Polynucleotide construct for chimeric antigen receptor expression and related viral vectors and methods

Abstract

Provided herein are polynucleotide constructs for increased expression of a chimeric antigen receptor system, as well as vectors, such as viral vectors, comprising same, cells comprising same, and methods of using same.

Description

260132003840POLYNUCLEOTIDE CONSTRUCT FOR CHIMERIC ANTIGEN RECEPTOR EXPRESSION AND RELATED VIRAL VECTORS AND METHODSCross-Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application No.63 / 738,507 filed on December 23, 2024, entitled “POLYNUCLEOTIDE CONSTRUCT FOR CHIMERIC ANTIGEN RECEPTOR EXPRESSION AND RELATED VIRAL VECTORS AND METHODS,” the contents of which are incorporated by reference in its entirety.Reference to an Electronic Sequence Listing

[0002] The present application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 260132003840SeqList.xml, created December 22, 2025, which is 144,881 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.Field

[0003] The present disclosure provides polynucleotide constructs for expression of a chimeric antigen receptor system, as well as vectors, such as viral vectors comprising same, cells comprising same, and methods of using same.Background

[0004] Chimeric antigen receptor (CAR) T cell therapies have demonstrated limited efficacy against solid tumors, in part due to challenges overcoming solid tumor heterogeneity and CAR T cell exhaustion associated with the immunosuppressive tumor microenvironment (TME). In addition to this challenge, there also are challenges in methods of delivering CAR-expressing cells to subjects in a manner that can provide increased expression for therapeutic efficacy of the CAR in the treatment of various diseases, including cancer. Provided herein are embodiments that address such needs.Summary260132003840

[0005] Provided herein is a polynucleotide construct comprising a nucleotide sequence encoding a protein fusion of a signal peptide and a chimeric antigen receptor (CAR). In some of any embodiments, the signal peptide is a modified CD8 signal peptide set forth by the amino acid sequence MALPVTALLLPLALLLHAARA (SEQ ID NO: 7).

[0006] In some of any embodiments, the CAR comprises in N to C terminal order an antigen binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain.

[0007] In some of any embodiments, the antigen binding domain comprises a means for binding an antigen. In some embodiments, the antigen is a tumor antigen, an antigen on an autoreactive B cell or T cell, a viral antigen, a small molecule, or a synthetic antigen. In some embodiments, the small molecule is a hapten. In some embodiments, the hapten is fluorescein or fluorescein isothiocyanate (FITC).

[0008] In some of any embodiments, the antigen binding domain is a single chain variable fragment (scFv) or a VHH single domain antibody.

[0009] In some of any embodiments, the spacer domain comprises a hinge region. In some embodiments, the hinge region is derived from an immunoglobulin, CD8, or CD28. In some embodiments, the CD8 comprises CD8a hinge.

[0010] In some of any embodiments, the transmembrane domain is a CD8 transmembrane domain or a CD28 transmembrane domain.

[0011] In some of any embodiments, the intracellular domain comprises a CD3 zeta (CD3Q domain. In some embodiments, the intracellular domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is a 4- IBB signaling domain or a CD28 signaling domain.

[0012] In some of any embodiments, one or more nucleotide sequences are codon optimized. In some of any embodiments, the polynucleotide construct is encoded in a bicistronic construct. In some of any embodiments, the polynucleotide construct is encoded in a multicistronic construct.

[0013] In some aspects, provided herein is a viral vector particle comprising the polynucleotide construct of any one of the preceding embodiments. In some embodiments, the viral vector particle is a lentiviral vector. In some embodiments, the lentiviral vector is a selfinactivating and replication-incompetent lentiviral vector particle. In some embodiments, the viral vector particle further comprises one or more surface T cell activating agents. In some260132003840embodiments, the one or more surface T cell activating agents comprise an extracellular portion of CD58 that binds to CD2, an anti-CD3 antibody or antigen binding fragment thereof, or CD80. In some embodiments, the viral vector particle comprises a multidomain fusion protein (MDF) displayed on its surface, wherein the multidomain fusion protein comprise an extracellular portion of CD58 that binds to CD2, an anti-CD3 antibody or antigen binding fragment thereof and a CD80 sequence, optionally wherein the MDF protein comprises an amino acid sequence that is identical to or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 115 or SEQ ID NO: 116. In some embodiments, the viral vector comprises a pseudotyped viral envelope protein, optionally wherein the pseudotyped viral envelope protein is cocal virus glycoprotein (G) (Cocal G), optionally wherein the Cocal G is a Cocal envelope variant containing a R354Q mutation or a K47Q mutation.

[0014] In some aspects, provided herein is a cell comprising the polynucleotide construct of any one of the preceding embodiments.

[0015] In some aspects, provided herein is a cell transduced by the viral vector particle of any one of the preceding embodiments. In some of any embodiments, the cell comprises a stem cell or a progenitor cell. In some embodiments, the stem cell comprises an induced pluripotent stem cell (iPSC). In some embodiments, the cell is a lymphocyte or lymphocyte progenitor. In some embodiments, the cell is a T cell. In some embodiments, the cell is a cytotoxic innate lymphocyte (CIL) cell. In some embodiments, the cell is a natural killer (NK) cell. In some embodiments, the cell is a monocyte or a macrophage.

[0016] In some aspects, provided herein is a method of transducing a cell comprising contacting a target cell with any of the polynucleotide constructs of any one of the preceding embodiments.

[0017] In some aspects, provided herein is a method of transducing a cell comprising contacting a target cell with the viral vector of any one of the preceding embodiments.

[0018] In some of any embodiments, the target cell comprises a stem cell or a progenitor cell. In some embodiments, the stem cell comprises an induced pluripotent stem cell (iPSC). In some embodiments, the target cell is a lymphocyte or lymphocyte progenitors. In some embodiments, the target cell comprises a T cell. In some embodiments, the T cell is a CD4+ or CD8+ T cell.260132003840

[0019] In some aspects, provided herein is a method of increasing expression of a polynucleotide construct expressing a chimeric antigen receptor (CAR), the method comprising transducing a cell with the polynucleotide construct of any one of the preceding embodiments or the viral vector particle of any one of the preceding embodiments.Brief Description of the Drawings

[0020] FIG. 1 shows CD3 and anti-CD22 CAR expression of T cells transduced with vector particles encoding one of the anti-CD22 CAR fusion proteins. Expression of CAR was measured using flow cytometry on Day 6 after transduction and the mean fluorescence intensity (MFI) is shown for each construct (e.g., i. CD22 CAR_1 (SEQ ID NO: 10), ii. CD22 CAR_4 (SEQ ID NO: 16), iii. CD22 CAR_2 (SEQ ID NO: 11), and iv. CD22 CAR_3 (SEQ ID NO: 12)).

[0021] FIG. 2 shows CD3+ and CAR+ expressing cells after transduction of lentiviral particles at various levels of MOI (MOIs: 0, 0.25, 0.5, 1, 2, and 5). The mean fluorescence intensity (MFI) for each condition is also shown. Cells were transduced with vector particles encoding an anti-CD22 CAR, including with an N-terminal variant CD8 signal peptide (SEQ ID NO: 7).

[0022] FIG.3A shows the number of integrations (e.g., vector copy number (VCN) per cell and per CAR) and FIG. 3B shows relative mRNA expression of cells transduced with vector particles encoding an anti-CD22 CAR with an N-terminal variant CD8 signal peptide (SEQ ID NO: 7). Various levels of MOI were tested (MOIs: 0, 0.25, 0.5, 1, 2, and 5).

[0023] FIG. 4 shows number of tumor cells after PBMCs were transduced with vector particles encoding an anti-CD22 CAR with the N-terminal variant CD8 signal peptide and as assessed by using a serial killing assay.

[0024] FIG. 5 shows the expansion and cytokine production of T cells transduced with vector particles encoding anti-CD22 CAR with the N-terminal unmodified CD8 signal peptide or anti-CD22 CAR with the N-terminal modified CD8 signal peptide.Detailed Description

[0025] The disclosure relates generally to a polynucleotide construct comprising a contiguous polynucleotide sequence encoding at least a chimeric antigen receptor (CAR) and methods for uses thereof. In some embodiments, the polynucleotide construct comprises a nucleotide sequence that encodes a fusion protein composed of a variant CD8 signal peptide260132003840(e.g., SEQ ID NO: 7) and a chimeric antigen receptor (CAR). In some embodiments, the polynucleotide construct is a multicistronic construct encoding a chimeric antigen receptor (CAR). In some aspects, the disclosure herein relates to the use of variant CD8 signal peptides in polynucleotide constructs (e.g., expressing a chimeric antigen receptor (CAR)) for improving cell surface expression of a CAR. In some embodiments, improving cell surface expression improves engineered cell functionality.

[0026] In some aspects, the provided polynucleotide constructs encoding CAR polypeptide sequences result in increased expression levels of the CAR when delivered to cells, such as T cells. In some embodiments, the increased expression levels are reflected in the RNA expression and protein expression of the encoded CAR polypeptide sequences. In some aspects, provided herein is a viral vector particle comprising any one of the polynucleotide constructs disclosed herein. In some embodiments, references to “viral vector” in the context of administration, transduction, dosing / titers, or particle-surface components are intended to refer to viral vector particles. In the present disclosure, viral vector is sometimes used as a shorthand for viral vector particles; for example, a lend viral vector particle is commonly referred to as an LVV. For clarity, a “viral vector” may refer to the engineered viral nucleic acid (DNA or RNA) comprising the transgene cassette and one or more vector elements, whereas a “viral vector particle” refers to a viral particle / virion comprising structural components (e.g., capsid and optionally an envelope) that packages a viral vector genome and is capable of delivering the viral vector to a cell (e.g., transducing a cell). In some embodiments, the increased expression levels in transduced cells are applicable at different levels of multiplicity of infection of the viral vector. In some embodiments, the increased expression levels in transduced cells are not dependent on the level of virus transduced. In some embodiments, the increased expression levels are related to the modified signal peptide encoded in the polynucleotide constructs provided herein.

[0027] In some aspects, provided herein is a cell transduced with any of the viral vectors disclosed herein.

[0028] In some aspects, provided herein is a method of transducing a cell comprising contacting a target cell with any one of the viral vectors disclosed herein. In some embodiments, by virtue of increasing CAR expression in cells, the viral vectors disclosed herein also improve the cytolytic activity of transduced cells. In some embodiments, cells transduced with the viral vectors disclosed herein display increased cytolytic killing. In some embodiments, the cells260132003840expressing the chimeric antigen receptor (CAR) comprising a modified signal peptide (e.g., SEQ ID NO: 7) have higher cytolytic killing than cells expressing the chimeric antigen receptor (CAR) comprising an unmodified signal peptide (e.g., SEQ ID NO: 6).

[0029] In some aspects, provided herein is a method of expressing a chimeric antigen receptor in a target cell comprising contacting the target cell with any one of the viral vectors disclosed herein.

[0030] In some aspects, provided herein is a cell produced by any of the methods disclosed herein.

[0031] 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 viral vectors disclosed herein.

[0032] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0033] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.I. POLYNUCLEOTIDE CONSTRUCTS

[0034] Provided herein are polynucleotide constructs encoding one or more separate proteins. In some embodiments, the protein is a chimeric antigen receptor (CAR). In some embodiments, the polynucleotide constructs comprise a nucleotide sequence that encodes a fusion protein of a modified CD8 signal peptide and the CAR. In some embodiments, the modified CD8 signal peptide comprises an amino acid sequence of MALPVTALLLPLALLLHAARA (SEQ ID NO: 7). In some embodiments, the modified CD8 signal peptide is set forth by the amino acid sequence of MALPVTALLLPLALLLHAARA (SEQ ID NO: 7). In some embodiments, the modified CD8 signal peptide is encoded by a nucleic acid encoding the amino acid sequence MALPVTALLLPLALLLHAARA (SEQ ID NO: 7).260132003840A. Chimeric Antigen Receptor

[0035] In some embodiments, the polynucleotide construct comprises a nucleotide sequence that encodes a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises a signal peptide and a spacer (e.g., hinge) domain. In some embodiments, the signal peptide is modified. In some embodiments, the spacer domain is modified. In some embodiments, the spacer domain is a shorter variant. In some embodiments, the CAR comprises the modified signal peptide sequence and a shorter hinge domain sequence. The one or more modified sequences may contain mutations such that the encoded sequence of the CAR may exhibit improved cell surface expression.

[0036] In some embodiments, the CAR construct contains an extracellular binding portion, a transmembrane domain and an intracellular signaling domain. In some embodiments, the intracellular signaling domain contains a costimulatory signaling domain and / or an activation signaling domain. In some embodiments, the CAR construct contains an extracellular binding portion, a transmembrane domain and an intracellular signaling domain comprising a costimulatory signaling domain. In some embodiments, the CAR construct contains an extracellular binding portion, a transmembrane domain and an intracellular signaling domain comprising an activation signaling domain. In some embodiments, the CAR construct contains an extracellular binding portion, a transmembrane domain and an intracellular signaling domain comprising a costimulatory signaling domain and an activation signaling domain. In some embodiments, the CAR comprises in N to C terminal order an antigen binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain.

[0037] In any embodiments described herein, the binding portion of the CAR can be, for example, a single chain fragment variable region (scFv) of an antibody, a Fab, Fv, Fc, or (Fab’)2 fragment, and the like.

[0038] In some embodiments, a costimulatory signaling domain serves to enhance the proliferation and survival of the lymphocytes upon binding of the CAR to a targeted moiety. The identity of the costimulatory signaling domain is limited only in that it has the ability to enhance cellular proliferation and survival activation upon binding of the targeted moiety by the CAR. Suitable costimulatory signaling domains include, but are not limited to: CD28 (see, e.g., Alvarez- Vallina, L. et al., Eur J Immunol. 1996. 26(10):2304-9); CD137 (4-1BB), a member of the tumor necrosis factor (TNF) receptor family (see, e.g., Imai, C. et al., Leukemia. 2004.18:676-84); and CD134 (0X40), a member of the TNFR- superfamily of receptors (see, e.g.,260132003840Latza, U. et al., Eur. J. Immunol. 1994. 24:677). A skilled artisan will understand that sequence variants of these costimulatory signaling domains can be used, where the variants have the same or similar activity as the domain on which they are modeled. In various embodiments, such variants have 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% sequence identity to the amino acid sequence of the domain from which they are derived.

[0039] In some embodiments of the invention, the CAR constructs comprise two costimulatory signaling domains. While the particular combinations include all possible variations of the four noted domains, specific examples include: 1) CD28+CD137 (4-1BB) and 2) CD28+CD134 (0X40).

[0040] In some embodiments, the activation signaling domain serves to activate cells upon binding of the CAR to a targeted moiety. The identity of the activation signaling domain is limited only in that it has the ability to induce activation of the selected cell upon binding of the targeted moiety by the CAR. Suitable activation signaling domains include the CD3(^ chain and Fc receptor y. In some embodiments, the signaling domain is a signaling domain of NKG2C or NKp44. The skilled artisan will understand that sequence variants of these noted activation signaling domains can be used without adversely impacting the invention, where the variants have the same or similar activity as the domain on which they are modeled. Such variants may have 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% sequence identity to the amino acid sequence of the domain from which they are derived.

[0041] In some embodiments, the CARs may include additional elements, for example, a transmembrane domain to ensure the fusion protein is maintained as an integral membrane protein, and a hinge domain that imparts flexibility to the recognition region and allows strong binding to the targeted moiety.

[0042] In some aspects, the polynucleotide constructs provided herein comprise a signal peptide, which ensures proper export of the fusion protein to the cells surface. In some embodiments, the signal peptide is modified. In some embodiments, the signal peptide is a CD8 signal peptide. In some embodiments, the CD8 signal peptide amino acid sequence is modified to MALPVTALLLPLALLLHAARA (SEQ ID NO: 7). In some embodiments, the modified signal peptide increases cleavage likelihood. In some embodiments, the increase in cleavage likelihood increases expression of the associated fusion protein.260132003840

[0043] In some embodiments, nucleotide sequence that encodes a CAR comprising an extracellular domain, optionally a hinge domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a costimulatory domain and an activation signaling domain. In some embodiments, the costimulatory and activation signaling 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 signaling domain. In some embodiments, the CAR comprises an extracellular domain, a CD8a hinge, a CD8a transmembrane domain, a 4- IBB costimulatory domain, and a CD3zeta signaling domain. In some embodiments, the CD8a hinge sequence is modified (e.g., mutated). In some embodiments, the CD8 alpha hinge is ten amino acids shorter than CD8 alpha hinge wildtype sequence. In some embodiments, the CD8a hinge is the shorter variant (e.g., the last ten amino acids are deleted; FVPVFLPAKP (SEQ ID NO: 1)). In some embodiments, the CD8a hinge is the wildtype variant (e.g., no changes to the amino acid sequence). In some embodiments, a nucleotide sequence encodes an extracellular domain, an CD28 hinge domain, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3zeta signaling domain. In some embodiments, the nucleotide sequence encodes an extracellular domain, an IgG4 hinge domain, a CD28 transmembrane domain, a 4- IBB co-stimulatory domain, and a CD3zeta signaling domain. In some embodiments, the nucleotide sequence encodes a CAR comprising an extracellular domain, a CD8a hinge, a CD28 transmembrane domain, a 4- IBB costimulatory domain, and a CD3zeta signaling domain.

[0044] Illustrative CAR constructs suitable for the provided polynucleotide constructs are provided below, including for use with the modified signal peptide (e.g., SEQ ID NO: 7):(1) SCFV-CD8TM-4-1BBIC-CD3^S (see, e.g., Liu E, Tong Y, Doth G, et al., Leukemia. 2018; 32: 520-531);(2) SCFV-CD28TM+IC-CD3^S (see, e.g., Han J, Chu J, Keung CW et al., Sci Rep. 2015; 5:11483; Kruschinski A, Moosmann A, Poschke I et al., Proc Natl Acad Sci USA. 2008; 105: 17481-17486; and Chu J, Deng Y, Benson DM et al., Leukemia. 2014; 28: 917-927);(3) SCFV-DAP12TM+IC (see, e.g., Muller N, Michen S, Tietze S et al., J Immunother. 2015; 38: 197-210);(4) SCFV-CD8TM-2B4IC-CD3^S (see, e.g., Xu Y, Liu Q, Zhong M et al., J Hematol Oncol. 2019; 12: 49);260132003840(5) SCFV-2B4TM+IC-CD3^S (see, e.g., Altvater B, Landmeier S, Pscherer S et al., Clin Cancer Res. 2009; 15: 4857-4866);(6) SCFV-CD28TM+IC-4-1BBIC-CD3^S (see, e.g., Kloss S, Oberschmidt O, Morgan M et al., Hum Gene Ther. 2017; 28: 897-913);(7) SCFV-CD16TM-2B4IC-CD3^S (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192);(8) SCFV-NKP44TM-DAP10IC-CD3^S (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192);(9) SCFV-NKP46TM-2B4IC-CD3^S (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192);(10) SCFV-NKG2DTM-2B4IC-CD3^S (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192);(11) SCFV-NKG2DTM-4-1BBIC-CD3^S (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192);(12) SCFV-NKG2DTM-2B4IC-DAP12IC-CD3^S (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192);(13) SCFV-NKG2DTM-2B4IC-DAP10IC-CD3^S (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192);(14) SCFV-NKG2DTM-4-1BBIC-2B4IC-CD3^S (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); and(15) SCFV-NKG2DTM-CD3^S (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192).1. Extracellular Binding Domain

[0045] In some embodiments, the binding portion of the CAR can be directed to any antigen that is desired to be targeted, such as due to its overexpression on cells or association with a disease or conditions like cancer. In some embodiments, the antigen binding domain comprises a means for binding an antigen. In some embodiments, the antigen is a tumor antigen, an antigen on an autoreactive B cell or T cell, a viral antigen, a small molecule, or a synthetic antigen. In some embodiments, the small molecule is a hapten. In some embodiments, the antigen binding domain is a single chain variable fragment (scFv) or a VHH single domain antibody.

