Viral particles having surface stimulatory molecules

Abstract

Viral particles for activating and transducing immune cells in vitro or in vivo, as well as compositions and methods for using said viral particles are provided. The present disclosure is based, at least in part, on the discovery that viral particles can be produced such that a cell surface protein is expressed on the viral envelope to simultaneously prime and transduce immune cells. In particular, as demonstrated herein, viral particles expressing a TCR targeting molecule and a costimulatory molecule enhance cell activation in vivo and transduction of nucleotides encoding a polypeptide of interest as compared to viral particles expressing only the TCR targeting molecule.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority and the benefit thereof based on U.S. Provisional Patent Application No. 63 / 339,332 filed on May 6, 2022, U.S. Provisional Patent Application No. 63 / 348,180 filed on June 2, 2022, U.S. Provisional Patent Application No. 63 / 351,064 filed on June 10, 2022, and U.S. Provisional Patent Application No. 63 / 487,734 filed on March 1, 2023, the entire contents of each of these U.S. Provisional Patent Applications being incorporated herein by reference in their entirety.

[0002] Incorporation by Reference of a Sequence Listing This application contains a sequence listing submitted via EFS - WEB in the.XML ST26 format, the entire contents of which are incorporated herein by reference. The said.XML copy created on May 4, 2023, is named 061479 - 505001WO_SeqList_ST26.xml and is 288 kilobytes in size.

Background Art

[0003] Background Cell therapy generally uses ex vivo transduction of immune cells to generate a population of therapeutic cells that are introduced into a patient. For example, T cells obtained from autologous or allogeneic sources can be transduced ex vivo with a vector encoding a chimeric antigen receptor. The resulting CAR T - cells are then injected into the patient.

[0004] Although it is desirable to generate therapeutic cells in vivo by delivering vectors to a patient, current methodologies for in vivo transduction of immune cells are plagued by technical, logistic, consistency, cost, and efficacy issues. The in vivo approach has not been widely explored because of the associated technical issues, and the main hurdles are the need to activate T cells in the body to effectively engineer them and the need to "control" the expansion of these engineered cells after transduction.

Summary of the Invention

Problems to be Solved by the Invention

[0005] Ex-vivo manufacturing of cell therapies requires a complex series of steps that begin with the collection of a patient's peripheral blood mononuclear cells by leukapheresis procedures and continue with the genetic modification of the patient's T cells in a cGMP facility, introducing delays, risks, and complex logistics into patient care. This is followed by the administration of lymphodepleting chemotherapy prior to the infusion of the final drug product. Therefore, there remains a need for improved cell therapies.

Means for Solving the Problems

[0006] Summary of the Invention The present disclosure is based, at least in part, on the discovery that viral particles can be produced to express cell surface proteins on the viral envelope to simultaneously prime and transduce immune cells. In particular, as demonstrated herein, viral particles expressing a TCR-targeting molecule and a costimulatory molecule enhance cell activation in vivo and transduction of nucleotides encoding a polypeptide of interest compared to viral particles expressing only the TCR-targeting molecule. Furthermore, it has been shown that including an adhesion molecule further enhances cell activation and transduction in vivo. Without wishing to be bound by theory, viral particles engineered to express a TCR-targeting molecule (e.g., a CD3-binding protein) and a costimulatory molecule (e.g., CD80 or CD86) activate signal 1 and signal 2 required for T cell activation. Furthermore, it is believed that the adhesion molecule stabilizes the interaction between immune cells and viral particles, thereby reconstructing an immune synapse that enables sufficient cell activation and nucleotide transduction. The present disclosure also shows in vivo activation and expansion of non-transduced T cells. Without wishing to be bound by theory, the particles described herein can drive the activation and expansion of tumor-infiltrating lymphocytes and tumor-reactive T cells present in tumor-draining regional lymph nodes or metastatic lymph nodes, indicating that such particles may be effective at low doses.

[0007] Accordingly, in some aspects, the present disclosure provides viral particles comprising a viral envelope comprising, on the surface thereof, at least one T cell adhesion molecule, at least one costimulatory protein or a combination thereof, and an immune cell activating protein. In some embodiments, the T cell adhesion molecule, costimulatory protein, and immune cell activating protein are each recombinant proteins.

[0008] In some or any of the foregoing or related aspects, at least one T cell adhesion molecule is selected from CD58, HHLA2, ICAM-1, OX40L, 4-1BBL, CD40, CD155, CD70, HVEM, GITRL, ICOS-L, CD30L, SLAM, Ly-9, CD84, Ly108, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, B7-H6, and any combination thereof. In some aspects, at least one T cell adhesion molecule is CD58.

