, as the term is used herein, specifically binds 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. The term adhesion molecule includes but is not limited to CD58, a CD58 extracellular portion, and functional fragments of CD58. As described above, the term “functional fragment” is used herein to describe a fragment of a polypeptide, or other molecule, that retains the desired function of the polypeptide. For example, a functional fragment of CD58 is a fragment of CD58 that specifically binds CD2. The adhesion molecule may be a protein, termed herein an “adhesion protein.”

[0128] In some embodiments, the costimulatory and / or adhesion molecule comprises an amino acid sequence 100% identical to a sequence in Table 1 or Table 2. In some embodiments, the costimulatory and / or adhesion molecule shares 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% identity to a sequence in Table 1 or Table 2. In some embodiments, the costimulatory and / or adhesion molecule shares less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, lessthan 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or less than 100% identity to a sequence in Table 1 or Table 2.

[0129] In some embodiments, the costimulatory and / or adhesion molecule comprises an amino acid sequence 100% identical to SEQ ID NO: 444. In some embodiments, the costimulatory and / or adhesion molecule shares 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% identity to SEQ ID NO:444. In some embodiments, the costimulatory and / or adhesion molecule shares less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or less than 100% identity to SEQ ID NO: 444.

[0130] In some embodiments, the costimulatory and / or adhesion molecule comprises an amino acid sequence 100% identical to SEQ ID NO: 445. In some embodiments, the costimulatory and / or adhesion molecule shares 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% identity to SEQ ID NO:445. In some embodiments, the costimulatory and / or adhesion molecule shares less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or less than 100% identity to SEQ ID NO: 445.

[0131] Polypeptide sequences of illustrative adhesion molecules are provided in Table 1, with the “start” and “end” positions of the extracellular portion of each. In each case, the adhesion molecule may comprise a polypeptide 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 any sequence in Table 1, or functional fragments thereof. Functional fragments may be or include any 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, or 600 (or any range thereof) amino acid portion that retains binding affinity to its cognate molecule, when measured using affinity assays such as biolayer interferometry or other assays that may be known in the art.Table 1Table 2

[0132] Some embodiments of a fusion molecule may comprise a binding domain from CD58 and a binding domain from CD80. In such an example, the CD80 domain may be the most membrane proximal and therefore the fusion molecule would comprise both the binding and transmembrane domains from CD80 as they appear in the endogenous protein.

[0133] In some embodiments, the adhesion molecule is CD58. CD58 is also known as lymphocyte function-associated antigen 3 (LFA-3). CD58 binds to CD2 (LFA-2) on T cells. The extracellular portion of CD58 is residues 29-215 of SEQ ID NO: 1 (SEQ ID NO: 10):FSQQIYGVVYGNVTFHVPSNVPLKEVLWKKQKDKVAELENSEFRAFSSFKNRVYLDTVSGSLTIYNLTS SDEDEYEMESPNITDTMKFFLYVLESLPSPTLTCALTNGSIEVQCMIPEHYNSHRGLIMYSWDCPMEQC KRNSTSIYFKMENDLPQKIQCTLSNPLFNTTSSIILTTCIPSSGHSRHR (SEQ ID NO: 10)

[0134] In some embodiments, the polypeptide sequence of CD58 shares at least 50%, 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% or at least 99% identity to SEQ ID NO: 248:FSQQIYGVVYGNVTFHVPSNVPLKEVLWKKQKDKVAELENSEFRAFSSFKNRVYLDTVSGSLTIYNLTS SDEDEYEMESPNITDTMKFFLYVLESL (SEQ ID NO: 248)

[0135] A crystal structure of CD58 is described in Ikemizu et al. PNAS USA 96(8):4289-94 (1999). The extracellular portion of CD58 has a ligand-binding domain and a second extracellular domain. In embodiments, the ligand-binding domain may be used as the functional fragment of CD58 — i.e., without the second extracellular domain.

[0136] In some embodiments, the adhesion molecule (or the fusion protein) comprises the polypeptide sequence of SEQ ID NO: 1 or 10, or a 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%, atleast 98% or at least 99% identity to SEQ ID NO: 1 or 10. In some embodiments, the adhesion molecule (or the fusion protein) comprises a sequence having less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or less than 100% identity to SEQ ID NO: 1 or 10. The adhesion molecule may encoded by a polynucleotide (e.g. a DNA or RNA polynucleotide).

[0137] The adhesion molecule may encoded by the polynucleotide sequence of CD58, SEQ ID NO: 11, or by a subsequence encoding the extracellular portion or a functional fragment. SEQ ID NO: 11 (5' to 3'): ATGGTTGCTGGGAGCGACGCGGGGCGGGCCCTGGGGGTCCTCAGCGTGGTCTGCCTGCTGCACTG CTTTGGTTTCATCAGCTGTTTTTCCCAACAAATATATGGTGTTGTGTATGGGAATGTAACTTTCCAT GTACCAAGCAATGTGCCTTTAAAAGAGGTCCTATGGAAAAAACAAAAGGATAAAGTTGCAGAACT GGAAAATTCTGAGTTCAGAGCTTTCTCATCTTTTAAAAATAGGGTTTATTTAGACACTGTGTCAGGT AGCCTCACTATCTACAACTTAACATCATCAGATGAAGATGAGTATGAAATGGAATCGCCAAATATT ACTGATACCATGAAGTTCTTTCTTTATGTGCTTGAGTCTCTTCCATCTCCCACACTAACTTGTGCATT GACTAATGGAAGCATTGAAGTCCAATGCATGATACCAGAGCATTACAACAGCCATCGAGGACTTA TAATGTACTCATGGGATTGTCCTATGGAGCAATGTAAACGTAACTCAACCAGTATATATTTTAAGA TGGAAAATGATCTTCCACAAAAAATACAGTGTACTCTTAGCAATCCATTATTTAATACAACATCAT CAATCATTTTGACAACCTGTATCCCAAGCAGCGGTCATTCAAGACACAGATATGCACTTATACCCA TACCATTAGCAGTAATTACAACATGTATTGTGCTGTATATGAATGGTATTCTGAAATGTGACAGAA AACCAGACAGAACCAACTCCAAT.

[0138] The polynucleotide sequence may be varied by codon-optimization or other methods to generate polynucleotide sequences having at least 50%, 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% or at least 99% identity to SEQ ID NO: 11, or a suitable subsequence, which may be used to express the adhesion molecule.

[0139] It will be appreciated that further variants of CD58 may be used. For example, homologs of CD58 from other species (mice, ape, horse, etc.) may be identified and tested for use in transducing human, or non-human, target cells. It is expected that at least some non-human homologs will retain adhesion molecule function when used with human target cells.

[0140] Further adhesion molecules useful in the practice of the present invention may include any molecule that specifically binds CD2, LFA-1, or DNAM-1. For example, the adhesion molecule may be a molecule that comprises an antibody, or antigen-binding fragment thereof, specific to CD2, LFA-1, or DNAM-1.

[0141] In some embodiments, the adhesion molecule binds to CD2. CD2 is also known as Til, LFA-2, and the erythrocyte rosette receptor. In its native state, CD2 is a surface protein expressed on T lymphocytes and NK cells. CD2 is a natural ligand for CD58. In addition to performing adhesion functions, engagement of CD2 by CD58 provides a costimulatory signal that may enhance activation and effector functions. In some embodiments, the particle comprises an adhesion molecule that binds to CD2, which may be CD58 or a fragment thereof. In some embodiments, the lentiviral particle comprises an antibody, single domain antibody, antibody fragment, and / or nanobody specific for CD2.

[0142] The foregoing description of CD58 and its derivatives as the adhesion molecule and CD2 as the cognate molecule may be extrapolated to the other adhesion molecules described herein. The adhesion molecule (or the fusion protein) may comprise any polypeptide sequence of in Table 1, to an extracellular portion thereof, or to a functional fragment thereof, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to a sequence in Table 1, to an extracellular portion thereof, or to a functional fragment thereof.Costimulatory Molecules

[0143] Disclosed herein, in some embodiments, are particles comprising costimulatory molecules. The costimulatory molecule may be included as part of a fusion molecule. The costimulatory molecule may be included as part of a particle (e.g., displayed on a particle surface).

[0144] The fusion molecule displayed on the particle may include a costimulatory molecule. However, in some embodiments, the fusion molecule does not include a costimulatory molecule. The particle may display a costimulatory molecule as a separate molecule on the surface of the particle, or the particle may lack any costimulatory molecule. The costimulatory molecule may be a protein, termed herein a “costimulatory protein.”

[0145] As used herein, the term “costimulatory molecule” refers to a molecule capable of providing a costimulatory signal to target cells, other than an adhesion molecule as defined herein. In a non-limiting example, interactions between CD58 and CD2 may also provide an activatory or costimulatory signal. In T cell biology, the binding of the T cell receptor by an antigen can provide the primary stimulatory signal to the cell. So-called costimulatory signals are provided by accessory molecules. An example costimulatory signal is the signal provided by binding of CD28 on T cells by a ligand. Some examples of ligands of CD28 include CD80 and CD86.

[0146] Illustrative costimulatory molecules include, but are not limited to, CD80 or CD86. Each of the foregoing may be employed as a costimulatory molecules as a full-length protein, an extracellular domain, or functional fragment.

[0147] Polypeptide sequences of illustrative costimulatory molecules are provided in Table 3, with the “start” and “end” positions of the extracellular portion of each. In each case, the costimulatory molecule may comprise a polypeptide 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 any sequence in Table 3, or functional fragments thereof. In some embodiments, the costimulatory molecule comprises a polypeptide having less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or 100% sequence identity to any sequence in Table 3, or a functional fragment thereof. Functional fragments may be or include any 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, or 600 amino acid portion that retains binding affinity to itscognate molecule, when measured using affinity assays such as biolayer interferometry or other assays known in the art.Table 3

[0148] In some embodiments, the costimulatory molecule is or includes CD80. In some embodiments, the costimulatory molecule is or includes a molecule that binds CD28. CD80 binds to CD28. The extracellular portion of CD80 includes residues 35-230 of SEQ ID NO: 12, which includes an Ig-like V-type domain (SEQ ID NO: 25) and an Ig-like C2-type domain (SEQ ID NO: 26), either or both of which may be included to form the costimulatory molecule.VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIV ILALRPSDEGTYECVVLKYEKDAFKREHLAEVT (SEQ ID NO: 25)PSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMC LIKYGHLRVNQTFN (SEQ ID NO: 26)

[0149] The crystal structure of CD80 (also known as B7-1) is described in Ikemizu et al. Immunity 12:51-60 (2000). The extracellular portion of CD80 has two domains, described above. In embodiments, one or both of the domains may be used as the functional fragment of CD80.

[0150] In some embodiments, the costimulatory molecule is or includes CD86. CD86 binds to CD28. The extracellular portion of CD86 includes residues 33-225 of SEQ ID NO: 13, which includes an Ig- like V-type domain (SEQ ID NO: 27) and an Ig-like C2-type domain (SEQ ID NO: 28), either or both of which may be included to form the costimulatory molecule.NETADLPCQFANSQNQSLSELWFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHN LQIKDKGLYQCIIHHKKPTGMIRIHQMNSELS (SEQ ID NO: 27)NVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGVMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCIL ETDKT (SEQ ID NO: 28)

[0151] The crystal structure of CD86 (also known as B7-1) is described in Schwartz et al. Nature 410: 604-608 (2001). The extracellular portion of CD86 has two domains, described above. In embodiments, one or both of the domains may be used as the functional fragment of CD86.

[0152] It will be appreciated that further variants of CD80 or CD86 may be used. For example, homologs of CD80 or CD86 from other species (mice, ape, horse, etc.) may be identified and tested for use in transducing human, or non-human, target cells. It is expected that at least some non-human homologs will retain costimulatory molecule function when used with human target cells.

[0153] In some embodiments, the costimulatory molecule (or the fusion protein) comprises the polypeptide sequence of one or more of SEQ ID NO: 12-13 and 25-28, or a 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% or at least 99% identity to one or more of SEQ ID NO: 12-13 and 25-28.

[0154] In some embodiments, the costimulatory molecule CD80 comprises the polypeptide sequence of SEQ ID NO: 250, or a 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% identity to SEQ ID NO: 250.

[0155] In some embodiments, the costimulatory molecule (or the fusion protein) comprises a polypeptide sequence having less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or less than 100% identity to one or more of SEQ ID NO: 12-13 and 25-28. The costimulatory molecule may encoded by a polynucleotide (e.g. a DNA or RNA polynucleotide). The costimulatory molecule may encoded by the polynucleotide sequence of CD80 (SEQ ID NO: 29) or CD86 (SEQ ID NO: 30), or by a subsequence encoding the extracellular portion or a functional fragment.ATGGGCCACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCATACCTCAATTTCTTTCAGCTC TTGGTGCTGGCTGGTCTTTCTCACTTCTGTTCAGGTGTTATCCACGTGACCAAGGAAGTGAAAGAA GTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGG CAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAA GAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATCCTGGCTCTGCGCCCATCTGAC GAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGGGAACACCTGGC TGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCAAC TTCTAATATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTCCTGGTTG GAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTA TGCTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTA TGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACAACCAAGCAAGAGCATTTTCCTGATA ACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGAC CTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTA CGCCCTGTA (SEQ ID NO: 29)ATGGATCCCCAGTGCACTATGGGACTGAGTAACATTCTCTTTGTGATGGCCTTCCTGCTCTCTGGTG CTGCTCCTCTGAAGATTCAAGCTTATTTCAATGAGACTGCAGACCTGCCATGCCAATTTGCAAACT CTCAAAACCAAAGCCTGAGTGAGCTAGTAGTATTTTGGCAGGACCAGGAAAACTTGGTTCTGAATGAGGTATACTTAGGCAAAGAGAAATTTGACAGTGTTCATTCCAAGTATATGGGCCGCACAAGTTTT GATTCGGACAGTTGGACCCTGAGACTTCACAATCTTCAGATCAAGGACAAGGGCTTGTATCAATGT ATCATCCATCACAAAAAGCCCACAGGAATGATTCGCATCCACCAGATGAACTCTGAACTGTCAGT GCTTGCTAACTTCAGTCAACCTGAAATAGTACCAATTTCTAATATAACAGAAAATGTGTACATAAA TTTGACCTGCTCATCTATACACGGTTACCCAGAACCTAAGAAGATGAGTGTTTTGCTAAGAACCAA GAACTCAACTATCGAGTATGATGGTGTTATGCAGAAATCTCAAGATAATGTCACAGAACTGTACG ACGTTTCCATCAGCTTGTCTGTTTCATTCCCTGATGTTACGAGCAATATGACCATCTTCTGTATTCT GGAAACTGACAAGACGCGGCTTTTATCTTCACCTTTCTCTATAGAGCTTGAGGACCCTCAGCCTCC CCCAGACCACATTCCTTGGATTACAGCTGTACTTCCAACAGTTATTATATGTGTGATGGTTTTCTGT CTAATTCTATGGAAATGGAAGAAGAAGAAGCGGCCTCGCAACTCTTATAAATGTGGAACCAACAC AATGGAGAGGGAAGAGAGTGAACAGACCAAGAAAAGAGAAAAAATCCATATACCTGAAAGGTCT GATGAAGCCCAGCGTGTTTTTAAAAGTTCGAAGACATCTTCATGCGACAAAAGTGATACATGTTTT (SEQ ID NO: 30)

[0156] The polynucleotide sequence may be varied by codon-optimization or other methods to generate polynucleotide sequences having at least 50%, 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% or at least 99% identity to SEQ ID NO: 29 or 30, or a suitable subsequence, which may be used to express the costimulatory molecule.

[0157] The foregoing description of CD80, CD86, and their derivatives as the costimulatory molecule and CD28 as the cognate molecule may be extrapolated to the other costimulatory molecules described herein, including but not limited to those listed in Table 3. The costimulatory molecule (or the fusion protein) may comprise any polypeptide sequence in Table 3, or an extracellular portion thereof, or a functional fragment thereof, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to a sequence in Table 3, or an extracellular portion thereof, or a functional fragment thereof.Activation Molecules

[0158] Disclosed herein, in some embodiments, are particles comprising activation molecules. The activation molecule may be included as part of a fusion molecule. The activation molecule may be included as part of a particle (e.g. displayed on a particle surface). An example of an activation molecule may include a TCR-binding molecule. An activation molecule is generally a molecule that is capable of activating an immune cell with a primary immune activation signal.

[0159] The fusion molecule displayed on the particle may include an activation molecule (e.g. a TCR- binding molecule) or other subunit that provides an activation signal to a target cell. However, in some embodiments, the fusion molecule does not include a TCR-binding molecule or other activation molecule. The particle may display a TCR-binding molecule as a separate molecule on the surface of the particle, or the particle may lack any TCR-binding molecule. The TCR-binding molecule may be a protein, termed herein a “TCR-binding protein.” The activation molecule may be or include an activation protein.

[0160] As used herein, the term “TCR-binding molecule” can refer to 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 orNK cell). The structure of the TCR, its components, and function is described in Susac et al. Cell 185(17):3201-3213.el9 (2022). 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, the activation molecule comprises an antibody, single domain antibody, antibody fragment, nanobody, or other binding protein specific for CD3. Illustrative antibodies include OKT3 (also known as Muromonab-CD3), otelixizumab, teplizumab and visilizumab. The complementarity determining regions of OKT3 are as follows:CDRH1: GYTFTRY (SEQ ID NO. 48)CDRH2: NPSRGY (SEQ ID NO. 49)CDRH3: YYDDHYCLDY (SEQ ID NO. 50)CDRL1: SASSSVSYMN (SEQ ID NO. 51)CDRL2: DTSKLAS (SEQ ID NO. 52)CDRL3: QQWSSNPFT (SEQ ID NO. 53)

[0161] The activation molecule (e.g. TCR-binding molecule) may be a single chain variable fragment (scFv) displayed on the particle as finked to a transmembrane region or an anchor. OKT3 in scFv format may be used.

[0162] In some embodiments, the activation molecule (e.g. TCR-binding molecule) is or includes an scFv comprising 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 the anti-CD3 scFv of SEQ ID NO: 31, which includes a variable light (VL) and variable heavy (VH) domain with a 3 x GGGS linker:DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTD YTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTSGGGGSGGGGSGGGGSQVQLVQSGGGVVQP GRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQ MDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSAAAKP (SEQ ID NO: 31)

[0163] In some embodiments, the activation molecule (e.g. TCR-binding molecule) is or includes an scFv comprising 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 the anti-CD3 scFv of SEQ ID NO: 249, which includes a variable light (VL) and variable heavy (VH) domain with a 3 x GGGS linker:DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTD YTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTSGGGGSGGGGSGGGGSQVQLVQSGGGVVQP GRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQ MDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSA (SEQ ID NO: 249)

[0164] The complementarity determining regions of the anti-CD3 scFv of SEQ ID NO: 249 scFv are as follows:CDRH1: RYTMH (SEQ ID NO: 54)CDRH2: YINPSRGYTNYNQKVKD (SEQ ID NO: 55)CDRH3: YYDDHYCLDY (SEQ ID NO: 56)CDRL1: SASSSVSYMN (SEQ ID NO: 57)CDRL2: DTSKLASG (SEQ ID NO: 58)CDRL3: QQWSSNPFT (SEQ ID NO: 59)

[0165] Other activation molecules and / or domains may comprise the binding regions of other proteins commonly found in the supramolecular activation complex (SMAC) between T lymphocytes and antigen presenting cells. For example, CD3, CD2, CD4, CD8, CD28, LFA-1, CD45, CD43, CD40, ICAM-1, CTLA-4, CD80, CD86, MHC, LFA-3, AND CD40L are proteins that may be present within the SMAC. The fusion proteins disclosed herein may comprise portions of these proteins or domains that bind to these proteins. For example, without wishing to be bound by theory, T cells may express one or both of CD4 and / or CD8 and fusion molecules disclosed herein may comprise domains that engage with either or both of CD4 and / or CD8.

[0166] When cells other than T cells are the intended target of the particles comprising a fusion molecule as disclosed herein, other binding domains may be more appropriate. For example, particles targeting NK cells may comprise domains that engage with proteins found on NK cells. In some embodiments, these proteins include CD2, CD16, NKp46, NKp30, and NKG2D. In some such embodiments, fusion proteins intended to target and / or activate NK cells may comprise domains that bind to CD2, CD16, NKp46, NKG2D, etc. Domains that bind to NKG2D may be derived from NKG2D ligands including, but not limited to: MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. In some embodiments, the fusion proteins described herein comprise a CD58 domain, a domain that binds NKG2D, and optionally a third domain which enhances activation of the target NK cell.

[0167] The activation molecule may be encoded by a polynucleotide (e.g. a DNA or RNA polynucleotide).

[0168] In some embodiments, the costimulatory, activation, and / or adhesion molecule is linked to a transmembrane domain. In some embodiments, the transmembrane domain may be the transmembrane domain of CD8, an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDlla, CD18), ICOS (CD278), 4-1 BB (CD137), 4-1 BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFT), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB- A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. As used herein, a transmembrane domain of a protein (e.g. a CAR, a synthetic cytokine receptor, and / or a surface protein on a particle) may be referred to as a TM domain, a TM region, or simply TM.

[0169] In some embodiments, the costimulatory, adhesion, and / or activation molecules are attached to a particle via a moiety that embeds in the particle envelope or membrane - e.g. a glycosylphosphatidylinositol (GPI) anchor. Exemplary anchors include lipid anchors, for example as in prenylated proteins, fatty acylated proteins, and GPI-linked proteins.

[0170] Without wishing to be bound by theory, reducing foreign junctions (i.e., between an adhesion molecule and a transmembrane domain) in the foreign nucleic acid incorporated into a lentiviral particle may reduce the immunogenicity of a subject to a lentiviral particle. Accordingly, in some embodiments, the transmembrane domain of multi-domain fusion polypeptide is derived from the same protein as the membrane proximal domain. For example, the MDF may include a fragment of CD56 that includes both the CD56 extracellular domain and CD56 transmembrane domain, e.g., as a one contiguous polypeptide sequence. In a variation, this CD56 fragment includes a linker or other insertion between the CD56 extracellular domain and the CD56 transmembrane domain. As another example, the MDF may include a CD80 or CD86 fragment that includes the CD80 or CD86 transmembrane domain, respectively, as a contiguous polypeptide sequence, or in a variation, a sequence with a linker or other insertion between said two domains.

[0171] For example, in some embodiments, the costimulatory and transmembrane domains are derived from CD80 and domains appear in series as they would in the endogenous protein (i.e. as a single sequence). In some embodiments wherein the costimulatory and / or adhesion domains are comprised within a fusion molecule, there may be multiple extracellular domains. For example, a fusion molecule as described hereinFusion Molecules

[0172] Disclosed herein, in some embodiments, are fusion molecules. The fusion molecule may include an adhesion molecule, a costimulatory molecule, or an activation molecule. The fusion molecule may include an adhesion molecule. The fusion molecule may include a costimulatory molecule. The fusion molecule may include an activation molecule. The fusion molecule may include an adhesion molecule, a costimulatory molecule, and an activation molecule. The fusion molecule may include an adhesion molecule and an activation molecule. The fusion molecule may include a costimulatory molecule and an activation molecule. The fusion molecule may be or include a fusion protein. The fusion molecule may be included as part of a particle. The fusion molecule may be used in a method described herein.

[0173] In some embodiments, the disclosure provides a fusion molecule comprising a combination of an adhesion molecule, a costimulatory molecule, and an activation molecule (e.g. a TCR-binding molecule), thereof each component linked directly or indirectly to the other components. In some embodiments, the fusion molecule comprises adhesion molecule, a costimulatory molecule, and an activation molecule (e.g. a TCR-binding molecule). In some embodiments, the fusion molecule comprises an adhesion molecule and a costimulatory molecule, but not a TCR-binding molecule. Insome embodiments, the fusion molecule comprises an adhesion molecule and an activation molecule (e.g. a TCR-binding molecule), but not a costimulatory molecule. The fusion molecule may further comprise one or more additional adhesion molecules, costimulatory molecules, or activation molecules (e.g. TCR-binding molecules).

[0174] “Fusion molecule” may refer to a molecule having multiple components linked together, directly or indirectly, covalently or non-covalently. A fusion molecule may be made up of several proteins. When those proteins are linked together into a single molecule by peptide bonds, the fusion molecule may be referred to as a “fusion protein.”