[0046] In some embodiments, the binding portion of the CAR is specific to a tumor antigen. The selection of the antigen binding domain will depend on the particular type of cancer to be260132003840treated. Tumor antigens are well known in the art and include, for example, a glioma- associated antigen, carcinoembryonic antigen (CEA), EGFRvIII, IL-llRa, IL-13Ra, EGFR, FAP, B7H3, Kit, CA EX, CS-1, MUC1, BCMA, bcr-abl, HER2, P-human chorionic gonadotropin, alphafetoprotein (AFP), ALK, CD19, CD123, cyclin Bl, lectin-reactive AFP, Fos-related antigen 1, ADRB3, thyroglobulin, EphA2, RAGE-1, RU1, RU2, SSX2, AKAP-4, LCK, OY-TES1, PAXS, SART3, CEL-1, fucosyl GM1, GloboH, MN-CA IX, EPCAM, EVT6-AML, TGS5, human telomerase reverse transcriptase, plysialic acid, PLAC1, RU1, RU2 (AS), intestinal carboxyl esterase, lewisY, sLe, LY6K, mut hsp70-2, M-CSF, MYCN, RhoC, TRP-2, CYPIBI, BORIS, prostase, prostate-specific antigen (PSA), PAX3, PAP, NY-ESO-1, LAGE-la, LMP2, NCAM, p53, p53 mutant, Ras mutant, gplOO, prostein, OR51E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR-beta, survivin and telomerase, legumain, HPV E6, E7, sperm protein 17, SSEA-4, tyrosinase, TARP, WT1, prostate-carcinoma tumor antigen- 1 (PCTA-1), ML-IAP, MAGE, MAGE-A1, MAD-CT-1, MAD-CT-2, MelanA / MART 1, XAGE1, ELF2M, ERG (TMPRSS2 ETS fusion gene), NAU, neutrophil elastase, sarcoma translocation breakpoints, NY-BR-1, ephnnB2, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD97, CD 171, CD 179a, androgen receptor, FAP, insulin growth factor (IGF)-I, IGFII, IGF-I receptor, GD2, o-acetyl-GD2, GD3, GM3, GPRCSD, GPR20, CXORF61, folate receptor (FRa), folate receptor beta, R0R1, Flt3, TAG72, TN Ag, Tie 2, TEM1, TEM7R, CLDN6, TSHR, UPK2, and mesothelin. Non-limiting examples of tumor antigens include the following: Differentiation antigens such as tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pi 5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP- 180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, IL13Ra2, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29VBCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG1 6, TA-90\Mac-2 binding protein\cyclophilm C-associated protein, TAAL6, TAG72,260132003840TLP, TPS, GPC3, MUC16, LMP1, EBMA-1, BARF-1, CS1, CD319, HER1, B7H6, L1CAM, IL6, and MET. In some embodiments, the CAR comprises binding domains that target two or more antigens as disclosed herein, in any combination. For example: CD19 and CD3, BCMA and CD3, GPRC5D and CD3, FCRL5 and CD3, CD38 and CD3, CD19 and CD20, CD19 and CD22, BCMA and GPRC5D, or CD20 and CD22. In some embodiments, the CAR comprises binding domains that target two or more antigens on the same target protein, for example two epitopes in BCMA. In some embodiments, the CAR comprises one or more binding domains that target CD22.

[0047] A skilled artisan is readily familiar with CARs against diverse tumor antigens. Any one of such CARs can be employed as the CAR. Numerous CARs have been incorporated into products approved by the FDA and include, but are not limited to, anti-CD19 and anti-BCMA CAR T cells such as tisagenlecleucel (Kymriah), axicabtagene ciloleucel (Yescarta), brexucabtagene autoleucel (Tecartus), lisocabtagene maraleucel (Breyanzi), idecabtagene vicleucel (Abecma), or ciltacabtagene autoleucel (Carvykti). It is within the level of a skilled artisan to generate similar constructs for specific targeting of a desired tumor antigen.

[0048] In some embodiments, the binding portion of the CAR can be directed to a universal antigen to target a wide variety of tumors without the need to prepare separate CAR constructs. The targeted moiety recognized by the CAR may also remain constant. In some embodiments, a ligand may be administered to the subject to allow interaction with target cells and interaction with the binding portion of the CAR. It is only the ligand portion of the small conjugate molecule that needs to be altered to allow the system to target cancer cells of different identity. Exemplary CAR systems are described in the section below.

[0049] In some embodiments, the CAR is an anti-CD19 CAR and the extracellular binding domain of the CD 19 CAR is specific to CD 19, for example, human CD 19. In some embodiments, the extracellular domain of the CD 19 CAR comprises an scFv derived from the FMC63 monoclonal antibody (FMC63), which comprises the heavy chain variable region (VH) and the light chain variable region (VL) of FMC63 connected by a linker. FMC63 and the derived scFv have been described in Nicholson et al., Mol. Immun. 34(16- 17): 1157-1165 (1997) and PCT Application Publication No. WO2018 / 213337, the entire contents of each of which are incorporated by reference herein. An exemplary anti-CD19 CAR is shown in Table 1 with its different portions including the extracellular domain.260132003840

[0050] In some embodiments, the CAR is an anti-CD20 CAR and the extracellular binding domain of the CD20 CAR is specific to CD20, for example, human CD20. In some embodiments, the extracellular binding domain of the CD20 CAR is derived from an antibody specific to CD20, including, for example, Leul6, IF5, 1.5.3, rituximab, obinutuzumab, ibritumomab, ofatumumab, tositumumab, odronextamab, veltuzumab, ublituximab, and ocrelizumab. In any of these embodiments, the extracellular binding domain of the CD20 CAR can comprise or consist of the VH, the VL, and / or one or more CDRs of any of the antibodies. An exemplary anti-CD20 CAR is shown in Table 2 and Table 3 with its different portions including its extracellular domain.

[0051] In some embodiments, the CAR is an anti-CD22 CAR and the extracellular binding domain of the CD22 CAR is specific to CD22, for example, human CD22. In some embodiments, the extracellular binding domain of the CD22 CAR is derived from an antibody specific to CD22, including, for example, epratuzumab, and inotuzumab ozogamicin. In any of these embodiments, the extracellular binding domain of the CD22 CAR can comprise or consist of the VH, the VL, and / or one or more CDRs of any of the antibodies. An exemplary anti-CD22 CAR is shown in Table 5 with its different portions including its extracellular domain.a. Universal CARs

[0052] In some embodiments, the CAR is a universal CAR that contains an antigen binding domain means for binding a universal tag. In some embodiments, the universal tag is a hapten. In some embodiments, the antigen binding domain of the CAR is a hapten-binding domain. In some embodiments, the hapten is fluorescein or fluorescein isothiocyanate and the CAR is an anti-FITC CAR. Antigen binding domains, including VL / VH pairs and scFvs, for innumerable haptens are known in the art or can be generated by conventional methods routinely.Accordingly, the present disclosure contemplates using any known hapten-binding domain. In some embodiments, the CAR is able to recognize a synthetic ligand composed of an agent that is a cell-targeting moiety that binds to a desired target cell (such as a cancer cell) and the hapten.

[0053] In some embodiments, the CAR is an anti-FITC CAR, and the ligand is composed of a fluorescein or fluorescein isothiocyanate (FITC) moiety conjugated to a cell targeting moiety that binds to a desired target cell (such as a cancer cell). Exemplary ligands are described below. In some embodiments, the ligand is FITC-folate.

[0054] An exemplary anti-FITC CAR is shown in Table 4 (see Section IB) and its different portions.260132003840

[0055] In some embodiments, the CAR comprises an scFv domain. In some embodiments, the scFv domain comprises anti-fluorescein isothiocyanate (FITC) E2. In some embodiments, the scFv domain comprises a light chain variable domain (VL), a linker, and a heavy chain variable domain (VH).

[0056] In some embodiments, the scFv VL comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 18 or 19. In some embodiments, the scFv VL comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NOS: 18 or 19. In some embodiments, the scFv VL comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NOS: 18 or 19. In some embodiments, the scFv VL comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NOS: 18 or 19. In some embodiments, the scFv VL comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NOS: 18 or 19. In some embodiments, the scFv VL comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NOS: 18 or 19. In some embodiments, the scFv VL comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NOS: 18 or 19. In some embodiments, the scFv VL comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NOS: 18 or 19. In some embodiments, the scFv VL comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NOS: 18 or 19. In some embodiments, the scFv VL comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NOS: 18 or 19. In some embodiments, the scFv VL comprises the nucleotide sequence of SEQ ID NOS: 18 or 19. In some embodiments, the scFv VL consists of the nucleotide sequence of SEQ ID NOS: 18 or 19.

[0057] In some embodiments, the scFv VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the scFv VL comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the scFv VL comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the scFv VL comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the scFv VL comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the scFv VL comprises an amino acid sequence at least 96%260132003840identical to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the scFv VL comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the scFv VL comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the scFv VL comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the scFv VL comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 2O.In some embodiments, the scFv VL comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the scFv VL consists the amino acid sequence of SEQ ID NO: 20.

[0058] In some embodiments, the scFv VH comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 21 or 22. In some embodiments, the scFv VH comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NOS: 21 or 22. In some embodiments, the scFv VH comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NOS: 21 or 22. In some embodiments, the scFv VH comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NOS: 21 or 22. In some embodiments, the scFv VH comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NOS: 21 or 22. In some embodiments, the scFv VH comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NOS: 21 or 22. In some embodiments, the scFv VH comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NOS: 21 or 22. In some embodiments, the scFv VH comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NOS: 21 or 22. In some embodiments, the scFv VH comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NOS: 21 or 22. In some embodiments, the scFv VH comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NOS: 21 or 22. In some embodiments, the scFv VH comprises the nucleotide sequence of SEQ ID NOS: 21 or 22. In some embodiments, the scFv VH consists of the nucleotide sequence of SEQ ID NOS: 21 or 22.

[0059] In some embodiments, the scFv VH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the scFv VH comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the scFv VH260132003840comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the scFv VH comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the scFv VH comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the scFv VH comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the scFv VH comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the scFv VH comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the scFv VH comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the scFv VH comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the scFv VH comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the scFv VH consists the amino acid sequence of SEQ ID NO: 23.

[0060] In some embodiments, the scFv linker comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 24 or 25. In some embodiments, the scFv linker comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NOS: 24 or 25. In some embodiments, the scFv linker comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NOS: 24 or 25. In some embodiments, the scFv linker comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NOS: 24 or 25. In some embodiments, the scFv linker comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NOS: 24 or 25. In some embodiments, the scFv linker comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NOS: 24 or 25. In some embodiments, the scFv linker comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NOS: 24 or 25. In some embodiments, the scFv linker comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NOS: 24 or 25. In some embodiments, the scFv linker comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NOS: 24 or 25. In some embodiments, the scFv linker comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NOS: 24 or 25. In some embodiments, the scFv linker comprises the260132003840nucleotide sequence of SEQ ID NOS: 24 or 25. In some embodiments, the scFv linker consists the nucleotide sequence of SEQ ID NOS: 24 or 25.

[0061] In some embodiments, the scFv linker comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the scFv linker comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the scFv linker comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the scFv linker comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the scFv linker comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the scFv linker comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the scFv linker comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the scFv linker comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the scFv linker comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the scFv linker comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the scFv linker comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the scFv linker consists the amino acid sequence of SEQ ID NO: 26.

[0062] In some embodiments, the scFv comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 27 or 28. In some embodiments, the scFv comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NOS: 27 or 28. In some embodiments, the scFv comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NOS: 27 or 28. In some embodiments, the scFv comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NOS: 27 or 28. In some embodiments, the scFv comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NOS: 27 or 28. In some embodiments, the scFv comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NOS: 27 or 28. In some embodiments, the scFv comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NOS: 27 or 28. In some embodiments, the scFv comprises a nucleotide sequence at least 98% identical to the260132003840nucleotide sequence of SEQ ID NOS: 27 or 28. In some embodiments, the scFv comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NOS: 27 or 28. In some embodiments, the scFv comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NOS: 27 or 28. In some embodiments, the scFv comprises the nucleotide sequence of SEQ ID NOS: 27 or 28. In some embodiments, the scFv consists of the nucleotide sequence of SEQ ID NOS: 27 or 28.

[0063] In some embodiments, the scFv comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the scFv comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the scFv comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the scFv comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the scFv comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the scFv comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the scFv comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the scFv comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the scFv comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the scFv comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the scFv consists of the amino acid sequence of SEQ ID NO: 29.

[0064] Various methods to target CARs and CAR-expressing cells have been described in the art, including, for example in US 2020 / 0123224, the disclosure of which is incorporated by reference herein. For example, a fluorescein or fluorescein isothiocyanate (FITC) moiety may be conjugated to an agent that binds to a desired target cell (such as a cancer cell), and thereby a CAR expressing an anti-fluorescein / FITC chimeric antigen receptor may be selectively targeted to the target cell labeled by the conjugate. In variations, other haptens recognized by CARs may be used in place of fluorescein / FITC. The CAR may be generated using various scFv sequences known in the art, or scFv sequences generated by conventional and routine methods. Further260132003840illustrative scFv sequences for fluorescein / FITC and for other haptens are provided in, for example, WO 2021 / 076788, the disclosure of which is incorporated by reference herein.

[0065] In one embodiment, the disclosure provides an illustration of this conjugate molecule / CAR system.

[0066] In some embodiments, the CAR system of the disclosure utilizes conjugate molecules as the bridge between CAR-expressing cells and targeted cancer cells. The conjugate molecules are conjugates comprising a hapten and a cell-targeting moiety, such as any suitable tumor cell-specific ligand. Illustrative haptens that can be recognized and bound by CARs, include small molecular weight organic molecules such as DNP (2,4-dinitrophenol), TNP (2,4,6-trinitrophenol), biotin, and digoxigenin, along with fluorescein and derivatives thereof, including FITC (fluorescein isothiocyanate), NHS-fluorescein, and pentafluorophenyl ester (PFP) and tetrafluorophenyl ester (TFP) derivatives, a knottin, a centyrin, and a DARPin. Suitable celltargeting moiety that may themselves act as a hapten for a CAR include knottins (see Kolmar H. et al., The FEBS Journal. 2008. 275(11):26684-90), centyrins, and DARPins (see Reichert, J.M. Ato 2009. 1(3): 190-209).

[0067] In some embodiments, the cell-targeting moiety is DUPA (DUPA-(99m) Tc), a ligand bound by PSMA-positive human prostate cancer cells with nanomolar affinity (KD = 14 nM; see Kularatne, S.A. et al., Mol Pharm. 2009. 6(3):780-9). In one embodiment, a DUPA derivative can be the ligand of the small molecule ligand linked to a targeting moiety, and DUPA derivatives are described in WO 2015 / 057852, incorporated herein by reference.

[0068] In some embodiments, the cell-targeting moiety is CCK2R ligand, a ligand bound by CCK2R-positive cancer cells (e.g., cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stroma, and colon; see Wayua. C. et al., Molecular Pharmaceutics. 2013. ePublication).

[0069] In some embodiments, the cell-targeting moiety is folate, folic acid, or an analogue thereof, a ligand bound by the folate receptor on cells of cancers that include cancers of the ovary, cervix, endometrium, lung, kidney, brain, breast, colon, and head and neck cancers; see Sega, E.I. et al., Cancer Metastasis Rev. 2008. 27(4):655-64).

[0070] In some embodiments, the cell-targeting moiety is an NK-1R ligand. Receptors for NK-1R the ligand are found, for example, on cancers of the colon and pancreas. In some embodiments, the NK-1R ligand may be synthesized according to the method disclosed in IntT Patent Appl. No. PCT / US2015 / 044229, incorporated herein by reference.260132003840

[0071] In some embodiments, the cell-targeting moiety may be a peptide ligand, for example, the ligand may be a peptide ligand that is the endogenous ligand for the NK1 receptor. In some embodiments, the small conjugate molecule ligand may be a regulatory peptide that belongs to the family of tachykinins which target tachykinin receptors. Such regulatory peptides include Substance P (SP), neurokinin A (substance K), and neurokinin B (neuromedin K), (see Hennig et al., International Journal of Cancer: 61, 786-792).

[0072] In some embodiments, the cell-targeting moiety is a CAIX ligand. Receptors for the CAIX ligand found, for example, on renal, ovarian, vulvar, and breast cancers. The CAIX ligand may also be referred to herein as CA9.

[0073] In some embodiments, the cell-targeting moiety is a ligand of gamma glutamyl transpeptidase. The transpeptidase is overexpressed, for example, in ovarian cancer, colon cancer, liver cancer, astrocytic gliomas, melanomas, and leukemias.

[0074] In some embodiments, the cell-targeting moiety is a CCK2R ligand. Receptors for the CCK2R ligand found on cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stroma, and colon, among others.

[0075] In some embodiments, the cell-targeting moiety is a PSMA ligand.

[0076] In some embodiments, the cell-targeting moiety is a FAP ligand.

[0077] In one embodiment, the cell-targeting moiety may have a mass of less than about 10,000 Daltons, less than about 9000 Daltons, less than about 8,000 Daltons, less than about 7000 Daltons, less than about 6000 Daltons, less than about 5000 Daltons, less than about 4500 Daltons, less than about 4000 Daltons, less than about 3500 Daltons, less than about 3000 Daltons, less than about 2500 Daltons, less than about 2000 Daltons, less than about 1500 Daltons, less than about 1000 Daltons, or less than about 500 Daltons. In another embodiment, the small molecule ligand may have a mass of about 1 to about 10,000 Daltons, about 1 to about 9000 Daltons, about 1 to about 8,000 Daltons, about 1 to about 7000 Daltons, about 1 to about 6000 Daltons, about 1 to about 5000 Daltons, about 1 to about 4500 Daltons, about 1 to about 4000 Daltons, about 1 to about 3500 Daltons, about 1 to about 3000 Daltons, about 1 to about 2500 Daltons, about 1 to about 2000 Daltons, about 1 to about 1500 Daltons, about 1 to about 1000 Daltons, or about 1 to about 500 Daltons.

[0078] In one illustrative embodiment, the linkage in a conjugate described herein can be a direct linkage (e.g., a reaction between the isothiocyanate group of FITC and a free amine group of a small molecule ligand) or the linkage can be through an intermediary linker. In one260132003840embodiment, if present, an intermediary linker can be any biocompatible linker known in the art, such as a divalent linker. In one illustrative embodiment, the divalent linker can comprise about 1 to about 30 carbon atoms. In another illustrative embodiment, the divalent linker can comprise about 2 to about 20 carbon atoms. In other embodiments, lower molecular weight divalent linkers (i.e., those having an approximate molecular weight of about 30 to about 300 Da) are employed. In another embodiment, linkers lengths that are suitable include, but are not limited to, linkers having 2, 3, 4, 5, 6, 7, 8, 9. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37. 38, 39 or 40, or more atoms.

[0079] In some embodiments, the hapten and the cell-targeting moiety can be directly conjugated through such means as reaction between the isothiocyanate group of FITC and free amine group of small ligands (e.g., folate, DUPA, and CCK2R ligand). However, the use of a linking domain to connect the two molecules may be helpful as it can provide flexibility and stability. Examples of suitable linking domains include: 1) polyethylene glycol (PEG); 2) polyproline; 3) hydrophilic amino acids; 4) sugars; 5) unnatural peptidoglycans; 6) polyvinylpyrrolidone; 7) Pluronic F-127. Linker lengths that are suitable include, but are not limited to, linkers having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, or more atoms.

[0080] In some embodiments, the linker may be a divalent linker that may include one or more spacers.