[0009] In some or any of the foregoing or related aspects, at least one costimulatory molecule is selected from CD45, CD2, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, OX40, 4-1BB, CD40L, and any combination thereof. In some aspects, at least one costimulatory molecule is CD80, CD86, or CD80 and CD86.

[0010] In some or any of the foregoing or related aspects, the immunocyte activation protein is a protein that specifically binds to CD2, CD3, CD28H, LFA-1, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, NKG2D, NKp46, NKp44, NKp30, CD244, TCR α chain, TCR β chain, TCR ζ chain, TCR γ chain, TCR δ chain, CD3ε TCR subunit, CD3γ TCR subunit, CD3δ TCR subunit, or NKp80. In some aspects, the immunocyte activation protein is a protein that specifically binds to CD3.

[0011] In some or any of the foregoing or related aspects, the immune cell activating protein is an antibody or an antigen-binding fragment thereof that binds to CD2, CD3, CD28H, LFA-1, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, NKG2D, NKp46, NKp44, NKp30, CD244 or NKp80. In some aspects, the immune cell activating protein is an antibody or an antigen-binding fragment thereof that binds to CD3. In some aspects, the antibody or antigen-binding fragment thereof that binds to CD3 is an anti-CD3 scFv.

[0012] In some or any of the foregoing or related aspects, the T cell adhesion molecule is CD58 and the co-stimulatory molecule is CD80. In other aspects, the T cell adhesion molecule is CD58 and the co-stimulatory molecule is CD86.

[0013] In some or any of the foregoing or related aspects, the T cell adhesion molecule is CD58, the immune cell activating protein is an anti-CD3 antibody or an antigen-binding fragment thereof, and the co-stimulatory molecule is CD80. In other aspects, the T cell adhesion molecule is CD58, the immune cell activating protein is an anti-CD3 antibody or an antigen-binding fragment thereof, and the co-stimulatory molecule is CD86.

[0014] In some or any of the foregoing or related aspects, the viral particle contains a payload. In some aspects, the payload is a nucleic acid. In some aspects, the nucleic acid is a non-coding nucleic acid, and optionally, the non-coding nucleic acid is siRNA, miRNA or shRNA. In some aspects, the nucleic acid contains a nucleotide sequence encoding a polypeptide of interest.

[0015] In some or any of the foregoing or related aspects, the viral particle contains a vector genome containing at least one nucleotide sequence encoding a polypeptide of interest.

[0016] In some embodiments, the present disclosure provides viral particles comprising: (i) a viral envelope on the surface of which are (a) an immunocyte-activating protein that binds to a T cell receptor, (b) a co-stimulatory molecule, and (c) a T cell adhesion molecule; and (ii) a vector genome comprising at least one nucleotide sequence encoding a polypeptide of interest. In some embodiments, (a) the immunocyte-activating protein is a protein that specifically binds to CD2, CD3, CD28H, LFA-1, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, NKG2D, NKp46, NKp44, NKp30, CD244, or NKp80; (b) the co-stimulatory molecule is selected from CD45, CD2, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, OX40, 4-1BB, CD40L, and any combination thereof; (c) the T cell adhesion molecule is selected from CD58, HHLA2, ICAM-1, OX40L, 4-1BBL, CD40, CD155, CD70, HVEM, GITRL, ICOSL, CD30L, SLAM, Ly-9, CD84, Ly108, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, B7-H6, and any combination thereof. In some embodiments, (a) the immunocyte-activating protein is an antibody or an antigen-binding fragment thereof that specifically binds to CD3; (b) the co-stimulatory molecule is CD80 or CD86; and (c) the T cell adhesion molecule is CD58.

[0017] In some or any of the foregoing or related embodiments, the viral envelope comprises a membrane-bound cytokine. In some embodiments, the membrane-bound cytokine is selected from IL-2, IL-7, IL-12, IL-15, IL-18, or IL-21.

[0018] In some or any of the foregoing or related aspects, the viral envelope comprises a viral envelope protein. In some aspects, the viral envelope protein is a VSV-G envelope protein, a measles virus envelope protein, a Nipah virus envelope protein, or a coxsackievirus G protein. In some aspects, the viral envelope comprises a coxsackievirus glycoprotein or a functional variant thereof. In some aspects, the coxsackievirus glycoprotein comprises an R354Q mutation as compared to SEQ ID NO: 5. In some aspects, the coxsackievirus glycoprotein comprises a K47Q mutation as compared to SEQ ID NO: 5. In some aspects, the coxsackievirus glycoprotein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 5, 13, and 19. In some aspects, the coxsackievirus glycoprotein comprises an amino acid sequence selected from SEQ ID NOs: 5, 13, and 19.