[0175] The fusion molecule may be made using various linkers, including chemical (covalent) bonds (e.g., by click chemistry) or by peptide bonds. When the fusion molecule is a fusion protein, the linker between each component of the fusion protein may be a single peptide bond (i.e., a direct C- to N- peptide bond in a polypeptide chain) or via a polypeptide linker. Illustrative polypeptide linkers may include, but are not limited to, the glycine-serine linkers, such as GGSGGS, GSSGSS, or others.

[0176] In some embodiments, the fusion molecule is or includes a fusion protein. The fusion protein may comprise an adhesion protein, one or more polypeptide linkers, and a costimulatory protein. In some embodiments, the fusion protein comprises an adhesion molecule, a costimulatory molecule, and an activation molecule.

[0177] In some embodiments of the fusion protein, the adhesion molecule is N-terminal to the costimulatory molecule. In some embodiments, the adhesion molecule is N-terminal to the activation molecule. In some embodiments, the adhesion molecule is C-terminal to the costimulatory molecule. In some embodiments, the adhesion molecule is C-terminal to the activation molecule.

[0178] In some embodiments of the fusion protein, the activation molecule is N-terminal to the costimulatory molecule. In some embodiments, the activation molecule is N-terminal to the adhesion molecule. In some embodiments, the activation molecule is C-terminal to the costimulatory molecule. In some embodiments, the activation molecule is C-terminal to the adhesion molecule.

[0179] In some embodiments of the fusion protein, the costimulatory molecule is N-terminal to the activation molecule. In some embodiments, the costimulatory molecule is N-terminal to the adhesion molecule. In some embodiments, the costimulatory molecule is C-terminal to the activation molecule. In some embodiments, the costimulatory molecule is C-terminal to the adhesion molecule.

[0180] Some embodiments of the fusion protein include a linker. Some embodiments include multiple linkers. In some embodiments, a linker directly connects the costimulatory molecule with the adhesion molecule. In some embodiments, a linker directly connects the costimulatory molecule with the activation molecule. In some embodiments, a linker directly connects the adhesion molecule with the activation molecule.

[0181] In some embodiments of the fusion protein, an N terminal end of the costimulatory molecule is juxtaposed (directly or via a linker) with an end of the adhesion molecule. In some embodiments of the fusion protein, a C terminal end of the costimulatory molecule is juxtaposed (directly or via a linker)with an end of the adhesion molecule. In some embodiments of the fusion protein, an N terminal end of the costimulatory molecule is juxtaposed (directly or via a linker) with an end of the activation molecule. In some embodiments of the fusion protein, a C terminal end of the costimulatory molecule is juxtaposed (directly or via a linker) with an end of the activation molecule.

[0182] In some embodiments of the fusion protein, an N terminal end of the activation molecule is juxtaposed (directly or via a linker) with an end of the adhesion molecule. In some embodiments of the fusion protein, a C terminal end of the activation molecule is juxtaposed (directly or via a linker) with an end of the adhesion molecule. In some embodiments of the fusion protein, an N terminal end of the activation molecule is juxtaposed (directly or via a linker) with an end of the costimulatory molecule. In some embodiments of the fusion protein, a C terminal end of the activation molecule is juxtaposed (directly or via a linker) with an end of the costimulatory molecule.

[0183] In some embodiments of the fusion protein, an N terminal end of the adhesion molecule is juxtaposed (directly or via a linker) with an end of the costimulatory molecule. In some embodiments of the fusion protein, a C terminal end of the adhesion molecule is juxtaposed (directly or via a linker) with an end of the costimulatory molecule. In some embodiments of the fusion protein, an N terminal end of the adhesion molecule is juxtaposed (directly or via a linker) with an end of the activation molecule. In some embodiments of the fusion protein, a C terminal end of the adhesion molecule is juxtaposed (directly or via a linker) with an end of the activation molecule. The fusion protein may comprise, in any order, a CD80, a CD80 extracellular portion, or a functional fragment of CD80; a CD58, a CD58 extracellular portion; or a functional fragment of CD58; an activation molecule (e.g. a TCR-binding molecule); and polypeptide linkers.

[0184] The fusion protein may comprise, in N- to C-terminal order, CD80, a CD80 extracellular portion, or a functional fragment of CD80; a polypeptide linker; and CD58, a CD58 extracellular portion; or a functional fragment of CD58.

[0185] The fusion protein may comprise, in N- to C-terminal order, CD58, a CD58 extracellular portion; or a functional fragment of CD58; a polypeptide linker; and CD80, a CD80 extracellular portion, or a functional fragment of CD80.

[0186] The fusion protein may comprise, in N- to C-terminal order, an activation molecule (e.g. a TCR- binding protein); a polypeptide linker; CD80, a CD80 extracellular portion, or a functional fragment of CD80; a polypeptide linker; and CD58, a CD58 extracellular portion; or a functional fragment of CD58.

[0187] The fusion protein may comprise, in N- to C-terminal order, CD80, a CD80 extracellular portion, or a functional fragment of CD80; a polypeptide linker; CD58, a CD58 extracellular portion; or a functional fragment of CD58; a polypeptide linker; and an activation molecule (e.g. a TCR-binding protein).

[0188] The fusion protein may comprise, in N- to C-terminal order, an activation molecule (e.g. a TCR- binding protein); a polypeptide linker; CD58, a CD58 extracellular portion; or a functional fragment of CD58; a polypeptide linker; and CD80, a CD80 extracellular portion, or a functional fragment of CD80.

[0189] The fusion protein may comprise, in N- to C-terminal order, CD58, a CD58 extracellular portion; or a functional fragment of CD58; a polypeptide linker; CD80, a CD80 extracellular portion, or a functional fragment of CD80; a polypeptide linker; and an activation molecule (e.g. a TCR-binding protein).

[0190] An illustrative fusion protein comprises a CD58 extracellular region and a-CD3 scFv fused to the N-terminus of a CD80 via a linker; this construct is an example of a tri-fusion polypeptide and is termed “498.” Some aspects of 498 are shown in FIG. 14A, any of which may be used in a fusion molecule herein. Some embodiments including 498, and studies exemplifying use of 498 are provided in WO2024097992 and W02024098028, which are incorporated herein by reference in their entirety.

[0191] An illustrative fusion protein comprises a CD58 extracellular region fused to the N-terminus of a CD80 via a linker; this construct is an example of a bi-fusion polypeptide and is termed “455.” In this construct, an aCD3 scFv is expressed as a separate polypeptide in the producer cells. Some aspects of 455 are shown in FIG. 14B, any of which may be used in a fusion molecule herein. Some embodiments including 455, and studies exemplifying use of 498 are provided in WO2024097992 and W02024098028, which are incorporated herein by reference in their entirety.

[0192] In each case, the polypeptide linker may be optional. It may be omitted by directly Unking protein molecule to the next via a peptide bound. Although one may generate fusion proteins through chemical synthesis, fusion proteins are made by expressing the fusion protein from a single polynucleotide comprising a polynucleotide sequence encoding the entire fusion protein. Methods for designing and cloning polynucleotides are known in the art.

[0193] The fusion molecule may be encoded by a polynucleotide (e.g. a DNA or RNA polynucleotide). In some embodiments, the disclosure provides polynucleotides encoding such fusion proteins. The polynucleotide may be an isolated polynucleotide, or it may be part of a vector (e.g., a plasmid) or it may be introduced into and propagated in a host cell.

[0194] Polypeptide sequences of illustrative triple CD58+CD80+aCD3scFv fusion proteins are provided in Table 4. In each case, the fusion protein may comprise a polypeptide 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 99%, or 100% sequence identity to any sequence in Table 4. In some embodiments, the fusion protein may comprise a polypeptide less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 99%, or less than 100% sequence identity to any sequence in Table 4. In each case, an optional signal peptide is shown in parentheses. The signal peptide is cleaved during expression of the sequence. Sequence identity to a reference sequence is determined without the optional residues. Some additional examples of fusion molecules are provided in WO2024097992 and W02024098028, which are incorporated herein by reference in their entirety. The fusion protein may include a linker such as the polypeptide sequence of GSSGGSGGGGSGGGGSGGGGS (SEQ ID NO: 34) or GSSGGSGGGGSGGGGSGGGGSSG (SEQ ID NO: 35).Table 4. Example fusion proteins

[0195] In some embodiments, the fusion molecule comprises a polypeptide encoded by SEQ ID NO: 246. In some embodiments, the fusion molecule comprises a polypeptide encoded by a polynucleotide 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%, or at least 99% identical to SEQ ID NO: 246. In some embodiments, the fusion molecule comprises a polypeptide encoded by a polynucleotide sequence that is at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to SEQ ID NO: 246. In someembodiments, the fusion molecule comprises a polypeptide encoded by a polynucleotide sequence that is less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, or less than 99% identical to SEQ ID NO: 246. In some embodiments, the fusion molecule comprises a polypeptide encoded by a polynucleotide sequence that is less than 99.1%, less than 99.2%, less than 99.3%, less than 99.4%, less than 99.5%, less than 99.6%, less than 99.7%, less than 99.8%, or less than 99.9% identical to SEQ ID NO: 246.

[0196] Some embodiments include a fusion molecule comprising: a CD58 extracellular domain, or a functional fragment thereof, a CD80 or CD86 extracellular domain, or a functional fragment thereof, and an anti-CD3 antibody or antigen-binding fragment thereof. In some embodiments, the CD58 extracellular domain comprises a polypeptide 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: 10. In some embodiments, the CD58 extracellular domain comprises a polypeptide 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: 10. In some embodiments, the CD80 extracellular domain comprises a polypeptide 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: 12. In some embodiments, the CD86 extracellular domain comprises a polypeptide 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: 13. An antigen-binding fragment may include a single chain variable fragment (scFv). For example, an anti-CD3 antigen-binding fragment may include an anti-CD3 scFv. In some embodiments, the anti-CD3 antigen-binding fragment comprises a polypeptide 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: 31.

[0197] Some embodiments include a fusion molecule comprising: a CD58 extracellular domain, or a functional fragment thereof, a CD80 or CD86 extracellular domain, or a functional fragment thereof, and an anti-CD3 antibody or antigen-binding fragment thereof.

[0198] Some embodiments include a CD58 extracellular domain sequence. The CD58 extracellular domain sequence may be or include a functional fragment of a CD58 extracellular domain. In some embodiments, the CD58 extracellular domain comprises a polypeptide 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: 10, 444 or 445. In some embodiments, the CD58 extracellular domain comprises a polypeptide 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: 444. In someembodiments, the CD58 extracellular domain comprises a polypeptide 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: 445.

[0199] Some embodiments include a CD80 or CD86 extracellular domain sequence. The CD80 or CD86 extracellular domain sequence may be or include a functional fragment of a CD80 or CD86 extracellular domain. The CD80 or CD86 extracellular domain sequence may be or include a CD80 extracellular domain sequence. The CD80 extracellular domain sequence may be or include a functional fragment of a CD80 extracellular domain. In some embodiments, the CD80 extracellular domain comprises a polypeptide 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: 12. The CD80 or CD86 extracellular domain sequence may be or include a CD86 extracellular domain sequence. The CD86 extracellular domain sequence may be or include a functional fragment of a CD86 extracellular domain. In some embodiments, the CD86 extracellular domain comprises a polypeptide 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: 13. In some embodiments, the anti-CD3 antigen-binding fragment comprises a polypeptide 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: 31.

[0200] In some embodiments, the fusion molecule comprises the polypeptide sequence of SEQ ID NO: 446. In some embodiments, the fusion molecule comprises a polypeptide 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%, or at least 99% identical to SEQ ID NO: 446. In some embodiments, the fusion molecule comprises a polypeptide sequence that is at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to SEQ ID NO: 446. In some embodiments, the fusion molecule comprises a polypeptide sequence that is less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, or less than 99% identical to SEQ ID NO: 446. In some embodiments, the fusion molecule comprises a polypeptide sequence that is less than 99.1%, less than 99.2%, less than 99.3%, less than 99.4%, less than 99.5%, less than 99.6%, less than 99.7%, less than 99.8%, or less than 99.9% identical to SEQ ID NO: 446.

[0201] In some embodiments, the fusion molecule includes a signal sequence. An example of a signal sequence may include the amino acid sequence of SEQ ID NO: 447. Some embodiments do not include the signal sequence. Some embodiments include a different signal sequence. Some embodiments omit a signal sequence.SEQ ID NO: 447: MVAGSDAGRALGVLSWCLLHCFGFISC

[0202] In some embodiments, the fusion molecule comprises the polypeptide sequence of SEQ ID NO: 44.8 In some embodiments, the fusion molecule comprises a polypeptide 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%, or at least 99% identical to SEQ ID NO: 448. In some embodiments, the fusion molecule comprises a polypeptide sequence that is at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to SEQ ID NO: 448. In some embodiments, the fusion molecule comprises a polypeptide sequence that is less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, or less than 99% identical to SEQ ID NO: 448. In some embodiments, the fusion molecule comprises a polypeptide sequence that is less than 99.1%, less than 99.2%, less than 99.3%, less than 99.4%, less than 99.5%, less than 99.6%, less than 99.7%, less than 99.8%, or less than 99.9% identical to SEQ ID NO: 448.

[0203] In some embodiments, the fusion molecule comprises the polypeptide sequence of SEQ ID NO: 449. In some embodiments, the fusion molecule comprises a polypeptide 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%, or at least 99% identical to SEQ ID NO: 449. In some embodiments, the fusion molecule comprises a polypeptide sequence that is at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to SEQ ID NO: 449. In some embodiments, the fusion molecule comprises a polypeptide sequence that is less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, or less than 99% identical to SEQ ID NO: 449. In some embodiments, the fusion molecule comprises a polypeptide sequence that is less than 99.1%, less than 99.2%, less than 99.3%, less than 99.4%, less than 99.5%, less than 99.6%, less than 99.7%, less than 99.8%, or less than 99.9% identical to SEQ ID NO: 449.SEQ ID NO: 448: FSQQIYGVVYGNVTFHVPSNVPLKEVLWKKQKDKVAELENSEFRAFSSFKNRVYLDTVSGSLTIYNLTS SDEDEYEMESPNTTDTMKFFLYVLESLPSPTLTCALTNGSIEVQCMIPEHYNSHRGLIMYSWDCPMEQC KRNSTSIYFKMENDLPQKIQCTLSNPLFNTTSSIILTTCIPSSGHSRHRGSGDIQMTQSPSSLSASVGDRVTI TCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQW SSNPFTFGQGTKLQITRTSGGGGSGGGGSGGGGSQVQLVQSGGGWQPGRSLRLSCKASGYTFTRYTM HWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYD DHYCLDYWGQGTPVTVSSAGSSGGSGGGGSGGGGSGGGGSSGVIHVTKEVKEVATLSCGHNVSVEEL AQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFK REHLAEVTLSVKADFPTPSISDFEIPTSNTRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAV SSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPR CRERRRNERLRRESVRPVSEQ ID NO: 449:MVAGSDAGRALGVLSVVCLLHCFGFISCFSQQIYGVVYGNVTFHVPSNVPLKEVLWKKQKDKVAELE NSEFRAFSSFKNRVYLDTVSGSLTIYNLTSSDEDEYEMESPNITDTMKFFLYVLESLPSPTLTCALTNGSI EVQCMIPEHYNSHRGLIMYSWDCPMEQCKRNSTSIYFKMENDLPQKIQCTLSNPLFNTTSSnLTTCIPSS GHSRHRGSGDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPS RFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTSGGGGSGGGGSGGGGSQVQL VQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFnS RDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSAGSSGGSGGGGSGGGGSGGGGSSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDI TNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRnCSTSGGF PEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQ EHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPVPayloads

[0204] The particle may be used to deliver a payload. The term “payload” refers to any molecule or combination of molecules whose delivery to a target cell is desired. Various payloads may be delivered using the particles desired herein, including but not limited to small molecules, polynucleotide and proteins. When the selected target cell is a T cell, the particles of the disclosure may be used to deliver a therapeutic agent targeting T cells, to genetically modified T cells, or to deliver a polynucleotide encoding a protein of interest to the T cell. Similarly, particles disclosed herein may be used to delivery payloads to NK cells.

[0205] Examples of payloads which may be included in a lentiviral particle are shown in FIG. 3B and FIG. 5. The payload in FIG. 3B includes a polynucleotide encoding an anti-CD22 CAR, RACR and free FRB. Any aspect shown in the figures may be included in a payload herein.

[0206] The payload may be a polynucleotide, such as a polynucleotide whose sequence encodes a protein or a non-coding nucleic acid (e.g., shRNA, microRNA, or siRNA). The polynucleotide may be an RNA, such as a messenger RNA (mRNA) or the vector genome of an RNA virus. It may be a DNA, such as the vector genome of a DNA virus.

[0207] The payload may comprise a polynucleotide encoding a chimeric antigen receptor (CAR). Illustrative CARs, and polynucleotide encoding them, are described herein. CARs including useful aspects that may be included in the present disclosure are also provided in in U.S. Pat. Nos. 7,741,465; 9,856,322 and 8,399,964. In some embodiments, the CAR is a CAR that specifically binds CD22. Some examples of payloads that encode anti-CD22 CARs are provided in WO2021197483 and US20230174654, which are incorporated herein by reference.

[0208] Some examples of payloads or polynucleotide inserts are provided in WO2024097992 and W02024098028, which are incorporated herein by reference in their entirety. An aspect provided in WO2024097992 or W02024098028 may be included in a payload or polynucleotide insert herein.

[0209] In some embodiments, the CAR may be encoded by a polynucleotide sequence that encodes a signal peptide to signal transport of the CAR in the cell. It is understood that typically the signal peptide is removed from the final CAR protein.

[0210] In some embodiments, the lentiviral particles of the present disclosure comprise a polynucleotide sequence encoding, in any order, on a polycistronic transcript: a promoter, a therapeutic protein (e.g. CAR), optionally a cytosolic FRB domain or a portion thereof, and optionally a synthetic cytokine polypeptide (e.g. RACR). In some embodiments, the polycistronic transcript comprises a promoter and a CAR. Illustrative promoters include, without limitation, a cytomegalovirus (CMV) promoter, a CAG promoter, an SV40 promoter, an SV40 / CD43 promoter, and a MND promoter.

[0211] In some embodiments, the MND promoter comprises a nucleic acid sequence that shares 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% identity to SEQ ID NO: 118.GAACAGAGAAACAGGAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCT CAGGGCCAAGAACAGTTGGAACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCC TGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCTAGA GAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAAC CAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCTATATAAGCAGAGCTCG TTTAGTGAACCGTCAGATCGCTAGC (SEQ ID NO: 118)

[0212] In some embodiments, the MND promoter comprises a nucleic acid sequence that shares 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% identity to SEQ ID NO: 172.AATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGGATCAAGGTCAGGAACAGAGAGACAGCAG AATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGTT GGAACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCC AAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCC AGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCG CTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCTATATAAGAGCCCACAACCCCTCACTCGGC (SEQ ID NO: 172)

[0213] In some embodiments, the CSF2RA signal sequence comprises a nucleic acid sequence that shares 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% identity to SEQ ID NO: 173.ATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCC (SEQ ID NO: 173)Polycistronic Constructs

[0214] Provided herein are polycistronic constructs encoding one or more separate proteins. In some embodiments, the polycistronic constructs comprise one, two, three, or four expression cassettes each encoding a separate protein. In some embodiments, the polycistronic constructs comprise four expression cassettes each encoding a separate protein. In some embodiments, the expression cassettes are separated by cleavable linkers.

[0215] The disclosure provides a polynucleotide construct comprising a contiguous polynucleotide sequence encoding at least two synthetic receptors and methods for uses thereof. In some embodiments, the polynucleotide construct is a polycistronic construct encoding a synthetic cytokine receptor, a synthetic chimeric antigen receptor (CAR), and a freely diffusible FRB, in which the cytokine receptor is responsive to rapamycin binding. Advantageously, FRB reduces the inhibitory effects of rapamycin on mTOR in cells engineered to express the polycistronic constructs provided herein. Expression of the freely diffusible FRB can promote consistent activation and proliferation of engineered cells.

[0216] In some embodiments, the polycistronic constructs provided herein comprise a nucleotide sequence encoding an FRB. In some embodiments, the polycistronic constructs provided herein comprise a nucleotide sequence encoding a chimeric antigen receptor (CAR). In some embodiments, the polycistronic constructs provided herein comprise a nucleotide sequence encoding a synthetic cytokine polypeptide. In some embodiments, the synthetic cytokine polypeptide comprises a synthetic cytokine gamma chain polypeptide and a synthetic cytokine beta chain polypeptide. In some embodiments, the synthetic cytokine gamma chain comprises interleukin 2 receptor subunit y (IL2RG). In some embodiments, the synthetic cytokine gamma chain further comprises FRB. In some embodiments, the synthetic cytokine beta chain comprises interleukin 2 receptor subunit 0 (IL2RB). In some embodiments, the synthetic cytokine gamma chain comprises further FKBP12. In other embodiments, the synthetic cytokine gamma chain comprises interleukin 2 receptor subunit y (IL2RG). In some embodiments, the synthetic cytokine gamma chain further comprises FKBP12. In some embodiments, the synthetic cytokine beta chain comprises interleukin 2 receptor subunit 0 (IL2RB). In some embodiments, the synthetic cytokine beta chain further comprises FRB.

[0217] In some embodiments, the polycistronic construct provided herein comprises nucleotide sequences encoding an FRB, a synthetic cytokine complex, and a CAR.

[0218] In some embodiments, the polycistronic construct comprises a nucleotide sequence encoding FRB, a nucleotide sequence encoding a synthetic cytokine polypeptide, and a nucleotide sequence encoding a CAR. In some embodiments, the nucleotide sequence encoding the synthetic cytokine complex comprises a first nucleotide sequence encoding FRB :IL2RG and a second nucleotide sequence encoding FKBP12:IL2RB. In some embodiments, the nucleotide sequence encoding the synthetic cytokine complex comprises a first nucleotide sequence encoding FKBP12:IL2RG and a second nucleotide sequence encoding FRB:IL2RB.Cytosolic FRB

[0219] In some embodiments, an expression cassette of the polycistronic construct encodes an independent FKBP-rapamycin-binding (FRB) polypeptide. The free FRB polypeptide may be or include a free FRB domain. The FRB domain is an approximately 270 base pair (bp) domain from the mammalian target of rapamycin (mTOR) protein kinase. It may be expressed in the cytosol as a freely diffusible soluble protein.

[0220] In some embodiments, the first expression cassette in the polycistronic construct comprises a nucleotide sequence encoding an independent FRB. In some embodiments, when the FRB is expressed, it is a freely diffusible soluble protein ("free FRB").

[0221] In some embodiments, the nucleotide sequence encoding the FRB is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOs: 256, 257, or 258. In some embodiments, the nucleotide sequence encoding the FRB is at least 100% identical to the nucleotide sequence of SEQ ID NOs: 256, 257, or 258. In some embodiments, the nucleotide sequence encoding the FRB comprises the nucleotide sequence of SEQID Nos: 256, 257, or 258. In some embodiments, the free FKBP12-rapamycin binding (FRB) polypeptide is encoded by a polynucleotide 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: 256, 257, or 258.

[0222] In some embodiments, the FRB comprises an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOs: 251, 252, or 260. In some embodiments, the FRB comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NOs: 251, 252, or 260. In some embodiments, the FRB comprises the amino acid sequence of SEQ ID NOs: 251, 252, or 260. In some embodiments, the lentiviral particle comprises a polynucleotide encoding a free FKBP12-rapamycin binding (FRB) domain polypeptide. In some embodiments, the free FRB polypeptide comprises a 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: 251, 252, or 260.

[0223] In some embodiments, synthetic cytokine receptor complex comprises a cytosolic polypeptide that binds to the ligand or a complex comprising the ligand.