[0081] An illustrative conjugate of the disclosure is FITC-FolateAn illustrative conjugate of the disclosure is FITC-CA9260132003840

[0082] Illustrative conjugates of the disclosure include the following molecules: FITC-(PEG)i2-Folate, FITC-(PEG)2o-Folate, FITC-(PEG)io8-Folate, FITC-DUPA, FITC-(PEG)I2-DUPA, FITC-CCK2R ligand, FITC-(PEG)I2-CCK2R ligand, FITC-(PEG)n-NKlR ligand and FITC-(PEG)2-CA9.

[0083] While the affinity at which the ligands and cancer cell receptors bind can vary, and in some cases low affinity binding may be preferable (such as about 1 pM), the binding affinity of the ligands and cancer cell receptors will generally be at least about 100 pM, 1 nM, 10 nM, or 100 nM, preferably at least about 1 pM or 10 pM, even more preferably at least about 100 pM.

[0084] Examples of conjugates and methods of making them are provided in U.S. patent applications US 2017 / 0290900, US 2019 / 0091308, and US 2020 / 0023009, all of which are incorporated herein by reference.2. Hinge Domain

[0085] In some embodiments, the CAR comprises a hinge domain. In some embodiments, the hinge domain comprises a short hinge or a medium hinge domain. In some embodiments, the hinge domain comprises a CD8 or an IgG. In some embodiments, the CD8 hinge comprises CD8a hinge. In some embodiments, the IgG hinge comprises an IgG4 hinge. In some embodiments, the IgG4 hinge is modified. In some embodiments, the IgG hinge comprises an IgGl hinge. In some embodiments, the hinge domain comprises a PD1 hinge. In some embodiments, the hinge domain comprises a CD28 hinge.

[0086] In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 30 or 31. In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NOS: 30 or 31. In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NOS: 30 or 31. In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NOS: 30 or 31. In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NOS: 30 or 31. In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NOS: 30 or 31. In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NOS: 30 or 31. In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 98% identical to the260132003840nucleotide sequence of SEQ ID NOS: 30 or 31. In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NOS: 30 or 31. In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NOS: 30 or 31. In some embodiments, the CD8a hinge comprises the nucleotide sequence of SEQ ID NOS: 30 or 31. In some embodiments, the CD8a hinge consists of the nucleotide sequence of SEQ ID NOS: 30 or 31.

[0087] In some embodiments, the CD8a hinge comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 32 or 33. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NOS: 32 or 33. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NOS: 32 or 33. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NOS: 32 or 33. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NOS: 32 or 33. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NOS: 32 or 33. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NOS: 32 or 33. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NOS: 32 or 33. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NOS: 32 or 33. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NOS: 32 or 33. In some embodiments, the CD8a hinge comprises the amino acid sequence of SEQ ID NOS: 32 or 33. In some embodiments, the CD8a hinge consists of the amino acid sequence of SEQ ID NOS: 32 or 33.

[0088] In some embodiments, the CD8 hinge comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the CD8 hinge comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the CD8 hinge comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the CD8 hinge comprises a nucleotide sequence at least 90%260132003840identical to the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the CD8 hinge comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the CD8 hinge comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the CD8 hinge comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the CD8 hinge comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the CD8 hinge comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the CD8 hinge comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the CD8 hinge comprises the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the CD8 hinge consists of the nucleotide sequence of SEQ ID NO: 3.

[0089] In some embodiments, the CD8 hinge lacks amino acids of SEQ ID NO: 1.

[0090] In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 51. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 51. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 51. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 51. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 51. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 51. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 51. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 51. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 51. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 51. In some embodiments, the modified IgG4 hinge comprises the amino acid sequence of SEQ ID NO: 51.260132003840In some embodiments, the modified IgG4 hinge consists of the amino acid sequence of SEQ ID NO: 51.

[0091] In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 52. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 52. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 52. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 52. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 52. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 52. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 52. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 52. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 52. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 52. In some embodiments, the modified IgG4 hinge comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the modified IgG4 hinge consists of the amino acid sequence of SEQ ID NO: 52.

[0092] In some embodiments, the IgGl hinge comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the IgGl hinge comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the IgGl hinge comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the IgGl hinge comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the IgGl hinge comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the IgGl hinge comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the260132003840IgGl hinge comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the IgGl hinge comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the IgGl hinge comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the IgGl hinge comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the IgGl hinge comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the IgGl hinge consists of the amino acid sequence of SEQ ID NO: 2.

[0093] In some embodiments, the PD1 hinge comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the PD1 hinge comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the PD1 hinge comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the PD1 hinge comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the PD1 hinge comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the PD1 hinge comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the PD1 hinge comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the PD1 hinge comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the PD1 hinge comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the PD1 hinge comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the PD1 hinge comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the PD1 hinge consists of the amino acid sequence of SEQ ID NO: 53.

[0094] In some embodiments, the CD28 hinge comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CD28 hinge comprises an amino acid260132003840sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CD28 hinge comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CD28 hinge consists of the amino acid sequence of SEQ ID NO: 4.3. Transmembrane Domain

[0095] In some embodiments, the CAR comprises a transmembrane domain. In some embodiments, the transmembrane domain comprises a CD8 transmembrane domain or a CD28 transmembrane domain. In some embodiments, the transmembrane domain comprises a CD8 domain. In some embodiments, the transmembrane domain comprises a CD28 domain. In some embodiments, the CD8 transmembrane domain comprises CD8a transmembrane domain. In some embodiments, the transmembrane domain is a CD8 transmembrane domain or a CD28 transmembrane domain.

[0096] In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 34. In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NO: 34. In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NO: 34. In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NO: 34. In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: 34. In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NO: 34. In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 97% identical to the nucleotide260132003840sequence of SEQ ID NO: 34. In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NO: 34. In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NO: 34. In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NO: 34. In some embodiments, the transmembrane domain comprises the nucleotide sequence of SEQ ID NO: 34. In some embodiments, the transmembrane domain consists of the nucleotide sequence of SEQ ID NO: 34.

[0097] In some embodiments, the transmembrane domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the transmembrane domain consists of the amino acid sequence of SEQ ID NO: 35.4. Intracellular Domain

[0098] In some embodiments, the CAR comprises an intracellular domain (e.g., endodomain). In some embodiments, the intracellular domain (e.g., endodomain) comprises a costimulatory molecule. In some embodiments, the intracellular domain comprises 4- IBB260132003840signaling domain, CD3 zeta (CD3Q domain, and / or CD28 signaling domain. In some embodiments, the intracellular domain comprises 4- IBB signaling domain. In some embodiments, the intracellular domain comprises CD3(^ domain. In some embodiments, the intracellular domain comprises CD28 signaling domain.

[0099] In some embodiments, the 4- IBB signaling domain comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 36 or 37. In some embodiments, the 4-1BB signaling domain comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NOS: 36 or 37. In some embodiments, the 4- IBB signaling domain comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NOS: 36 or 37. In some embodiments, the 4- IBB signaling domain comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NOS: 36 or 37. In some embodiments, the 4-1BB signaling domain comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NOS: 36 or 37. In some embodiments, the 4- IBB signaling domain comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NOS: 36 or 37. In some embodiments, the 4- IBB signaling domain comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NOS: 36 or 37. In some embodiments, the 4- IBB signaling domain comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NOS: 36 or 37. In some embodiments, the 4-1BB signaling domain comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NOS: 36 or 37. In some embodiments, the 4- IBB signaling domain comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NOS: 36 or 37. In some embodiments, the 4- IBB signaling domain comprises the nucleotide sequence of SEQ ID NOS: 36 or 37. In some embodiments, the 4- IBB signaling domain consists the nucleotide sequence of SEQ ID NOS: 36 or 37.

[0100] In some embodiments, the 4- IBB signaling domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 38. In some embodiments, the 4- IBB signaling domain comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 38. In some embodiments, the 4-1BB signaling domain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 38. In some embodiments, the 4- IBB signaling domain comprises an amino acid sequence at least 90% identical to the amino acid260132003840sequence of SEQ ID NO: 38. In some embodiments, the 4- IBB signaling domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 38. In some embodiments, the 4- IBB signaling domain comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 38. In some embodiments, the 4-1BB signaling domain comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 38. In some embodiments, the 4- IBB signaling domain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 38. In some embodiments, the 4- IBB signaling domain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 38. In some embodiments, the 4-1BB signaling domain comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 38. In some embodiments, the 4- IBB signaling domain comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the 4- IBB signaling domain consists of the amino acid sequence of SEQ ID NO: 38.

[0101] In some embodiments, the CD3(^ domain comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 39, 40, 41, or 42. In some embodiments, the CD3(^ domain comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NOS: 39, 40, 41, or 42. In some embodiments, the CD3(^ domain comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NOS: 39, 40, 41, or 42. In some embodiments, the CD3(^ domain comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NOS: 39, 40, 41, or 42. In some embodiments, the CD3(^ domain comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NOS: 39, 40, 41, or 42. In some embodiments, the CD3(^ domain comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NOS: 39, 40, 41, or 42. In some embodiments, the CD3(^ domain comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NOS: 39, 40, 41, or 42. In some embodiments, the CD3(^ domain comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NOS: 39, 40, 41, or 42. In some embodiments, the CD3(^ domain comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NOS: 39, 40, 41, or 42. In some embodiments, the CD3(^ domain comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NOS: 39, 40, 41, or 42. In some embodiments, the CD3(^ domain comprises the nucleotide sequence of SEQ260132003840ID NOS: 39, 40, 41, or 42. In some embodiments, the CD3(^ domain consists of the nucleotide sequence of SEQ ID NOS: 39, 40, 41, or 42.

[0102] In some embodiments, the CD3(^ domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 43. In some embodiments, the CD3(^ domain comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 43. In some embodiments, the CD3(^ domain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 43. In some embodiments, the CD3(^ domain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 43. In some embodiments, the CD3(^ domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 43. In some embodiments, the CD3(^ domain comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 43. In some embodiments, the CD3(^ domain comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 43. In some embodiments, the CD3(^ domain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 43. In some embodiments, the CD3(^ domain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 43. In some embodiments, the CD3(^ domain comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 43. In some embodiments, the CD3(^ domain comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, the CD3(^ domain consists of the amino acid sequence of SEQ ID NO: 43.B. Exemplary CARs

[0103] In some embodiments, the CAR is an anti-CD19 CAR. In some embodiments, the anti-CD19 CAR comprises an extracellular binding domain with means for binding CD 19. In some embodiments, the CD 19 CAR may comprise a signal peptide, an extracellular binding domain that specifically binds CD19, a spacer (e.g., hinge) domain, a transmembrane domain, an intracellular domain comprising an intracellular costimulatory domain and an intracellular activation signaling domain. In some embodiments, the intracellular costimulatory domain is a signaling domain of CD28 or 4- IBB. In some embodiments, the intracellular activation signaling domain is a CD3 zeta signaling domain. In some embodiments, the signal peptide is modified. In some embodiments, the signal peptide is a modified CD8 signal peptide. In some embodiments, the modified CD8 signal peptide comprises an amino acid sequence of260132003840MALPVTALLLPLALLLHAARA (SEQ ID NO: 7). In some embodiments, the polynucleotide construct encodes an anti-CD19 CAR with features set forth in Table 1.

[0104] In some embodiments, the CAR is an anti-CD20 CAR. In some embodiments, the anti-CD20 CAR comprises an extracellular binding domain with means for binding CD20. In some embodiments, the CD20 CAR may comprise a signal peptide, an extracellular binding domain that specifically binds CD20, a spacer (e.g., hinge) domain, a transmembrane domain, an intracellular domain comprising an intracellular costimulatory domain, and an intracellular activation signaling domain. In some embodiments, the intracellular costimulatory domain is a signaling domain of CD28 or 4- IBB. In some embodiments, the intracellular activation signaling domain is a CD3 zeta signaling domain. In some embodiments, the signal peptide is modified. In some embodiments, the signal peptide is a modified CD8 signal peptide. In some embodiments, the modified CD8 signal peptide comprises an amino acid sequence of MALPVTALLLPLALLLHAARA (SEQ ID NO: 7). In some embodiments, the polynucleotide260132003840construct encodes an anti-CD20 CAR with features set forth in Table 2 (anti-CD20 CAR with flag) or Table 3 (anti-CD20 CAR without flag).260132003840

[0105] In some embodiments, the polynucleotide construct encodes a CAR with features set forth in Table 4. In some embodiments, the CAR is an anti-FITC CAR. In some embodiments, the anti-FITC CAR comprises a signal peptide. In some embodiments, the signal peptide is modified. In some embodiments, the signal peptide is a modified CD8 signal peptide. In some embodiments, the modified CD8 signal peptide comprises an amino acid sequence of MALPVTALLLPLALLLHAARA (SEQ ID NO: 7).260132003840

[0106] In some embodiments, the CAR is an anti-CD22 CAR. In some embodiments, the anti-CD22 CAR comprises an extracellular binding domain with means for binding CD22. In some embodiments, the CD22 CAR comprises a signal peptide, an extracellular binding domain that specifically binds CD22, a spacer (e.g., hinge) domain, a transmembrane domain, an intracellular domain comprising an intracellular costimulatory domain, and an intracellular activation signaling domain. In some embodiments, the intracellular costimulatory domain is a signaling domain of CD28 or 4- IBB. In some embodiments, the intracellular activation signaling domain is a CD3 zeta signaling domain. In some embodiments, the signal peptide is modified. In some embodiments, the signal peptide is a modified CD8 signal peptide. In some embodiments, the modified CD8 signal peptide comprises an amino acid sequence of MALPVTALLLPLALLLHAARA (SEQ ID NO: 7). In some embodiments, the polynucleotide construct encodes an anti-CD22 CAR with sequences set forth in Table 5. In some particular embodiments, the CAR is a CAR set forth in SEQ ID NO: 14 and encoded by a sequence set forth in SEQ ID NO: 11 or degenerates thereof. In other particular embodiments, the CAR is a CAR set forth in SEQ ID NO: 15 and encoded by a sequence set forth in SEQ ID NO: 12 or degenerates thereof.260132003840

[0107] In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 44, 45, 46, or 47. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NOS: 44, 45, 46, or 47. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NOS: 44, 45, 46, or 47. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NOS: 44, 45, 46, or 47. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NOS: 44, 45, 46, or 47. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NOS: 44, 45, 46, or 47. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NOS: 44, 45, 46, or 47. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NOS: 44, 45, 46, or 47. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NOS: 44, 45, 46, or 47. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NOS: 44, 45, 46, or 47. In some embodiments, the polynucleotide construct comprises the nucleotide sequence of SEQ ID NOS: 44, 45, 46, or 47. In some embodiments, the polynucleotide construct consists of the nucleotide sequence of SEQ ID NOS: 44, 45, 46, or 47.260132003840

[0108] In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 44, 45, or 47. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NOS: 44, 45, or 47. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NOS: 44, 45, or 47. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NOS: 44, 45, 47. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NOS: 44, 45, 47. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NOS: 44, 45, 47. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NOS: 44, 45, 47. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NOS: 44, 45, 47. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NOS: 44, 45, 47. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NOS: 44, 45, 47. In some embodiments, the polynucleotide construct comprises the nucleotide sequence of SEQ ID NOS: 44, 45, 47. In some embodiments, the polynucleotide construct consists of the nucleotide sequence of SEQ ID NOS: 44, 45, 47.

[0109] In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 48, 49, or 50. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 80% identical to the nucleotide sequence of SEQ ID NOS: 48, 49, or 50. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 85% identical to the nucleotide sequence of SEQ ID NOS: 48, 49, or 50. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 90% identical to the nucleotide sequence of SEQ ID NOS: 48, 49, or 50. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 95% identical to the nucleotide sequence of SEQ ID NOS: 48, 49, or 50. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 96% identical to the nucleotide sequence of260132003840SEQ ID NOS: 48, 49, or 50. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 97% identical to the nucleotide sequence of SEQ ID NOS: 48, 49, or 50. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 98% identical to the nucleotide sequence of SEQ ID NOS: 48, 49, or 50. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 99% identical to the nucleotide sequence of SEQ ID NOS: 48, 49, or 50. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 100% identical to the nucleotide sequence of SEQ ID NOS: 48, 49, or 50. In some embodiments, the polynucleotide construct encodes an amino acid sequence comprising the sequence of SEQ ID NOS: 48, 49, or 50. In some embodiments, the polynucleotide construct encodes an amino acid sequence consisting of the sequence of SEQ ID NOS: 48, 49, or 50.

[0110] In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 48 or 50. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 80% identical to the nucleotide sequence of SEQ ID NOS: 48 or 50. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 85% identical to the nucleotide sequence of SEQ ID NOS: 48 or 50. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 90% identical to the nucleotide sequence of SEQ ID NOS: 48 or 50. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 95% identical to the nucleotide sequence of SEQ ID NOS: 48 or 50. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 96% identical to the nucleotide sequence of SEQ ID NOS: 48 or 50. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 97% identical to the nucleotide sequence of SEQ ID NOS: 48 or 50. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 98% identical to the nucleotide sequence of SEQ ID NOS: 48 or 50. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 99% identical to the nucleotide sequence of SEQ ID NOS: 48 or 50. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 100% identical to the nucleotide sequence of SEQ ID NOS: 48 or 50. In some embodiments, the polynucleotide construct encodes an amino acid sequence comprising the sequence of SEQ ID NOS: 48 or 50. In some embodiments, the260132003840polynucleotide construct encodes an amino acid sequence consisting of the sequence of SEQ ID NOS: 48 or 50.

[0111] In further embodiments, an illustrative nucleotide sequence encoding a CAR may comprise SEQ ID NO: 46, and an illustrative CAR amino acid sequence may comprise SEQ ID NO: 49.

[0112] An illustrative nucleotide insert may comprise SEQ ID NO: 72.

[0113] In some embodiments, the CAR may be encoded by a nucleic acid sequence that encodes a signal peptide to signal transport of the CAR in the cell. An illustrative CAR amino acid sequence signal peptide may comprise SEQ ID NO: 7. It is understood that typically the signal peptide is removed from the protein.

[0114] In some embodiments, when expressed by a cell, a chimeric antigen receptor polypeptide comprising SEQ ID NO: 73 is contemplated. In some embodiments, SEQ ID NO: 73 can comprise or consist of human or humanized amino acid sequences.

[0115] In some embodiments, variant nucleic acid sequences or amino acid sequences having 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% sequence identity to SEQ ID NO: 73 are contemplated.

[0116] While the affinity at which the CARs, expressed by the lymphocytes, bind to the targeted moiety can vary, and in some cases low affinity binding may be preferable (such as about 50 nM), the binding affinity of the CARs to the targeted ligand will generally be at least about 100 nM, 1 pM, or 10 pM, preferably at least about 100 pM, 1 fM or 10 fM, even more preferably at least about 100 fM.

[0117] In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the construct encodes a260132003840polypeptide comprising an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the construct encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the construct encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 13.

[0118] In some embodiments, the construct comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 10. In some embodiments, the construct comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NO: 10. In some embodiments, the construct comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NO: 10. In some embodiments, the construct comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NO: 10. In some embodiments, the construct comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: 10. In some embodiments, the construct comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NO: 10. In some embodiments, the construct comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NO: 10. In some embodiments, the construct comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NO: 10. In some embodiments, the construct comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NO: 10. In some embodiments, the construct comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NO: 10. In some embodiments, the construct comprises the nucleotide sequence of SEQ ID NO: 10. In some embodiments, the construct consists of the nucleotide sequence of SEQ ID NO: 10.

[0119] In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the construct encodes a polypeptide260132003840comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the construct encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the construct encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 14.

[0120] In some embodiments, the construct comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the construct comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the construct comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the construct comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the construct comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the construct comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the construct comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the construct comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the construct comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID260132003840NO: 11. In some embodiments, the construct comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the construct comprises the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the construct consists of the nucleotide sequence of SEQ ID NO: 11.

[0121] In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the construct encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, the construct encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 15.