[0019] In some or any of the foregoing or related aspects, the antibody or antigen-binding fragment thereof that binds to anti-CD3 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2 or 12. In some aspects, the antibody or antigen-binding fragment thereof that binds to anti-CD3 comprises SEQ ID NO: 2 or SEQ ID NO: 12.

[0020] In some or any of the foregoing or related aspects, the viral particle comprises a nucleotide sequence encoding a multipartite cell surface receptor. In some aspects, the multipartite cell surface receptor comprises an FKBP-rapamycin complex binding domain (FRB domain) and an FK506 binding protein domain (FKBP). In some aspects, the multipartite cell surface receptor is a rapamycin-activated cell surface receptor.

[0021] In some or any of the foregoing or related aspects, the viral particle comprises a nucleotide sequence encoding a chimeric antigen receptor (CAR). In some aspects, the viral particle comprises a nucleotide sequence encoding a rapamycin-activated cell surface receptor and a nucleotide sequence encoding a CAR. In some aspects, the viral particle comprises a vector genome comprising, from 5' to 3': a nucleotide sequence encoding a CAR and a nucleotide sequence encoding a multipartite cell surface receptor. In some aspects, the nucleotide sequences are operably linked. In some aspects, the CAR comprises an antigen-binding domain specific for a cancer-related antigen, and the multipartite cell surface receptor is a rapamycin-activated cell surface receptor.

[0022] In some or any of the foregoing or related aspects, the CAR comprises an antigen-binding domain specific for a cancer-related antigen. In some aspects, the cancer-related antigen is CD19, BCMA, GPRC5D, ROR1, FcRL5 / FcRH5, alpha-fetoprotein or Her2. In other aspects, the CAR is a universal CAR. In some aspects, the CAR comprises a hapten-binding domain.

[0023] In some aspects, the present disclosure provides a viral particle comprising: (i) a viral envelope on the surface of which are (a) an immunocyte-activating protein that specifically binds to CD3, (b) a costimulatory molecule that binds to CD28, and (c) a T cell adhesion molecule; and (ii) a vector genome comprising (a) a nucleotide sequence encoding a rapamycin-activated cell surface receptor and (b) a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain specific for a cancer-related antigen, and optionally, the nucleotide sequences are operably linked.

[0024] In some or any of the foregoing or related aspects, CD58 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 17. In some aspects, CD58 comprises the amino acid sequence of SEQ ID NO: 17.

[0025] In some or any of the foregoing or related aspects, CD80 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 20. In some aspects, CD80 comprises the amino acid sequence of SEQ ID NO: 20.

[0026] In some or any of the foregoing or related aspects, CD86 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 23. In some aspects, CD86 comprises the amino acid sequence of SEQ ID NO: 23.

[0027] In some or any of the foregoing or related aspects, the multipartite cell surface receptor comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 77, 78, or both 77 and 78. In some aspects, the multipartite cell surface receptor comprises the amino acid sequence of SEQ ID NO: 77, 78, or both 77 and 78. In some aspects, the multipartite cell surface receptor is encoded by a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 83, 84, or both 83 and 84. In some aspects, the multipartite cell surface receptor is encoded by the nucleotide sequence of SEQ ID NO: 83, 84, or both 83 and 84.

[0028] In some or any of the foregoing or related aspects, the vector genome comprises a promoter. In some aspects, the promoter is an MND promoter, a CAG promoter, an SV40 promoter, an SV40 / CD43 promoter or an EF-1α promoter. In some aspects, the promoter is an inducible promoter.

[0029] In some or any of the foregoing or related aspects, the viral particles are lentiviral particles.

[0030] In some or any of the foregoing or related aspects, the viral particles transduce T cells in vivo. In other aspects, the viral particles transduce T cells ex vivo. In some aspects, the viral particles activate a T cell population comprising at least 50% CD25(+) cells, at least 70% CD25(+) cells or at least 90% CD25(+) cells.

[0031] In some embodiments, the present disclosure provides a pharmaceutical composition comprising the viral particles described herein and a pharmaceutically acceptable carrier.

[0032] In some embodiments, the present disclosure provides a method of transducing a population of T cells in vivo in a subject, the method comprising administering to the subject a viral particle or a pharmaceutical composition thereof, wherein the viral particle comprises a nucleotide sequence encoding a polypeptide of interest, and wherein the polypeptide of interest is expressed in the population of T cells after administration. In some embodiments, the population of T cells secretes (i) at least 2×10 4 pg / ml of TNFα, (ii) at least 2×10 4 pg / ml of IL-2, (iii) at least 2×10 5 pg / ml of IFNγ, or (iv) any combination of (i) to (iii) at least 3 days after administration of the lentiviral particles.

[0033] In other embodiments, the present disclosure provides a method of generating immune cells expressing a chimeric antigen receptor in a subject in need thereof, the method comprising administering to the subject the viral particles or pharmaceutical composition described herein, wherein the viral particle comprises a nucleotide sequence encoding the chimeric antigen receptor.