[0224] Without wishing to be bound by theory, the cytosolic FRB may confer resistance to the immunosuppressive effect of the non-physiological ligand (e.g., rapamycin or rapalog). For example, in cells lacking the cytosolic FRB protein, rapamycin may provide an immunosuppressive effect. Cytosolic FRB present in cells transduced by certain embodiments of the lentiviral particles disclosed herein may enable the transduced cells to be resistant to the suppressive effects of rapamycin, or analogs thereof. This may be accomplished by providing a decoy binding domain for rapamycin such that rapamycin cannot engage with mTOR and induce the natural immunosuppressive effects seen in cells lacking the exogenously provided cytosolic FRB protein.Synthetic Cytokine Receptor

[0225] Some embodiments include a polynucleotide encoding a small molecule-activated cytokine receptor. The small molecule-activated cytokine receptor may be or include a rapamycin-activated cytokine receptor (RACR). Some embodiments include a polynucleotide encoding a chimeric antigen receptor (CAR) that specifically binds cluster of differentiation-22 (CD22) and a polynucleotide encoding a small molecule-activated cytokine receptor. In some embodiments, the polynucleotide encoding the CAR and polynucleotide encoding the small molecule-activated cytokine receptor are included together as part of one polynucleotide molecule. In some embodiments, the polynucleotide encoding the CAR and polynucleotide encoding the small molecule-activated cytokine receptor are separate polynucleotide molecules. The small molecule-activated cytokine receptor may activate T cells. The small molecule-activated cytokine receptor may activate NK cells.

[0226] The small molecule-activated cytokine receptor may be activated by a small molecule. The small molecule may include rapamycin, a rapalog, tacrolimus, coumermycin, gibberellin, abscisic acid,methotrexate, cyclosporin A, FKCsA, trimethoprim- synthetic ligand for FKBP, or a derivative thereof. The small molecule may include rapamycin or a rapalog. The small molecule may include rapamycin. The small molecule may include a rapalog. Binding of the extracellular domains to the small molecule may be sufficient for the intracellular cytokine receptor signaling domains of the two polypeptide chains to activate cytokine signal transduction. In some embodiments, the small molecule-activated cytokine receptor does not include an antigen binding domain.

[0227] In some embodiments, an expression cassette of the polycistronic construct encodes a synthetic cytokine receptor. The synthetic cytokine receptors of the present disclosure may comprise a synthetic gamma chain and a synthetic beta chain, each comprising a dimerization domain. The dimerization domains controllably dimerize in the presence of a non-physiological ligand, thereby activating signaling of the synthetic cytokine receptor. For example, in some embodiments, the synthetic cytokine receptors disclosed herein may comprise an FRB domain and an FKBP12 binding domain on separate polypeptides such that the polypeptides dimerize in the presence of rapamycin or an analog thereof.

[0228] The synthetic gamma chain polypeptide comprises a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain. The dimerization domain may be extracellular (N-terminal to the transmembrane domain) or intracellular (C-terminal to the transmembrane domain and N- or C-terminal to the IL-2G intracellular domain.

[0229] The synthetic beta chain polypeptide comprises a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain. The dimerization domain may be extracellular (N-terminal to the transmembrane domain) or intracellular (C-terminal to the transmembrane domain and N- or C-terminal to the IL-2RB or IL-7RB intracellular domain).

[0230] In some embodiments, the polycistronic construct provided herein comprises one or more nucleotide sequences encoding a synthetic cytokine receptor. In some embodiments, the one or more nucleotide sequences correspond to one or more expression cassettes. In some embodiments, the polynucleotide construct provided herein comprises one expression cassette encoding IL2RG and a second expression cassette encoding IL2RB.

[0231] In some embodiments, the synthetic gamma chain polypeptide is encoded by a nucleic acid sequence that encodes a signal peptide. In some embodiments, the synthetic beta chain polypeptide is encoded by a nucleic acid sequence that encodes a signal peptide. A skilled artisan is readily familiar with signal peptides that can provide a signal to transport a nascent protein in the cells. Any of a variety of signal peptides can be employed.

[0232] In some embodiments, the nucleotide encoding the synthetic cytokine gamma chain polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOs: 261, 262, or 263. In some embodiments, thenucleotide encoding the synthetic cytokine gamma chain polypeptide is at least 100% identical to the nucleotide sequence of SEQ ID NOs: 261, 262, or 263. In some embodiments, the nucleotide encoding the synthetic cytokine gamma chain polypeptide comprises the nucleotide sequence of SEQ ID NOs: 261, 262, or 263.

[0233] In some embodiments, the synthetic cytokine gamma chain polypeptide comprises interleukin 2 receptor subunit y (IL2RG). In some embodiments, the IL2RG comprises an amino add sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino add sequence of SEQ ID NOs: 264 or 265. In some embodiments, the IL2RG comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NOs: 264 or 265. In some embodiments, the IL2RG comprises the amino acid sequence of SEQ ID NOs: 264 or 265.

[0234] In some embodiments, the second expression cassette further comprises a nucleotide sequence encoding an FRB domain. In some embodiments, the nucleotide sequence encoding the FRB is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 257. In some embodiments, the nucleotide sequence encoding the FRB is at least 100% identical to the nucleotide sequence of SEQ ID NO: 257. In some embodiments, the nucleotide sequence encoding the FRB comprises the nucleotide sequence of SEQ ID NO: 257.

[0235] In some embodiments, the FRB comprises an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 252. In some embodiments, the FRB comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 252. In some embodiments, the FRB comprises the amino acid sequence of SEQ ID NO: 252.

[0236] In some embodiments, the second expression cassette is codon optimized.

[0237] In some embodiments, the second expression cassette comprises a nucleotide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 266. In some embodiments, the second expression cassette comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NO: 266. In some embodiments, the second expression cassette comprises the nucleotide sequence of SEQ ID NO: 266.

[0238] In some embodiments, the second expression cassette encodes an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 267. In some embodiments, the second expression cassette encodes an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 267. In some embodiments, the second expression cassette encodes an amino acid sequence comprising the sequence of SEQ ID NO: 267.

[0239] In some embodiments, the second expression cassette further comprises a nucleotide sequence encoding an FKBP12 domain or functional fragment thereof. FKBP12 is also known as FKBP1A or FK506 binding protein. In some embodiments, the nucleotide sequence encoding the FKBP12 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotidesequence of SEQ ID NOs: 268 or 269. In some embodiments, the nucleotide sequence encoding the FKBP12 is at least 100% identical to the nucleotide sequence of SEQ ID NOs: 268 or 269. In some embodiments, the nucleotide sequence encoding the FKBP12 comprises the nucleotide sequence of SEQ ID NOs: 268 or 269.

[0240] In some embodiments, the FKBP12 comprises an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 253. In some embodiments, the FKBP12 comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 253. In some embodiments, the FKBP12 comprises the amino acid sequence of SEQ ID NO: 253.GVQVETISPGDGRTFPKRGQTCWHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMS VGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKL (SEQ ID 253)

[0241] In some embodiments, the nucleotide encoding the synthetic cytokine beta chain polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOs: 270 or 271. In some embodiments, the nucleotide encoding the synthetic cytokine beta chain polypeptide is at least 100% identical to the nucleotide sequence of SEQ ID NOs: 270 or 271. In some embodiments, the nucleotide encoding the synthetic cytokine beta chain polypeptide comprises the nucleotide sequence of SEQ ID NOs: 270 or 271.

[0242] In some embodiments, the synthetic cytokine beta chain polypeptide comprises interleukin 2 receptor subunit 0 (IL2RB).

[0243] In some embodiments, the IL2RB comprises an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOs: 272. In some embodiments, the IL2RB comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NOs: 272. In some embodiments, the IL2RB comprises the amino acid sequence of SEQ ID NOs: 272.

[0244] In some embodiments, the third expression cassette further comprises a nucleotide sequence encoding FKBP12.

[0245] In some embodiments, the nucleotide sequence encoding the FKBP12 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 274. In some embodiments, the nucleotide sequence encoding the FKBP12 is at least 100% identical to the nucleotide sequence of SEQ ID NO: 274. In some embodiments, the nucleotide sequence encoding the FKBP12 comprises the nucleotide sequence of SEQ ID NO: 274.

[0246] In some embodiments, the FKBP12 comprises an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 275. In some embodiments, the FKBP12 comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 275. In some embodiments, the FKBP12 comprises the amino acid sequence of SEQ ID NO: 275.

[0247] In some embodiments, the third expression cassette is codon optimized.

[0248] In some embodiments, the third expression cassette comprises a nucleotide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 276. In some embodiments, the third expression cassette comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NO: 276. In some embodiments, the third expression cassette comprises the nucleotide sequence of SEQ ID NO: 276.

[0249] In some embodiments, the third expression cassette encodes an amino add sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 277. In some embodiments, the third expression cassette encodes an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 277. In some embodiments, the third expression cassette encodes an amino acid sequence comprising the sequence of SEQ ID NO: 277.

[0250] In some embodiments, the third expression cassette further comprises a nucleotide sequence encoding FRB. In some embodiments, the nucleotide sequence encoding the FRB is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 257. In some embodiments, the nucleotide sequence encoding the FRB is at least 100% identical to the nucleotide sequence of SEQ ID NO: 257. In some embodiments, the nucleotide sequence encoding the FRB comprises the nucleotide sequence of SEQ ID NO: 257.Intracellular Domain

[0251] In some embodiments, the intracellular signaling domain of the first transmembrane receptor protein comprises an interleukin-2 receptor subunit gamma (IL2Rg) domain.

[0252] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain, a first dimerization domain, a second transmembrane receptor protein comprising an IL-2RB intracellular domain, and a second dimerization domain.

[0253] In some embodiments, the synthetic beta chain comprises an interleukin-2 receptor subunit beta (IL2RB) intracellular domain. IL2RB is also known as IL15RB or CD122. Thus, when referred to herein, IL2RB can also mean IL15RB. That is, the terms are used interchangeably in the present disclosure.

[0254] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain, a first dimerization domain, a second transmembrane receptor protein comprising an IL-7RB intracellular domain, and a second dimerization domain.

[0255] In some embodiments, the synthetic beta chain comprises an interleukin-7 receptor subunit beta (IL7RB) intracellular domain.

[0256] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain, a first dimerization domain, a secondtransmembrane receptor protein comprising an IL-21RB intracellular domain, and a second dimerization domain.

[0257] In some embodiments, the synthetic beta chain comprises an interleukin-21 receptor subunit beta (IL21RB) intracellular domain.Dimerization Domain

[0258] The dimerization domains may be heterodimerization domains, including but not limited to FK506-Binding Protein of size 12 kD (FKBP12, also referred to herein as FKBP) and a FKBP12- rapamycin binding (FRB) polypeptide, which dimerize in the presence of rapamycin or a rapalog. The FRB polypeptide may be or include an FRB domain.

[0259] Alternatively, the first dimerization domain and the second dimerization domain may be a FK506-Binding Protein of size 12 kD (FKBP) and a caldneurin domain, which dimerize in the presence of FK506 or an analogue thereof.

[0260] In some embodiments the dimerization domains are homodimerization domains selected from: i) FK506-Binding Protein of size 12 kD (FKBP); ii) cyclophilinA (CypA); or iii) gyrase B (CyrB); with the corresponding non-physiological ligands being, respectively i) FK1012, AP1510, AP1903, or AP20187; ii) cyclosporin-A (CsA); or iii) coumermycin or analogs thereof.

[0261] In some embodiments, the first and second dimerization domains of the transmembrane receptor proteins are a FKBP domain and a cyclophilin domain.

[0262] In some embodiments, the first and second dimerization domains of the transmembrane receptor proteins are a FKBP domain and a bacterial dihydrofolate reductase (DHFR) domain.

[0263] In some embodiments, the first and second dimerization domains of the transmembrane receptor proteins are a calcineurin domain and a cyclophilin domain.

[0264] In some embodiments, the first and second dimerization domains of the transmembrane receptor proteins are PYRl-like 1 (PYL1) and abscisic acid insensitive 1 (ABI1).

[0265] In some embodiments, the lentiviral particle includes a polynucleotide encoding a rapamycin activated cytokine receptor (RACR), or a functional fragment thereof. In some embodiments, the lentiviral particle comprises a polynucleotide encoding a FRB polypeptide, or a functional fragment thereof. In some embodiments, the FRB polypeptide comprises a polypeptide 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: 252. In some embodiments, the FRB polypeptide is encoded by a polynucleotide 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: 257.

[0266] In some embodiments, the lentiviral particle comprises a polynucleotide encoding a cytokine gamma chain polypeptide. In some embodiments, the cytokine gamma chain polypeptide 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%, at least 99%, or 100% identical to SEQ ID NO: 264 or 265. In some embodiments, the cytokine gamma chain polypeptide is encoded by a polynucleotide 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: 261, 262, or 263. In some embodiments, the FRB polypeptide and the cytokine gamma chain polypeptide are fused together.

[0267] In some embodiments, the lentiviral particle comprises a polynucleotide encoding a FKBP polypeptide. In some embodiments, the FKBP polypeptide comprises a polypeptide 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: 253. In some embodiments, the FKBP polypeptide is encoded by a polynucleotide 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: 268.

[0268] In some embodiments, the lentiviral particle comprises a polynucleotide encoding a cytokine beta chain polypeptide. In some embodiments, the cytokine beta chain polypeptide 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%, at least 99%, or 100% identical to SEQ ID NO: 272. In some embodiments, the cytokine beta chain polypeptide is encoded by a polynucleotide 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: 270 or 271. In some embodiments, the FKBP polypeptide and the cytokine beta chain polypeptide are fused together.

[0269] In some embodiments, the FRB polypeptide and cytokine beta chain polypeptide are fused together and the FKBP polypeptide and the cytokine gamma chain polypeptide are fused together. In other embodiments, the FRB polypeptide and cytokine gamma chain polypeptide are fused together and the FKBP polypeptide and the cytokine gamma chain polypeptide are fused together.Transmembrane Domain

[0270] The transmembrane domain of a synthetic cytokine receptor is the sequence of the synthetic cytokine receptor that spans the membrane. The transmembrane domain (also referred to herein as “TM” or “TM domain”) may comprise a hydrophobic alpha helix. In some embodiments, the transmembrane domain is a human protein.

[0271] In some embodiments, the TM domain and the intracellular signaling domain are from the same cytokine receptor. In some embodiments, the synthetic gamma chain polypeptide contains an IL-2RG TM domain and an IL-2RG intracellular domain. In some embodiments, the synthetic beta chainpolypeptide contains an IL-2RB TM domain and an IL-2RB intracellular domain. In some embodiments, the synthetic beta chain polypeptide contains an IL-7RB TM domain and an IL-7RB intracellular domain. In some embodiments, the synthetic beta chain polypeptide contains an IL-21RB TM domain and an IL-21RB intracellular domain.

[0272] In some embodiments, one or more additional contiguous amino acids of the ectodomain directly adjacent to the TM domain of the cytokine receptor also can be included as part of the polypeptide sequence of a chain of the synthetic cytokine receptor. In some embodiments, 1-20 contiguous amino acids of the ectodomain adjacent to the TM domain of the cytokine receptor is included as part of the polypeptide sequence of a chain of the synthetic cytokine receptor. The portion of the ectodomain may be a contiguous sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids directly adjacent (e.g. N-terminal to) the TM sequence.

[0273] In some of any embodiments, a method comprising the synthetic cytokine receptors disclosed herein further includes administering a non-physiological ligand to the subject. In some embodiments, the non-physiological ligand is able to bind to the synthetic cytokine receptor and induce cytokine signaling in the cell. In some embodiments, the nonphysiological ligand includes rapamycin or a rapamycin analog.

[0274] In some embodiments, the synthetic cytokine receptor is able to be bound by the non- physiological ligand rapamycin or a rapamycin analog. In some embodiments, the synthetic cytokine receptor is responsive to the non-physiological ligand rapamycin or a rapamycin analog, in which binding of the non-physiological ligand to the dimerization domains of the synthetic cytokine receptor induces cytokine receptor-mediated signaling in the cell, such as via the JAK / STAT pathway.Illustrative Polycistronic Constructs

[0275] In some embodiments, the polycistronic construct comprises in 5' to 3' order a nucleotide sequence encoding FRB, a nucleotide sequence encoding a synthetic cytokine polypeptide, and a nucleotide sequence encoding a CAR. In some embodiments, the nucleotide sequence encoding the synthetic cytokine polypeptide comprises in 5' to 3' order a first nucleotide sequence encoding FRB operably linked to IL2RG and a second nucleotide sequence encoding FKBP12 operably linked to IL2RB. In some embodiments, the nucleotide sequence encoding the synthetic cytokine polypeptide comprises in 5' to 3' order a first nucleotide sequence encoding FKBP12 operably linked to IL2RG and a second nucleotide sequence encoding sFRB operably linked to IL2RB.

[0276] In some embodiments, the lentiviral particles of the present disclosure comprise a polynucleotide sequence encoding, in 5' to 3' order on a polycistronic transcript: MND promoter - FRB - [2A and ER signal sequence] - RACRg - [2A and ER signal sequence] - RACRb - [2A and hCSF2R signal sequence] - anti-CD22 CAR. In some embodiments, the lentiviral particles of the present disclosure comprise a polynucleotide sequence encoding, in 5' to 3' order on a polycistronic transcript: MND promoter - [CD8a signal peptide] - anti-CD22 CAR. In any of the foregoing or subsequent embodiments, the polynucleotide sequence encoding the anti-CD22 CAR may comprise, in 5’-3’ order,a polynucleotide sequence encoding an anti-CD22 VH, a polynucleotide sequence encoding a G4S linker, a polynucleotide sequence encoding an anti-CD22 VL, a polynucleotide sequence encoding a CD8 hinge and transmembrane domain, a polynucleotide sequence encoding a 41BB intracellular signaling domain, and a polynucleotide sequence encoding a CD3z signaling domain. In any of the foregoing or subsequent embodiments, the polynucleotide sequence encoding the anti-CD22 CAR may alternatively comprise, in 5 ’-3’ order, a polynucleotide sequence encoding an anti-CD22 VL, a polynucleotide sequence encoding a G4S linker, a polynucleotide sequence encoding an anti-CD22 VH, a polynucleotide sequence encoding a CD8 hinge and transmembrane domain, a polynucleotide sequence encoding a 4 IBB intracellular signaling domain, and a polynucleotide sequence encoding a CD3z signaling domain.

[0277] In some embodiments, the lentiviral particle comprises a 2A nucleic acid sequence, which may induce ribosomal skipping. 2A sequences may be used in polycistronic polynucleotides and vectors. In some embodiments, the lentiviral particle comprises a 2A nucleic acid sequence that shares 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% identity to SEQ ID NO: 278. GAGGGCCGAGGCAGCCTGCTGACCTGCGGTGATGTGGAAGAAAACCCGGGCCCC (SEQ ID NO: 278)

[0278] In some embodiments, the lentiviral particle comprises an ER signal sequence nucleic acid sequence that shares 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% identity to SEQ ID NO: 279.ATGCCCTTGCCCGTGACCGCGTTGCTCCTGCCCTTGGCTCTACTGCTGCACGCCGCTAGACCC (SEQ ID NO: 279)

[0279] In some embodiments, the lentiviral particle comprises a 2A nucleic add sequence that shares 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% identity to SEQ ID NO: 280.GCCACCAATTTCAGCCTCCTGAAACAAGCCGGTGACGTTGAAGAGAACCCCGGCCCC (SEQ ID NO: 280)

[0280] In some embodiments, the lentiviral particle comprises an ER signal sequence nucleic acid sequence that shares 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% identity to SEQ ID NO: 281.ATGCCCCTGGGGTTGCTGTGGTTGGGACTCGCCCTCCTCGGCGCCCTGCACGCTCAAGCC (SEQ ID NO: 281).

[0281] In some embodiments, the lentiviral particle comprises a 2A nucleic add sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 282.GCAACAAACTTTTCTCTGCTGAAGCAGGCCGGCGATGTGGAAGAAAACCCTGGACCT (SEQ ID NO: 282)

[0282] In some embodiments, the lentiviral particles of the present disclosure comprise a polynucleotide sequence encoding, in 5' to 3' order on a polycistronic transcript:(a) a MND promoter;(b) a CAR;(c) a cytosolic FRB domain or a portion thereof;(d) a RACR cell-surface receptor; and(e) a WPRE sequence

[0283] In some embodiments, the lentiviral particles of the present disclosure comprise a polynucleotide sequence encoding, in 5' to 3' order:(a) a CAR;(b) a cytosolic FRB domain or a portion thereof; and(c) a RACR cell-surface receptor.

[0284] In some embodiments, the lentiviral particle comprises a nucleic acid sequence that shares 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% identity to SEQ ID NO: 119.GAACAGAGAAACAGGAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCT CAGGGCCAAGAACAGTTGGAACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCC TGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCTAGA GAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAAC CAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCTATATAAGCAGAGCTCG TTTAGTGAACCGTCAGATCGCTAGC (SEQ ID NO: 119)

[0285] In some embodiments, the lentiviral particle comprises a polypeptide sequence that shares 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% identity to SEQ ID NO: 120.MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLI YHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSG EGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSA LKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSESKYGPPCPP CPMFWVLVWGGVLACYSLLVTVAFnFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDV EENPGPEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEW CRKYMKSGNVKDLLQAWDLYYHVFRRISKGSGATNFSLLKQAGDVEENPGPMPLGLLWLGLALLGA LHAQAGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEG VAQMSVGQRAKLTISPDYAYGATGHPGnPPHATLVFDVELLKLGEGSNTSKENPFLFALEAWISVGS MGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKG GALGEGPGASPCNQHSPYWAPPCYTLKPETGSGATNFSLLKQAGDVEENPGPMALPVTALLLPLALLL HAARPILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGKDTIPWLGHLLVGLSGAFGFHLVYLLINCRNTGP WLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDK VPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLS GEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDL VDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV (SEQ ID NO: 120)

[0286] In some embodiments, the lentiviral particle comprises a nucleic acid sequence that shares 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% identity to SEQ ID NO: 121.

[0287] In some embodiments, the lentiviral particles of the present disclosure comprises a polynucleotide sequence encoding, in 5' to 3' order on a polycistronic transcript:(a) a MND promoter;(b) a cytosolic FRB domain or a portion thereof;(c) a RACR cell-surface receptor;(d) a CAR; and(e) a WPRE sequence.

[0288] In some embodiments, the lentiviral particles of the present disclosure comprises a polynucleotide sequence encoding, in 5' to 3' order:(a) a cytosolic FRB domain or a portion thereof;(b) a RACR cell-surface receptor; and(c) a CAR.