[0122] In some embodiments, the construct comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the construct comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the construct comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the construct comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the construct260132003840comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the construct comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the construct comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the construct comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the construct comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the construct comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the construct comprises the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the construct consists of the nucleotide sequence of SEQ ID NO: 12.

[0123] In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the construct encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, the construct encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 17.260132003840

[0124] In some embodiments, the construct comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 16. In some embodiments, the construct comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NO: 16. In some embodiments, the construct comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NO: 16. In some embodiments, the construct comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NO: 16. In some embodiments, the construct comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: 16. In some embodiments, the construct comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NO: 16. In some embodiments, the construct comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NO: 16. In some embodiments, the construct comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NO: 16. In some embodiments, the construct comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NO: 16. In some embodiments, the construct comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NO: 16. In some embodiments, the construct comprises the nucleotide sequence of SEQ ID NO: 16. In some embodiments, the construct consists of the nucleotide sequence of SEQ ID NO: 16.IL VECTORS AND ENGINEERED RETROVIRAL PARTICLES

[0125] Also provided herein are viral vectors and engineered viral vectors, including engineered retroviral vectors, containing any of the polynucleotide constructs described herein, such as any described in Section I. These include viral vector particles, such as engineered retroviral vector particles, comprising and / or packaging any of the polynucleotide constructs and that may also be engineered with additional particle- surface proteins.

[0126] In some embodiments, the polynucleotide constructs can be inserted into a nucleic acid vector. As used herein, the term “nucleic acid vector” is intended to mean any nucleic acid that functions to carry, harbor, or express a nucleic acid of interest. Nucleic acid vectors can have specialized functions such as expression, packaging, pseudotyping, transduction or sequencing, for example. Nucleic acid vectors also can have, for example, manipulatory functions such as a cloning or shuttle vector. The structure of the vector can include any desired form that is feasible to make and desirable for a particular use. Such forms include, for example, circular forms such as plasmids and phagemids, as well as linear or branched forms. A nucleic260132003840acid vector can be composed of, for example, DNA or RNA, as well as contain partially or fully, nucleotide derivatives, analogs and mimetics. Such nucleic acid vectors can be obtained from natural sources, produced recombinantly or chemically synthesized.

[0127] Non-limiting examples of vector systems of the present disclosure include a retrovirus, a lentivirus, a foamy virus, and a Sleeping Beauty transposon.

[0128] In some embodiments, the polynucleotide is incorporated into a viral vector. As it is well known in the art, a viral particle is a tool that allows or facilitates the transfer of an entity from one environment to another. In accordance with the disclosure, and by way of example, some viral particles used in recombinant DNA techniques allow entities, such as a segment of DNA, to be transferred into a host cell. Examples of vectors used in recombinant DNA techniques include but are not limited to, plasmids, chromosomes, artificial chromosomes, or viruses. The term "expression vector" means a construct capable of in vivo or in vitro / ex vivo expression.

[0129] Further, in some aspects, provided herein is a virus particle encapsulating the polynucleotide constructs disclosed herein. In some embodiments, any of the polynucleotide constructs can be provided as a payload in the generation of a viral particle. Also provided herein are viral particles, such as lentiviral vectors, incorporating any of the provided polynucleotide constructs for delivery of components of the CAR to a target cell. In further embodiments, the virus particles can be engineered to express one or more surface T cell activating agents. In some embodiments, the one or more surface T cell activating agents comprise a T cell surface receptor. In some embodiments, the T cell surface receptor comprises CD58, anti-CD3, or CD80.

[0130] Further, in some aspects, the virus particles encapsulating nucleotide vectors provided herein can comprise engineered viral envelopes. In some embodiments, the viral envelope comprises a transduction enhancer. In some embodiments, the viral envelope comprises at an immune cell- activating protein. In some embodiments, the viral envelope comprises a co- stimulation molecule. In some embodiments, the viral envelope comprises an immune cell-activating protein, and a co- stimulation molecule.A. Retroviral Vectors

[0131] Also provided herein are retroviral particles, such as lentiviral vectors, incorporating any of the provided polynucleotide constructs for delivery of the CAR to a target cell. In further embodiments, the virus particles can be engineered to express one or more surface T cell260132003840activating agents. In some embodiments, the one or more surface T cell activating agents comprise a T cell surface receptor. In some embodiments, the T cell surface receptor comprises CD58, anti-CD3, or CD80.

[0132] Retroviruses include lentiviruses, gamma-retroviruses, and alpha-retroviruses, each of which may be used to deliver polynucleotides to cells using methods known in the art.Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. The higher complexity enables the virus to modulate its life cycle, as in the course of latent infection. Some examples of lentivirus include the Human Immunodeficiency Viruses (HIV-1 and HIV-2) and the Simian Immunodeficiency Virus (SIV). Retroviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted, making the vector biologically safe.

[0133] In some embodiments, the virus particle comprises a retroviral particle. In some embodiments, the virus is a retrovirus. A large number of different retroviruses have been identified. Examples of retrovirus include but are not limited to: murine leukemia virus (MLV), human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus (AEV). A detailed list of retroviruses may be found in Coffin et al., 1997, "Retroviruses", Cold Spring Harbor Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763.

[0134] A lentiviral vector (LVV) of the disclosure may be derived from or may be derivable from any suitable lentivirus. A recombinant retroviral vector particle is capable of transducing a recipient cell with a nucleotide of interest (NOI). Once within the cell, the RNA genome from the vector particle is reverse transcribed into DNA and integrated into the DNA of the recipient cell. In some embodiments of the disclosure, at least part of one or more protein coding regions essential for replication may be removed from the virus. This makes the viral vector replication defective. Portions of the viral genome may also be replaced by an NOI in order to generate a vector comprising an NOI which is capable of transducing a target non-dividing host cell and / or integrating its genome into a host genome.260132003840

[0135] Illustrative lentiviral vectors include those described in Naldini et al. (1996) Science 272:263-7; Zufferey et al. (1998) J. Virol. 72:9873-9880; Dull et al. (1998) J. Virol. 72:8463-8471; U.S. Pat. No. 6,013,516; and U.S. Pat. No. 5,994,136, which are each incorporated herein by reference in their entireties. In general, these vectors are configured to carry the essential sequences for selection of cells containing the vector, for incorporating foreign nucleic acid into a lentiviral particle, and for transfer of the nucleic acid into a target cell.

[0136] In some embodiments, the LVV is replication incompetent. In some embodiments, the LVV is self-inactivating and replication incompetent. Such LVV particles are desirable for safety reasons for embodiments that include introducing cells transduced with such LVV particles into a subject. When replication incompetent retroviral particles are used to transduce a cell, LVV particles are not produced from the transduced cell. Modifications to the retroviral genome are known in the art to assure that retroviral particles that include the genome are replication incompetent. However, it will be understood that in some embodiments for any of the aspects provided herein, replication competent recombinant retroviral particles can be used.

[0137] In some embodiments, the replication incompetent LVV particle can be derived from HIV, SIV, or FIV. In some embodiments, the replication incompetent LVV particle can be derived from the human immunodeficiency virus (HIV).

[0138] A commonly used lentiviral vector system is the so-called third- generation system. Third-generation lentiviral vector systems include four plasmids. The “transfer plasmid” encodes the polynucleotide sequence that is delivered by the lentiviral vector system to the target cell. The transfer plasmid generally has one or more transgene sequences of interest flanked by long terminal repeat (LTR) sequences, which facilitate integration of the transfer plasmid sequences into the host genome. For safety reasons, transfer plasmids are generally designed to make the resulting vector replication incompetent. For example, the transfer plasmid lacks gene elements necessary for generation of infective particles in the host cell. In addition, the transfer plasmid may be designed with a deletion of the 3' LTR, rendering the virus “selfinactivating” (SIN). See Dull et al. (1998) J. Virol. 72:8463-71; Miyoshi et al. (1998) J. Virol.72:8150-57. The viral particle may also comprise a 3' untranslated region (UTR) and a 5' UTR. The UTRs comprise retroviral regulatory elements that support packaging, reverse transcription, and integration of a proviral genome into a cell following contact of the cell by the retroviral particle.260132003840

[0139] In some embodiments, the LVV genome comprises a self-inactivating (SIN) long terminal repeat (LTR) configuration. The 3' LTR may include a U3 deletion sufficient to abrogate LTR promoter / enhancer activity following reverse transcription and integration. In certain embodiments, the U3 deletion is about 100-400 bp, optionally about 200-300 bp, and is copied to the 5' LTR during reverse transcription, thereby reducing the risk of enhancer-mediated activation of host genes.

[0140] In some embodiments, the LVV is replication-incompetent by virtue of split packaging, wherein gag-pol, rev, and envelope functions are provided in trans from helper plasmids or stable producer cell lines and are excluded from the transfer vector genome. In certain embodiments, accessory genes (e.g., vif, vpr, vpu, nef) are omitted, mutated, or otherwise inactivated to reduce the likelihood of recombination or residual replicative capacity. Third-generation systems also generally include two “packaging plasmids” and an “envelope plasmid.” The “envelope plasmid” generally encodes an Env gene operatively linked to a promoter. In an illustrative third-generation system, the Env gene is VSV-G and the promoter is the CMV promoter. The third-generation system uses two packaging plasmids, one encoding gag and pol and the other encoding rev as a further safety feature; an improvement over the single packaging plasmid of so-called second-generation systems. Although safer, the third-generation system can be more cumbersome to use and result in lower viral titers due to the addition of an additional plasmid. Illustrative packing plasmids include, without limitation, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI. Many retroviral vector systems rely on the use of a “packaging cell line.” In general, the packaging cell line is a cell line whose cells are capable of producing infectious retroviral particles when the transfer plasmid, packaging plasmid(s), and envelope plasmid are introduced into the cells. Various methods of introducing the plasmids into the cells may be used, including transfection or electroporation. In some cases, a packaging cell line is adapted for high-efficiency packaging of a retroviral vector system into retroviral particles.

[0141] As used herein, the terms “retroviral vector” or “lentiviral vector” is intended to mean a nucleic acid that encodes a retroviral or lentiviral cis nucleic acid sequence required for genome packaging and one or more polynucleotide sequence to be delivered into the target cell. Retroviral particles and lentiviral particles generally include an RNA genome (derived from the transfer plasmid), a lipid-bilayer envelope in which the Env protein is embedded, and other accessory proteins including integrase, protease, and matrix protein. As used herein, the terms260132003840“retroviral particle” and “lentiviral particle” refers a viral particle that includes an envelope, has one or more characteristics of a lentivirus, and is capable of invading a target host cell. Such characteristics include, for example, infecting non-dividing host cells, transducing non-dividing host cells, infecting or transducing host immune cells, containing a retroviral or lentiviral virion including one or more of the gag structural polypeptides, containing a retroviral or lentiviral envelope including one or more of the env encoded glycoproteins, containing a genome including one or more retrovirus or lentivirus cis-acting sequences functioning in replication, proviral integration or transcription, containing a genome encoding a retroviral or lentiviral protease, reverse transcriptase or integrase, or containing a genome encoding regulatory activities such as Tat or Rev. The transfer plasmids may comprise a cPPT sequence, as described in U.S. Patent No. 8,093,042.

[0142] The efficiency of the system is an important concern in vector engineering. The efficiency of a retroviral or lentiviral vector system may be assessed in various ways known in the art, including measurement of vector copy number (VCN) or vector genomes (vg) such as by quantitative polymerase chain reaction (qPCR), or titer of the virus in infectious units per milliliter (lU / mL). For example, the titer may be assessed using a functional assay performed on the cultured tumor cell line HT1080 as described in Humbert et al. Development of third-generation Cocal Envelope Producer Cell Lines for Robust Retroviral Gene Transfer into Hematopoietic Stem Cells and T-cells. Molecular Therapy 24:1237-1246 (2016). When titer is assessed on a cultured cell line that is continually dividing, no stimulation is required and hence the measured titer is not influenced by surface engineering of the retroviral particle. Other methods for assessing the efficiency of retroviral vector systems are provided in Gaererts et al. Comparison of retroviral vector titration methods. BMC Biotechnol. 6:34 (2006).

[0143] In some embodiments, the retroviral particles and / or lentiviral particles of the disclosure comprise a polynucleotide comprising a sequence encoding a heterologous protein CAR that specifically binds to the target antigens. In some embodiments, a sequence encoding a heterologous protein CAR that specifically binds to the target antigens is operatively linked to a promoter. Illustrative promoters include, without limitation, a cytomegalovirus (CMV) promoter, a CAG promoter, an SV40 promoter, an SV40 / CD43 promoter, and a MND promoter.

[0144] In some embodiments, the retroviral particles comprise transduction enhancers. In some embodiments, the retroviral particles comprise tagging proteins.260132003840

[0145] In some embodiments, each of the retroviral particles comprises a polynucleotide comprising, in 5' to 3' order: (i) a 5' long terminal repeat (LTR) or untranslated region (UTR), (ii) a promoter, (iii) a sequence of a polynucleotide construct as described herein, and (iv) a 3' LTR or UTR. In some embodiments, the polynucleotide construct sequence encodes a synthetic cytokine receptor RACR system. In some embodiments, the polynucleotide construct sequence encodes a synthetic cytokine RACR system and a CAR.

[0146] In some embodiments, the transfer vector (genomic cassette packaged into the LVV particle) comprises, in 5'^-3' order: a 5' LTR (with inactive U3 after reverse transcription), a primer binding site (PBS), a packaging signal (T), an Rev-responsive element (RRE), an optional central polypurine tract / central termination sequence (cPPT / CTS), a payload transgene sequence (e.g. a CAR) including an internal promoter driving the payload transgene, optional post- transcriptional regulatory elements (e.g., WPRE or functional equivalents), and a 3' SIN LTR. In some embodiments, a polyadenylation signal (e.g., bGH or SV40 polyA) is included to stabilize transcripts.

[0147] The disclosure further provides various retroviral particles, including but not limited to gamma-retroviral particles, alpha-retroviral particles, and lentiviral particles. In some embodiments, the particle may be a viral particle, a retroviral particle, a lentiviral particle, a gamma-retroviral particle. In some embodiments, the viral particle comprises a VSV G-protein or functional variant thereof. In some embodiments, the viral particle comprises a Cocal G-protein or functional variant thereof.B. Engineered Viral Envelope

[0148] In some embodiments, the virus particles that encapsulate polynucleotide constructs provided herein can comprise engineered viral envelopes.

[0149] As contemplated by the present disclosure, a particle may be engineered to display on its surface a pseudotyped envelope glycoprotein and in some cases also a separate targeting domain and / or transduction enhancer, including any as described herein. The targeting domain and / or transduction enhancer may be a multidomain fusion protein (MDF) exposed on the outside surface of the particle. Each surface protein, such as the pseudotyped envelope glycoprotein or other surface protein, may be associated with the particle, when the particle comprises a lipid envelope, directly or via a transmembrane portion or anchor. In each case, a protein may be displayed as a full-length form, including its native transmembrane portion.260132003840Alternatively, the extracellular portion of a protein may be displayed with a heterologous transmembrane portion (e.g., a transmembrane portion from another membrane protein) or a membrane anchor (e.g., a glycosylphosphatidylinositol anchor). The term “display” is understood to mean the position of a protein on the surface of the particle such that the molecule may contact cognate molecules on a target cell, such as a T cell. It is further contemplated that for particles lacking an envelope (such as a non-enveloped viral particle) the fusion molecule may be displayed on the particle either by association with a component of the particle (e.g., a capsid protein) or by a direct linkage with the particle (e.g., as a fusion protein comprises a capsid protein).

[0150] The ex vivo efficiency of a retroviral or lend viral particle system may be assessed in various ways known in the art, including measurement of vector copy number (VCN) or vector genomes (vg) such as by quantitative polymerase chain reaction (qPCR), digital droplet PCR (ddPCR) or titer of the virus in infectious units per milliliter (lU / mL). For example, the titer may be assessed using a functional assay performed on the cultured tumor cell line HT1080 as described in Humbert et al. Development of Third-generation Cocal Envelope Producer Cell Lines for Robust Retroviral Gene Transfer into Hematopoietic Stem Cells and T-cells.Molecular Therapy 24:1237-1246 (2016). When titer is assessed on a cultured cell line that is continually dividing, no stimulation is required and hence the measured titer is not influenced by surface engineering of the retroviral particle. Other methods for assessing the efficiency of retroviral vector systems are provided in Gaererts et al. Comparison of retroviral vector titration methods. BMC Biotechnol. 6:34 (2006).

[0151] Exemplary features of an engineered viral envelope are described in the following subsections.I. Pseudotyped Viral Envelope Protein

[0152] In some embodiments, the viral vector may comprise a heterologous viral envelope glycoprotein giving a pseudotyped viral vector. For example, the viral envelope glycoprotein may be derived from RD114 or one of its variants, VSV-G, Gibbon-ape leukemia virus (GALV), or is the Amphotropic envelope, Measles envelope or baboon retroviral envelope glycoprotein. In some embodiments, the cell-surface receptor is a VSV G protein from the Cocal strain or a functional variant thereof. In some embodiments, the viral envelope comprises a viral envelope protein. In some embodiments, a viral envelope protein is a VSV-G envelope protein, a measles virus envelope protein, a nipha virus envelope protein, or a cocal virus G protein. In260132003840some embodiments, the viral particle comprises a modified VSV G protein that lacks LDLR binding affinity. In some embodiments, these mutations comprise mutations at positions 47 (for example, K47Q) and / or 354 (for example, R354A).

[0153] In some embodiments, the viral envelope protein is a protein from the Cocal strain (Cocal glycoprotein). In some embodiments, the protein is a Cocal envelope protein containing a mutation at position 354 (R354). In some embodiments, the protein is a Cocal envelope protein containing a mutation at position 47 (K47). In some embodiments, the protein is a Cocal envelope variant containing a R354Q mutation. In some embodiments, the protein is a Cocal envelope variant containing a K47Q mutation. In some embodiments, this variant may be referred to as “blinded” Cocal envelope. Illustrative Cocal envelope variants are provided in, e.g., US 2020 / 0216502 Al, which is incorporated herein by reference in its entirety.

[0154] Various fusion glycoproteins can be used to pseudotyped lentiviral vectors. While the most commonly used example is the envelope glycoprotein from vesicular stomatitis virus (VSVG), many other viral proteins have also been used for pseudotyping of lentiviral vectors. See Joglekar et al. Human Gene Therapy Methods 28:291-301 (2017). The present disclosure contemplates substitution of various fusion glycoproteins. Notably, some fusion glycoproteins result in higher vector efficiency.

[0155] In some embodiments, the retroviral particles comprise a surface protein that binds to a surface marker on a target host cell, allowing host cell transduction. In some embodiments, the cell surface receptor is a T cell surface receptor.

[0156] In some embodiments, pseudotyping a fusion glycoprotein or functional variant thereof facilitates targeted transduction of specific cell types, including, but not limited to, innate lymphoid cells, cytotoxic innate lymphoid cells, or NK cells. In some embodiments, the fusion glycoprotein or functional variant thereof is / are full-length polypeptide(s), functional fragment(s), homolog(s), or functional variant(s) of Human immunodeficiency virus (HIV) gpl60, Murine leukemia virus (MLV) gp70, Gibbon ape leukemia virus (GALV) gp70, Feline leukemia virus (RD114) gp70, Amphotropic retrovirus (Ampho) gp70, 10A1 MLV (10A1) gp70, Ecotropic retrovirus (Eco) gp70, Baboon ape leukemia virus (BaEV) gp70, Measles virus (MV) H and F, Nipah virus (NiV) H and F, Rabies virus (RabV) G, Mokola virus (MOKV) G, Ebola Zaire virus (EboZ) G, Lymphocytic choriomeningitis virus (LCMV) GP1 and GP2, Baculovirus GP64, Chikungunya virus (CHIKV) El and E2, Ross River virus (RRV) El and E2, Semliki Forest virus (SFV) El and E2, Sindbis virus (SV) El and E2, Venezualan equine260132003840encephalitis virus (VEEV) El and E2, Western equine encephalitis virus (WEEV) El and E2, Influenza A, B, C, or D HA, Fowl Plague Virus (FPV) HA, Vesicular stomatitis virus VSV-G, or Chandipura virus and Piry virus CNV-G and PRV-G.

[0157] In some embodiments, the fusion glycoprotein or functional variant thereof is a full-length polypeptide, functional fragment, homolog, or functional variant of the G protein of Vesicular Stomatitis Alagoas Virus (VSAV), Carajas Vesiculovirus (CJSV), Chandipura Vesiculovirus (CHPV), Cocal Vesiculovirus (COCV), Vesicular Stomatitis Indiana Virus (VSIV), Isfahan Vesiculovirus (ISFV), Maraba Vesiculovirus (MARAV), Vesicular Stomatitis New Jersey virus (VSNJV), Bas-Congo Virus (BASV). In some embodiments, the fusion glycoprotein or functional variant thereof is the Cocal virus G protein.

[0158] In some embodiments, the viral particle comprises a polypeptide comprising a Cocal glycoprotein that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 98.

[0159] In some embodiments, the viral particle comprises a nucleic acid sequence encoding a Cocal glycoprotein that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 99.

[0160] In some embodiments, the viral particle comprises a nucleic acid sequence encoding a Cocal glycoprotein that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 100.2. Transduction and Targeting Proteins

[0161] In some embodiments, the viral envelope comprises more than one polypeptide on the surface. In some embodiments, the viral envelope comprises a transduction enhancer and / or a one or more targeting proteins. In some embodiments, the viral envelope comprises a transduction enhancer. In some embodiments, the viral envelope comprises at an immune cellactivating protein. In some embodiments, the viral envelope comprises a co-stimulation molecule. In some embodiments, the viral envelope comprises an immune cell- activating protein, and a co-stimulation molecule.

[0162] In some embodiments, the viral envelope may be a multidomain fusion protein (MDF) composed of a multiple binding domain protein, wherein each binding domain binds to a different target molecule. In some embodiments, each binding domain binds to a target molecule on a target cell that is an immune cell, such as a T cell. In some embodiments, the more than one polypeptide binds to a target immune cell and replicates an immunological synapse. In some260132003840embodiments, the binding domains of the multiple binding protein are connected by a linker. In some embodiments, the lentiviral particle is engineered to express a MDF protein comprising a T cell activation domain (or activation molecule) and at least one costimulatory molecule. In some embodiments, the at least one costimulatory molecule may also be an adhesion molecule or the MDF may further comprise an adhesion molecule. In some embodiments, the viral envelope comprises an immune cell-activating protein, a co-stimulatory molecule, and an adhesion molecule, wherein the immune cell-activating protein, co-stimulatory molecule, and adhesion molecule each bind a target immune cell. The T cell activation molecule and at least one costimulatory molecule, and optionally a further adhesion molecule, may each be proteins and may collectively be fused into one (or more) fusion proteins. In some embodiments, surface engineering of a viral particle with the MDF protein may generate a macromolecular complex at the interface of the particle and the cell that acts as an artificial supramolecular activation cluster. In some embodiments, such a fusion protein displayed on the surface of a viral vector particle is able to bind, activate, and permit transduction of target cells (e.g., T cells), wherein transduction of the target cell is enhanced relative to transduction with a similar viral particle lacking the displayed transduction enhancer fusion protein.

[0163] It is understood that, in certain embodiments, the transduction enhancer, such as a MDF, comprised within the viral envelope may include a protein embedded in the lipid bilayer such that the transduction enhancer is displayed on the surface of the viral particle. In some embodiments, the MDF contains a transmembrane domain or a GPI anchor for incorporation into the viral envelope. In some embodiments, a lentiviral particle that is engineered to display, on its surface, the MDF exhibits enhanced transduction. In some embodiments, the surface engineered MDF is a transduction enhancer.

[0164] In some embodiments, the MDF comprises multiple binding domains that bind to two or more target molecules selected from an immune cell activating receptor (e.g. a T cell activating receptor), and at least one T cell costimulatory receptor, and in some cases also an adhesion molecule. The T cell activation domain, the at least one costimulatory molecule or the adhesion molecule each independently comprises a binding domain that may be an antibody or antigen-binding fragment or may be a ligand binding protein derived from a native cell surface ligand. In some embodiments, the ligand binding domain includes at least a functional fragment or portion of a native cell surface ligand that is able to bind to a cognate receptor. For instance, the ligand binding domain includes an extracellular binding domain portion of a native cell260132003840surface ligand. As a results, it is understand that the term “functional fragment” is used herein to a fragment of a polypeptide, or other molecule, that retains the desired function of the polypeptide such as the ability to bind to a cognate receptor. For example, a functional fragment of CD58 is a fragment of CD58 that specifically binds CD2.

[0165] In some embodiments, the viral envelope comprises one or more transduction enhancers. In some embodiments, the transduction enhancers include binding domains directed against T cell activation receptors or NK cell activation receptors, include and / or co-stimulatory molecules. In some embodiments, one or more transduction enhancers comprise one or more of anti-CD3scFv, CD86, CD80, and / or CD58. In some embodiments, the transduction enhancer is a membrane-bound multidomain fusion (MDF) containing two or more of an immune cell activation domain. In some embodiments, the transduction enhancers comprise at least an anti-CD3 scFv, and CD58. In some embodiments, the transduction enhancers comprise at least an anti-CD3 scFv, and CD80. In some embodiments, the transduction enhancers comprise at least an anti-CD3 scFv, and CD86. In some embodiments, the transduction enhancers comprise at least an anti-CD3 scFv, a CD80, and CD58. In some embodiments, the transduction enhancers comprise at least an anti-CD3 scFv, a CD86, and CD58.

[0166] In provided embodiments, a molecule may be displayed with a native transmembrane portion. Alternatively, the extracellular portion of the molecule may be displayed with a heterologous transmembrane portion (e.g., a transmembrane portion from another membrane protein) or a membrane anchor (e.g., a glycosylphosphatidylinositol anchor). Each fusion molecule may be associated with the particle, when the particle comprises a lipid envelope, directly or via the transmembrane portion or anchor connected to one of the other molecules in the fusion molecule. For example, a T cell activation domain, at least one costimulatory molecule, and in some cases a further adhesion 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 some embodiments, an engineered lend viral particle displaying a multi-domain fusion polypeptide will comprise a transmembrane domain from the polypeptide domain which is membrane proximal. In a variant, the fusion molecule comprises or is associated with another membrane- associated molecule, thereby displaying the fusion molecule on the particle.

[0167] 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 multidomain260132003840protein. 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.a. Immune Cell Activating Agents

[0168] In some embodiments, the transduction enhancer comprises a mitogenic stimulus, which is incorporated into a retroviral or lentiviral capsid, such that the virus both activates and transduces T cells. This removes the need to add vector and mitogen. In some embodiments, the transduction enhancer comprises a mitogenic transmembrane protein and / or one or more costimulatory molecules, which get(s) incorporated into the retrovirus when it buds from the producer / p ackaging cell membrane. In some embodiments, the transduction enhancers are expressed as separate cell surface molecules on the producer cell rather than being part of the viral envelope glycoprotein.

[0169] In some embodiments, the viral vector described herein comprises a mitogenic transduction enhancer in the viral envelope. In some embodiments, the mitogenic transduction enhancer is derived from the host cell during retroviral vector production. In some embodiments, the mitogenic transduction enhancer is made by the packaging cell and expressed at the cell surface. When the nascent retroviral vector buds from the host cell membrane, the mitogenic transduction enhancer may be incorporated in the viral envelope as part of the packaging cell-derived lipid bilayer. In some embodiments, the mitogenic enhancer is an antibody or fragment thereof. In some embodiments, the mitogenic enhancer is a single domain antibody, for example, a camelid antibody. In some embodiments, the mitogenic enhancer is an scFv. In some embodiments, the mitogenic enhancer is a nanobody.

[0170] In some embodiments, the transduction enhancer is host-cell derived. The term “hostcell derived” indicates that the mitogenic transduction enhancer is derived from the host cell as described above and is not produced as a fusion or chimera from one of the viral genes, such as gag, which encodes the main structural proteins; or env, which encodes the envelope protein.

[0171] Envelope proteins are formed by two subunits, the transmembrane (TM) that anchors the protein into the lipid membrane and the surface (SU) which binds to the cellular receptors. In some embodiments, the packaging-cell derived mitogenic transduction enhancer of the present invention does not comprise the surface envelope subunit (SU).260132003840

[0172] In some embodiments, the mitogenic transduction enhancer has the structure: M-S-TM, in which M is a mitogenic domain; S is an optional spacer domain and TM is a transmembrane domain.

[0173] The mitogenic domain is the part of the mitogenic transduction enhancer which causes T-cell activation. It may bind or otherwise interact, directly or indirectly, with a T cell, leading to T cell activation. In some embodiments, the mitogenic domain binds a T cell surface antigen, such as CD3, CD28, CD134 and CD137.

[0174] CD3 is a T-cell co-receptor. It is a protein complex composed of four distinct chains. In mammals, the complex contains a CD3y chain, a CD35 chain, and two CD3e chains. These chains associate with the T-cell receptor (TCR) and the z-chain to generate an activation signal in T lymphocytes. The TCR, z-chain, and CD3 molecules together comprise the TCR complex. In some embodiments, the mitogenic domain binds to a CD3e chain.

[0175] In some embodiments, the T cell activation domain is an immune cell-activating agent that comprises a mitogenic stimulus, which is incorporated into a retroviral or lend viral capsid, such that the virus both activates and transduces T cells. In some embodiments, the T cell activation domain comprises a binding domain that binds to a component of the TCR-CD3 complex. In some embodiments, the T cell activation domain comprises a binding domain that binds to CD3, TCRa, TCRp, TCRy, or TCR6. CD3 is a T-cell co-receptor. It is a protein complex composed of four distinct chains, CD3e, CD3y, CD36, CD3(^. These chains associate with the T-cell receptor (TCR) and the z-chain to generate an activation signal in T lymphocytes. The TCR, z-chain, and CD3 molecules together comprise the TCR complex. In some embodiments, the T cell activating domain specifically binds CD3y, CD36, or CD3e. In particular embodiments, the T cell activation domain comprises a binding domain that binds to CD3e.

[0176] In some embodiments, the mitogenic domain comprises all or part of an antibody or other molecule which specifically binds a T-cell surface antigen. In some embodiments, the antibody activates the TCR or CD28. In some embodiments, the antibody binds the TCR, CD3 or CD28. Examples of such antibodies include: OKT3, 15E8 and TGN1412. Other suitable antibodies include: Anti-CD28: CD28.2, 10F3, Anti-CD3 / TCR: UCHT1, and YTH12.5, TR66.

[0177] In some embodiments, the mitogenic domain comprises the binding domain from OKT3, 15E8, TGN1412, CD28.2, 10F3, UCHT1, YTH12.5 or TR66.260132003840

[0178] In some embodiments, the mitogenic domain comprises all or part of a costimulatory molecule such as OX40L and 41BBL. For example, the mitogenic domain may comprise the binding domain from OX40L or 41 BBL.

[0179] OKT3, also known as Muromonab-CD3 is a monoclonal antibody targeted at the CD3e chain. It is clinically used to reduce acute rejection in patients with organ transplants. It was the first monoclonal antibody to be approved for clinical use in humans.

[0180] In some embodiments, the viral envelope comprises an immune cell- activating protein. In some embodiments, the immune cell- activating protein specifically binds a receptor on an immune cell. In some embodiments, the immune cell- activating protein provides signal one for T cell activation.

[0181] In some embodiments, the immune cell- activating protein specifically binds CD2, CD3, CD28H, LFA-1, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Lyl08, NKG2D, NKp46, NKp44, NKp30, CD244, or NKp80. In some embodiments, the immune cell- activating protein specifically binds CD3y, CD36, or CD3e. In some embodiments, the immune cell-activating protein specifically binds CD3y, CD36, CD3e, CD9, CD5, CD22, CD33, CD37, CD64, CD45, CD28H, LFA-1, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Lyl08, CD16, CD56, NKG2D, NKp46, NKp44, NKp30, CD244, NKp80, TCRa chain, TCRP chain, TCRy chain, or TCR6 chain. In some embodiments, the immune cellactivating protein specifically binds CD3y, CD36, or CD3e. In some embodiments, the immune cell- activating protein specifically binds CD3.

[0182] In some embodiments, the immune cell-activating protein is an antibody or antigen binding fragment thereof that specifically binds a receptor on an immune cell. In some embodiments, the immune cell- activating protein is an antibody or antigen binding fragment thereof that specifically binds CD28, CD2, CD3, CD28H, LFA-1, 0X40, 4-1BB, CD40L, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Lyl08, NKG2D, NKp46, NKp44, NKp30, CD244, or NKp80. In some embodiments, the immune cell-activating protein is an antibody or antigen binding fragment thereof that specifically binds CD28, CD2, CD3y, CD36, CD3e, CD4, CD8, CD9, CD5, CD22, CD33, CD37, CD64, CD45, CD28H, LFA-1, 0X40, 4-1BB, CD40L, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Lyl08, CD16, CD56, NKG2D, NKp46, NKp44, NKp30, CD244, NKp80, TCRa chain, TCRP chain, TCRy chain, or TCR6 chain, some embodiments, the immune cell- activating protein is an antibody or antigen binding fragment thereof that specifically binds CD3y, CD36, or CD3e. In260132003840some embodiments, immune cell-activating protein is an antibody or antigen binding fragment thereof that specifically binds CD3.

[0183] Antibodies targeting the polypeptides described herein are known to those of skill in the art. Methods for generating antibodies are known to those of skill in the art.

[0184] In some embodiments, the T cell activating domain is an antibody or an antigenbinding fragment thereof, or a ligand binding domain. In some embodiments, the T cell activating domain is an antibody, or antigen-binding fragment thereof, that specifically binds CD3e. The antibody or antigen-binding fragment may be a Fab, Fv, scFv, or a single domain antibody such as a VHH. In some embodiments, the T cell binding domain is a single chain variable fragment (scFv) that specifically binds CD3e.

[0185] In some embodiments, the viral envelope comprises an anti-CD3e antibody, or antigen-binding fragment thereof. In some embodiments, the anti-CD3e antibody, or antigenbinding fragment thereof is coupled to a transmembrane domain. Examples of such antibodies include: OKT3, 15E8, TGN1412, UCHT1, YTH12.5, or TR66. An illustrative anti-CD3e antibody is OKT3. OKT3, also known as Muromonab-CD3, is a monoclonal antibody targeted at the CD3e chain. For instance, OKT3, also known as Muromonab-CD3 is a monoclonal antibody targeted at the CD3e chain. It is clinically used to reduce acute rejection in patients with organ transplants. It was the first monoclonal antibody to be approved for clinical use in humans.

[0186] In some embodiments, the T cell activating domain is a anti-CD3e antibody or antigen-binding fragment, such as an scFv, that comprises a variable light (VL) domain comprising complementarity determining regions: L- CDR1, L-CDR2 and L-CDR3, wherein: L-CDR1 comprises the sequence SASSSVSYMN (SEQ ID NO: 101); L-CDR2 comprises the sequence DTSKLASG (SEQ ID NO: 102); and L- CDR3 comprises the sequence QQWSSNPFT (SEQ ID NO: 103); and an antibody VH domain comprising H-CDR1, H-CDR2 and H-CDR3, wherein: H-CDR1 comprises the sequence RYTMH (SEQ ID NO: 104); H-CDR2 comprises the sequence YINPSRGYTNYNQKVKD (SEQ ID NO: 105); and H-CDR3 comprises the sequence YYDDHYCLDY (SEQ ID NO: 106).

[0187] In some embodiments, the viral envelope comprises a single chain fragment variable fragment (scFv) of an anti-CD3 antibody.

[0188] In some embodiments, the T cell activating domain is a anti-CD3e antibody or antigen-binding fragment, such as an scFv, that comprises a variable light (VL) domain260132003840comprising the sequence set forth in SEQ ID NO: 107 or a sequence that exhibits at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 107, and comprises a variable heavy (VH) domain comprising the sequence set forth in SEQ ID NO: 108 or a sequence that exhibits at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 108. In some embodiments, the T cell activating domain is a anti- CD3e antibody or antigen-binding fragment, such as an scFv, that comprises a variable light (VL) domain comprising the sequence set forth in SEQ ID NO: 107 and a variable heavy (VH) domain comprising the sequence set forth in SEQ ID NO: 108.

[0189] In some embodiments, the anti-CD3 scFv comprises VH and VL regions linked by a flexible (G4S)n linker. In some embodiments, the linker comprises GGGGSGGGGSGGGGS (SEQ ID NO: 109)

[0190] In some embodiments, the T cell activating domain is a anti-CD3e scFv comprising the sequence set forth in SEQ ID NO: 110 or a sequence that exhibits at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 110. In some embodiments, the T cell activating domain is a anti-CD3e scFv comprising the sequence set forth in SEQ ID NO: 110.b. Co-Stimulatory Molecules

[0191] In some embodiments, the viral envelope comprises at least one co- stimulatory molecule. In some embodiments, the co-stimulatory molecule specifically binds a receptor on an immune cell. In some embodiments, the co-stimulatory provides signal two for cell activation.

[0192] As used herein, the term “costimulatory molecule” refers to a molecule capable of generating a costimulatory signal to T cells. Lymphocytes, such as T cells and natural killer (NK) cells, typically require several signals and interactions with antigen presenting cells (APCs) for optimal priming to gain full effector functions. For T cells these include signaling through the T cell receptor (TCR), costimulatory molecules (such as CD28 and CD2), cytokines, as well as various adhesion molecules necessary to allow sufficient time for proper synapse formation and signal transduction. NK cells require similar types of stimulation but may rely on different activating receptors, such as NKG2D, NKp46, and DNAM-1. For T cells, proper costimulation, in addition to TCR stimulation, is especially important for effective priming and many studies have shown that TCR stimulation alone can lead to functional anergy and unresponsiveness. Costimulatory signals augment T and NK cell function by enhancing cell260132003840metabolism, cytokine production, differentiation, and long-term persistence. Costimulation is an important factor for cell proliferation, differentiation and survival. In some embodiments, costimulatory molecules include, but are not limited to, CD45, CD2, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

[0193] In some embodiments, the costimulatory molecule includes, but is not limited to, binding agents, such as scFvs, antibodies, single-domain antibodies, antibody fragments, nanobodies that bind to any of the costimulatory molecules described herein. In some embodiments, these binding agents may include anti-CD28, anti-CD2, anti-CD45, anti-CD4, anti-CD5, anti-CD8, anti-CD9, anti-CD16, anti-CD22, anti-CD33, anti-CD37, anti-CD64, anti-CD80, anti-CD86, anti-CD137, anti-CD154, anti-CD28H, anti-LFA-1, anti-OX40, anti-4-lBB, anti-CD40L, anti-DNAM-1, anti-CD27, anti-ICOS, anti-LIGHT, anti-GITR, anti-CD30, anti-SLAM, anti-Ly-9, anti-CD84, anti-Lyl08, anti-NKG2D, anti-NKp46, anti-NKp44, anti-NKp30, anti-CD244, anti-NKp80, anti-TCRa chain, anti-TCRp chain, anti-TCRy chain, and anti-TCR6 chain agents.

[0194] In some embodiments, the at least one costimulatory molecule comprises a binding domain that binds to a T cell costimulatory receptor. In some embodiments, the at least one costimulatory molecule comprises costimulatory protein that is a costimulatory ligand of the T cell receptor or is a portion thereof comprising an extracellular domain that binds to a T cell costimulatory receptor. In some embodiments, the T cell costimulatory receptor is selected from CD28, ICOS, 4-1BB (CD137), 0X40 (CD134), GITR (CD357), CD27, CD30, HVEM (TNFRSF14), CD2, DNAM-1 (CD226), 2B4 (CD244), LFA-1, CD28H (TMIGD2), SLAM family receptors (SLAM / CD150, Ly-9 / CD229, CD84, LylO8 / SLAMF6), and NKG2D.