[0289] In some embodiments, a lentiviral particle comprises a polypeptide sequence that shares 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% identity to SEQ ID NO: 122.MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKY MKSGNVKDLLQAWDLYYHVFRRISKGSGATNFSLLKQAGDVEENPGPMPLGLLWLGLALLGALHAQ AGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQM SVGQRAKLTISPDYAYGATGHPGnPPHATLVFDVELLKLGEGSNTSKENPFLFALEAWISVGSMGLIIS LLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGE GPGASPCNQHSPYWAPPCYTLKPETGSGATNFSLLKQAGDVEENPGPMALPVTALLLPLALLLHAARPI LWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCR KYMKSGNVKDLLQAWDLYYHVFRRISKGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKV LKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPAS LSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAY CTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPP PELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLVGSGAT NFSLLKQAGDVEENPGPMLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKY LNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGG TKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLE WLGVIWGSETTYYNSALKSRLTnKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQ GTSVTVSSESKYGPPCPPCPMFWVLVWGGVLACYSLLVTVAFnFWVKRGRKKLLYIFKQPFMRPVQT TQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG GKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 122)

[0290] In some embodiments, a lentiviral particle comprises a nucleic add sequence that shares 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% identity to SEQ ID NO: 123. ATGGAGATGTGGCACGAGGGACTGGAGGAGGCAAGCAGACTGTACTTTGGCGAGAGGAACGTGA AGGGCATGTTCGAGGTGCTGGAGCCACTGCACGCAATGATGGAGAGGGGACCACAGACCCTGAAG GAGACATCTTTCAACCAGGCATACGGAAGGGACCTGATGGAGGCACAGGAGTGGTGCCGGAAGTA TATGAAGAGCGGCAATGTGAAGGACCTGCTGCAGGCCTGGGATCTGTACTATCACGTGTTTCGGA GAATCTCCAAGGGCTCTGGCGCCACCAACTTCTCCCTGCTGAAGCAGGCCGGCGATGTGGAGGAG AATCCTGGACCAATGCCACTGGGACTGCTGTGGCTGGGACTGGCCCTGCTGGGCGCCCTGCACGCC CAGGCCGGCGTGCAGGTGGAGACAATCAGCCCTGGCGACGGACGCACCTTTCCAAAGAGGGGACA GACATGCGTGGTGCACTACACCGGCATGCTGGAGGATGGCAAGAAGTTCGACAGCTCCAGAGATA GGAATAAGCCCTTTAAGTTCATGCTGGGCAAGCAGGAAGTGATCAGGGGATGGGAGGAGGGAGT GGCACAGATGTCTGTGGGACAGCGGGCCAAGCTGACAATCAGCCCAGACTATGCATACGGAGCAA CCGGACACCCTGGAATCATCCCACCTCACGCCACACTGGTGTTTGATGTGGAGCTGCTGAAGCTGG GCGAGGGCAGCAACACCTCCAAGGAGAATCCATTTCTGTTCGCCCTGGAGGCCGTGGTCATCTCTG TGGGCAGCATGGGCCTGATCATCTCCCTGCTGTGCGTGTACTTTTGGCTGGAGCGCACAATGCCAC GGATCCCCACCCTGAAGAACCTGGAGGACCTGGTGACCGAGTACCACGGCAATTTCTCCGCCTGGT CTGGCGTGAGCAAGGGACTGGCAGAGTCTCTGCAGCCAGATTATAGCGAGCGGCTGTGCCTGGTG AGCGAGATCCCACCCAAGGGAGGCGCCCTGGGAGAGGGACCAGGAGCCTCCCCTTGCAACCAGCA CTCTCCTTACTGGGCCCCTCCATGTTATACCCTGAAGCCAGAGACAGGCAGCGGAGCTACTAACTT CTCCCTGCTGAAGCAAGCAGGCGACGTGGAAGAAAATCCTGGACCAATGGCACTGCCAGTGACCG CCCTGCTGCTGCCTCTGGCCCTGCTGCTGCACGCAGCCAGACCCATCCTGTGGCACGAAATGTGGC ATGAAGGCCTGGAGGAGGCAAGCAGGCTGTACTTTGGCGAGCGGAATGTGAAAGGAATGTTTGAA GTGCTGGAGCCTCTGCACGCCATGATGGAGAGGGGCCCTCAGACCCTGAAGGAGACATCCTTTAA CCAGGCCTACGGCAGAGACCTGATGGAGGCCCAGGAGTGGTGCAGGAAGTATATGAAGTCTGGAA ATGTGAAAGACCTGCTGCAGGCCTGGGATCTGTATTATCACGTGTTCAGGCGCATCTCTAAGGGCA AGGATACAATCCCTTGGCTGGGACACCTGCTGGTGGGACTGAGCGGAGCCTTTGGCTTCATCATCC TGGTGTATCTGCTGATCAACTGCCGCAATACAGGCCCATGGCTGAAGAAGGTGCTGAAGTGTAAC ACCCCCGACCCTTCCAAGTTCTTTTCTCAGCTGTCTAGCGAGCACGGCGGCGATGTGCAGAAGTGG CTGTCCTCTCCATTTCCCAGCTCCTCTTTCAGCCCAGGAGGACTGGCACCAGAGATCTCCCCACTGG AGGTGCTGGAGAGGGACAAGGTGACCCAGCTGCTGCTGCAGCAGGATAAGGTGCCTGAGCCAGCC TCCCTGAGCTCCAACCACTCCCTGACCTCTTGCTTTACAAATCAGGGCTACTTCTTTTTCCACCTGC CAGACGCACTGGAGATCGAGGCATGTCAGGTGTATTTCACATACGATCCCTATAGCGAGGAGGAC CCTGATGAGGGAGTGGCCGGCGCCCCAACCGGATCTAGCCCACAGCCTCTGCAGCCACTGAGCGG AGAGGACGATGCATATTGTACATTTCCTTCCCGCGACGATCTGCTGCTGTTCTCTCCAAGCCTGCTG GGAGGACCAAGCCCACCTTCCACCGCACCAGGCGGCTCCGGGGCAGGGGAGGAGCGGATGCCAC CCTCTCTGCAGGAGAGAGTGCCAAGGGACTGGGATCCACAGCCACTGGGACCTCCAACCCCTGGA GTGCCAGACCTGGTGGATTTCCAGCCCCCTCCAGAGCTGGTGCTGAGAGAGGCAGGAGAGGAGGT GCCTGACGCAGGACCAAGAGAGGGCGTGAGCTTTCCTTGGTCCAGGCCACCTGGACAGGGAGAGT TCAGAGCCCTGAACGCCAGGCTGCCCCTGAATACAGACGCCTACCTGTCTCTGCAGGAGCTGCAG GGCCAGGATCCTACACACCTGGTCGGATCTGGCGCCACCAACTTTAGCCTGCTGAAGCAGGCAGG CGACGTGGAAGAGAACCCTGGACCAATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAGCTGC CACACCCTGCCTTCCTGCTGATCCCAGATATCCAGATGACACAGACCACATCCTCTCTGTCCGCCTC TCTGGGCGACAGAGTGACCATCTCTTGTAGGGCCAGCCAGGATATCTCCAAGTACCTGAACTGGTA TCAGCAGAAGCCTGACGGCACAGTGAAGCTGCTGATCTACCACACCTCTAGGCTGCACAGCGGAG TGCCATCCCGGTTCAGCGGATCCGGATCTGGAACAGACTATTCTCTGACCATCAGCAACCTGGAGC AGGAGGATATCGCCACATACTTTTGCCAGCAGGGCAATACCCTGCCATATACATTCGGCGGAGGA ACCAAGCTGGAGATCACCGGAAGCACATCCGGATCTGGCAAGCCAGGATCCGGAGAGGGATCTAC AAAGGGAGAGGTGAAGCTGCAGGAGAGCGGACCAGGACTGGTGGCACCCAGCCAGTCCCTGTCT GTGACCTGTACAGTGTCTGGCGTGAGCCTGCCCGATTACGGCGTGTCCTGGATCAGACAGCCACCA AGGAAGGGACTGGAGTGGCTGGGCGTGATCTGGGGCTCTGAGACCACATACTATAATAGCGCCCT GAAGTCCCGGCTGACCATCATCAAGGACAACAGCAAGTCCCAGGTGTTTCTGAAGATGAATAGCC TGCAGACCGACGATACAGCCATCTACTATTGCGCCAAGCACTACTATTACGGCGGCTCCTACGCCA TGGATTATTGGGGCCAGGGCACCTCCGTGACAGTGAGCTCCGAGTCTAAGTATGGCCCTCCATGCC CCCCTTGTCCTATGTTCTGGGTGCTGGTGGTGGTGGGAGGCGTGCTGGCCTGTTACTCCCTGCTGGT GACCGTGGCCTTTATCATCTTCTGGGTGAAGCGCGGCCGGAAGAAGCTGCTGTATATCTTTAAGCA GCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAGGACGGCTGCAGCTGTAGGTTTCCAGAGG AGGAGGAGGGAGGATGCGAGCTGCGCGTGAAGTTCTCTCGGAGCGCCGATGCCCCTGCCTACCAGCAGGGACAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGG ATAAGAGGAGGGGAAGAGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGG GCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATTCCGAGATCGGCATGAAGGGA GAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTGAGCACCGCCACAAAGGACA CCTATGATGCCCTGCACATGCAGGCCCTGCCACCCAGGTGA (SEQ ID NO: 123)

[0291] In some embodiments, the lentiviral particles of the present disclosure comprise a polynucleotide sequence encoding, in 5' to 3' order on a polycistronic transcript:(a) a MND promoter;(b) a cytosolic FRB domain or a portion thereof;(c) a CAR;(d) TGF- DN domain or portion thereof; and(e) a WPRE sequence.

[0292] In some embodiments, the lentiviral particles of the present disclosure comprise a polynucleotide sequence encoding, in 5' to 3' order:(a) a cytosolic FRB domain or a portion thereof;(b) a CAR; and(c) a TGF- DN domain or portion thereof.

[0293] In some embodiments, the lentiviral particle comprises a polypeptide sequence that shares 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% identity to SEQ ID NO: 124.MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKY MKSGNVKDLLQAWDLYYHVFRRISKGSGATNFSLLKQAGDVEENPGPMLLLVTSLLLCELPHPAFLLI PDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGT DYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAP SQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTnKDNSKSQVFLKMN SLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSESKYGPPCPPCPMFWVLWVGGVLACYSL LVTVAHIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQG QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMGRGLLRGLWPLHTV LWTRIASTn’PHVQKSVNNDMTVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVC VAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNnFSEEYN TSNPDLLLVIFQVTGISLLPPLGVAISVinFYCYRVNRQQKRRR (SEQ ID NO: 124)

[0294] In some embodiments, the lentiviral particle comprises a nucleic acid sequence that shares 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% identity to SEQ ID NO: 125.ATGGAGATGTGGCACGAGGGACTGGAGGAGGCATCCAGACTGTACTTCGGCGAGAGGAACGTGA AGGGCATGTTTGAGGTGCTGGAGCCACTGCACGCCATGATGGAGAGAGGCCCCCAGACCCTGAAG GAGACATCTTTCAACCAGGCCTATGGAAGGGACCTGATGGAGGCACAGGAGTGGTGCCGGAAGTA CATGAAGAGCGGCAATGTGAAGGACCTGCTGCAGGCCTGGGATCTGTACTATCACGTGTTCCGGA GAATCAGCAAGGGCTCCGGCGCCACCAACTTTAGCCTGCTGAAGCAGGCAGGCGACGTGGAGGAG AATCCAGGACCTATGCTGCTGCTGGTGACATCCCTGCTGCTGTGCGAGCTGCCACACCCAGCCTTC CTGCTGATCCCCGATATCCAGATGACCCAGACCACAAGCTCCCTGAGCGCCTCCCTGGGCGACAGG GTGACAATCTCTTGTCGGGCCAGCCAGGATATCTCCAAGTATCTGAATTGGTACCAGCAGAAGCCC GACGGCACCGTGAAGCTGCTGATCTATCACACATCTAGACTGCACAGCGGCGTGCCTTCCAGGTTTTCTGGCAGCGGCTCCGGCACCGACTACTCTCTGACAATCAGCAACCTGGAGCAGGAGGATATCGC CACCTATTTCTGCCAGCAGGGCAATACCCTGCCTTACACATTTGGCGGCGGCACAAAGCTGGAGAT CACCGGCTCTACAAGCGGATCCGGCAAGCCAGGATCCGGAGAGGGATCTACCAAGGGAGAGGTG AAGCTGCAGGAGAGCGGACCTGGACTGGTGGCACCATCTCAGAGCCTGTCCGTGACCTGTACAGT GTCTGGCGTGAGCCTGCCAGATTATGGCGTGAGCTGGATCAGGCAGCCACCTAGGAAGGGACTGG AGTGGCTGGGCGTGATCTGGGGCTCCGAGACCACATACTATAACAGCGCCCTGAAGTCCCGCCTG ACCATCATCAAGGACAACTCTAAGAGCCAGGTGTTCCTGAAGATGAATTCCCTGCAGACCGACGA TACAGCCATCTACTATTGCGCCAAGCACTACTATTACGGCGGCTCTTATGCCATGGATTACTGGGG CCAGGGCACCAGCGTGACAGTGTCTAGCGAGTCCAAGTACGGCCCACCCTGCCCTCCATGTCCCAT GTTTTGGGTGCTGGTGGTGGTGGGAGGCGTGCTGGCCTGTTATTCCCTGCTGGTGACCGTGGCCTT CATCATCTTTTGGGTGAAGCGCGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAG ACCCGTGCAGACCACACAGGAGGAGGACGGCTGCAGCTGTAGGTTCCCAGAGGAGGAGGAGGGA GGATGCGAGCTGAGGGTGAAGTTTTCCCGGTCTGCCGATGCCCCTGCCTATCAGCAGGGCCAGAAT CAGCTGTACAACGAGCTGAATCTGGGCAGGCGCGAGGAGTACGACGTGCTGGATAAGAGGAGAG GAAGGGACCCTGAGATGGGAGGCAAGCCAAGGCGCAAGAACCCTCAGGAGGGCCTGTATAATGA GCTGCAGAAGGACAAGATGGCCGAGGCCTACTCCGAGATCGGCATGAAGGGAGAGCGGAGAAGG GGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACCGCCACAAAGGACACCTACGATGCACT GCACATGCAGGCCCTGCCACCTAGAGGATCTGGAGCCACAAACTTCAGCCTGCTGAAGCAGGCCG GCGATGTGGAGGAGAATCCTGGACCAATGGGAAGAGGACTGCTGAGGGGACTGTGGCCACTGCAC ATCGTGCTGTGGACCAGGATCGCCTCTACAATCCCACCCCACGTGCAGAAGAGCGTGAACAATGA CATGATCGTGACCGATAACAATGGCGCCGTGAAGTTTCCCCAGCTGTGCAAGTTCTGTGACGTGCG CTTTTCCACCTGTGATAACCAGAAGTCCTGCATGTCTAATTGTAGCATCACATCCATCTGCGAGAA GCCTCAGGAGGTGTGCGTGGCCGTGTGGCGGAAGAACGACGAGAATATCACCCTGGAGACAGTGT GCCACGATCCCAAGCTGCCTTATCACGACTTCATCCTGGAGGATGCCGCCTCTCCTAAGTGTATCA TGAAGGAGAAGAAGAAGCCAGGCGAGACCTTCTTTATGTGCAGCTGTTCCTCTGACGAGTGCAAC GATAATATCATCTTCTCCGAGGAGTACAACACCTCTAATCCTGACCTGCTGCTGGTCATCTTTCAGG TGACAGGCATCTCCCTGCTGCCTCCACTGGGCGTGGCCATCTCTGTGATCATCATCTTTTATTGTTA CAGAGTGAACAGGCAGCAGAAGCGCCGGCGCTAG (SEQ ID NO: 125)

[0295] In some embodiments, the FRB domain (e.g. free FRB domain) comprises a polypeptide sequence that shares 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 99%, or 100% identity to SEQ ID NO: 251.MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKY MKSGNVKDLLQAWDLYYHVFRRISK (SEQ ID NO: 251)

[0296] In some embodiments, the FRB domain comprises a polypeptide sequence that shares 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 99%, or 100% identity to SEQ ID NO: 252.ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCR KYMKSGNVKDLLQAWDLYYHVFRRISK (SEQ ID NO: 252)

[0297] In some embodiments, the IL-2 receptor gamma domain comprises a polypeptide sequence that shares 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 99%, or 100% identity to SEQ ID NO: 264.GSNTSKENPFLFALEAVVISVGSMGLnSLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKG LAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET (SEQ ID NO: 264)

[0298] In some embodiments, the FKBP domain comprises a polypeptide sequence that shares 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 99%, or 100% identity to SEQ ID NO: 253.GVQVETISPGDGRTFPKRGQTCWHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMS VGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKL (SEQ ID NO: 253)

[0299] In some embodiments, the IL-2 receptor beta domain comprises a polypeptide sequence that shares 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 99%, or 100% identity to SEQ ID NO: 272.GKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLS SPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEA CQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGG SGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRP PGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV (SEQ ID NO: 272)

[0300] In some embodiments, the rapamycin-Activated Cell-Surface Receptor (RACR) and FRB domain complex comprises a polypeptide sequence that shares 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 99%, or 100% identity to SEQ ID NO: 254.MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKY MKSGNVKDLLQAWDLYYHVFRRISKASRRKRGSGEGRGSLLTCGDVEENPGPMPLPVTALLLPLALLL HAARPILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEA QEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGSNTSKENPFLFALEAVVISVGSMGLnSLLCVYFW LERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPC NQHSPYWAPPCYTLKPETGSGATNFSLLKQAGDVEENPGPMPLGLLWLGLALLGALHAQAGVQVETI SPGDGRTFPKRGQTCWHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAK LTISPDYAYGATGHPGIIPPHATLVFDVELLKLGEGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGP WLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDK VPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLS GEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDL VDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV (SEQ ID NO: 254)

[0301] In some embodiments, the rapamycin-activated cell-surface receptor (RACR) and FRB domain complex and anti-CD22 CAR comprises a polypeptide sequence that shares 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 99%, or 100% identity to SEQ ID NO: 255.MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKY MKSGNVKDLLQAWDLYYHVFRRISKASRRKRGSGEGRGSLLTCGDVEENPGPMPLPVTALLLPLALLL HAARPILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEA QEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGSNTSKENPFLFALEAVVISVGSMGLnSLLCVYFW LERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPC NQHSPYWAPPCYTLKPETGSGATNFSLLKQAGDVEENPGPMPLGLLWLGLALLGALHAQAGVQVETI SPGDGRTFPKRGQTCWHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAK LTISPDYAYGATGHPGIIPPHATLVFDVELLKLGEGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGP WLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDK VPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLS GEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDL VDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV GSGATNFSLLKQAGDVEENPGPLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQD ISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTF GGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPR KGLEWLGVIWGSETTYYNSALKSRLTnKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDY WGQGTSVTVSSGAVHTRGLDFACDFWVLVWGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFM RPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPR (SEQ ID NO: 255)

[0302] Some embodiments relate to or include an expression vector that comprises a gene encoding the free FRB linked to another protein. The linkage may include a 2A sequence (e.g. SEQ ID NO: 278 or similar to SEQ ID NO: 278). An example may include SEQ ID NO: 283 or sequence similar to SEQ ID NO: 283 which encodes a free FRB and includes a 2A sequence (e.g. 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: 283).ATGGAGATGTGGCACGAGGGACTGGAGGAGGCAAGCAGACTGTACTTTGGCGAGAGGAACGTGA AGGGCATGTTCGAGGTGCTGGAGCCACTGCACGCAATGATGGAGAGGGGACCTCAGACACTGAAG GAGACCTCCTTCAACCAGGCCTATGGCAGAGACCTGATGGAGGCCCAGGAGTGGTGCAGGAAGTA CATGAAGTCTGGCAATGTGAAGGACCTGCTGCAGGCCTGGGATCTGTACTATCACGTGTTTCGGAG AATCAGCAAGGCTAGCAGAAGAAAGAGAGGCAGCGGCGAGGGCCGAGGCAGCCTGCTGACCTGC GGTGATGTGGAAGAAAACCCGGGCCCC (SEQ ID NO: 283)

[0303] Some embodiments relate to or include an expression vector that comprises an ER signal sequence, a FRB sequence, a IL2RG sequence, and an optional 2A sequence. An example may include SEQ ID NO: 285 or sequence similar to SEQ ID NO: 285 (e.g. 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: 285). Such a sequence may encode the sequence of SEQ ID NO: 286, or a similar sequence to SEQ ID NO: 286 (e.g. 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: 286). Some embodiments include an ER signal-encoding sequence 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: 279. Some embodiments include an ER signal sequence 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: 287. Some embodiments include a FRB- encoding sequence 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: 257. Some embodiments include an FRB sequence that encodes a polypeptide 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: 252. The FRB sequence may include a T2098L mutation, which may be relative to a wild type or control FRB sequence. Some embodiments include an IL2RG-encoding sequence 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: 261. Some embodiments include an IL2RG sequence 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: 264. Some embodiments include a 2A sequence 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: 280.ATGCCCTTGCCCGTGACCGCGTTGCTCCTGCCCTTGGCTCTACTGCTGCACGCCGCTAGACCCATCC TGTGGCACGAGATGTGGCACGAGGGCCTGGAGGAGGCTAGCAGACTGTACTTCGGCGAGAGAAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCCCTGCACGCCATGATGGAGAGAGGCCCTCAGACCCT GAAGGAGACAAGCTTCAACCAAGCCTACGGCAGAGACCTGATGGAGGCCCAAGAGTGGTGCAGA AAGTACATGAAGAGCGGCAACGTGAAGGACCTGCTGCAAGCCTGGGACCTGTACTACCACGTGTT CAGAAGAATCAGCAAGGGCAGCAATACAAGCAAGGAAAACCCCTTCCTGTTCGCCCTGGAGGCCG TGGTGATCAGCGTGGGCAGCATGGGCCTGATCATCAGCCTGCTGTGCGTGTACTTCTGGCTGGAGA GAACCATGCCTAGAATCCCCACCCTGAAGAACCTGGAGGACCTGGTGACCGAGTACCACGGCAAC TTCAGCGCCTGGAGCGGCGTGAGCAAGGGCCTGGCCGAGAGCCTGCAGCCCGACTACAGCGAGCG ACTGTGCCTGGTGAGCGAGATTCCCCCTAAGGGCGGGGCCTTGGGTGAGGGACCCGGGGCAAGCC CGTGCAATCAGCACAGCCCCTACTGGGCCCCCCCCTGTTACACCCTGAAGCCCGAGACCGGCAGC GGAGCCACCAATTTCAGCCTCCTGAAACAAGCCGGTGACGTTGAAGAGAACCCCGGCCCC (SEQ ID 285)MPLPVTALLLPLALLLHAARPILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLK ETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGSNTSKENPFLFALEAVVIS VGSMGLnSLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIP PKGGALGEGPGASPCNQHSPYWAPPCYTLKPET (SEQ ID 286)

[0304] Some embodiments relate to or include an expression vector that comprises an ER signal sequence, a FKBP sequence, and a IL2Rb sequence. An example may include SEQ ID NO: 288 or sequence similar to SEQ ID NO: 288 (e.g. 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: 288). Such a sequence may encode the sequence of SEQ ID NO: 289, or a similar sequence to SEQ ID NO: 289 (e.g. 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: 289). Some embodiments include an ER signal-encoding sequence 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: 281. Some embodiments include an ER signal sequence 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: 290. Some embodiments include a FKBP-encoding sequence 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: 268. Some embodiments include a FKBP sequence 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: 253. Some embodiments include an IL2Rb-encoding sequence 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: 270. Some embodiments include IL2Rb sequence 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: 272.

[0305] Some embodiments relate to or include a small molecule-activated cytokine receptor. The small molecule-activated cytokine receptor may include a first and second polypeptide. The first polypeptide of the small molecule-activated cytokine receptor may include an FRB sequence and a IL2RG sequence. The first polypeptide of the small molecule-activated cytokine receptor may include 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%, at least 99%, or 100% identical to SEQ ID NO:451. Some such peptides are encoded by a polynucleotide 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: 450. Some such polynucleotides are included in a lentivirus herein.

[0306] The second polypeptide of the small molecule-activated cytokine receptor may include an FKBP sequence and a IL2RB sequence. The second polypeptide of the small molecule-activated cytokine receptor may include 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%, at least 99%, or 100% identical to SEQ ID NO: 453. Some such peptides are encoded by a polynucleotide 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: 452. Some such polynucleotides are included in a lentivirus herein.Compositions and formulations

[0307] In some embodiments, the disclosure provides a pharmaceutical composition comprising a particle according to the disclosure and a pharmaceutically acceptable carrier. The formulations and compositions of the present disclosure may comprise a combination of any number of viral particles, and optionally one or more additional pharmaceutical agents (polypeptides, polynucleotides, compounds etc.) formulated in pharmaceutically acceptable or physiologically-acceptable compositions for administration to a cell, tissue, organ, or an animal, either alone, or in combination with one or more other modalities of therapy. In some embodiments, the one or more additional pharmaceutical agents further increases transduction efficiency of viral particles.

[0308] In some embodiments, the formulations and compositions of the present disclosure may comprise a combination of any number of viral particles.

[0309] The present disclosure also provides pharmaceutical compositions comprising an expression cassette or vector (e.g., therapeutic vector) disclosed herein and one or more pharmaceutically acceptable carriers, diluents or excipients. In some embodiments, the pharmaceutical composition comprises a lentiviral vector comprising an expression cassette disclosed herein, e.g., wherein the expression cassette comprises one or more polynucleotide sequences encoding one or more chimeric antigen receptor (CARs) and variants thereof.

[0310] The pharmaceutical compositions that contain the expression cassette or vector genome may be in any form that is suitable for the selected mode of administration, for example, for intraventricular, intramyocardial, intracoronary, intravenous, intra-arterial, intra-renal, intraurethral, epidural, intrathecal, intraperitoneal, or intramuscular administration. The vector genome can be administered, as sole active agent, or in combination with other active agents, in a unit administration form, as a mixture with pharmaceutical supports, to animals and human beings. In some embodiments, thepharmaceutical composition comprises cells transduced ex vivo with any of the vector genomes according to the present disclosure.