[0195] In some embodiments, the costimulatory molecule includes, but is not limited to, binding agents, such as scFvs, antibodies, single-domain antibodies, antibody fragments, nanobodies that bind to any of the costimulatory molecules described herein. In other embodiments, the costimulatory molecule includes a ligand binding domain (e.g., extracellular domain) of a costimulatory protein ligand.

[0196] In some embodiments, the at least one costimulatory molecule is independently a costimulatory ligand selected from CD80, CD86, ICOSL, 4-1BBL, OX40L, GITRL, CD40L, OX40L, CD27, CD70, CD30L, LIGHT (TNFSF14), LTalpha, CD58 (LFA-3), ICAM-1, CD112 (Nectin-2), CD155 (PVR), CD48, HHLA2, MICA, MICB, and ULBP1-6, or a portion of any thereof comprising an extracellular domain that binds to a T cell costimulatory receptor. For260132003840instance, the portion thereof is an extracellular domain or any of the foregoing or a functional fragment thereof that is able to bind to a T cell costimulatory receptor.

[0197] In some embodiments, the co-stimulation molecule is a ligand for CD28. CD28 is one of the proteins expressed on T cells that provide co-stimulatory signals required for T cell activation and survival. T cell stimulation through CD28 in addition to the T-cell receptor (TCR) can provide a potent signal for the production of various interleukins (IL-6 in particular). In some embodiments, the co-stimulation molecule is an antibody, or fragment thereof, that binds to CD28. Examples of such antibodies include: 15E8 and TGN1412. Other suitable antibodies include: CD28.2 and 10F3.

[0198] In some embodiments, the co-stimulation molecule is CD86. CD86, also known as B7-2, is a ligand for CD28. In some embodiments, the ligand for CD28 is CD86. In some embodiments, the co-stimulation molecule is CD80. CD80 is an additional ligand for CD28. In some embodiments, the ligand for CD28 is CD80. In some embodiments, the ligand for CD28 is an anti-CD28 antibody or an anti-CD28 scFv. In some embodiments, the anti-CD28 antibody or an anti-CD28 scFv is coupled to a transmembrane domain for display on the surface of the viral envelope.

[0199] In some embodiments, the costimulatory molecule comprises a CD86, a CD86 extracellular domain thereof, or a functional fragment of CD86.

[0200] In some embodiments, the co-stimulation molecule is a CD86 polypeptide comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, the costimulation molecule is a CD86 polypeptide comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 74.

[0201] In some embodiments, the CD86 polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 76. In some embodiments, the CD86 polypeptide is encoded by a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 76.

[0202] In some embodiments, the at least one costimulatory molecule comprises a CD80, a CD80 extracellular domain thereof, or a functional fragment of CD80. An exemplary CD80 is human CD80 set forth in Uniprot P33681. In some embodiments, the co-stimulation molecule is a CD80 polypeptide comprising the amino acid sequence of SEQ ID NO: 75. In some embodiments, the co-stimulation molecule is a CD80 polypeptide comprising an amino acid260132003840sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 75. The extracellular portion of CD80 includes residues 35-242 which includes an Ig-like V-type domain (amino acids 35-135) and an Ig-like C2-type (amino acids 145-230), either or both of which may be included to form the costimulatory molecule. In embodiments, one or both of the domains may be used as the functional fragment of CD80. In some embodiments, the at least one costimulatory molecule comprises a CD80 that comprises a CD80 extracellular domain and also the native transmembrane domain of CD80, and optionally contiguous sequences of the CD80 cytoplasmic domain. Thus, in some embodiments, the CD80 component of the MDF comprises a mature CD80 polypeptide that includes both its extracellular immunoglobulin-like domains and its native transmembrane and optionally in some cases also cytoplasmic regions, thereby providing a natural anchoring mechanism within the LVV membrane. The inclusion of the cytoplasmic tail of CD80 may facilitate correct trafficking of the MDF protein through the secretory pathway and promote incorporation into the viral membrane. In alternative embodiments, truncated or chimeric CD80 variants retaining the transmembrane region may be employed to modulate surface expression or intracellular signaling.

[0203] In some embodiments, the CD80 polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 77. In some embodiments, the CD80 polypeptide is encoded by a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 77.

[0204] In some embodiments, CD80 of the MDF comprises the sequence set forth in SEQ ID NO: 111 or a sequence that exhibits at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 111.

[0205] CD134, also known as 0X40, is a member of the TNFR- superfamily of receptors which is expressed on activated T cells. 0X40 may promote cell division and survival. 0X40 is a secondary costimulatory molecule, expressed after 24 to 72 hours following activation; its ligand, OX40L, is also not expressed on resting antigen presenting cells, but is following their activation. In some embodiments, the viral particle comprises a ligand for 0X40, or functional fragment thereof, coupled to its native transmembrane domain or a heterologous transmembrane domain.

[0206] CD 134, also known as 0X40, is a member of the TNFR-superfamily of receptors which is expressed on activated T cells. 0X40 may promote cell division and survival. 0X40 is260132003840a secondary costimulatory molecule, expressed after 24 to 72 hours following activation; its ligand, OX40L, is also not expressed on resting antigen presenting cells, but is following their activation. In some embodiments, the viral particle comprises a ligand for 0X40, or functional fragment thereof, coupled to its native transmembrane domain or a heterologous transmembrane domain.

[0207] CD137, also known as 4-1BB, is a member of the tumor necrosis factor (TNF) receptor family. CD137 is expressed on activated T cells. In addition, CD137 expression is found on dendritic cells, follicular dendritic cells, natural killer cells, granulocytes and cells of blood vessel walls at sites of inflammation. The best characterized activity of CD 137 is its costimulatory activity for activated T cells. Crosslinking of CD137 enhances T cell proliferation, IL-2 secretion survival and cytolytic activity. In some embodiments, the viral particle comprises a ligand for 4- IBB, or functional fragment thereof, coupled to its native transmembrane domain or a heterologous transmembrane domain. 4-1BBL is a cytokine that belongs to the tumor necrosis factor (TNF) ligand family. This transmembrane cytokine is a bidirectional signal transducer that acts as a ligand for 4- IBB, which is a costimulatory receptor molecule in T lymphocytes. 4-1BBL has been shown to reactivate anergic T lymphocytes in addition to promoting T lymphocyte proliferation.

[0208] Viral particles comprising one or more activation or co- stimulation molecule(s) may be made by engineering the packaging cell line by methods provided by WO 2016 / 139463; or by expression of the T-cell activation or co-stimulation molecule(s) from a polynucleotide helper vector as described in IntT Pat. Pub. No. WO 2020 / 106992 Al, both of which are incorporated herein by reference in their entireties.c. Adhesion Molecules

[0209] In some embodiments, the viral particle comprises an adhesion molecule. In some embodiments, the at least one costimulatory molecule also acts as an adhesion molecule. In some embodiments, the MDF additionally includes an adhesion molecule. As used herein, the term “adhesion molecule” refers to a subset of cell surface molecules involved in the binding of cells with other cells. Adhesion cells may help to form more stable interactions, such as an immunological synapse, between immune cells. The immunological synapse is a stable adhesive junction between a polarized immune effector cell and an antigen-bearing cell. In some embodiments, the adhesion molecule may provide a costimulatory signal to the target cell.Without being bound by theory, surface engineering of a particle with an adhesion molecule, a260132003840co-stimulation molecule, and an activation molecule (such as an anti-CD3 antibody fragment) may generate a macromolecular complex at the interface of the particle and cell that acts as artificial supramolecular activation cluster. In some embodiments, the adhesion molecule comprises CD58, ICAM-1, ICAM-2, ICAM- 3, ICAM-4, ICAM-5, JAM-A, CD155 or CD112; an extracellular domain thereof; or a functional fragment thereof. In some embodiments, the adhesion molecule comprises an antibody or antigen- binding fragment thereof. In some embodiments, the adhesion molecule specifically binds CD2, LFA-1, or DNAM-1.

[0210] In some embodiments, the adhesion molecule may provide a costimulatory signal to the target cell. In some embodiments, adhesion molecules include, but are not limited to, CD58, HHLA2, ICAM-1, OX40L, 4-1BBL, CD40, CD155, CD70, HVEM, GITRL, ICOSL, CD30L, SLAM, Ly-9, CD84, Lyl08, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and B7-H6. In some embodiments, the adhesion molecule includes, but is not limited to, binding agents, such as scFvs, antibodies, single-domain antibodies, antibody fragments, and nanobodies that bind to any of the adhesion or costimulatory molecules described herein. In other embodiments, the adhesion molecule, which also may act as a costimulatory molecule, includes a ligand binding domain (e.g., extracellular domain) of a protein ligand.

[0211] In some embodiments, these binding agents may include anti-CD28, anti-CD2, anti-CD28H, anti-LFA-1, anti-OX40, anti-4-lBB, anti-CD40L, anti-DNAM-1, anti-CD27, anti-ICOS, anti-LIGHT, anti-GITR, anti-CD30, anti-SLAM, anti-Ly-9, anti-CD84, anti-LylO8, anti-NKG2D, anti-NKp46, anti-NKp44, anti-NKp30, anti-CD244, anti-NKp80, anti-TCRa chain, anti-TCRp chain, anti-TCRy chain, and anti-TCR6 chain agents.

[0212] In some embodiments, the adhesion molecule binds to CD2. CD2 is also known as Til, LFA-2, and the erythrocyte rosette receptor and 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 provides a costimulatory signal that may enhance activation and effector functions. In some embodiments, the lentiviral particle comprises a molecule that binds to CD2. In some embodiments, the lentiviral particle comprises an antibody, single domain antibody, antibody fragment, and / or nanobody specific for CD2. In some embodiments, the lentiviral particle comprises CD58, or a functional portion thereof that binds to CD2.

[0213] In some embodiments, at least one costimulatory molecule is CD58, which also acts as an adhesion molecule. In some embodiments, the adhesion molecule is CD58. In some260132003840embodiments, the at least one costimulatory molecule comprises a CD58, a CD58 extracellular domain thereof, or a functional fragment of CD58. In some embodiments, the at least one costimulatory molecule comprises an extracellular domain portion of CD58 that comprises a CD58 extracellular domain able to bind to CD2. An exemplary CD58 is human CD58 set forth in Uniprot P19256. In some embodiments, the extracellular domain of CD58 corresponds to amino acids 29-215 of Uniprot P19256.

[0214] In some embodiments, the co-stimulation molecule is a CD58 polypeptide comprising the amino acid sequence of SEQ ID NO: 78. In some embodiments, the costimulation molecule is a CD58 polypeptide comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 78.

[0215] In some embodiments, the CD58 polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 79. In some embodiments, the CD58 polypeptide is encoded by a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 79.

[0216] In some embodiments, CD58 of the MDF comprises the sequence set forth in SEQ ID NO: 112 or a sequence that exhibits at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 112. 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: 113.d. Exemplary Multidomain Fusion Transduction Enhancer

[0217] In some embodiments, the viral envelope comprises a fusion molecule that comprises a CD58 extracellular domain, or a functional fragment thereof, a CD80 or CD86 extracellular domain, or a functional fragment thereof, and an activation domain, for example, an antigenbinding fragment of an anti-CD3 antibody.

[0218] In some embodiments, the membrane-bound multidomain fusion (MDF) protein comprises, in contiguous linkage, a CD58 extracellular domain, an anti-CD3 single-chain variable fragment (scFv) that functions as a T cell activation domain, and a CD80 domain that includes its extracellular, transmembrane, and cytoplasmic regions. These domains may be connected in series by one or more flexible polypeptide linkers, which provide spatial separation260132003840and conformational flexibility to maintain proper folding, orientation, and biological function of each domain when displayed on the surface of the LVV particle.

[0219] In certain embodiments, the MDF protein comprises, in N- to C-terminal order, a CD58 extracellular domain, a polypeptide linker, an anti-CD3 scFv, a second polypeptide linker, and a CD80 domain comprising the mature CD80 extracellular domain in contiguous linkage with its transmembrane and cytoplasmic tail. This arrangement positions the CD58 domain at the N-terminus for initial contact with the T cell CD2 receptor, followed by the anti-CD3 scFv that provides TCR-dependent activation, and the membrane- anchored CD80 domain that offers costimulatory signaling through CD28 or CTLA-4 engagement. Without wishing to be bound by theory, this domain configuration is believed to promote cooperative engagement of multiple T cell receptors while maintaining appropriate orientation and surface anchoring.

[0220] In other embodiments, the order of these domains may be varied. The fusion protein may comprise, in N- to C-terminal order, (i) an activation molecule such as an anti-CD3 scFv, followed by a linker, then CD58 or a functional fragment thereof, followed by a linker, and finally CD80 or a functional fragment thereof. In other embodiments, the sequence may be reversed or rearranged, for example: CD80-linker-CD58-linker- activation molecule, or activation molecule-linker-CD80-linker-CD58. Such variations may be selected according to the desired spatial presentation of the individual binding domains on the LVV particle surface or the preferred signaling hierarchy upon T cell engagement.

[0221] In some embodiments, the linker is a flexible linker, such as a GS linker. In some embodiments, the use of defined flexible linkers between domains is believed to allow optimal receptor clustering without over-activation, thereby improving functional infectivity and reducing the risk of activation-induced T cell exhaustion. Each polypeptide linker independently may comprise a flexible, hydrophilic sequence such as GSG, GSSGGSGGGGSGGGGSGGGGSSG (SEQ ID NO: 114), or other glycine- and serine-rich linkers known in the art. In some embodiments, linkers are about 3 to 25 amino acids in length, optionally about 5 to 20 amino acids, and provide sufficient spacing to prevent steric hindrance among the adjacent domains. In certain embodiments, linkers are designed to promote independent folding of each domain and preserve functional binding affinity for the respective ligands (e.g., CD2, CD3, and CD28). Variants of these linkers that include additional glycine residues, short spacers, or protease-resistant motifs may also be used.260132003840

[0222] Each of the domains of the MDF protein (CD58, anti-CD3 scFv, and CD80) may independently be a full-length sequence, extracellular portion, or functional fragment thereof, provided that the extracellular portion or fragment retains its binding or activation capability.

[0223] In some embodiments, the lentiviral particles described herein display a CD58-anti-CD3 scFv-CD80 tri-fusion polypeptide and transduce T cells in vivo generating CAR T cells. Without wishing to be bound by theory, the combination of CD58, anti-CD3, and CD80 within a single MDF molecule enables simultaneous engagement of CD2, CD3 / TCR, and CD28 on the surface of T cells. This multimodal engagement mimics the cooperative signaling of immune synapse formation while being tunable by surface density on the EVV particle.

[0224] In some embodiments, the MDF protein comprises the sequence set forth in SEQ ID NO: 115 or a sequence that exhibits at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 115. In some embodiments, the MDF protein comprises the sequence set forth in SEQ ID NO: 116 or a sequence that exhibits at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 116.3. Additional Non-Viral Proteins

[0225] In some embodiments, the viral particle comprises at least one non-viral protein. In some embodiments, the viral particle comprises at least one non-viral protein in addition to those described supra.

[0226] In some embodiments, the viral particle comprises a targeting ligand. In some embodiments, the viral particle comprises CD22, or a functional fragment thereof, coupled to its native transmembrane domain or a heterologous transmembrane domain. In some embodiments, the viral particle comprises CD 19, or a functional fragment thereof, coupled to its native transmembrane domain or a heterologous transmembrane domain. In some embodiments, CD 19 acts as a ligand for blinatumomab, thus providing an adapter for coupling the particle to T-cells via the anti-CD3 moiety of blinatumomab. In some embodiments, another type of particle surface ligand can serve to couple an appropriately surface engineered lentiviral particle to a T-cell using a multispecific antibody comprising a binding moiety for the particle surface ligand. In some embodiments, the multispecific antibody is a bispecific antibody, for example, a Bispecific T-cell engager (BiTE).

[0227] In some embodiments, the non-viral protein is a cytokine. In some embodiments, the cytokine may be selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18, IL-21,260132003840and any combination thereof. Where the non- viral protein used is a soluble protein (such as an scFv or a cytokine) it may be tethered to the surface of the viral particle by fusion to a transmembrane domain, such as the transmembrane domain of CD8. Alternatively, it may be indirectly tethered to the lentiviral particle by use of a transmembrane protein engineered to bind the soluble protein. Further inclusion of one or more cytoplasmic residues may increase the stability of the fusion protein.

[0228] The mitogenic transduction enhancer and / or cytokine- based transduction enhancer may comprise a “spacer sequence” to connect the antigen-binding domain with the transmembrane domain. A flexible spacer allows the antigen-binding domain to orient in different directions to facilitate binding. As used herein, the term “coupled to” refers to a chemical linkage, a direct C-terminal to N-terminal fusion of two protein; chemical linkage to a non-peptide space; chemical linkage to a polypeptide space; and C-terminal to N-terminal fusion of two protein via peptide bonds to a polypeptide spacer, e.g., a spacer sequence.

[0229] The spacer sequence may, for example, comprise an IgGl Fc region, an IgGl hinge or a human CD8 stalk or the mouse CD8 stalk. The spacer may alternatively comprise an alternative linker sequence which has similar length and / or domain spacing properties as an IgGl Fc region, an IgGl hinge or a CD8 stalk. A human IgGl spacer may be altered to remove Fc binding motifs. In some embodiments, the spacer sequence may be derived from a human protein.

[0230] In some embodiments, the spacer sequence comprises a CD8 derived hinge.

[0231] In some embodiments, the spacer sequence comprises a ‘short’ hinge. The short hinge is described as hinge region comprising fewer nucleotides relative to CAR hinge regions known in the art.

[0232] The transmembrane domain is the sequence of the mitogenic transduction enhancer and / or cytokine-based transduction enhancer that spans the membrane. The transmembrane domain may comprise a hydrophobic alpha helix. The transmembrane domain may be derived from CD28. In some embodiments, the transmembrane domain is derived from a human protein.

[0233] The viral particle of the present invention may comprise a cytokine-based transduction enhancer in the viral envelope. In some embodiments, the cytokine-based transduction enhancer is derived from the host cell during viral particle production. In some embodiments, the cytokine-based transduction enhancer is made by the host cell and expressed at the cell surface. When the nascent viral particle buds from the host cell membrane, the260132003840cytokine-based transduction enhancer may be incorporated in the viral envelope as part of the packaging cell-derived lipid bilayer.

[0234] The cytokine-based transduction enhancer may comprise a cytokine domain and a transmembrane domain. It may have the structure C-S-TM, where C is the cytokine domain, S is an optional spacer domain (e.g., a spacer sequence) and TM is the transmembrane domain. The spacer domain and transmembrane domains are as defined above.

[0235] The cytokine domain may comprise a T-cell activating cytokine, such as from IL2, IL7 and IL15, or a functional fragment thereof. As used herein, a “functional fragment” of a cytokine is a fragment of a polypeptide that retains the capacity to bind its particular receptor and activate T-cells.