[0311] The pharmaceutical compositions of the present disclosure, formulation of pharmaceutically acceptable excipients and carrier solutions may be useful to those of skill in the art, such as for development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, intraperitoneal, and intramuscular administration and formulation.

[0312] In some embodiments, the present disclosure provides formulations or compositions suitable for the delivery of viral vector systems (i.e., viral-mediated transduction) including, but not limited to, retroviral (e.g., lentiviral) vectors.Kits

[0313] Disclosed herein, in some embodiments, are kits. 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 fusion molecule. In some embodiments, the kit includes a particle. In some embodiments, the kit includes a composition described herein. The kit may include instructions for use, such as instructions for use in a method herein.

[0314] In some embodiments, the disclosure provides a kit comprising the particle and instructions for use in transduction of target cells and / or treatment of a subject. The kit may include a pharmaceutically acceptable carrier and / or an injection device. The kit may further include suitable tubing for administering the particles.

[0315] In some aspects, provided herein is a lentiviral vector comprising any one of the polycistronic constructs disclosed herein. In some aspects, provided herein is a cell comprising any of the lentiviral vectors disclosed herein. The lentivirus or cell may be included in a kit.Methods of Use

[0316] The compositions described herein such as fusion proteins or particles described may be used in vitro or ex vivo. The lentiviral particles described may be used ex vivo, in a cell manufacturing process or at a bedside as described, e.g., in Int’l Pat. Pub. No. WO 2022 / 072885, Int’l Pat. Pub. No. 2019 / 217954, Int’l Pat. Pub. No. 2020 / 123649, and Int’l Pat. Pub. No. 2009 / 072003. In some embodiments, the disclosure provides an ex vivo method of transducing target cells, comprising contacting the target cells with the particle according to the present disclosure. In some embodiments, the particles described herein may be used to transduce cells that have not been previously activated. For example, the particles described herein may be useful for transducing cells that have not been previously contacted with cell activation beads or activation reagents (e.g. Dynabeads or other reagents comprising anti-CD3 and / or anti-CD28 antibodies or binding fragments thereof). Where a methodherein describes use of a lentiviral particle, use of another particle is contemplated where appropriate and feasible. Where a method herein describes use of a lentiviral particle, use of a composition or fusion molecule is also contemplated where appropriate and feasible. For example, a fusion molecule, contained on the surface of a lentiviral particle, or a pharmaceutical composition may be administered to or contacted with a cell such as an immune cell (e.g. T cell).

[0317] In some aspects, provided herein is a method of transducing a cell comprising contacting a target cell with any of the polycistronic constructs disclosed herein. In some aspects, provided herein is a method of expressing a chimeric antigen receptor and / or a synthetic cytokine receptor in a target cell. In some aspects, provided herein is a cell produced by any of the methods disclosed herein. In some aspects, provided herein is a method of administering to a subject any of the cells disclosed herein. In some aspects, provided herein is a method of administering to a subject any of the lentiviral vectors disclosed herein.

[0318] Non-limiting examples of cells that can be the target of the lentiviral particle described herein include T lymphocytes, dendritic cells (DC), Tregcells, B cells, Natural Killer cells, and macrophages.Ex-Vivo Manufacturing

[0319] In some aspects, the disclosure provides a method of delivering a nucleic acid to a cell ex vivo. In some embodiments, the disclosure provides a method of delivering a nucleic acid to an immune cell ex vivo. In some embodiments, the lentiviral particles of the disclosure activate and transduce an immune cell ex vivo. In some embodiments, the disclosure provides a method of delivering a nucleic acid to a cell in an ex-vivo closed-loop manufacturing process. In some embodiments, an ex-vivo manufacturing process is an extracorporeal process. In exemplary embodiments, the lentiviral vectors disclosed herein permit delivery of a nucleic acid to a target cell during a closed-loop process. Exemplary methods of closed-loop and / or extracorporeal processes are disclosed in US Patent Publication No. 2021 / 0244871 and WO2022072885, both of which are incorporated herein in their entirety. In some embodiments, the lentiviral vectors as disclosed herein may be used to transduce cells ex vivo. For example, in exemplary closed-loop manufacturing processes, cells are obtained from a subject, washed, incubated and / or contacted with lentiviral particles, optionally washed again, and infused into the subject in a closed-loop system. In such embodiments, the lentiviral particles as disclosed herein are useful even without prior activation of the cells and are capable of binding to the cells in a short incubation and / or contacting step. In some embodiments, the incubation and / or contacting step is approximately or less than one hour. In some embodiments, the incubation and / or contacting step is approximately or less than one hour, approximately or less than two hours, approximately or less than three hours, approximately or less than four hours, or approximately or less than five hours. In some embodiments, the incubation and / or contacting step is less than 12 hours or less than 24 hours. In some embodiments, a nucleic acid is delivered to a cell by transduction with a lentiviral vector such that the nucleic acid enters the cell ex-vivo. In some embodiments, a nucleic acidis delivered to a cell by contacting the lentiviral vector to the surface of the cell. In such embodiments, the nucleic acid may enter the cell ex-vivo or in vivo after the cells (complexed with the lentiviral vector) are infused back into the subject.

[0320] In some embodiments, provided herein are bedside systems and methods for performing cellbased therapies and treatments in a subject-connected, closed-loop continuous-flow manner, including cellular modifications and treatments, e.g., to produce chimeric antigen receptor T (CAR T) cells. In some embodiments of a system described herein, blood is removed from a subject, processed, customized, and returned to the subject in a closed-loop, continuous-flow manner. An arrangement of modules and units are used sequentially for separation and collection of target cells from whole blood, employing for example, leukapheresis and / or other cell enrichment techniques, optionally including cell enrichment, purification and / or washing using an elutriation device, followed by one or more cell customization procedures, e.g., to generate CAR-T cells, optionally followed by cell enrichment, purification, fractionation, and / or washing, after which the processed and modified fraction comprising CAR-T cells are returned to the subject by means of an outlet conduit. One exemplary system is manufactured by Lupagen™ and is a closed-loop, continuous-flow system. Such systems and methods are disclosed in WO2019217964, which is incorporated herein by reference in its entirety.

[0321] In some embodiments, the lentiviral vectors as disclosed herein eliminate the need for an ex- vivo activation step. In such embodiments, the isolated cells could be transduced directly after leukapheresis, washing, or selection. It is contemplated that the surface engineering described herein may be useful for allowing the lentiviral particles disclosed herein to activate and transduce cells in a single step. In such embodiments, the lentiviral particles disclosed herein may enable a short or truncated manufacturing process, reducing the time spent in ex-vivo manufacturing by eliminating one or more unit operations (e.g. activation prior to transduction) and / or reducing the amount of time that may be necessary in post-transduction cell culture. Without wishing to be bound by theory, in some embodiments the lentiviral vectors as described herein, in particular those particles comprising a fusion multidomain protein, bind to target cells with a higher avidity than lentiviral particles not comprising a fusion multidomain protein. In such embodiments, the fusion multidomain protein may allow the described lentiviral particles to bind target cells more tightly, reducing the incubation time for transduction and increasing transduction frequency and efficiency. In some embodiments, the time to effectively bind a lentiviral particle to a target cell may be one hour or less.

[0322] It is contemplated that the present disclosure provides an ex vivo method of generating an engineered cell comprising contacting a target cell with a particle comprising a fusion molecule comprising an adhesion molecule linked to a costimulatory molecule, a fusion molecule comprising an adhesion molecule linked to an activation molecule, or a fusion molecule comprising an adhesion molecule linked to a costimulatory molecule and an activation molecule wherein the contacting step is performed for approximately one hour, for approximately two hours, approximately three hours, approximately four hours, approximately five hours, approximately six hours, approximately 12 hours,approximately 24 hours, approximately 12-24 hours (inclusive of endpoints), or longer. This method may require the contacting step to be performed in a closed-loop manufacturing or extracorporeal process as described herein. Alternatively, this method may require the contacting step to be performed in a traditional ex-vivo engineered cell manufacturing process. For example, in a perfusion incubator or a centrifuge (such as a Sepax or Rotea machine).Methods of Use in Vivo

[0323] Disclosed herein, in some embodiments, are methods comprising administering the lentiviral particle to a subject. In some embodiments, the subject has cancer or is in need of cancer treatment. In some embodiments, the administration treats the cancer. In some embodiments, the cancer comprises CD22+ cancer cells. In some embodiments, the cancer is or includes a B-cell malignancy. In some embodiments, the subject has cancer or is in need of treatment for an autoimmune disease. In some embodiments, the administration treats or alleviates symptoms of the cancer. In some embodiments, the administration treats or alleviates symptoms of the autoimmune disease. In some embodiments, the autoimmune disease is caused or exacerbated by B cells, in exemplary embodiments, CD22+ cells.

[0324] In some embodiments, the lentiviral particles described herein transduce target cells in vivo. In some embodiments, the target cells are immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the lentiviral particles described herein transduce T cells in vivo. In some embodiments, the lentiviral particles described herein transduce T cells in vivo generating CAR T cells. In some embodiments, the lentiviral particles described herein display a CD58-CD80-anti-CD3 scFv tri-fusion polypeptide and transduce T cells in vivo generating CAR T cells. Where a method herein describes use of a lentiviral particle, use of another particle is contemplated where feasible.

[0325] An example CAR T cell generated by a lentiviral particle herein is shown in FIG. 3C, which includes an anti-CD22 CAR. The figure also shows an FRB and RACR encoded by a viral payload in the T cell. Any aspect shown in the figure may be included in CAR T cell generated using a method herein. The CAR T cell may be generated in vivo, through, for example, transducing a subject in vivo via administering a lentiviral particle or a composition comprising the lentiviral particle.

[0326] In some embodiments, the disclosure provides an in vivo method of transducing target cells in a subject in need thereof, comprising administering to the subject a particle or pharmaceutical composition of the disclosure. The particle may be administered by any appropriate method including intranodal, intravenous, or subcutaneous injection. In some embodiments, the viral particle is administered via a route selected from the group consisting of intranodal, extracorporeal, parenteral, intravenous, intramuscular, subcutaneous, intratumoral, intraperitoneal, and intralymphatic. In some embodiments, the viral particle is administered multiple times. In some embodiments, the viral particle is administered by intralymphatic injection of the viral particle. In some embodiments, the viral particle is administered by intraperitoneal injection of the viral particle. In some embodiments, the viral particle is administered by intra-nodal injection - that is, the viral particle may be administered via injectioninto one or more lymph nodes. In some embodiments, the lymph nodes for administration are the inguinal lymph nodes. In some embodiments, the viral particle is administered by injection of the viral particle into tumor sites (i.e. intratumoral). In some embodiments, the viral particle is administered subcutaneously. In some embodiments, the viral particle is administered systemically. In some embodiments, the viral particle is administered intravenously. In some embodiments, the viral particle is administered intra-arterially. In some embodiments, the viral particle is a lentiviral particle.

[0327] In some embodiments, the lentiviral particle is administered by intraperitoneal, subcutaneous, or intranodal injection. In some embodiments, the lentiviral particle is administered by intraperitoneal injection. In some embodiments, the lentiviral particle is administered by subcutaneous injection. In some embodiments, the lentiviral particle is administered by intranodal injection.

[0328] The present disclosure provides a method of treatment comprising administering a therapeutically effective dose of lentiviral particles to a subject in need thereof. In some embodiments, a therapeutically effective dose of the lentiviral particles described herein are administered. In some embodiments, a therapeutically effective dose comprises about O.lxlO6transducing units (TUs), about 0.2xl06TUs, about 0.3xl06TUs, about 0.4xl06TUs, about 0.5xl06TUs, about 0.6xl06TUs, about 0.7xl06TUs, about 0.8xl06TUs, about 0.9xl06TUs, about lxlO6TUs, about 1.2xl06TUs, about 1.4xl06TUs, about 1.6xl06TUs, about 1.8xl06TUs, about O.lxlO6TUs, about O.lxlO6TUs, about O.lxlO6TUs, about O.lxlO6TUs, about 2xl06TUs, about 2.5xl06TUs, about 3xl06TUs, about 4xl06TUs, about 5xl06TUs, about 6xl06TUs, about 7xl06TUs, about 8xl06TUs, about 9xl06TUs, about lxlO7TUs, about 2xl07TUs, about 3xl07TUs, about 4xl07TUs, about 5xl07TUs, about 6xl07TUs, about 7xl07TUs, about 8xl07TUs, about 9xl07TUs, about lxlO8TUs, about 2xl08TUs, about 3xl08TUs, about 4xl08TUs, about 5xl08TUs, about 6xl08TUs, about 7xl08TUs, about 8xl08TUs, about 9xl08TUs, about lxlO9TUs, or about 2xl09TUs. In some embodiments, the therapeutically effective dose is about 1.2 x 108TU. In some embodiments, the therapeutically effective dose is about 3.5 x 108TU. In some embodiments, the therapeutically effective dose is about 1.0 x 109TU. In some embodiments, the therapeutically effective dose is about 3.0 x 109TU. In some embodiments, the therapeutically effective dose is about 9.0 x 109TU. In some embodiments, the therapeutically effective dose is between about 1.2 x 108TU and 9.0 x 109TU, inclusive.

[0329] Dose level may be measured in transducing units, determined by any known method. Exemplary methods include cellular culture or colony formation tests (wherein a target cell population is exposed to the particles and the number of transduced cells are counted), digital polymerase chain reaction (dPCR), or any other method known in the art.

[0330] In some embodiments, the transduced immune cells comprising the polynucleotide of the present disclosure is administered to the subject.

[0331] Some embodiments include administration of a composition herein (e.g. a lentiviral particle) as a monotherapy. Some examples include administration of a composition herein in combination with another compound. The other compound may include a compound that activates a cell surface receptor.For example, some embodiments include a small molecule that activates a small molecule-activated cell-surface receptor. Some embodiments include rapamycin for activation of a rapamycin-activated cell-surface receptor. Some examples include administration of a composition herein (e.g. a lentiviral particle) in combination with rapamycin.

[0332] A subject who is treated herein, or otherwise administered a composition herein may be relapsed / refractory to a prior treatment. The subject may have received a prior treatment. The subject may have failed a prior treatment. The prior treatment may include a viral or gene therapy treatment. The prior treatment may include a CAR T cell administration. The CAR T cells may have included an anti-CD19 CAR. The prior treatment may include a viral or gene therapy treatment. The prior treatment may include a cancer treatment. The prior treatment may include a chemotherapy.Treating cancer

[0333] Disclosed herein, in some embodiments, are methods of treating cancer. The method may include administering a composition or lentiviral particle herein to a subject. In some embodiments, the subject has cancer, is in need of cancer treatment, has been identified as having cancer, or has been identified as in need of cancer treatment.

[0334] The disclosure provides a method of treating a condition in a subject, comprising administering to the subject the lentiviral particles or pharmaceutical composition of the disclosure. The condition may include cancer. The cancer may be relapsed / refractory. The cancer may be CD22+. The cancer may include CD22+ B cells. The cancer may include malignant CD22+ B cells. In some embodiments, the condition is a B-cell malignancy, a myeloma, or a solid tumor malignancy. The disclosure provides a method of treating diffuse large B-cell lymphoma (DLBCL), Burkitt’s type large B-cell lymphoma (B-LBL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cell lymphoma (MCL), hematological malignancy, colon cancer, lung cancer, liver cancer, breast cancer, renal cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, brain cancer, squamous cell carcinoma, leukemia, myeloma, B cell lymphoma, kidney cancer, uterine cancer, adenocarcinoma, pancreatic cancer, chronic myelogenous leukemia, glioblastoma, neuroblastoma, medulloblastoma, or sarcoma in a subject, comprising administering to the subject the lentiviral particles or pharmaceutical composition of the disclosure.

[0335] In some embodiments, the disclosure provides a method of treating a B-cell malignancy in a subject comprising administering to the subject a lentiviral particle comprising a cocal-pseudotyped lentiviral envelope with a membrane-bound multidomain fusion protein and a transgene encoding an anti-CD22 chimeric antigen receptor (CAR) comprising an anti-CD22 scFv and 4-1BB and CD3z intracellular signaling domains. In some embodiments, the disclosure provides a method of treating a B-cell malignancy in a subject comprising administering to the subject a lentiviral particle comprising a cocal-pseudotyped lentiviral envelope with a membrane-bound multidomain fusion protein and a transgene encoding, in any order, (i) an anti-CD22 chimeric antigen receptor (CAR) comprising an anti-CD22 scFv and 4- IBB and CD3z intracellular signaling domains; (ii) an inducible T-cell proliferative signaling system (e.g. a synthetic cytokine receptor system or RACR as described herein); and, optionally, (iii) a human FRB domain.

[0336] In some embodiments, the disclosure provides a method of treating a lymphoma in a subject comprising administering to the subject a lentiviral particle comprising a cocal-pseudotyped lentiviral envelope with a membrane-bound multidomain fusion protein and a transgene encoding an anti-CD22 chimeric antigen receptor (CAR) comprising an anti-CD22 scFv and 4-1BB and CD3z intracellular signaling domains. In some embodiments, the disclosure provides a method of treating a lymphoma in a subject comprising administering to the subject a lentiviral particle comprising a cocal-pseudotyped lentiviral envelope with a membrane-bound multidomain fusion protein and a transgene encoding, in any order, (i) an anti-CD22 chimeric antigen receptor (CAR) comprising an anti-CD22 scFv and 4- IBB and CD3z intracellular signaling domains; (ii) an inducible T-cell proliferative signaling system (e.g. a synthetic cytokine receptor system or RACR as described herein); and, optionally, (iii) a human FRB domain.Treating autoimmune disorders

[0337] Disclosed herein, in some embodiments, are methods of treating an autoimmune disorder. The method may include administering a composition or lentiviral particle herein to a subject. In some embodiments, the subject has an autoimmune disorder, is in need of autoimmune disorder treatment, has been identified as having the autoimmune disorder, or has been identified as in need of autoimmune disorder treatment.

[0338] Some embodiments relate to a method of treating an autoimmune disorder. The method may include administering a composition or lentiviral particle herein to the subject. In some embodiments, the subject has a disorder comprising lupus. The lupus may include lupus erythematosus or lupus nephritis. In some embodiments, the disorder is or includes systemic lupus erythematosus (SLE). In some embodiments, the disorder is or includes lupus nephritis (LN).

[0339] Some embodiments relate to a method of treating a condition or disorder. In some embodiments, the condition is an autoimmune disease or disorder. The disclosure also provides a method of treating an autoimmune disease in a subject, comprising administering to the subject the lentiviral or pharmaceutical composition of the disclosure. Some examples of an autoimmune disease may include systemic lupus erythematosus, Sjogren’s syndrome, ANCA-associated vasculitis and autoimmune hemolytic anemia, rheumatoid arthritis, systemic sclerosis, multiple sclerosis, neuromyelitis optica spectrum disorder, chronic inflammatory demyelinating polyradiculoneuropathy, immune-mediated necrotizing myopathy, pemphigus vulgaris, dermatomyositis, adult-onset Still’s disease, inflammatory bowel disease, type 1 diabetes mellitus, graft vs. host disease, a myasthenia gravis, multiple sclerosis, immune dysregulation, polyendocrinopathy enteropathy X-linked (IPEX) or autoimmune arthritis.

[0340] In some embodiments, the disclosure provides a method of treating an autoimmune disorder in a subject comprising administering to the subject a lentiviral particle comprising a cocal-pseudotyped lentiviral envelope with a membrane-bound multidomain fusion protein and a transgene encoding an anti-CD22 chimeric antigen receptor (CAR) comprising an anti-CD22 scFv and 4-1BB and CD3z intracellular signaling domains. In some embodiments, the disclosure provides a method of treating an autoimmune disorder in a subject comprising administering to the subject a lentiviral particle comprising a cocal-pseudotyped lentiviral envelope with a membrane-bound multidomain fusion protein and a transgene encoding, in any order, (i) an anti-CD22 chimeric antigen receptor (CAR) comprising an anti-CD22 scFv and 4-1BB and CD3z intracellular signaling domains; (ii) an inducible T-cell proliferative signaling system (e.g. a synthetic cytokine receptor system or RACR as described herein); and, optionally, (iii) a human FRB domain.

[0341] Systemic lupus erythematosus (SLE) is an autoimmune disorder that affects millions of people, predominantly women. Despite advances in SLE therapeutics, including B cell-depleting therapies such as rituximab and belimumab, patients with lupus often have end organ disease with poor outcomes. Manifestations may include lupus nephritis (LN), which may lead to kidney disease. Patients with Class I and II LN generally have a favorable renal prognosis and do not require immunosuppressive therapy in the absence of extra-renal manifestations. Patients with Class in and IV LN first receive induction therapy that includes anti-inflammatory and immunosuppressive agents (e.g., combination therapy with glucocorticoids and either mycophenolate mofetil or cyclophosphamide), followed by long-term maintenance with prednisolone and immunosuppressive therapy (e.g., daily mycophenolate mofetil or azathioprine). However, induction of LN remission is often unsuccessful and treatment toxicities from high-dose corticosteroids, alkylating agents, and immunomodulatory agents are common. A significant unmet medical need persists for patients with this debilitating and sometimes fatal disease.

[0342] Due to their role in the pathogenesis of SLE, B cells are a useful target in the development of therapies for SLE treatment. B cells, as precursors of plasma cells that generate expression of autoantibodies, play a role in pathogenesis of SLE by secreting pro-inflammatory cytokines such as interferon gamma (fFN-y) and interleukin 6 (IL-6), acting as antigen-presenting cells, and activating T cells. Improved B-cell-targeted therapies are needed. CAR T-cell therapy may be useful to enhance eradication of B cells from circulation.

[0343] In some embodiments, the disclosure provides a method of treating systemic lupus erythematosus (SLE) in a subject comprising administering to the subject a lentiviral particle comprising a cocal-pseudotyped lentiviral envelope with a membrane-bound multidomain fusion protein and a transgene encoding an anti-CD22 chimeric antigen receptor (CAR) comprising an anti- CD22 scFv and 4-1BB and CD3z intracellular signaling domains. In some embodiments, the disclosure provides a method of treating SLE in a subject comprising administering to the subject a lentiviral particle comprising a cocal-pseudotyped lentiviral envelope with a membrane-bound multidomain fusion protein and a transgene encoding, in any order, (i) an anti-CD22 chimeric antigen receptor (CAR)comprising an anti-CD22 scFv and 4-1BB and CD3z intracellular signaling domains; (ii) an inducible T-cell proliferative signaling system (e.g. a synthetic cytokine receptor system or RACR as described herein); and, optionally, (iii) a human FRB domain.

[0344] SLE symptoms may include painful or swollen joints, fever, chest pain, hair loss, mouth ulcers, swollen lymph nodes, feeling tired, or a red rash such as a rash affecting the face. Symptoms may include periods of illness, called flares, or periods of remission during which there are few symptoms. Any of said symptoms may be improved upon a treatment provided herein.

[0345] In some embodiments, the lupus or SLE develops into LN. In some embodiments, the lupus or SLE does not develop into LN. Some embodiments include treating a subject in need of SLE treatment. Some embodiments include treating a subject in need of SLE treatment, wherein the administration is given before development of LN, or in the absence of LN. Some embodiments include treating a subject in need of SLE treatment, after the SLE develops into or includes LN. Some embodiments relate to a method of treating LN, comprising administering to a subject in need of LN treatment a composition described herein.Manufacturing Methods

[0346] 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 fusion molecule (or fusion protein) as described herein. The fusion molecule (or fusion protein) and the vector genome are expressed by the host cell. The host cell packages the vector genome into a lentiviral particle comprising the fusion molecule (or fusion protein).

[0347] Lentiviral particles of the present disclosure may enhance in vivo activity. Lentiviral particles of the present disclosure may resist serum inactivation. Lentiviral particles of the present disclosure provide efficient targeting of activated T cells. Lentiviral particles of the present disclosure may require low physical particle per transducing unit compared to two component glycoproteins. Lentiviral particles of the present disclosure retain potential to transduce a broad range of non-T effector cells. Lentiviral particles of the present disclosure enhance particle to T cell binding. Lentiviral particles of the present disclosure enhance T cell activation. Lentiviral particles of the present disclosure enhance immune cell expansion. Lentiviral particles of the present disclosure enhance immune cell transduction. Lentiviral particles of the present disclosure enhance anti-tumor potency. Lentiviral particles of the present disclosure enhance immune cell persistence.

[0348] Some embodiments include a method of making an adhesion molecule, a costimulatory molecule, an activation molecule, or a fusion molecule. The method may include transcribing or translating a nucleic acid (such as a DNA or RNA) that encodes a protein comprising the adhesion molecule, costimulatory molecule, activation molecule, or fusion molecule.Definitions

[0349] 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.

[0350] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0351] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and 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 the breadth of the range.

[0352] The singular forms “a”, “an”, and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0353] 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).

[0354] As used herein, the term “about” a number refers to that number plus or minus 15% of that number. The term “about” a range refers to that range minus 15% of its lowest value and plus 15% of its greatest value.

[0355] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.

[0356] The terms “subject,” “individual,” or “patient” are often used interchangeably herein. A “subject” can be an animal. The subject can be a mammal. The mammal can be a human. The subject may be diagnosed or suspected of being at risk for a disease.

[0357] As used herein, the term “similar” may refer to a polynucleotide or polypeptide sequence at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% to a reference sequence.

[0358] A percent sequence identity may be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in thecomparison window may comprise additions or deletions (i.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 window of comparison and multiplying the result by 100 to yield the % sequence identity. A sequence identity may include a sequence identity to a reverse complement. 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. Any discrepancies between the written description and

[0359] Any discrepancies between the written description and a sequence listing submitted herein may be resolved in favor of the written description.

[0360] As used herein, the terms “treatment” or “treating” may be used in reference to a pharmaceutical 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 refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the 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.

[0361] A “transduction unit” or “TU” as it relates to a viral particle indicates 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 a CAR flow cytometry analytical method that measures functional CD22 CAR lentiviral particle titer.EXAMPLES

[0362] The following Examples describe how embodiments of the invention may be made, evaluated, and used. The Examples are intended to be illustrative and non-limiting.Example 1

[0363] This Example shows in vitro data characterizing particles containing an anti-CD22 CAR payload (Product A) and a RACR-anti-CD22 CAR (Product B). Diagrams of some aspects of Product A and Product B with a comparison of their payloads are included in FIG. 5.

[0364] Product A is a 3rdgeneration, self-inactivating (SIN), replication-incompetent lentiviral vector (LW) product comprising: a cocal-pseudotyped lentiviral envelope with a membrane-bound multidomain fusion protein (MDF) (FIG. 1), and a transgene encoding an anti-CD22 chimeric antigen receptor (CAR) comprising an anti-CD22 single chain variable fragment and 4- IBB and CD3^ intracellular signaling domains (FIG. 2). Example amino acid sequences for an MDF protein as used lin Product may include SEQ ID NO: 446 or 448. Product A is an example of a lentiviral particle, comprising: a polynucleotide encoding a chimeric antigen receptor (CAR) that specifically binds cluster of differentiation-22 (CD22). Product A includes the nucleotide sequence of SEQ ID NO: 291, encoding an anti-CD22 CAR that includes the amino acid sequence of SEQ ID NO: 292. Product A selectively binds, activates and transduces T cells in vivo to generate CAR T cells that express anti-CD22 CAR. Expression of the anti-CD22 CAR mediates antigen specific killing against CD22-positive target cells. Product A can be administered intranodally into superficial inguinal lymph nodes under ultrasound guidance or administered intravenously. Product A may be given without the need for lymphodepletion.

[0365] Product B is similar to A, but further includes a rapamycin-activated cytokine receptor (RACR) and other aspects in this paragraph. Product B is an example of a lentiviral particle comprising: a polynucleotide encoding a chimeric antigen receptor (CAR) that specifically binds cluster of differentiation-22 (CD22), an inducible T-cell proliferative signaling system (e.g. rapamycin-activated cytokine receptor, RACR) and a human protein domain (e.g. FRB) derived from the mammalian target of rapamycin (mTOR) complex that binds intracellular rapamycin-FKBP complexes to confer rapamycin resistance to transduced cells. Product B includes the nucleotide sequence of SEQ ID NO: 291, encoding an anti-CD22 CAR that includes the amino acid sequence of SEQ ID NO: 292. Product B further includes the nucleotide sequence of SEQ ID NO: 283, encoding an FRB protein sequence of SEQ ID NO: 251, and including a 2A sequence of SEQ ID NO: 278. Product B further includes the nucleotide sequence of SEQ ID NO: 285, encoding the amino add sequence of SEQ ID NO: 286 (which includes an ER signal sequence of SEQ ID NO: 287, a T2098L FRB sequence of SEQ ID NO: 252, and a IL2RG sequence of SEQ ID NO: 264), and including a 2A sequence of SEQ ID NO: 280. Product B further includes the nucleotide sequence of SEQ ID NO: 288, encoding the amino add sequence of SEQ ID NO: 289 (which includes an ER signal sequence of SEQ ID NO: 290, a FKBP sequence of SEQ ID NO: 253, and an IL2Rb sequence of SEQ ID NO: 272). The nucleotide sequences of SEQ ID NO: 291, 283, 285, and 288 are finked as part of a single polynucleotide vector in Product B. Product B selectively binds, activates and transduces T cells in vivo to generate CAR T cells that express anti- CD22 CAR. Expression of the anti-CD22 CAR mediates antigen specific killing against CD22-positive target cells. Like Product A, Product B can be administered intranodally into superficial inguinal lymph nodes under ultrasound guidance or administered intravenously. Product B may be given without the need for lymphodepletion.

[0366] This example demonstrates that both Product A and Product B potently and specifically bind and activate T cells in vitro, resulting in the generation of functional CAR T-cells. CAR T-cells generated from Product A and Product B demonstrate potent cytotoxicity and cytokine production in vitro in an antigen-specific manner. Finally, CAR-T cells generated from RACR-containing Product A enrich and expand when treated with RACR-stimulating agents in vitro.Vector Production and Characterization

[0367] Suspension 293T cells were grown to a density of 2E6 cells / mL and transfected with relevant vector production plasmids. Two days post transfection, lentiviral vector was harvested from the supernatant, clarified, and purified via a Mustang Q capsule (Cytiva). Eluted samples were buffer exchanged by tangential flow filtration (Sartorius Vivaflow50, VF05P4) and sterile filtered with a 0.2 mm PES syringe filter (Millipore). Vector titer was assessed using a SUP-T1 (ATCC, CRL-1942) transduction assay. SUP-T1 gDNA was analyzed for payload integration using ddPCR primers specific to the lentiviral psi packaging signal (Fwd primer: 5’ ACTrGAAAGCGAAAGGGAAAC-3’, Rev primer: 5’- CACCCATCTCTCTCCTTCTAGCC-3’, and psi FAM probe: 5’- FAM / AGCTCTCTC / ZEN / GACGCAGGACTCGGC / 3IABkFQ-3’) and normalized to RPP30 housekeeping gene (Fwd Primer: 5’- GCTrCCAAGAAAGCCAAGTG-3’, Rev Primer: 5’- GGGAAGAAGGGAGTGCTGAC-3’, and RPP30 HEX Probe: 5’-HEX / TGAGGGCTG / ZEN / AAAAGAATGCCCCAGTCTCT / 3IABkFQ-3’) on the QX200 Droplet Generator Digital system (Bio-Rad). Vector particle number was quantified using a Videodrop (Myriade).T cell Binding, Activation and transduction

[0368] To test the ability of Product A and Product B particles to bind T cells in a dose dependent manner PBMCs and particles were cultured together for 1 hour at 10E6 cells / mL at various multiplicities of infection (MOI) at room temperature (FIG 6). Cocal (particle binding) on T cells was evaluated by flow cytometry using anti-Cocal antibody clone 20D10 (generated in cooperation with Genscript).

[0369] PBMCs were transduced at a density of 2E6 cells / mL by adding vector directly to the well at various MOIs. 1 day later the cells are washed and plated in fresh X-Vivo media supplemented with IL- 2 (250 lU / ml). Activation is assessed on day 3 and transduction on day 7 by flow cytometry. For flow cytometry cells were washed with PBS, stained with a fixable viability dye, washed with FACS buffer (Thermo Fisher Scientific), and then stained for 30min in FACS buffer containing the relevant antibody cocktail used at 1:100 dilution. Cells were enumerated with Cellaca MX (Nexcelom). Data were acquired on Attune Flow Cytometers (Thermo Fisher Scientific). Flow cytometry analysis showed Product A and Product B particles bound CD3+ cell similarly indicating that Product A and Product B are capable of binding to T cells (FIG. 7).

[0370] To test the ability Product A and Product B particles to activate and transduce T cells in a dose dependent manner unstimulated PBMCs from 3 healthy doners were incubated with Product A or Product B particles at various MOIs for 24hrs (beginning at day 0). Product A or Product B particles were added to PBMCs from 3 healthy donors at several MOIs at a concentration of 10e6 PBMCs / ml. After a room temperature incubation for 1 hour, flow cytometry was used to identify viral cocal glycoprotein bound to CD3+ T cells. The majority of T cells stained positively for the cocal glycoprotein, even at MOI=1, the lowest dose tested. These data indicate Product A and Product B potently bind T cells. Three days after particle addition, T cell activation was assessed using flow cytometry for the activation surface marker CD25. Consistent with the binding data, particles of Product A and Product B activated T cells, and this occurred in a dose-dependent manner. Seven days after adding the particles, flow cytometry was used to detect the G4S flexible linker, a component of the anti- CD22 CAR, in T cells to measure transduction efficiency. On Day 7, expression of the CD22 CAR was assessed by flow cytometry (FIG. 6). Flow cytometry analysis of CD22 CAR+ T cells showed that Product A and Product B particles efficiently transduce T cells in a dose dependent manner (FIG. 8). Overall, these data show that Product A and Product B particles bind, activate, and transduce T cells in vitro, without the need for prior stimulation.CAR T-cell Function

[0371] To assess CAR T-cell cytotoxicity an Incucyte® killing assay was used which involves coculturing CAR+ T cells with fluorescent tumor cell lines. The fluorescence intensity is then assessed over time to measure tumor cell control. This study utilized two different CD22+ tumor cell fines, Nalm6 and Raji which express differing levels of CD22 protein. Seven days after transduction CAR+ T-cells were measured using flow cytometry and killing assays were set up at multiple CAR T- celktumor effector: target (E:T) ratios. As shown herein, anti-CD22 CAR T-cells were potently cytotoxic at all E:T ratios tested. (FIG. 9).

[0372] To test whether the anti CD22 CAR T killing is CD22 antigen dependent, T cells transduced with Product A or Product B particles were cocultured with wildtype and CD22 knockout Raji cells. Seven days after transduction with Product A or Product B particles CAR-T cells were cocultured with wildtype and CD22 knockout Raji cells at 0.25:1 E:T ratio (CAR: Tumor Cell). Growth of the tumor cells was monitored based on tumor cell fluorescence using an Incucyte. Wildtype Raji cells were killed by Product A and Product B transduced T cells but the CD22 knockout Raji cells were not killed by either CAR T cell (FIG. 10A). This example shows that the killing of CD22+ tumor cells by Product A and Product B transduced CAR-T cells is antigen dependent.

[0373] Next the ability of Product A and Product B particles to generate CAR T-cells that increase cytokine production upon stimulation with CD22+ target cells was tested. Seven days after particle addition to PBMCs, CAR T-cells were quantified by flow cytometry and cultured with various tumorcell lines for 24 hours at a 1 : 1 E:T ratio. The supernatant was collected and quantified for levels of IFN- y, IL-2, and TNF-a using a Meso Scale Discovery (MSD) assay. As shown herein, when cultured with CD22 positive Nahn6 and Raji cells production of all three T cell activation cytokines was observed (BIG. 11). Cytokine production was not observed when the anti-CD22 CAR T-cells were cultured with CD22 negative K562 or Raji CD22 KO cells, indicating the cytokine production is antigen-dependent.Characterization ofrapamycin activated cytokine receptor (RACR)

[0374] In addition to the anti-CD22 CAR, Product B particles package the Rapamycin-activated cytokine receptor (RACR). This system combines the rapamycin binding proteins FKBP12 and the FRB component of mTOR with the IL-2 / IL-15 signaling domain. When rapamycin is added to the RACR positive cells it induces an IL-2 / IL-15-like proliferation and survival signal. To assess the activity of the rapamycin activated cytokine receptor (RACR) in Product B, CAR-T cells were generated by transducing PBMCs with Product B particles as described above. On day 7 (after transduction) 5xl05anti-CD22+ CAR T cells were grown in culture with IL-2 only (50IU / ml), Rapamycin only (lOnM), AP21967 only (50nM) and IL-2 (50IU / ml) + Rapamycin (lOnM) for 21 days. On day 7, 14 and 21 the percentage of anti-CD22+ CAR T cells of CD3+ cells was quantified via flow cytometry. IL-2 alone did not increase the percentage of anti-CD22+ CAR T cells. Rapamycin only (lOnM) resulted in an increase in the percentage of anti-CD22+ CAR T cells showing that the RACR construct is active and stimulated by rapamycin. AP21967 only (50nM), a rapamycin analog, resulted in an increase in the percentage of anti-CD22+ CAR T cells showing that the RACR construct is active and stimulated by a rapamycin analog. IL-2 (50IU / ml) + Rapamycin (lOnM) resulted in in an increase in the percentage of anti-CD22+ CAR T cells showing that the RACR construct is active and stimulated by a rapamycin when the T cell is also stimulated with IL-2 (FIG. 12).

[0375] To test the ability of RACR expressing anti-CD22 CAR T cells to expand when stimulated through RACR, CAR-T cells were generated by transducing PBMCs with Product B particles. On day 7 (after transduction) 5xl05anti-CD22+ CAR T cells were grown in culture with IL-2 only (50IU / ml), Rapamycin only (lOnM), AP21967 only (50nM) and IL-2 (50IU / ml) + Rapamycin (lOnM) for 21 days. On day 7, 14 and 21 the number of anti-CD22+ CAR T cells was quantified using flow cytometry. Treatment with IL-2 alone resulted in a 5.6-fold increase in the number of anti-CD22+ CAR T cells. Treatment with Rapamycin only (lOnM) resulted in a 6-fold increase in the number of anti-CD22+ CAR T cells. Treatment with AP21967 only (50nM) resulted in a 20-fold increase in the number of anti-CD22+ CAR T cells. Treatment with IL-2 (50IU / ml) + Rapamycin (lOnM) resulted in a 17-fold increase in the number of anti-CD22+ CAR T cells (FIG. 13).

[0376] This example shows Product A and Product B particles containing anti-CD22 CAR payload selectively bind and activate T cells. Product A and Product B particles containing anti-CD22 CAR payload are also able to efficiently transduce T cells. Anti-CD22 CAR T cells generated by infection with Product A or Product B particles exhibit antitumor function in a CD22-dependent manner (killingof tumor cells and cytokine production). Anti-CD22 CAR T cells generated by infection with Product B, which contains the RACR construct, are enriched and expand when exposed to rapamycin or rapamycin analogs.Example 2 - Clinical Trial with Product A

[0377] Product A is described in Example 1. This example describes a Phase 1 study of Product A in relapsed / refractory (R / R) CD22+ large B-cell lymphoma (LBCL) or systemic lupus erythematosus (SLE). Subjects eligible for this study may be CAR-T naive (that is, have not previously received CAR- T therapy) or CAR-T exposed (that is, have previously received CAR-T therapy). This example refers to often to Product A. The methods provided in this example may be applicable to Product B, except where the distinguishing features of Product A over Product B are relevant.

[0378] Dose levels: Table 5 outlines possible dose levels (DL) for Product A.Table 5: Dose Levels for Product A

[0379] Intermediate dose levels can be added between DLs in Table 5. Safe intermediate DLs can be calculated using the Bayesian Optimal Interval (BOIN) design. Normal saline may be used as the diluent for Product A.

[0380] Administration of Product A: Product A can be administered intranodally (IN) or intravenously (IV).

[0381] Subject monitoring: Following administration of Product A, the subject will be monitored for any infusion reaction per institutional guidelines. If the infusion is stopped prior to the total volume of Product A being administered the volume that was administered can be recorded.

[0382] Useful sites for intranodal injections: Intranodal injections can be performed in the inguinal lymph nodes. The superolateral, superomedial, the inferolateral or the inferomedial groups of lymph nodes can be used for intranodal injections of Product A.

[0383] Visualization of target lymph nodes: Visualization of target lymph nodes may be performed using ultrasound. A high frequency transducer may be used for thin subject (10-15 MHz) and a lower frequency probe (less than 10 MHz) may be used for larger subjects.Example 3 - Clinical Trial with Product B

[0384] Product B is described in Example 1. This example describes a Phase 1, open-label study of Product B in combination with rapamycin in relapsed / refractory (R / R) CD22+ large B-cell lymphoma. Subjects eligible for this study may be CAR-T naive (that is, have not previously received CAR-Ttherapy) or CAR-T exposed (that is, have previously received CAR-T therapy). The methods provided in this example may be applicable to Product A, except where the distinguishing features of Product B over Product A are relevant.Study rationale

[0385] Product B particles can be administered intranodally (IN) to generate CAR T cells, which are designed to bind to the CD22 antigen present on B-cells.

[0386] For this example, Product B will be administered IN and its potential benefit to subjects will be evaluated. IN administration provides access to a high concentration of naive and memory T cells, which may enhance CAR T cell persistence; moreover, depending on proximity to the tumor, IN administration of Product B may stimulate tumor antigen-specific T cells and expand the pool of T cells engaged to target tumor.

[0387] Rapamycin will be administered to subjects receiving Product B in the final step of dose finding for the final combination therapy. Rapamycin may have multiple potential benefits to patients in the context of Product B administration. First, it will interact with the rapamycin-activated cytokine receptor (RACR) payload elements (RACRy, RACR , FKBP-12) expressed in CAR T cells to provide proliferative signals for CAR T cell enrichment. Second, it may suppress the activity of non-transduced cells. Overall, these effects are intended to enhance enrichment of CAR T cells in the absence of lymphodepleting chemotherapy (LDC).

[0388] The dosing interval between Product B and rapamycin is designed to balance in vivo T cell activation / transduction and profiferation of CAR T cells through the RACR system by suppressing nontransduced cells, enhancing enrichment and expansion of transduced cells, and dampening anti-CAR T cell immune suppression. The timing of rapamycin dosing seeks to minimize safety concerns by allowing a “window” between Product B dosing and rapamycin initiation.Study Design

[0389] This is a Phase 1 study of the safety, efficacy, and PK of Product B in combination with rapamycin in adult subjects diagnosed with relapsed refractory (R / R) CD22+ LBCL. LBCL subjects with aggressive lymphoma such as diffuse large B-cell lymphoma (DLBCL), transformed DLBCL (tDLBCL), follicular lymphoma grade 3B (FL3B), and primary mediastinal B-cell lymphoma (PMBCL) will be enrolled. Subjects may have received / failed a CD19-directed CAR cell treatment.

[0390] The objective of the Phase 1 portion of this study is to determine the MTD / MAD and RP2D(s) of Product B + / - rapamycin using intranodal (IN) route of administration (ROA).

[0391] Eligible subjects will initially be enrolled in the dose finding (DF) phase of the study at doses anticipated to be safe and potentially biologically active. Once doses are cleared for safety per the Bayesian optimal interval (BOIN) study design, while DF continues, subjects may be backfilled to the dose(s) that demonstrate promising anti-tumor activity.

[0392] DF consists of two stages with stage 1 aiming to identify the MTD / MAD of Product B as a monotherapy and stage 2 aiming to identify the MTD / MAD of Product B in combination with rapamycin.

[0393] The subjects will receive a single dose of Product B at the assigned dose level on day 1 of this study. For subjects receiving rapamycin, commercially available rapamycin may be started within days 4-6 of this study (approximately 72 to 120 hours after the administration of Product B) and continue for a total of 60 days, as tolerated.Methodology

[0394] Subjects will be consented and screened. Eligible subjects will receive Product B on day 1 of this study. Rapamycin will be added once Product B DF is complete. Rapamycin dosing will be once daily targeting a trough level of 10 ng / mL and within in the range for trough levels starting within day 4 of this study for 60 days total, as tolerated. In the Phase 1 portion of the study, dose limiting toxicides (DLTs) will be assessed for 28 days from Product B treatment or 14 days from last dose of rapamycin, whichever is later. Safety will be assessed throughout the study for both Phase 1 and Phase 2. Efficacy will be evaluated at approximately Months 1, 3, 6, 9, 12, 18, and 24 following Product B administration.

[0395] After treatment, subjects will be followed on this study for 2 years for collection of safety data, disease status, and survival. All Product B treated subjects that complete this study and those that choose to withdraw from the study early, will be asked to enroll in a long-term follow-up (LTFU) study that will continue to follow subjects for survival and safety for up to 15 years following treatment with Product B.Eligibility

[0396] Inclusion criteria: LBCL subjects with aggressive lymphoma such as diffuse large B-cell lymphoma (DLBCL), transformed DLBCL (tDLBCL), follicular lymphoma grade 3B (FL3B), and primary mediastinal B-cell lymphoma (PMBCL) will be enrolled. Patients will be age 18 or older at time of consent and have measurable disease according to Lugano 2014 criteria. Subjects who have previously had CD22-directed therapy will have a biopsy confirming presence of CD22 expression since completing the prior CD22-directed therapy.Dosage of Product B

[0397] Product B will be dosed based on a subject’s assigned DL. Dosing of Product B may be based on Transcription Units (TUs), defined by digital polymerase chain reaction (dPCR), which measures functional Product B particle titer.

[0398] Rapamycin dosing will be once daily, as tolerated, targeting a trough level of 10 ng / mL and within in the range for trough levels approved per country.Administration

[0399] Product B: The initial proposed route of administration (ROA) for Product B is direct delivery into a lymph node chain (IN).

[0400] Rapamycin: Rapamycin will be administered once daily starting within days 4-6 of this study (i.e., 3 - 5 calendar days after dosing with Product B). Trough level targeting approximately 10 ng / mL (as ascertained by standard commercial assay at treating institutions and within trough level range of country PI) may be checked daily until therapeutics trough is achieved, then bi-weekly until discontinued.

[0401] The following guidance may be used for dose adjustments to achieve the target trough level. For trough level < 4-5 ng / mL, dose may be increased 10-25% (to nearest half pill, or more precisely if using Equid formulation) and checked trough after two doses at new dose. For trough levels > 12-15 ng / mL, the next dose may be held and reduced by 10-25% (to nearest half piU, or more precisely if using Equid formulation) and check trough after 2 doses at new dose.Duration of Treatment

[0402] Product B wiE be dosed as a single injection on day 1 of this study.

[0403] For those receiving rapamycin, it wiE start within the range of days 4-6 of this study (assuming they meet treatment initiation criteria) and continue for 60 days, as tolerated. If a dose is not tolerated oraEy (defined as vomiting any / aE administered dose within 30 minutes of oral administration), then a dose may be repeated at earliest cEnical convenience.Retreatment with Product B and / or Rapamycin

[0404] If a subject achieved a partial response (PR) and did not experience a dose limiting toxicity (DLT) while receiving rapamycin during their initial treatment course, the subject may receive another course of rapamycin for 60-days, as tolerated.

[0405] If a subject achieved a CR at any response assessment after their initial treatment with Product B + / - rapamycin and experienced disease progression (either determined cEnicaEy or by imaging study), and the subject did not experience a DLT in their initial treatment course, the subject may receive another dose of Product B (Retreatment Day 1) at the same DL that the subject previously received foEowed by rapamycin (if previously received) for 60 days (starting within the range of Retreatment Days 4 - 6), as tolerated. Post treatment assessments wEl be performed after their first dose of retreatment.Study Assessments and ProceduresPharmacokinetics (PK) and Pharmacodynamics (Pd) Testing

[0406] PK: Peripheral blood will be obtained to assess Pk of Product B particles in peripheral blood. PK may also evaluate maximum CAR T cell concentration (Cmax), time of maximum concentration (Tmax), total exposure as measured by total area under the curve from Day 1 to Day 29 (AUCO-28). Commercial assays for evaluation of rapamycin PK may be used to monitor trough levels and adjust dosing accordingly, as per rapamycin administration instruction above.