[0236] IL2 is one of the factors secreted by T cells to regulate the growth and differentiation of T cells and certain B cells. IL2 is a lymphokine that induces the proliferation of responsive T cells. It is secreted as a single glycosylated polypeptide, and cleavage of a signal sequence is required for its activity. Solution NMR suggests that the structure of IL2 comprises a bundle of 4 helices (termed A-D), flanked by 2 shorter helices and several poorly defined loops. Residues in helix A, and in the loop region between helices A and B, are important for receptor binding. III. METHODS AND USES THEREOF

[0237] In some embodiments, provided herein is a method of transducing a cell comprising contacting a target cell with a particle, such as a viral particle produced from a viral vector, containing any of the polynucleotide constructs provided herein. In some embodiments, the provided methods deliver the polynucleotide constructs to cells for expression of the provided systems including a CAR. In some embodiments, the polynucleotide constructs are contained within viral particles produced from viral vectors encoding the polynucleotide constructs provided herein. In some embodiments, the viral particles are used to transduce target cells. In some embodiments, the provided methods are carried out ex vivo or in vitro to engineer target cells with the polynucleotide construct. In some embodiments, the engineered cells are administered to a subject. In some embodiments, the provided methods are carried out in vivo and a viral vector containing the polynucleotide construct is introduced to a subject for in vivo targeted delivery of the polynucleotide vector to a target cell, such as a T cell.260132003840A. Methods of Transducing a Cell

[0238] In some embodiments, provided herein is a method of transducing a cell comprising contacting a target cell with a particle, such as a viral vector, containing any of the polynucleotide constructs provided herein. In some embodiments, the target cell comprises a stem cell. In some embodiments, the stem cell comprises an induced pluripotent stem cell (iPSC). In some embodiments, the target cell comprises a progenitor cell. In some embodiments, the progenitor cell comprises a peripheral blood mononuclear cell (PBMC). In some embodiments, the PBMC comprises lymphocytes. In some embodiments, the target cell comprises a T cell. In some embodiments, the T cell comprises a CD4+ or CD8+ T cell. In some embodiments, the target cell comprises a cytotoxic innate lymphocyte (CIL) cell. In some embodiments, the target cell comprises a natural killer (NK) cell. In some embodiments, the target cell comprises a white blood cell. In some embodiments, the white blood cell comprises a monocyte. In some embodiments, the white blood cell comprises a macrophage. In some embodiments, the method further comprises contacting the target cell with a (i) a guide RNA (gRNA) targeting a target site in an endogenous gene, and (ii) an RNA-guided endonuclease, thereby inserting the nucleotide sequence into the endogenous gene.

[0239] In some aspects, the polynucleotides described herein may be delivered to cells in vivo. In some embodiments, a polynucleotide encoding the elements of the polynucleotide construct disclosed herein is administered to a subject directly via administration of a particle comprising the polynucleotide. In some embodiments, the particle is a viral particle. In some embodiments, the viral particle comprises an anti-CD3 scFv and a cocal glycoprotein and is capable of delivering the polynucleotide to cells in vivo. In some embodiments, the polynucleotide encodes a chimeric antigen receptor (CAR). The polynucleotides disclosed herein may be administered to the subject which allows the production of the construct components (e.g., CAR) in vivo. In some embodiments, the administration of such polynucleotide generates similar effect in vivo as direct administration of ex vivo engineered cells expressing the CAR. In some embodiments, the administration of such polynucleotide improves the in vivo transduction efficiency of a particle. In some embodiments, the polynucleotide is an mRNA.

[0240] The polynucleotides described herein may also be delivered to cells ex vivo. Viral particles described herein may be used ex vivo, either in convention cell manufacturing processes or in an extracorporeal or bedside process as described, e.g., in Int’l Pat. Pub. No.260132003840WO 2022 / 072885 Al. In one embodiment, 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).

[0241] In some embodiments, the disclosure provides a method of delivering a nucleic acid to a cell in an ex vivo CAR T manufacturing process. Such methods typically involve the isolation of PBMCs from a patient via leukapheresis. These cells are washed and optionally further purified via one or more selection steps to isolate particular T cell populations of interest. In some aspects, these might include CD4+ and / or CD8+ T cells. The washed and / or purified cells may be optionally activated and then transduced using a lentiviral vector. The activation step may comprise contacting the cells with an exogenous activation agent such as anti-CD3 and anti-CD28 antibodies bound to a substrate or using unbound antibodies. Illustrative activation agents include anti-CD3 and anti-CD28-presenting beads and / or soluble polymers. After transduction, the cells may be optionally further washed and cultured until harvest. Methods of manufacturing engineered cell therapies, including CAR T cells, are known in the art (see e.g., Abou-el-Enein, M. et al. Blood Cancer Discov (2021), Vol 2(5): 408-422; Arcangeli, S. et al. Front. Immunol (19 Jun 2020), Vol. 11 (1217) 1-13; Ghassemi, S. et al. Nat Biomed Eng (Feb 2022), Vol 6(2): 118-128; Vormittag, P. et al. Curr Opin Biotechnol (Oct 2018), Vol. 54: 164-181; each of which is herein incorporated by reference). Illustrative methods of autologous CAR T manufacturing are disclosed in US Patent Publication Nos. 2019 / 0269727, 2016 / 0122782, 2021 / 0163893, and US 2017 / 0037369, each of which is incorporated herein in its entirety.

[0242] 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 manufacturing260132003840processes, 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 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 acid is 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.

[0243] 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 enables 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 necessary in post-transduction cell culture.B. Methods of Expressing a Chimeric Antigen Receptor

[0244] In some embodiments, provided herein is a method of expressing a chimeric antigen receptor in a target cell. In some embodiments, the methods include contacting a target cell with a particle, such as a viral vector, containing any of the polynucleotide constructs provided herein. In some embodiments, the contacting is carried out ex vivo or in vitro. In some embodiments, the contacting is carried out in vivo in a subject by administering the polynucleotide constructs or particles, such as viral vectors, containing the same to a subject.

[0245] In some embodiments, the target cell comprises a stem cell. In some embodiments, the stem cell comprises an induced pluripotent stem cell (iPSC).260132003840

[0246] In some embodiments, the target cell comprises a progenitor cell. In some embodiments, the progenitor cell comprises a peripheral blood mononuclear cell (PBMC). In some embodiments, the PBMC comprises lymphocytes.

[0247] In some embodiments, the target cell comprises a T cell. In some embodiments, the T cell comprises a CD4+ or CD8+ T cell.

[0248] In some embodiments, the target cell comprises a cytotoxic innate lymphocyte (CIL) cell.

[0249] In some embodiments, the target cell comprises a natural killer (NK) cell.

[0250] In some embodiments, the target cell comprises a white blood cell. In some embodiments, the white blood cell comprises a monocyte. In some embodiments, the white blood cell comprises a macrophage.

[0251] In some embodiments, the method is performed ex vivo or in vitro.

[0252] In some embodiments, the method is performed in vivo.

[0253] In some embodiments, provided herein is a cell produced by any of the methods provided herein.

[0254] In some embodiments, the target cells expressing the chimeric antigen receptor, including the modified CD8 signal peptide, can be assessed for expression using flow cytometry. In some embodiments, the target cells expressing the chimeric antigen receptor, including the modified CD8 signal peptide, are assessed for proliferation. In some embodiments, the target cells expressing the chimeric antigen receptor, including the modified CD8 signal peptide, are assessed for cytokine release. In some embodiments, a CAR T cell cytokine release assay is used. In some embodiments, the target cells expressing the chimeric antigen receptor, including the modified CD8 signal peptide, are assessed for tumor killing activity. In some embodiments, a CAR T serial killing assay is used to evaluate CAR T cells killing of tumor targets. In some embodiments, the target cells expressing the chimeric antigen receptor, including the modified CD8 signal peptide, are assessed for RNA expression, including mRNA expression, of the CAR. In some embodiments, the target cells expressing the chimeric antigen receptor, including the modified CD8 signal peptide, are assessed for vector copy number of the viral particles.C. Methods of Administering and treatment

[0255] Also provided herein are methods of administering any of the provided cells engineered with a provided polynucleotide construct to a subject. Also provided herein are methods of administering to a subject any of the provided particles, such as viral vectors, for260132003840example a lentiviral vector. In some embodiments, the subject has a disease or condition, and the methods of administering are for treating a disease or condition. In any of subject methods, the cells or particles are administered as pharmaceutical compositions. In some embodiments, the compositions are for use in treating a disease or condition. Also provided are uses of provided compositions for treating a disease or condition in a subject. Such methods and uses include therapeutic methods and uses, for example, involving administration of the engineered cells or particles (e.g., viral vectors), or compositions containing the same, to a subject having a disease of condition. In some cases, the disease or condition is a tumor or cancer. In some embodiments, the cells or pharmaceutical composition thereof is administered in an effective amount to effect treatment of the disease or disorder. Uses include uses of the cells or pharmaceutical compositions thereof in such methods and treatments, and in the preparation of a medicament in order to carry out such therapeutic methods. In some embodiments, the methods thereby treat the disease or condition or disorder in the subject.

[0256] In some embodiments, engineered cells that contain any of the provided polynucleotide constructs that encode a chimeric antigen receptor (CAR) may be administered to a subject to treat a disease or condition. In some embodiments, particles, such as viral vectors (e.g., lentiviral vectors) may be directly administered to a subject for in vivo targeted delivery of the polynucleotide construct to target cells for the production of the various construct components (e.g., CAR) in vivo. The disclosed cells or particles (e.g., viral vectors) may be administered in a number of ways depending upon whether local or systemic treatment is desired.

[0257] In the case of adoptive cell therapy, methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions.

[0258] In general, administration may be topical, parenteral, or enteral. The compositions of the disclosure are typically suitable for parenteral administration. As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue, thus generally resulting in the direct administration into the blood stream, into muscle, or into an internal organ. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a260132003840surgical incision, by application of the composition through a tissue- penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrastemal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intratumoral, intrasynovial injection or infusions; and kidney dialytic infusion techniques. In an embodiment, parenteral administration of the compositions of the present disclosure comprises intravenous administration. 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 injection into a lymph node, such as an inguinal lymph node. 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 intraarterially. In some embodiments, the viral particle is a lentiviral particle.

[0259] Formulations of a pharmaceutical composition suitable for parenteral administration typically generally comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. Parenteral formulations also include aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water. Illustrative parenteral administration forms include solutions or suspensions in sterile aqueous260132003840solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, or in a liposomal preparation. Formulations for parenteral administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In some embodiments, the solution intended for subcutaneous administration includes hyaluronidase.

[0260] The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and / or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

[0261] The present polynucleotide constructs may be administered in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.260132003840

[0262] In certain embodiments, in the context of infusing differentiated cells or transgenic differentiated cells according to the disclosure, a subject is administered the range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges, and / or such a number of cells per kilogram of body weight of the subject. For example, in some embodiments the administration of the cells or population of cells can comprise administration of about 103to about 109cells per kg body weight including all integer values of cell numbers within those ranges.

[0263] In some embodiments, provided herein is a method of administering to a subject any of the cells provided herein.

[0264] In some embodiments, provided herein is a method of administering to a subject any of the viral vectors provided herein.

[0265] In some embodiments, the CAR encoded by the provided polynucleotide construct is targeted to an antigen associated with a disease or condition and the methods include administering the cells (e.g., by adoptive cell therapy) or viral vector, such as lentiviral vectors, to a subject that has or is suspected of having the disease or condition. In some embodiments, among provided methods are methods for treating a subject suffering from cancer, including the step of administering a provided cell engineered with any of the provided polynucleotide constructs of the disclosure to the subject, wherein the cancer is treated in the subject. In some embodiments, among provided methods are methods for treating a subject suffering from cancer, including the step of administering a provide viral vector, such as lentiviral particle, incorporating any of the provided polynucleotide constructs of the disclosure to the subject, wherein the cancer is treated in the subject.260132003840

[0266] In some embodiments, the cancer is a solid tumor, such as a melanoma, non-small cell lung cancer, or breast cancer. The methods of the present disclosure may include treating any cancer, including, without limitation, acute granulocytic leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, adenocarcinoma, adenosarcoma, adrenal cancer, adrenocortical carcinoma, anal cancer, anaplastic astrocytoma, angiosarcoma, appendix cancer, astrocytoma, basal cell carcinoma, B-cell lymphoma, bile duct cancer, bladder cancer, bone cancer, bone marrow cancer, bowel cancer, brain cancer, brain stem glioma, brain tumor, breast cancer, carcinoid tumors, cervical cancer, cholangiocarcinoma, chondrosarcoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngioma, cutaneous lymphoma, cutaneous melanoma, diffuse astrocytoma, ductal carcinoma in situ, endometrial cancer, ependymoma, epithelioid sarcoma, esophageal cancer, Ewing sarcoma, extrahepatic bile duct cancer, eye cancer, fallopian tube cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid cancer, gastrointestinal stromal tumors, general, germ cell tumor, gestational trophoblastic disease, glioblastoma multiforme, glioma, hairy cell leukemia, head and neck cancer, hemangioendothelioma, Hodgkin lymphoma, Hodgkin lymphoma, Hodgkin's disease, hypopharyngeal cancer, infiltrating ductal carcinoma, infiltrating lobular carcinoma, inflammatory breast cancer, intestinal cancer, intrahepatic bile duct cancer, invasive / infiltrating breast cancer, islet cell cancer, jaw cancer, Kaposi sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, leptomeningeal metastases, leukemia, lip cancer, liposarcoma, liver cancer, lobular carcinoma in situ, low-grade astrocytoma, lung cancer, lymph node cancer, lymphoma, male breast cancer, medullary carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesenchymal chondrosarcoma, mesenchymal mesothelioma, metastatic breast cancer, metastatic melanoma, metastatic squamous neck cancer, mixed gliomas, mouth cancer, mucinous carcinoma, mucosal melanoma, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, nasal cavity cancer, nasopharyngeal cancer, neck cancer, neuroblastoma, neuroendocrine tumors, non-Hodgkin lymphoma, non-small cell lung cancer, oat cell cancer, ocular cancer, ocular melanoma, oligodendroglioma, oral cancer, oral cavity cancer, oropharyngeal cancer, osteogenic sarcoma, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian primary peritoneal carcinoma, ovarian sex cord stromal tumor, Paget's disease, pancreatic cancer, papillary carcinoma, paranasal sinus cancer, parathyroid cancer, pelvic cancer, penile cancer, peripheral nerve cancer, peritoneal cancer,260132003840pharyngeal cancer, pheochromocytoma, pilocytic astrocytoma, pineal region tumor, pineoblastoma, pituitary tumors, primary central nervous system, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, bone sarcoma, soft-tissue sarcoma, uterine, sinus cancer, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cancer, spinal column cancer, spinal cord cancer, spinal tumor, squamous cell carcinoma, stomach cancer, synovial sarcoma, T-cell lymphoma, testicular cancer, throat cancer, thymoma / thymic carcinoma, thyroid cancer, tongue cancer, tonsil cancer, transitional cell cancer, triple-negative breast cancer, tubal cancer, tubular carcinoma, undiagnosed cancer, ureteral cancer, uterine adenocarcinoma, uterine cancer, uterine sarcoma, vaginal cancer, and vulvar cancer.

[0267] In some embodiments, the CAR encoded by the provided polynucleotide construct is a CAR that targets a ligand that is able to bind to an antigen on the surface of a cell associated with a disease or condition. In some embodiments, the CAR of any of the provided embodiments is an anti-CD22 CAR directed against CD22. In some embodiments, the CAR of any of the provided embodiments is an anti-CD22 CAR directed against CD22 comprises a modified CD8 signal peptide. In some embodiments, the CAR of any of the provided embodiments is an anti-CD22 CAR directed against CD22 comprises a shorter CD8 alpha hinge. In some embodiments, the CAR of any of the provided embodiments is an anti-CD22 CAR directed against CD22 comprises a modified CD8 signal peptide and a shorter CD8 alpha hinge.IV. DEFINITIONS

[0268] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0269] As used herein, the singular forms “a”, “an”, and “the” are include the plural forms as well, unless the context indicates otherwise. The conjugation “and / or” denotes all possible combinations of one or more of listed items.

[0270] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or260132003840parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0271] It is understood that aspects and embodiments of the invention described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments.

[0272] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance does or does not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally substituted group means that the group is unsubstituted or is substituted.

[0273] The term “composition” refers to any mixture of two or more products, substances, or compounds, including cells or antibodies. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof. The preparation is generally in such form as to permit the biological activity of the active ingredient (e.g., antibody) to be effective.

[0274] A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0275] As used herein, combination refers to any association between or among two or more items. The combination can be two or more separate items, such as two compositions or two collections, can be a mixture thereof, such as a single mixture of the two or more items, or any variation thereof. The elements of a combination are generally functionally associated or related.

[0276] As used herein, a kit is a packaged combination that optionally includes other elements, such as additional agents and instructions for use of the combination or elements thereof, for a purpose including, but not limited to, therapeutic uses.

[0277] “Subject” as used herein refers to the recipient of polynucleotide construct or other agent. The term includes mammal, such as primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep or a pig, preferably a human.

[0278] ‘ ‘Treat,” “treating” or “treatment” as used herein refers to any type of action or administration that imparts a benefit to a subject that has a disease or disorder, including improvement in the condition of the patient (z.e., improvement, reduction, or amelioration of one or more symptoms, and partial or complete response to treatment).

[0279] The term “effective amount” refers to an amount effective to generate a desired biochemical, cellular, or physiological response. The term “therapeutically effective amount” refer to the amount, dosage, or dosage regime of a therapy effective to cause a desire treatment260132003840effect. As used herein, an “individual” or a “subject” is a mammal. A “mammal” for purposes of treatment includes humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, etc. In some embodiments, the individual or subject is human.

[0280] “Polynucleotide” as used herein refers to a biopolymer composed of two or more nucleotide monomers covalently bonded through ester linkages between the phosphoryl group of one nucleotide and the hydroxyl group of the sugar component of the next nucleotide in a chain. DNA and RNA are non-limiting examples of polynucleotides.

[0281] “Polypeptide” as used herein refers to a polymer consisting of amino acid residues chained together by peptide bonds, forming part of (or the whole of) a protein.

[0282] It will be understood by a skilled person that numerous different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed.

[0283] Nucleic acids may comprise DNA or RNA. They may be single- stranded or doublestranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. For the purposes of the use as described herein, it is to be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of interest.

[0284] The term “variant” means a polynucleotide or polypeptide having at least one substitution, insertion, or deletion in its sequence compared to a reference polynucleotide or polypeptide. A “functional variant” is a variant that retains one or functions of the reference polynucleotide or polypeptide.

[0285] As used herein the term “sequence identity”, or “identity” in relation to polynucleotides or polypeptide sequences, refers to the extent to which two optimally aligned polynucleotides or polypeptide sequences match at each position in the alignment across the full length of the reference sequence. The “percent identity” is the number of matched positions in260132003840the optimal alignment, divided by length of the reference sequence plus the sum of the lengths of any gaps in the reference sequence in the alignment. The optimal alignment is the alignment that results in the maximum percent identity. Alignment of sequences to determine percent identity can be accomplished by a number of well-known methods, including for example by using mathematical algorithms, such as, for example, those in the BLAST suite or Clustal Omega sequence analysis programs. Unless noted otherwise, the term “sequence identity” in the claims refers to sequence identity as calculated by BLAST version 2.12.0 using default parameters. And, unless noted otherwise, the alignment is an alignment of all or a portion of the polynucleotide or polypeptide sequences of interest across the full length of the reference sequence.

[0286] The term “cell population” refers to mixture of cells suspended in solution, attached to a substrate, or stored in a container. The characteristics of a cell population as a whole can be studied with bulk measurements of sample volumes having a plurality of cells. Flow cytometry methods may be employed to reduce problems with background fluorescence which are encountered in bulk cell population measurements.