[0407] Pd: Peripheral blood will be obtained to assess Pd of Product B and B cell aplasia.

[0408] If bone marrow biopsy and lymph node biopsies are done per standard of care (SOC), tissue samples will be requested to assess Pd in those tissues as well.

[0409] Based on evaluation of potential immunogenicity and risk assessment to subject safety, immunogenicity evaluations will be obtained to evaluate antibodies to cocal glycoprotein (Product B pseudotyping) and anti-CD22 CAR protein (payload protein) starting Day 1, at restaging, and every response assessment thereafter.Disease Assessment

[0410] Disease Assessments will be performed at Baseline during the screening period, then at Months 1, 3, 6, 9, 12, 18, and 24. Assessments will utilize the Lugano 2014 criteria.

[0411] Additionally, peripheral blood (and bone marrow, when indicated for a given indication) may be collected for possible minimal residual disease testing and correlative analyses to safety / efficacy outcomes.Statistical Methods

[0412] The Bayesian Optimal Interval (BOIN) design will be used to find the MTD / MAD of Product B + / - rapamycin. The BOIN design is implemented in a manner similar to the traditional 3+3 design but is more flexible and possesses superior operating characteristics that are comparable to those of the more complex model-based designs, such as the continual reassessment method (CRM). BOIN received the fit-for-purpose designation from the FDA (2021) as a tool for drug development.

[0413] The DF consists of 2 stages: stage 1 aiming to identify the MTD / MAD of Product B as a monotherapy, and stage 2 aiming to identify the MTD / MAD of Product B in combination with rapamycin. The target toxicity rate for the MTD is = 0.25 and the maximum sample size is 33, of which 21 is for stage 1 monotherapy DF and 12 is for stage 2 combination DF. Subjects will be enrolled and treated in cohorts of n= 3. DLTs are defined in the safety section above, and only those DLTs that occur within the first 28 days from Product B treatment or 14 days from last dose of rapamycin, whichever is later, will be used to make decisions for dose escalation / de-escalation. The BOIN design uses the following rule, optimized to minimize the probability of incorrect dose assignment, to guide dose escalation / de-escalation:1. if the observed DLT rate at the current dose is < 0.197, escalate to the next higher dose level;2. if the observed DLT rate at the current dose is > 0.298, de-escalate to the next lower dose level;3. otherwise, stay at the current dose level.Table 6: Dose Escalation / De-escalation Rules Per the BOIN Design

[0414] To better characterize and optimize the RP2D, depending on the toxicity and PK / Pd data, backfill (up to 6-9 additional subjects per dose) could start once the given been deemed tolerable per the BOIN design.

[0415] After the MTD / MAD of Product B in combination with rapamycin is determined, DE with up to up to 12 subjects per dose may be conducted at doses demonstrating safety (i.e., no higher than the MTD / MAD) to gain more experience with toxicity, tolerability, PK, and anti-tumor activity of the doses.

[0416] During the dose expansion, safety will be monitored using the BOIN de-escalation boundary after 6 and 12 patients, i.e., the dose expansion will be halted if [the number of patients with toxicities] / [the number of patients treated] >= 2 / 6 and 4 / 12.

[0417] Data obtained from subjects enrolled into backfill and dose expansion will be utilized to aid in the determination of the RP2D. The RP2D will be chosen based on considerations of the MTD estimated per the BOIN, and on an overall assessment of safety data from subsequent cycles and efficacy / PK information collected at all different doses tested. The RP2D is required to be no higher than the MTD.Data Analysis

[0418] Data from the DF and DE portions will be combined for analyses. Analysis of primary, secondary, and exploratory endpoints will be descriptive and may include summary statistics such as means, standard deviations, and 95% confidence intervals (if applicable). AEs / SAEs, AESIs, and laboratory abnormalities will be described and summarized by doses.

[0419] Categorical efficacy endpoints (e.g., ORR) will be summarized by frequency and 95% confidence intervals, if applicable. Kaplan-Meier curves and median time-to-event data will be presented for time-to-event variables (e.g., DOR, PFS, and OS).Example 4

[0420] Experiments in Example 4 and Example 5 were conducted using lentiviral particles with payloads encoding an anti-CD20 CAR to demonstrate aspects such as lentiviral administration, in vivo transduction, and in vivo CAR T cell generation. These examples are expected to be applicable to similar particles with payloads encoding an anti-CD22 CAR.

[0421] Example 4 describes the efficacy of lymph node administration of engineered viral particles in non-human primates. T cell transduction and activation with viral particles surface engineered to display a CD58 and CD80 di-fusion polypeptide and anti-CD3 scFv showed in vivo CAR T cell generation. The viral particles were engineered to display an anti-CD3 scFv that binds NHP CD3 and a payload comprising a human-specific anti-CD20 CAR which cross-reacts with NHP CD20 (BIG. 15A). Similar viral particles are contemplated which may be engineered to display an anti-CD3 scFv that binds NHP CD3 and a payload comprising a human-specific anti-CD22 CAR, which may cross-react with NHP CD22.Table 7: Abbreviations

[0422] The objectives of the study included analyzing the ability of engineered viral particles to transduce T cells and generate functional CAR T cells in vivo in a large animal model. Another objective of the study was to assess lymph node injection as a viable route of administration for engineered viral particles. Another objective of the study was to analyze the function of the generated CAR T cells on B cell depletion as well the persistence of the CAR T cells and / or B cell depletion.

[0423] In the present NHP study, the subjects of the study were Pig-tailed macaques (Macaca nemestrina) that were either male or female, a minimum size of 3.5-5 kg, no age restriction (other than to ensure sufficient size). Three animals were studied. A summary of the animals treated is shown in FIG. 15C.

[0424] Engineered viral particles were generated with surface molecules that are cross-reactive with pig-tailed macaque (Human CD58-Human CD80 fusion polypeptide and an NHP-specific anti-CD3scFv) (BIG. 15A) and anti-CD20-CAR and low-affinity nerve growth factor receptor (LNGFR) molecule payload (FIG. 15B).

[0425] The particles were dosed in transducing units per kg (TU / kg) as determined by ddPCR titration on SupTl cells. FIG. 16 shows the study design and timeline for the study.

[0426] Vector formulations were maintained at -80°C until the day of animal treatment. Vector was kept on ice for transport to the animal facility and equilibrated at room temperature for approximately 5-15 minutes prior to lymph node injection in macaques using a single-use sterile needle and 1 ml syringe. Vector was injected within 2 hours of preparation.

[0427] Animals were individually housed (i) during the initial pre-study period in which the animals were acclimated to the jacket and tether and (ii) for at least 4 weeks following engineered viral particle injection. Animals had ad libitum access to water and food was only restricted prior to sedation or anesthesia.Study Procedure

[0428] Blood was drawn pre-study for isolation of (i) PBMCs, (ii) serum, and (iii) gDNA. Serum and gDNA were isolated and stored at -80 °C for later analyses. PBMCs were cryopreserved, analyzed by a pre-injection flow cytometry panel and transduced with the same engineered viral particles that were injected in vivo.

[0429] In addition, in the two-week period prior to lymph node injection, up to two blood draws were taken to determine baseline values for the assays below.

[0430] Day O:Prophylactic diphenhydramine (IV injection, one dose)Surgical placement of central catheter (if on tether)Surgical placement of telemetry device to monitor body temperatureOptional: blood drawUltrasound-guided Lymph Node injection of engineered viral particles

[0431] Animals with a central intravenous catheter implanted received a continuous IV antibiotic infusion via the catheter to prevent septicemia.

[0432] Animal Monitoring and Blood Collection: Blood was drawn throughout the study with biweekly or triweekly blood draws planned for the first 3-4 weeks of the experiment and weekly thereafter. The number of draws will depend on assay results and the clinical condition of each animal. Blood draw schedule may be adjusted dependent on maximum blood draw allowance of lOml / kg body weight every two weeks.

[0433] Animals were observed daily for general health, appetite, activity level, responsiveness, and fecal production. Body temperature was measured continually using a telemetry device. Animals were monitored for clinical signs of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) based on an established set of criteria. Clinical signs of CRS or ICANS were confirmed by CBCs, serum chemistries, serum IL-6, serum ferritin and serum CRP levels.Animals treated with engineered viral particle product in the present study did not display any clinical signs of CRS.

[0434] Study Endpoint: The animals were monitored and have blood drawn periodically following injection of engineered viral particles.Analytical Assays:A. Cell Surface Flow CytometryB. Serum IL-6C. Serum ferritinD. Serum CRPSafety Efficacy Criteria:

[0435] Safety and Efficacy was assessed in each animal using a combination of analytics comparing results pre- and post-injection.A. Depletion of B Cells (Flow Cytometry). The fraction of starting CD20+ cells were plotted longitudinally starting with pre-injection samples and extending throughout the study.B. Clinical Symptoms of CRS. The serum concentration of IL-6, ferritin, and CRP were plotted longitudinally starting with pre-injection samples and extending throughout the study.

[0436] This study is a pilot however, despite the small study size, meaningful trends in regards to B cell depletion in vivo were detected.Safety Criteria:

[0437] Safety was assessed using a combination of analytics.A. CRS Monitoring a. Body temperature b. CBCs c. Serum IL-6 d. Serum ferritin e. Serum CRP f. Serum chemistries (kidney (creatinine, BUN, total protein, electrolytes) and liver (AST, ALT, ALP, total bilirubin, albumin) function or damage)B. Clinical and Physical Assessments - including general health, appetite, activity level, responsiveness, hydration, and fecal productionC. Necropsy and Histopathology on a subset of animals a. Gross findings b. Histopathology of tissue sections c. qPCR of tissue sections d. RNA-ISH of tissue positive by qPCR.Results

[0438] Depletion of B Cells was analyzed throughout the study in the fraction of starting CD20+ cells. In two of the three animals studied, B cell reduction was observed throughout the course of the study (BIG. 17). In vivo transduction with the engineered viral particles was well-tolerated in the animals as seen in minor increases in inflammatory markers following injection (FIG. 18). The safety data confirm none of the animals showed clinical symptoms of Cytokine release syndrome (CRS). Thus, this study supports a finding that a viral particle comprising a dual-fusion polypeptide along with a separately expressed anti-CD3 targeting protein can effectively transduce and generate functional CAR T cells in vivo via an intranodal pathway administration. Similar data to this example are expected for viral particles that include a payload encoding anti-CD22.Example 5

[0439] Example 5 describes in vivo T cell transduction and activation with viral particles surface engineered to display a CD58, CD80, and anti-CD3 scFv tri-fusion polypeptide. The methods depict successful in vivo CAR T cell generation. This non-human primate (NHP) study was conducted in M. nemestrina and showed generation of anti-CD20 CAR T cells and was well tolerated in all animals. The viral particles were engineered to display an anti-CD3 scFv that binds NHP CD3 and a payload comprising a human-specific anti-CD20 CAR which cross-reacts with NHP CD20 (FIG. 19A). Study Design and study methods are in large part the same as those described in Example 4.

[0440] The objectives of the study included analyzing the ability of engineered viral particles to transduce T cells and generate functional CAR T cells in a large animal model. Another objective of the study was to assess lymph node injection as a viable route of administration for engineered viral particles.

[0441] In the present NHP study, the subjects of the study were Pig-tailed macaques (Macaca nemestrina) that were either male of female, a minimum size of 3.5-5 kg, no age restriction (other than to ensure sufficient size). Six animals were studied: 4 test article treated + 2 control (PBS or empty particle) treated. Pigtailed macaques are well suited for lentiviral studies due to TRIM5a allele that permits efficient transduction. TRIM5a in rhesus and cynomolgus macaques restricts retroviral transduction. A summary of the animals treated is shown in FIG. 21B.

[0442] Engineered viral particles were generated with surface molecules that are cross-reactive with pig-tailed macaque (Human CD58-NHP-specific anti-CD3 scFv-Human CD80 multi-domain fusion (MDF) polypeptide) (FIG. 19 A) and aCD20-CAR-Flag payload (FIG. 19B). The particles showed efficient transduction of pig-tailed macaque PBMCs in vitro at MOI=0.2 (FIG. 20).

[0443] The particles were dosed in transducing units per kg (TU / kg) as determined by ddPCR titration on SupTl cells. The payload was designed to express a Flag-tagged aCD20-CAR since there is no available antibody that recognizes the aCD20-CAR.

[0444] FIG. 21A shows a study design and timeline for the study.

[0445] Vector formulations were maintained at -80°C until the day of animal treatment. Vector was kept on ice for transport to the animal facility and equilibrated at room temperature for approximately 5-15 minutes prior to lymph node injection in macaques using a single-use sterile needle and 1 ml syringe. Vector was injected within 2 hours of preparation. Control animals had an equivalent volume of vehicle (PBS) or equivalent number of surface engineered particles that did not contain a viral payload (empty particle) injected into the lymph node.

[0446] Animals were individually housed (i) during the initial pre-study period in which the animals were acclimated to the jacket and tether and (ii) for at least 4 weeks following engineered viral particle injection. Animals had ad libitum access to water and food was only restricted prior to sedation or anesthesia.Study Procedure

[0447] Blood was drawn pre-study for isolation of (i) PBMCs, (ii) serum, and (iii) gDNA. Serum and gDNA were isolated and stored at -80 °C for later analyses. PBMCs were cryopreserved, analyzed by a pre-injection flow cytometry panel and transduced with the same engineered viral particles that were injected in vivo.

[0448] Criteria for exclusion from study include:(i) inability of engineered viral particles to activate T cells,(ii) inability of engineered viral particles to transduce T cells, and(iii) inability of CAR expressing T cells to kill target cells and produce cytokines.

[0449] Pre-Study Blood Draws: In the two-week period prior to lymph node injection, up to two blood draws were taken to determine baseline values for the assays below.

[0450] Day O:Prophylactic diphenhydramine (IV injection, one dose)Surgical placement of central catheter (if on tether)Surgical placement of telemetry device to monitor body temperatureOptional: blood drawUltrasound-guided LN injection of engineered viral particles

[0451] Animals with a central intravenous catheter implanted received a continuous IV antibiotic infusion via the catheter to prevent septicemia.

[0452] Animal Monitoring and Blood Collection: Blood was drawn throughout the study with biweekly or triweekly blood draws planned for the first 3-4 weeks of the experiment and weekly thereafter. The number of draws will depend on assay results and the clinical condition of each animal. Blood draw schedule may be adjusted dependent on maximum blood draw allowance of lOml / kg body weight every two weeks.

[0453] Animals were observed daily for general health, appetite, activity level, responsiveness, and fecal production. Body temperature was measured continually using a telemetry device. Animals were monitored for clinical signs of cytokine release syndrome (CRS) and immune effector cell-associatedneurotoxicity syndrome (ICANS) based on an established set of criteria. Clinical signs of CRS or ICANS will be confirmed by CBCs, serum chemistries, serum IL-6, serum ferritin and serum CRP levels. Animals treated with engineered viral particle product in the present study did not display any clinical signs of CRS.

[0454] Study Endpoint: The animals were monitored and have blood drawn periodically following injection of engineered viral particles.

[0455] A. Cell Surface Flow Cytometry1. NHP PBMC Screening PanelTable 82. qPCR Assay on gDNA extracted from peripheral blood to detect transduced cells3. MSD Assay to measure cytokines using serum isolated from peripheral blood4. CBC5. Serum chemistries6. Serum IL-67. Serum ferritin8. Serum CRPEfficacy Criteria:

[0456] Efficacy was assessed in each animal using a combination of analytics comparing results pre- and post-injection.1. Depletion of B Cells (Flow Cytometry). The number of B cells per pl present were plotted longitudinally starting with pre-injection samples and extending throughout the study.2. Cytokine Release (MSD Assay). The serum concentration of each cytokine were plotted longitudinally starting with pre-injection samples and extending throughout the study.3. Detection of CAR T Cells in Peripheral Blood (Flow Cytometry, qPCR). The number of Flag-expressing T cells (Flow Cytometry) present were plotted longitudinally starting with pre-injection samples and extending throughout the study.

[0457] With a total of 4 animals treated significant trends in regards to CAR T cell generation and B cell depletion in vivo were detected.Safety Criteria:

[0458] Safety will be assessed using a combination of analytics.1. CRS Monitoring i. Body temperature ii. CBCs iii. Serum IL-6 iv. Serum ferritin v. Serum CRP vi. Serum chemistries (kidney (creatinine, BUN, total protein, electrolytes) and liver (AST, ALT, ALP, total bilirubin, albumin) function or damage)

[0459] Clinical and Physical Assessments: including general health, appetite, activity level, responsiveness, hydration, and fecal production

[0460] Necropsy and Histopathology on a subset of animals:1. Gross findings2. Histopathology of tissue sections3. qPCR of tissue sections4. RNA-ISH of tissue positive by qPCR.Results

[0461] Intranodal injection of engineered viral particles generated functional CAR T cells in Animal #1 (FIG. 22A-22B). Flow cytometry analysis showed a recurring expansion of the CAR T cells as well as persistence and the emergence of a population of memory CAR T cells at Day 35 (BIG. 22A-22B). CAR T cell kinetics were analyzed through flow cytometry analysis of cell staining with the activation marker CD25 (FIG. 23). CAR T cell activation peaked on Day 7 and Day 49 and such kinetics are consistent with antigen-engagement and B cell eradication. The presence of CD25_CAR T cells at Day 49 indicates antigen-driven expansion from a persistent memory T cell population. The generation of functional CAR T cells was further evidenced by sustained B cell aplasia for approximately 70 days (FIG. 24A-24B). The depletion of B cells coincides with the increase in CAR+ T cells (FIG. 25). In vivo transduction with the engineered viral particles was well-tolerated in Animal #1. FIG. 26A shows a timeline of observed clinical symptoms. Increase in inflammatory markers and the onset of clinical symptoms of mild CRS (FIG. 26B peaks) and neurotoxicity (FIG. 26B first peak) coincided with CAR T cell expansion (FIG. 26B). Animal #1 responded rapidly to invention with a single dose of each medication. In summary, the data show that intranodal administration of engineered viral particles waswell-tolerated in Animal #1. The engineered viral particles show potent in vivo CAR T cell generating activity as demonstrated by flow cytometric detection of CAR T cells and sustained B cell depletion.

[0462] A dose de-escalation study was performed with Animal #2 wherein the animal was dosed with a half-log lower dose (BIG. 27A-27B). Animal #2 did not show symptoms of CRS: no fever, decreased appetite, or decreased activity.

[0463] Animal #3 showed symptoms of mild CRS: fever, decreased appetite, and decreased activity on Day 3. Animal #3 responded rapidly to invention with Tocilizumab and Anakinra. Expansion of CAR+ T cells on Day 7 appeared concomitantly with B cell aplasia (FIG. 28A-28B). B cell aplasia was complete and persistent through at least day 50 of the study.

[0464] Animal #4 did not show symptoms of CRS or ICAN. Animal #4 was prophylactically treated with Anakinra between Day 7 and Day 10 (FIG. 29A-29B). Similar to Animals #1 and #3, Animal #4 exhibited peak expansion of CAR+ T cells around Day 10 with corresponding complete and persistent B cell aplasia through at least Day 50 of the study.

[0465] In summary, viral particles displaying multi-domain fusion (MDF) protein surface engineering showed potent in vivo anti-CD20 CAR T cell generation. Consistent generation of anti-CD20 CAR T cells that drive persistent B cell aplasia was observed in three out of three animals at the full dose (Animals 1, 3, and 4 of this Example). Typically, circulating CAR T cells are not detectable by industry standard flow cytometry. However, surprisingly, substantial circulating CAR-T cells generated by the viral particles described were also detectable by an industry standard flow cytometry assay. Further, none of the animals in the studies underwent lymphodepletion or supportive cytokine treatment prior to administration. The engineered viral particles were well-tolerated and toxicity associated with particle administration was either mild or not observed during peak CAR T expansion in most animals studied.

[0466] In conclusion, these findings demonstrate in vivo CAR T cell generation was achieved with CAR+ T cell expansion peaking around Day 10 of the study. The generation of functional CAR T cells was further evidenced B cell aplasia which coincided with the increase in CAR+ T cells beginning at Day 7 of the study. Thus, this study supports a finding that a viral particle comprising a tri-fusion polypeptide comprising binding domains from CD58 and CD80, and an anti-CD3 binding domain, can effectively transduce and generate functional CAR T cells in vivo via an intranodal route of administration. Similar data to this example are expected for viral particles that include a payload encoding anti-CD22.Example 6: Toxicology StudyStudy Summary

[0467] A non-GLP pilot toxicology study was initiated to evaluate safety and pharmacology of Product B with and without rapamycin following a single intravenous (IV) injection in a CD34-humanized NSG™ mouse model (donor n = 2). Treatment with Product B resulted in a dose-dependent increase in T-cell activation in animals on day 3 of this study. B-cell depletion was observed beginning on day 3of this study and remained durable through the last timepoint on day 24 of this study, both with and without rapamycin. Depletion began earlier and is more robust in Donor 1 animals compared to Donor 2 animals. Peak CAR+ T-cell expansion occurred on day 18 of this study. Increased CAR+ T-cell frequencies and concentrations were identified in Donor 1 animals treated with rapamycin compared to their no rapamycin counterparts. No test-article related adverse events were (through day 45 of this study).Study Design

[0468] The study included a pilot toxicology study in CD34-humanized NSG™ mice. A study diagram and details regarding 8 treatment groups for this study are shown in FIG.30A-30B. Treatments included Product B administered at a concentration of 6.98e8 TU / mL. The toxicology study plan included the following:• In-life safety: clinical signs, body weight, mortality, local irritation of the dosing site• Flow blood for B cells & CAR+ T cells (pre-dose, post-dose weeks 1-4, week 9, endpoint)1. Collect plasma for cytokine analysis• Flow spleen, bone marrow, and liver from representative animals at endpoint for enumeration of B cells and CAR+ T cells• Collect all major organs (blood, bone marrow (femur), spleen, liver, heart, lung, kidney, brain, injection site, ovary, adrenal gland, spinal cord) half in PFA and half snap frozen for potential followup evaluation of histopathological and biodistribution endpointsStudy Results

[0469] Treatment with Product B resulted in a dose-dependent increase in T-cell activation in animals from both donors on day 3 of this study. B-cell depletion was observed beginning on day 3 of this study and remained durable through the last timepoint on day 24. Depletion began earlier and was more robust in Donor 1 animals. Peak CAR+ T-cell expansion occurred on day 18 of this study. Increased CAR+ T-cell frequencies and numbers were observed in Donor 1 animals treated with rapamycin compared to their no rapamycin counterparts. No Product B related changes in body weight were observed.

[0470] FIG. 31 shows frequencies of human B and T cell levels on day -3 of this study (before Product B administration). Compared to Donor 2, Donor 1 had lower B cell levels, but higher T cell levels.

[0471] FIG. 32 shows dose-dependent B-cell depletion. B cell depletion was evident as early as study day 3 at the max feasible dose (140 x 106 TU) in Donor 1. B cell depletion began earlier in the study and was more robust in Donor 1 animals compared to Donor 2. Rapamycin appeared to enhance B-cell depletion at the low dose (6 x 106 TU) in Donor 1 and at all doses in Donor 2, where starting T cell levels were low. Rapamycin increased B-cell depletion in vehicle-treated control animals (consistent with an immunosuppressive MOA).

[0472] FIG. 33 shows that peak CAR+ T cell frequencies on study day 18. In Donor 1 animals there was an increase in frequencies of CAR+ T cells in groups treated with rapamycin compared to no rapamycin controls. Donor 2 animals had lower CAR+ T cell frequencies compared to Donor 1.

[0473] FIG. 34 shows data on circulating CAR+ T cell numbers. Peak CAR+ T cell numbers were observed on Study Day 18. Donor 1 animals treated with rapamycin had increased numbers of CAR+ T cells compared to their no rapamycin treated counterparts. Low concentration of total T cells in Donor 2 limited calculation of CAR+ T cell concentrations in peripheral blood.Conclusions

[0474] These results indicate that Product B was able to generate functional CAR T-cells and was administered safely to animals in this model study.Example 7: Pharmacology StudyStudy Summary

[0475] Product B was evaluated in a dose-escalation study with or without rapamycin combination treatment using PBMC-humanized NSG MHC I / n DKO mice bearing systemic Nahn6 tumors.Study Objectives

[0476] The purpose of the study was to evaluate the pharmacology of Product B + / - rapamycin in PBMC humanized mice bearing systemic Nahn6 tumor. Objectives included: evaluation of Product B T cell activation, CAR T cell transduction, and CAR T cell proliferation; evaluation of kinetics and magnitude of tumor control; evaluation of the impact of rapamycin on CAR T cell enrichment, expansion, and benefit to tumor clearance; and evaluation of treatment effects and dose-dependent impacts on survival.Study Design

[0477] Animals were engrafted with 0.25 x 10e Nalm6 tumor via tail vein injection on Study Day -4. Animals were humanized with 20 x 10e PBMCs Study Day -1 via intraperitoneal (IP) injection. Successful tumor engraftment was assessed by in vivo imaging on day -1. Study details are provided in Table 9 and FIG. 35.Table 9Study Results

[0478] Product B transduced T cells in a dose-dependent manner and the addition of rapamycin enhanced CAR T-cell transduction and promoted CAR T-cell enrichment. Treatment with Product B alone resulted in a limited level of T-cell transduction, with circulating CAR T-cell concentrations peaking between Study Days 6 and 13 and decreased thereafter. Rapamycin promoted CAR T-cell enrichment and expansion, with CAR T cells expanding through day 20 and persisting from day 20 to 56. Consistent with prior studies, Control CAR T-cell expansion peaked at day 13, contracted and remained at a low steady state following tumor control.

[0479] Both monotherapy and combination therapy cohorts demonstrated dose-dependent generation and expansion of CAR T cells, with elevated concentrations of T cells in circulation following administration of mid- / high doses of Product B (50 x 10A6 / 100 x 10A6) compared to low doses (10 x 10A6). However, no major differences in CAR T-cell concentrations were demonstrated between the mid / high dose groups.T-cell activation was evaluated with the first analysis timepoint (Study Day 6), which was too late to capture acute / transient T-cell activation.

[0480] Combination treatinent with Product B and rapamycin corresponded with improved kinetics and magnitude of tumor clearance compared to monotherapy. Product B monotherapy cohorts demonstrated slowed tumor growth kinetics but failed completely clear tumor. The addition of rapamycin allowed for complete clearance of the tumor by day 7 in the mid and high dose groups, with slower clearance in the low dose group. Addition of rapamycin to vehicle-treated animals demonstrated increased tumor growth, which indicates that the presence of rapamycin did not hinder Nahn6 tumor growth capability in vivo; moreover, despite increased tumor burden in the presence of rapamycin, treatment with Product B still demonstrated dose-dependent CAR T-cell generation and subsequent tumor control.Treatment with Control demonstrated rapid control and clearance of Nalm6 tumors.

[0481] Summary of Clinical Observations and Survival. All mice treated with Product B demonstrated improved survival compared to vehicle treated mice, regardless of dose level. All vehicle- treated mice ± rapamycin were humanely euthanized due to tumor burden. Low and high doses of Product B in the monotherapy cohort demonstrated increased survival rates compared to mice in the mid-dose cohort (50 x 10A6 TU). Combination treatment with Product B and rapamycin improved survival, with low dose cohorts demonstrating 100% survival and mid / high doses of Product B demonstrating increased survival benefit over vehicle controls and without rapamycin groups. Control- treated mice demonstrated 100% survival.ConclusionsThis study evaluated treatment effects of Product B ± rapamycin across a range of doses in PBMC humanized MHC I / n DKO mice bearing Nahn6 tumors. Product B monotherapy demonstrated dosedependent CAR T-cell generation between the low (10 x 10A6 TU) and mid / high doses (50 x 10A6 / 100 x 10A6 TU) between Study Days 6 and 13 and contracted thereafter. Low dose Product B CAR T cells did not expand to quantities above vehicle controls (< 10 CAR T-cells / pl blood), and median concentrations [across all doses] of 9 CAR T cells / pl blood (maximum = 45 CAR T cells / pl blood) were demonstrated in mid / high dose Product B monotherapy cohorts. Product B monotherapy-treated mice controlled tumor in a dose-dependent manner, between the low (10 x 10A6 TU) versus mid / high (50 x 10A6 / 100 x 10A6 TU) doses. Combination treatment with rapamycin increased Product B CAR T-cell enrichment and expansion compared to monotherapy cohorts, independent of dose. Peak CAR T-cell concentrations were demonstrated on Study Day 27 with median concentration [across all doses] of 216 CAR T cells / pl blood (maximum = 1429 CAR T cells / pl blood) and were maintained throughout the remainder of the study. Addition of rapamycin resulted in complete tumor control by Study Day 7 in the highest dose cohort (100 x 10A6 TU) and Study Day 28 in the lowest dose tested (10 x 10A6 TU).Example 8: Lupus Treatment

[0482] Studies will be performed similar to those in Example 3, except that instead of treating subjects with lymphoma, subjects with systemic lupus erythematosus (SLE) or lupus nephritis (LN) will be treated. Product B will be provided to subjects with SLE or LN to evaluate the safety, efficacy, and PK / Pd of Product B in adult subjects with SLE or LN, and outcomes related to SLE and LN will be identified in the subjects following treatment.Example 9: Development of a Surface Engineered Lentiviral Vector for In Vivo Generation of CD22-Directed CAR T CellsBackground

[0483] Advances in autologous CD19 CAR T therapies have transformed treatment for B-cell malignancies (BCM). Despite the transformational benefit, many challenges remain for autologous CAR T therapies, including operational, logistical, and cost burdens, plus the need for alternative treatment strategies for patients who are refractory to approved CD19 CAR T therapies. CD22 is a B- cell specific antigen expressed on most BCMs and autoreactive B cells and is a validated target for CD22-directed CAR T products in BCMs. Product B includes an off-the-shelf, multidomain fusion (MDF) protein surface engineered lentiviral vector designed to generate CD22-directed CAR T cells in vivo without requiring lymphodepleting chemotherapy. In addition to a CD22-CAR, Product B also encodes a rapamycin-activated cytokine receptor (RACR) as part of the payload, which was designed to enrich and expand CAR T cells in vivo in the presence of rapamycin. Described here are preclinical evaluations of Product B ± rapamycin.Methods

[0484] Product B includes an investigational lentiviral vector (LVV) drug product designed to selectively bind, activate, and transduce T cells in vivo, resulting in in vivo anti-CD22 CAR T-cell generation.

[0485] Some aspects of Product B are shown in FIG. 3A, which includes a 3rd generation, SIN, replication incompetent LW that includes (1) a membrane-bound MDF including human CD58, anti- CD3 scFv, and human CD80 sequences, and (2) an envelope pseudotyped with cocal fusion glycoprotein. The MDF protein confers selective binding and activation of T cells and the cocal glycoprotein (cocal) facilitates vector internalization and delivery of the payload into the cytoplasm. Some further aspects of Product B as used herein are shown in FIG. 3B-3C, including a payload depiction of a payload and molecules encoded by the payload in a T cell acting upon a target CD22+ B cell and being further activated by rapamycin and resistant to rapamycin. The Product B payload encodes a fully human anti-CD22 CAR (anti-CD22 scFv, 4-1BB, and CD3Q and RACR, which may allow rapamycin-activated enrichment and expansion of CAR T cells through delivery of IL-2 / IL-15 signaling to transduced cells.

[0486] Product B lots were manufactured internally by a transient transfection of suspension 293T cells. The transgene payload includes the RACR system and the CD22-directed CAR with 4-1BB and CD3 intracellular signaling domains. In vitro studies with Product B utilized healthy donor and patient PBMCs. Functional assays were performed by co-culturing Product B-generated CAR T cells with target cell lines. Some aspects of the in vitro studies in this example are also included in Example 1.

[0487] An in vivo pre-clinical evaluation of Product B ± rapamycin in Nahn6 tumor-bearing NSG MHC I / n DKO mice was performed. An experimental outline that was followed is shown in FIG. 35, and is also described in Example 7. In vivo studies were performed in PBMC-humanized NSG MHC- I / II DKO mice bearing systemic tumor, and analyses were performed using in vivo imaging and flow cytometry. NSG MHC I / n double knockout (DKO) mice were engrafted with 0.25 x 106 Nahn6 tumor cells (IV) on Study Day -4. Mice were subsequently humanized with 20 x 106 healthy donor PBMCs (IP) on Study Day -1. Product B was administered to mice via single injection IP on Study Day 0 with one of the following doses: 0 [vehicle], 10 x 106, 50 x 106, or 100 x 106 transducing units (TU) ± rapamycin (sirolimus). Rapamycin treatment was initiated on Study Day 6 in indicated cohorts with a dose and regimen intended to achieve recommended trough levels of rapamycin in vivo (based on sirolimus USPI). Mice were serially bled and imaged throughout the study to measure cell composition and CAR T cell persistence and tumor progression / control, respectively.Results

[0488] In vitro treatment of PBMCs with Product B demonstrated MDF surface engineering mediated selective binding and activation of T cells and subsequent transduction of T cells resulting in expressionof RACR and anti-CD22 CAR. Product B generated CAR T cells mediated antigen-specific tumor cell killing and cytokine secretion in response to CD22hi (Raji) and CD221o (Nalm6) cells, but not CD22- negative cells (K562, Raji-CD22KO).

[0489] Product B binding, activation, and transduction efficiencies were evaluated following 1 hour incubation of the indicated cell types with Product B across a range of multiplicities of infection (MOIs). Results indicated that Product B particles efficiently and specifically bound, activated, and transduced T cells. BIG. 36A shows frequencies of cocal-positive T cells (left) and other PBMC types (right) (see also FIG. 7). FIG. 36B shows mean frequency (left) and gMFT of CD25 (right) as an indicator of cell activation (see also FIG. 8). FIG.36C and FIG. 8 show frequencies of CAR+ T cells and representative flow cytometry dot plots for nontransduced (NTD) and transduced (MOI = 1) T cells (FIG. 8). In FIG. 36A-36C, n = 3 healthy donor PBMC lots; points indicate mean and whiskers represent SEM.

[0490] Antigen-specific functional activities of Product B generated CAR T cells were evaluated following stimulation with Raji (CD22high), Nahn6 (CD221ow), K562 (CD22neg), and Raji CD22 KO (CD22neg) cells for 24-72 hours at an E:T ratio = 1:1. Results indicated that Product B generated CAR T cells mediated CD22 antigen-specific cytotoxicity and cytokine secretion. The left panel of FIG. 10A includes histograms showing expression of CD22 on parental Raji and Raji CD22 KO cell fines. FIG. 10B includes graphs showing tumor cell growth in cultures with tumor only or tumor with Product B CAR T cells (see also the middle and right panels of FIG. 10A). FIG. 11 includes IFN-y, IL-2, and TNF-a cytokine secretion data following 24-hour in vitro cell stimulation.

[0491] Durability of tumor control by Product B was impacted by antigen expression and was augmented with rapamycin-RACR engagement. Product B generated CAR T cells were serially stimulated with Nalm6 (CD221ow) or Raji tumor cells (CD22high) in the presence or absence of 10 nM rapamycin. CAR T cells were re-challenged with tumor every 2-3 days. Tumor cells were transduced to express near infrared or NucLight Orange, and cell counts were measured over time with a live-cell imaging platform. Data are shown in FIG. 37. n = 3 healthy donor PBMC lots for CAR T generation. In the figure, points indicate the mean of technical duplicates and whiskers represent SD.

[0492] In humanized mouse studies Product B generated CAR T cells that mediated anti-tumor activity, and combination treatment with rapamycin further enriched and expanded CAR T cells in vivo and corresponded with complete tumor cell eradication and prolonged animal survival. For example, in vivo administration of Product B drove dose-dependent generation of CAR T cells, and expansion of CAR T cells was potentiated by rapamycin-RACR. Circulating CAR T cells were evaluated by flow cytometry (FIG. 38). CAR+ T cellular concentrations were determined with counting beads. Product B monotherapy and Product B + rapamycin combination therapy plots are shown on left and right panels, respectively. Points indicate mean; bars indicate standard error (n = 5-6 mice per group). Cell concentrations per pL blood are represented on a logarithmic scale.

[0493] Combination treatment with Product B and rapamycin demonstrated dose-dependent, RACR- expanded CAR T cell mediated tumor control. Tumor burden was evaluated via bioluminescent imagingin PBMC-humanized mice bearing Nalm6 tumor (FIG. 39A). Product B monotherapy and Product B + rapamycin combination therapy plots are shown on left and right panels, respectively (n = 5-6 mice per group). Kaplan-Meier survival curves are shown in FIG. 39B for each of the treatment groups.Conclusions

[0494] Use of Product B resulted in specific and efficient transduction of T cells resulting in generation of anti-CD22 CAR T cells equipped with the RACR system. The transduction of T cells corresponded with generation of CD22-directed CAR T cells capable of killing both CD22hi and CD221o tumor cells in vitro and in vivo. Moreover, the RACR system and administration of rapamycin promoted enrichment and expansion of CAR T in vivo resulting in complete tumor clearance. Product B generated CAR T cells demonstrated antigen-specific cell killing and cytokine secretion, and CAR T cells were responsive to both CD22high and CD221ow tumor cell lines, which can be further augmented through combination treatment with rapamycin to engage the RACR system. Administration of Product B ± rapamycin to tumor bearing, PBMC-humanized mice results in in vivo generation of CAR T cells, and rapamycin-RACR may drive enrichment and expansion of CAR T cells. Results included complete tumor clearance and improved survival. These nonclinical data support planned studies in R / R LBCL post CD19-directed CAR T therapy.* * *

[0495] 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.

[0496] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0497] The singular forms “a”, “an”, and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0498] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated fisted items, as well as the lack of combinations when interpreted in the alternative (or).

[0499] All publications and patents mentioned herein are hereby incorpo...

Claims

CLAIMS1. A lentiviral particle, comprising: a polynucleotide encoding a chimeric antigen receptor (CAR) that specifically binds cluster of differentiation-22 (CD22), and (a) or (b):(a) a surface protein comprising: a heterologous viral glycoprotein, a CD58 extracellular domain sequence, a CD80 or CD86 extracellular domain sequence, an anti-CD3 antibody or antigen-binding fragment, or a combination thereof; or(b) a polynucleotide encoding a small molecule-activated cytokine receptor.

2. The lentiviral particle of claim 1, wherein the CAR comprises fight chain CDRs selected from any of the following groups:LCDR1 having the sequence of SEQ ID NO: 413, LCDR2 having the sequence set forth in SEQ ID NO: 414 and LCDR3 having the sequence set forth in SEQ ID NO: 415,LCDR1 having the sequence set forth in SEQ ID NO: 401, LCDR2 having the sequence set forth in SEQ ID NO: 402 and LCDR3 having the sequence set forth in SEQ ID NO: 403,LCDR1 having the sequence set forth in SEQ ID NO: 407, LCDR2 having the sequence set forth in SEQ ID NO: 408 and LCDR3 having the sequence set forth in SEQ ID NO: 409, orLCDR1 having the sequence set forth in SEQ ID NO: 441, LCDR2 having the sequence set forth in SEQ ID NO: 442 and LCDR3 having the sequence set forth in SEQ ID NO: 443; and wherein the CAR comprises heavy chain CDRs selected from any of the following groups:HCDR1 having the sequence set forth in SEQ ID NO: 416, HCDR2 having the sequence set forth in SEQ ID NO: 417 and HCDR3 having the sequence set forth in SEQ ID NO: 418,HCDR1 having the sequence set forth in SEQ ID NO: 404, HCDR2 having the sequence set forth in SEQ ID NO: 405 and HCDR3 having the sequence set forth in SEQ ID NO: 406,HCDR1 having the sequence set forth in SEQ ID NO: 410, HCDR2 having the sequence set forth in SEQ ID NO: 411 and HCDR3 having the sequence set forth in SEQ ID NO: 412, orHCDR1 having the sequence set forth in SEQ ID NO: 438, HCDR2 having the sequence set forth in SEQ ID NO: 439 and HCDR3 having the sequence set forth in SEQ ID NO: 440.

3. The lentiviral particle of claim 1, wherein the CAR comprises a variable light (VL) polypeptide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 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: 299, 419, 421, or 423.

4. The lentiviral particle of claim 3, wherein the polynucleotide encoding the CAR comprises a VL domain-encoding 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: 298.

5. The lentiviral particle of claim 1, wherein the CAR comprises a variable heavy (VH) polypeptide 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: 295, 420, 422, or 424.

6. The lentiviral particle of claim 5, wherein the polynucleotide encoding the CAR comprises a VH domain-encoding 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: 294.

7. The lentiviral particle of claim 1, wherein the CAR comprises a cluster of differentiation 8 (CD8) hinge and transmembrane domain 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: 301.

8. The lentiviral particle of claim 7, wherein the polynucleotide encoding the CAR comprises a CD8 hinge- and transmembrane domain-encoding 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: 300.

9. The lentiviral particle of claim 1, wherein the CAR comprises a 4-1BB 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: 84 or 201.

10. The lentiviral particle of claim 9, wherein the polynucleotide encoding the CAR comprises a 4-lBB-encoding 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: 200 or 302.

11. The lentiviral particle of claim 1, wherein the CAR comprises a CD3zeta 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: 82 or 203.

12. The lentiviral particle of claim 11, wherein the polynucleotide encoding the CAR comprises a CD3zeta-encoding 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: 202 or 303.

13. The lentiviral particle of claim 1, wherein the CAR comprises a polypeptide 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 any one of SEQ ID NOs: 292, 435-437 or 456.

14. The lentiviral particle of claim 13, wherein the polynucleotide encoding the CAR comprises a 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: 454 or 455.

15. The lentiviral particle of claim 1, comprising the heterologous viral glycoprotein.

16. The lentiviral particle of claim 15, wherein the viral glycoprotein comprises a cocal glycoprotein.

17. The lentiviral particle of claim 16, wherein the cocal glycoprotein comprises a polypeptide 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: 74.

18. The lentiviral particle of claim 1, comprising the CD58 extracellular domain sequence.

19. The lentiviral particle of claim 18, wherein the CD58 extracellular domain sequence 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: 10, 444 or 445.

20. The lentiviral particle of claim 1, comprising the CD80 extracellular domain sequence.

21. The lentiviral particle of claim 20, wherein the CD80 extracellular domain sequence 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: 12 or 250.

22. The lentiviral particle of claim 1, comprising the CD86 extracellular domain sequence.

23. The lentiviral particle of claim 22, wherein the CD86 extracellular domain sequence 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: 13.

24. The lentiviral particle of claim 1, comprising the anti-CD3 antibody or antigenbinding fragment.

25. The lentiviral particle of claim 24, wherein the anti-CD3 antibody or antigen-binding fragment comprises complementarity-determining regions (CDRs) selected from (i) or (ii):(i) HCDR1 having the sequence set forth in SEQ ID NO: 48, HCDR2 having the sequence set forth in SEQ ID NO: 49 and HCDR3 having the sequence set forth in SEQ ID NO: 50, LCDR1 having the sequence of SEQ ID NO: 51, LCDR2 having the sequence set forth in SEQ ID NO: 52 and LCDR3 having the sequence set forth in SEQ ID NO: 53, or(ii) HCDR1 having the sequence set forth in SEQ ID NO: 54, HCDR2 having the sequence set forth in SEQ ID NO: 55 and HCDR3 having the sequence set forth in SEQ ID NO: 56, LCDR1 having the sequence set forth in SEQ ID NO: 57, LCDR2 having the sequence set forth in SEQ ID NO: 58 and LCDR3 having the sequence set forth in SEQ ID NO: 59.

26. The lentiviral particle of claim 24, wherein the anti-CD3 antibody or antigen-binding fragment comprises the anti-CD3 antigen-binding fragment, and wherein the anti-CD3 antigenbinding fragment comprises a single chain variable fragment (scFv).

27. The lentiviral particle of claim 26, wherein the anti-CD3 scFv comprises a polypeptide 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: 31 or 249.

28. The lentiviral particle of claim 1, further comprising a fusion protein comprising: the CD58 extracellular domain sequence, the CD80 or CD86 extracellular domain sequence, and the anti-CD3 antibody or antigen-binding fragment.

29. The lentiviral particle of claim 28, wherein the fusion protein comprises a polypeptide 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 any one of SEQ ID NOs: 446 or 448-449.

30. The lentiviral particle of claim 1, comprising the polynucleotide encoding the small molecule-activated cytokine receptor.

31. The lentiviral particle of claim 30, wherein the small molecule comprises rapamycin or a rapalog.

32. The lentiviral particle of claim 31, wherein the small molecule-activated cytokine receptor comprises a FKBP12-rapamycin binding (FRB) polypeptide.

33. The lentiviral particle of claim 32, wherein the FRB polypeptide comprises a 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: 252.

34. The lentiviral particle of claim 33, wherein the polynucleotide encoding the small molecule-activated cytokine receptor comprises an FRB polypeptide-encoding 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: 257.

35. The lentiviral particle of claim 31, wherein the small molecule-activated cytokine receptor comprises a cytokine gamma chain polypeptide.

36. The lentiviral particle of claim 35, wherein the cytokine gamma chain polypeptide comprises 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: 264 or 265.

37. The lentiviral particle of claim 36, wherein the polynucleotide encoding the small molecule-activated cytokine receptor comprises a cytokine gamma chain-encoding 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: 261, 262, or 263.

38. The lentiviral particle of claim 31, wherein the small molecule-activated cytokine receptor comprises a FRB polypeptide fused with a cytokine gamma chain polypeptide.

39. The lentiviral particle of claim 31, wherein the small molecule-activated cytokine receptor comprises a FK506-Binding Protein of size 12 kD (FKBP) polypeptide.

40. The lentiviral particle of claim 39, wherein the FKBP polypeptide comprises a 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: 253.

41. The lentiviral particle of claim 40, wherein the polynucleotide encoding the small molecule-activated cytokine receptor comprises a FKBP polypeptide-encoding 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: 268.

42. The lentiviral particle of claim 31, wherein the small molecule-activated cytokine receptor comprises a cytokine beta chain polypeptide.

43. The lentiviral particle of claim 42, wherein the cytokine beta chain polypeptide comprises 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: 272.

44. The lentiviral particle of claim 43, wherein the polynucleotide encoding the small molecule-activated cytokine receptor comprises a cytokine beta chain polypeptide-encoding 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: 270 or 271.

45. The lentiviral particle of claim 31, wherein the small molecule-activated cytokine receptor comprises a FKBP polypeptide fused with a cytokine beta chain polypeptide.

46. The lentiviral particle of claim 31, wherein the lentiviral particle further comprises a polynucleotide encoding a free FRB polypeptide.

47. The lentiviral particle of claim 46, wherein the free FRB polypeptide comprises a 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: 251, 252, or 260.

48. The lentiviral particle of claim 47, wherein the polynucleotide encoding the free FRB 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%, at least 99%, or 100% identical to SEQ ID NO: 256, 257, or 258.

49. A lentiviral particle, comprising: a polynucleotide comprising a sequence that is at least 95% identical to SEQ ID NO: 292 and encodes a chimeric antigen receptor (CAR) that specifically binds cluster of differentiation-22 (CD22); a fusion molecule comprising: a CD58 extracellular domain sequence, a CD80 or CD86 extracellular domain sequence, and an anti-CD3 antibody or antigen-binding fragment; and a cocal glycoprotein.

50. The lentiviral particle of claim 49, further comprising a polynucleotide sequence encoding a synthetic cytokine receptor comprising: a first polypeptide comprising a sequence that is at least 95% identical to SEQ ID NO: 451, and a second polypeptide comprising a sequence that is at least 95% identical to SEQ ID NO: 453.

51. The lentiviral particle of claim 50, further comprising a polynucleotide sequence encoding a free FRB polypeptide that comprises a sequence at least 95% identical SEQ ID NO: 251.

52. A method, comprising administering the lentiviral particle of any one of the previous claims to a subject.

53. The method of claim 52, wherein the administration treats cancer in the subject.

54. The method of claim 53, wherein the cancer comprises CD22+ cancer cells.

55. The method of claim 52, wherein the administration treats an autoimmune disorder in the subject.

56. The method of claim 55, wherein the autoimmune disorder comprises lupus.

Images