[0287] As used herein, the term “engineered” refers to a cell that has been stably transduced with a heterologous polynucleotide or subjected to gene editing to introduce, delete, or modify polynucleotides in the cell, or cells transiently transduced with a polynucleotide in a manner that causes a stable phenotypic change in the cell.V. EXEMPLARY EMBODIMENTS

[0288] Among the provided embodiments are:1. A polynucleotide construct comprising a nucleotide sequence encoding a protein fusion of a signal peptide and a chimeric antigen receptor (CAR), wherein the signal peptide is a modified CD8 signal peptide set forth by the amino acid sequence MALPVTALLLPLALLLHAARA (SEQ ID NO: 7).2. The polynucleotide construct of embodiment 1, wherein the CAR comprises in N to C terminal order an antigen binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain.3. The polynucleotide construct of embodiment 2, wherein the antigen binding domain comprises a means for binding an antigen.2601320038404. The polynucleotide construct of embodiment 3, wherein the antigen is a tumor antigen, an antigen on an autoreactive B cell or T cell, a viral antigen, a small molecule, or a synthetic antigen.5. The polynucleotide construct of embodiment 4, wherein the small molecule is a hapten.6. The polynucleotide construct of embodiment 5, wherein the hapten is fluorescein or fluorescein isothiocyanate (FITC).7. The polynucleotide construct of any one of embodiments 2 to 6, wherein the antigen binding domain is a single chain variable fragment (scFv) or a VHH single domain antibody.8. The polynucleotide construct of any one of embodiments 2 to 7, wherein the spacer domain comprises a hinge region.9. The polynucleotide construct of embodiment 8, wherein the hinge region is derived from an immunoglobulin, CD8, or CD28.10. The polynucleotide construct of embodiment 9, wherein the CD8 comprises CD8a hinge.11. The polynucleotide construct of any one of embodiments 2 to 10, wherein the transmembrane domain is a CD8 transmembrane domain or a CD28 transmembrane domain.12. The polynucleotide construct of any one of embodiments 2 to 11, wherein the intracellular domain comprises a CD3 zeta (CD3Q domain.13. The polynucleotide construct of any one of embodiments 2 to 12, wherein the intracellular domain comprises a costimulatory signaling domain.14. The polynucleotide construct of embodiment 13, wherein the costimulatory signaling domain is a 4- IBB signaling domain or a CD28 signaling domain.15. The polynucleotide construct of any one of embodiments 1 to 14, wherein one or more nucleotide sequences are codon optimized.16. The polynucleotide construct of any one of embodiments 1 to 15, wherein the polynucleotide construct is encoded in a bicistronic construct.17. The polynucleotide construct of any one of embodiments 1 to 16, wherein the polynucleotide construct is encoded in a multicistronic construct.18. A viral vector particle comprising the polynucleotide construct of any one of embodiments 1 to 17.26013200384019. The viral vector particle of embodiment 18, wherein the viral vector particle is a lentiviral vector particle, optionally wherein the lentiviral vector is a self-inactivating and replication-incompetent lentiviral vector particle.20. The viral vector particle of embodiment 18 or embodiment 19, wherein the viral vector particle further comprises one or more surface T cell activating agents.21. The viral vector particle of embodiment 20, wherein the one or more surface T cell activating agents comprise an extracellular portion of CD58 that binds to CD2, an anti-CD3 antibody or antigen binding fragment thereof, or CD80.22. The viral vector particle of any one of embodiments 18-21, wherein the viral vector particle comprises a multidomain fusion protein (MDF) displayed on its surface, wherein the multidomain fusion protein comprise an extracellular portion of CD58 that binds to CD2, an anti-CD3 antibody or antigen binding fragment thereof and a CD80 sequence, optionally wherein the MDF protein comprises an amino acid sequence that is identical to or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 115 or SEQ ID NO: 116.23. The viral vector particle of any one of embodiments 18-22, wherein the viral vector comprises a pseudotyped viral envelope protein, optionally wherein the pseudotyped viral envelope protein is cocal virus glycoprotein (G) (Cocal G), optionally wherein the Cocal G is a Cocal envelope variant containing a R354Q mutation or a K47Q mutation.24. A cell comprising the polynucleotide construct of any one of embodiments 1 to 17.25. A cell transduced by the viral vector particle of any one of embodiments 18 to 23.26. The cell of embodiment 24 or embodiment 25, wherein the cell comprises a stem cell or a progenitor cell.27. The cell of embodiment 26, wherein the stem cell comprises an induced pluripotent stem cell (iPSC).28. The cell of embodiment 24 or embodiment 253, wherein the cell is a lymphocyte or lymphocyte progenitor.29. The cell of embodiment 24 or embodiment 25, wherein the cell is a T cell.30. The cell of embodiment 24 or embodiment 25, wherein the cell is a cytotoxic innate lymphocyte (CIL) cell.26013200384031. The cell of embodiment 24 or embodiment 25, wherein the cell is a natural killer (NK) cell.32. The cell of embodiment 24 or embodiment 25, wherein the cell is a monocyte or a macrophage.33. A method of transducing a cell comprising contacting a target cell with any of the polynucleotide constructs of any one of embodiments 1 to 17.34. A method of transducing a cell comprising contacting a target cell with the viral vector of any one of embodiments 18 to 21.35. The method of embodiment 33 or embodiment 34, wherein the target cell comprises a stem cell or a progenitor cell.36. The method of embodiment 35, wherein the stem cell comprises an induced pluripotent stem cell (iPSC).37. The method of embodiment 33 or embodiment 34, wherein the target cell is a lymphocyte or lymphocyte progenitors.38. The method of embodiment 33 or embodiment 34, wherein the target cell comprises a T cell.39. The method of embodiment 38, wherein the T cell is a CD4+ or CD8+ T cell. 40. A method of increasing expression of a polynucleotide construct expressing a chimeric antigen receptor (CAR), the method comprising transducing a cell with the polynucleotide construct of any one of embodiments 1 to 17 or the viral vector particle of any one of embodiments 18-23.VI. EXAMPLES

[0289] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.Example 1 Identification of signal peptide for increased transgene expression

[0290] This example describes the identification of signal peptide sequences to increase cell surface expression levels of recombinant proteins in cells. The identified signal peptides can be used in polynucleotide constructs for improving cell surface expression of certain proteins, for example, components of the rapamycin activated cytokine receptor (RACR) system, chimeric antigen receptors (CARs), and other recombinant proteins.260132003840

[0291] In this example, a polynucleotide construct encoding one of the polypeptide chains of an exemplary rapamycin activated cytokine receptor (RACR) was designed with alternative signal peptides and assessed for predicted cleavage by in silico computational strategies. RACR is composed of two polypeptides: a first synthetic cytokine polypeptide composed of a FKBP12 domain and either an IL2Ry signaling domain or a beta chain signaling domain, and a second synthetic cytokine polypeptide composed of an FRB domain and the other of the IL2Ry domain or a beta chain signaling domain (e.g., signaling domain from IL2RP).

[0292] Computational techniques were used to predict cleavage likelihood of the different signal peptide variants. For more details, see, e.g., Teufel, F., Almagro Armenteros, J.J., Johansen, A.R. et al. SignalP 6.0 predicts all five types of signal peptides using protein language models. Nat Biotechnol 40, 1023-1025 (2022).

[0293] In one experiment, polynucleotide constructs encoding a fusion protein composed of an N-terminal LCN-2 signal peptide (SEQ ID NO: 5) and a heterologous FKBP12-containing polypeptide, or a fusion protein composed of an N-terminal LCN-2 signal peptide (SEQ ID NO: 5) and an FRB -containing polypeptide were designed in silico and assessed for probability of cleavage.

[0294] Results indicated that constructs encoding an FKBP12-containing polynucleotide were predicted to have a high cleavage probability using an LCN2 signal peptide, but constructs encoding the FRB-containing polynucleotide with the same LCN2 signal peptide did not result in optimal cleavage of the signal peptides (Table El). These results support that signal peptides are not compatible with every protein or protein domain.

[0295] To identify signal peptides compatible with FRB-containing polypeptides, polynucleotide constructs encoding a fusion protein composed of an FRB-containing polypeptide and either an N-terminal CD4 signal peptide, an N-terminal CD8 signal peptide, or modified versions thereof were designed in silico and assessed for predicted cleavage. CD4 and CD8 signal peptides also have an advantage of being compatible with T cells.260132003840

[0296] Table E2 depicts results of assessment of predicted cleavage in constructs encoding the polypeptide with an N-terminal wildtype CD8 signal peptide (SEQ ID NO: 6) or modified CD8 signal peptide sequences. As shown in Table E2, modifying the last amino acid of the CD8 signal peptide sequence by substituting proline (P) for the neutral amino acid alanine (A) in the last position (designated “P21A”, SEQ ID NO: 7) improved cleavage likelihood compared to the wild-type CD8 signal peptide. In contrast, mutating proline to arginine (R) in the last amino acid of the signal peptide (designated “P21R”, SEQ ID NO: 8) decreased the cleavage likelihood and substituting the last amino acid with tryptophan (W) (designated “P21W”, SEQ ID NO: 9) eliminated likelihood of cleavage.

[0297] These data identify that certain modified CD8 signal peptides with alanine in the last position (e.g., P21A) improve predicted cleavage of FRB-containing polypeptides and likelihood of increased expression.Example 2 Generation and Assessment of CAR Constructs with CD8 signal peptide

[0298] This example describes the generation of polynucleotide constructs encoding a fusion protein composed of either an N-terminal wild-type CD8 signal peptide (SEQ ID NO: 6) or the P21A variant CD8 signal peptide (SEQ ID NO: 7), as described in Example 1, and a chimeric antigen receptor (CAR), and assessment of expression and functional characterization of cells expressing the CAR. All polynucleotide constructs encoded a fusion protein composed of an N- terminal signal peptide and an anti-CD22 CAR polypeptide sequence containing the following components in N-terminal to C-terminal order: an anti-CD22 scFv, a hinge spacer (CD8 alpha hinge), a transmembrane domain, and an intracellular domain with a costimulatory signaling domain (4- IBB) and a CD3zeta signaling domain (Z).

[0299] Polynucleotides constructs were generated encoding three exemplary fusion proteins sequences with an N-terminal CD8 signal peptide or modified version and a heterologous anti- CD22 CAR as summarized in Table E3. The fusion protein designated CD22 CAR_1 contained260132003840a modified CD8 signal peptide with a proline (P) in the last position of the CD8 signal peptide, while both the fusion proteins designated CD22 CAR_2 and CD22 CAR_3 contained the modified P21A variant CD8 signal peptide with an alanine (A) instead of a proline (P). The sequence of the fusion protein designated CD22 CAR 3 also included an anti-CD22 CAR with a CD8 alpha hinge that was ten amino acids shorter than CD8 alpha hinge in the other anti-CD22 CARs. All constructs were codon optimized.

[0300] For lentiviral vector production, 293T producer cells were transfected with plasmids expressing viral vector proteins (gag / pol, rev) and a transfer plasmid encoding each of the above polynucleotide constructs. The lentiviral vectors also were engineered to encode CD58, anti-CD3, and CD80 surface proteins to mediate T cell activation. Following viral vector production, the cell culture was centrifuged to pellet the cells and the supernatant containing crude virus was collected. Lenti-X concentrator was used to concentrate the lentiviral vector particles.

[0301] T-cells were transduced with concentrated lentivirus containing vector particles encoding one of the anti-CD22 CAR fusion proteins. Expression of CAR was measured using flow cytometry on Day 6 after transduction. FIG. 1 shows CD3 and anti-CD22 CAR expression. The mean fluorescence intensity (MFI) is shown for each construct on FIG. 1 and Table E4. Greater expression of CD22-CAR positive cells was detected in T cells transduced with lentivirus encoding an anti-CD22 CAR with the N-terminal variant CD8 signal peptide as260132003840demonstrated by the increase in MFI (e.g., Table E4 and FIG. 1, iii. constructs CD22 CAR_2 and iv. CD22 CAR_3).

[0302] To further assess expression, T cells were transduced at different levels of multiplicity of infection (MOI) of lentivirus and CAR expression was monitored. Briefly, on day 0, PBMCs from three healthy donors were seeded and either were not transduced (0) or were transduced at MOI of 0.25, 0.5, 1, 2, and 5. On day 1, PBMCs were washed and replated. On day 7, cells were stained to assess CAR expression using flow cytometry (FIG. 2). In addition, on day 7, a portion of the cells were frozen for vector copy number (VCN) analysis, or were further processed for RNA extraction for analysis of expression of mRNA encoding the CAR by qPCR using primers specific to the CAR CD3z region. Further, transduced CAR-expressing cells harvested on day 7 also were assessed for cytolytic activity in a cell killing assay, proliferation, and cytokine production.

[0303] Cells transduced with lentivirus encoding an anti-CD22 CAR with the N-terminal variant CD8 signal peptide had higher levels of CD3+CAR+ cells at all levels of MOI tested, as determined by flow cytometry (FIG. 2). Either a 2L or 10L culture was used for lentivirus manufacturing. Cells transduced with lentivirus encoding an anti-CD22 CAR with the N-terminal variant CD8 signal peptide also had reduced number of integrations (FIG. 3A) and higher mRNA expression of the CAR (FIG. 3B).

[0304] Functionality of cells transduced with lentivirus encoding one of the anti-CD22 CAR fusion proteins was assessed using a serial killing assay. PBMCs were transduced with lentivirus as described above. For the killing assay, CAR-transduced effector T cells and CD22-expressing tumor target cells were initially seeded at a 0.5:1 CAR-expressing cells:tumor cells ratio (effector:target; 20k CAR-expressing cells:40k tumor cells) with no IL-2. Every 2-3 days 40,000260132003840CD22-expressing tumor cells were added, and the number of living cells was monitored using Incucyte. As shown in FIG. 4, improved cytolytic killing was observed by cells that had been transduced with lentivirus encoding an anti-CD22 CAR with the N-terminal variant CD8 signal peptide, as evidenced by a drastic decrease in the number of tumor target cells in cells transduced with polynucleotide constructs encoding the CD22_CAR_2 and CD22_CAR_3 (FIG. 4).

[0305] To monitor proliferation and cytokine production, 2.5xl05CAR-expressing T cells were added to CD22-coated plates in absence of IL-2. Three days later, cells were re-plated in media with IL-2. At Day 9, the fold expansion and levels of IFN-y and TNF-a in the supernatant were then assessed. The cells had been transduced with lentivirus encoding an anti-CD22 CAR with the N-terminal variant CD8 signal peptide (e.g., CD22_CAR_2 and CD22_CAR_3) produced CAR T cells with improved ability to proliferate and cytokine production compared to T cells transduced with the anti-CD22 CAR with the N-terminal unmodified CD8 signal peptide (CD22_CAR_1) (FIG. 5).

[0306] Results showed that the variant CD8 signal peptide can be used in polynucleotide constructs for improving cell surface expression of a CAR, for example, to improve engineered cell functionality.

[0307] The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.260132003840SEQUENCES260132003840260132003840260132003840260132003840260132003840260132003840260132003840260132003840260132003840260132003840260132003840260132003840260132003840260132003840260132003840260132003840260132003840260132003840260132003840260132003840260132003840

Claims

260132003840CLAIMS1. A polynucleotide construct comprising a nucleotide sequence encoding a protein fusion of a signal peptide and a chimeric antigen receptor (CAR), wherein the signal peptide is a modified CD8 signal peptide set forth by the amino acid sequence MALPVTALLLPLALLLHAARA (SEQ ID NO: 7).

2. The polynucleotide construct of claim 1, wherein the CAR comprises in N to C terminal order an antigen binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain.

3. The polynucleotide construct of claim 2, wherein the antigen binding domain comprises a means for binding an antigen.

4. The polynucleotide construct of claim 3, wherein the antigen is a tumor antigen, an antigen on an autoreactive B cell or T cell, a viral antigen, a small molecule, or a synthetic antigen.

5. The polynucleotide construct of claim 4, wherein the small molecule is a hapten.

6. The polynucleotide construct of claim 5, wherein the hapten is fluorescein or fluorescein isothiocyanate (FITC).

7. The polynucleotide construct of any one of claims 2 to 6, wherein the antigen binding domain is a single chain variable fragment (scFv) or a VHH single domain antibody.

8. The polynucleotide construct of any one of claims 2 to 7, wherein the spacer domain comprises a hinge region.

9. The polynucleotide construct of claim 8, wherein the hinge region is derived from an immunoglobulin, CD8, or CD28.

10. The polynucleotide construct of claim 9, wherein the CD8 comprises CD8a hinge.

11. The polynucleotide construct of any one of claims 2 to 10, wherein the transmembrane domain is a CD8 transmembrane domain or a CD28 transmembrane domain.26013200384012. The polynucleotide construct of any one of claims 2 to 11, wherein the intracellular domain comprises a CD3 zeta (CD3Q domain.

13. The polynucleotide construct of any one of claims 2 to 12, wherein the intracellular domain comprises a costimulatory signaling domain.

14. The polynucleotide construct of claim 13, wherein the costimulatory signaling domain is a 4- IBB signaling domain or a CD28 signaling domain.

15. The polynucleotide construct of any one of claims 1 to 14, wherein one or more nucleotide sequences are codon optimized.

16. The polynucleotide construct of any one of claims 1 to 15, wherein the polynucleotide construct is encoded in a bicistronic construct.

17. The polynucleotide construct of any one of claims 1 to 16, wherein the polynucleotide construct is encoded in a multicistronic construct.

18. A viral vector particle comprising the polynucleotide construct of any one of claims 1 to 17.

19. The viral vector particle of claim 18, wherein the viral vector particle is a lentiviral vector particle, optionally wherein the lentiviral vector is a self-inactivating and replication-incompetent lentiviral vector particle.

20. The viral vector particle of claim 18 or claim 19, wherein the viral vector particle further comprises one or more surface T cell activating agents.

21. The viral vector particle of claim 20, wherein the one or more surface T cell activating agents comprise an extracellular portion of CD58 that binds to CD2, an anti-CD3 antibody or antigen binding fragment thereof, or CD80.

22. The viral vector particle of any one of claims 18-21, wherein the viral vector particle comprises a multidomain fusion protein (MDF) displayed on its surface, wherein the multidomain fusion protein comprise an extracellular portion of CD58 that binds to CD2, an anti-CD3 antibody or antigen binding fragment thereof and a CD80 sequence, optionally wherein the MDF protein comprises an amino acid sequence that is identical to or has at least26013200384085%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 115 or SEQ ID NO: 116.

23. The viral vector particle of any one of claims 18-22, wherein the viral vector comprises a pseudotyped viral envelope protein, optionally wherein the pseudotyped viral envelope protein is cocal virus glycoprotein (G) (Cocal G), optionally wherein the Cocal G is a Cocal envelope variant containing a R354Q mutation or a K47Q mutation.

24. A cell comprising the polynucleotide construct of any one of claims 1 to 17.

25. A cell transduced by the viral vector particle of any one of claims 18 to 23.

26. The cell of claim 24 or claim 25, wherein the cell comprises a stem cell or a progenitor cell.

27. The cell of claim 26, wherein the stem cell comprises an induced pluripotent stem cell (iPSC).

28. The cell of claim 24 or claim 25, wherein the cell is a lymphocyte or lymphocyte progenitor.

29. The cell of claim 24 or claim 25, wherein the cell is a T cell.

30. The cell of claim 24 or claim 25, wherein the cell is a cytotoxic innate lymphocyte (CIL) cell.

31. The cell of claim 24 or claim 25, wherein the cell is a natural killer (NK) cell.

32. The cell of claim 24 or claim 25, wherein the cell is a monocyte or a macrophage.

33. A method of transducing a cell comprising contacting a target cell with any of the polynucleotide constructs of any one of claims 1 to 17.

34. A method of transducing a cell comprising contacting a target cell with the viral vector of any one of claims 18 to 21.

35. The method of claim 33 or claim 34, wherein the target cell comprises a stem cell or a progenitor cell.26013200384036. The method of claim 35, wherein the stem cell comprises an induced pluripotent stem cell (iPSC).

37. The method of claim 33 or claim 34, wherein the target cell is a lymphocyte or lymphocyte progenitors.

38. The method of claim 33 or claim 34, wherein the target cell comprises a T cell.

39. The method of claim 38, wherein the T cell is a CD4+ or CD8+ T cell.

40. A method of increasing expression of a polynucleotide construct expressing a chimeric antigen receptor (CAR), the method comprising transducing a cell with the polynucleotide construct of any one of claims 1 to 17 or the viral vector particle of any one of claims 18-23.

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