[0034] In a further embodiment, the present disclosure provides a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject the viral particles or pharmaceutical composition described herein, wherein the viral particle comprises a nucleotide sequence encoding a therapeutic polypeptide.

[0035] In some or any of the foregoing or related embodiments, the viral particles are administered by intraperitoneal, subcutaneous, or intranodal injection. In some embodiments, the viral particles are administered via the inguinal lymph node by intranodal injection.

[0036] In some or any of the foregoing or related aspects, the subject in need thereof has a disease or disorder, which includes B cell malignancies, relapsed / refractory CD19-expressing malignancies, diffuse large B cell lymphoma (DLBCL), Burkitt's large B cell lymphoma (B-LBL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cell lymphoma (MCL), hematological malignancies, colon cancer, lung cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, brain cancer, squamous cell carcinoma, leukemia, myeloma, B cell lymphoma, kidney cancer, uterine cancer, adenocarcinoma, pancreatic cancer, chronic myeloid leukemia, glioblastoma, neuroblastoma, medulloblastoma, sarcoma, and any combination thereof.

[0037] In some aspects, the disclosure provides a kit comprising a container containing the viral particles described herein and, optionally, a pharmaceutically acceptable carrier, and instructions for transducing T cells in vivo in a subject, the instructions including the step of administering the viral particles to the subject. In some aspects, the subject has a disease or disorder. In some aspects, the instructions include the step of administering the viral particles by intraperitoneal, subcutaneous, or intranodal injection.

[0038] In other aspects, the disclosure provides a kit comprising a container containing the viral particles described herein and, optionally, a pharmaceutically acceptable carrier, and instructions for treating a subject in need of treatment, the instructions including the step of administering the viral particles to the subject. In some aspects, the subject has a disease or disorder. In some aspects, the instructions include the step of administering the viral particles by intraperitoneal, subcutaneous, or intranodal injection.

[0039] In some embodiments, the present disclosure provides the viral particles described herein for use in a method of in vivo transducing T cells in a subject, the method comprising the step of administering the viral particles to the subject. In other embodiments, the present disclosure provides the viral particles described herein for use in a method of treating a subject having a disease or disorder, the method comprising the step of administering the viral particles to the subject.

[0040] In some embodiments, the present disclosure provides the use of the viral particles described herein for the manufacture of a medicament for in vivo transducing T cells in a subject, the method comprising the step of administering the viral particles to the subject. In other embodiments, the present disclosure provides the use of the viral particles described herein for the manufacture of a medicament for treating a subject having a disease or disorder, the method comprising the step of administering the viral particles to the subject.

[0041] In some embodiments, the present disclosure provides a method of ex vivo transducing a population of T cells in a subject, the method comprising contacting the population of T cells with a viral particle or a pharmaceutical composition thereof, wherein the viral particle comprises a nucleotide sequence encoding a polypeptide of interest, the polypeptide of interest is expressed in the population of T cells after administration, and the contacting step is performed ex vivo. In some embodiments, the contacting step is performed during a closed-loop manufacturing process. In some embodiments, the T cells have not previously been contacted with an exogenous activator during the manufacturing process. BRIEF DESCRIPTION OF THE DRAWINGS

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BRIEF DESCRIPTION OF THE DRAWINGS

[0082] DETAILED DESCRIPTION In some embodiments, the present disclosure provides virus particles comprising a viral envelope containing an immunocyte activation protein, a costimulatory molecule, a T cell adhesion molecule, and any combination thereof. In some embodiments, the virus particles are delivery agents for a molecule of interest. In some embodiments, the molecule is a nucleotide sequence encoding a polypeptide of interest. In some embodiments, the molecule is a non-coding nucleic acid. In some embodiments, the non-coding nucleic acid is cDNA, shRNA, microRNA, or siRNA.

[0083] In some embodiments, the virus particles described herein activate and transduce immunocytes in vivo. In some embodiments, the virus particles described herein activate and transduce immunocytes in vitro. In some embodiments, the virus particles activate and transduce immunocytes simultaneously.

[0084] In some embodiments, the viral particles and methods provided herein obviate the need for pre-activation of immune cells prior to administration of the viral particles. In some embodiments, the method does not include pre-activation of immune cells in a subject prior to administration of the viral particles (e.g., no pre-activation within about 1, 2, 3, 4, 5, 6, or 7 days or within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks prior to administration of the viral particles). In some embodiments, pre-activation of immune cells optionally includes activating CD3 and / or CD28 signaling in immune cells (e.g., T cells) by administering anti-CD3 and / or anti-CD28 antibodies, respectively. Thus, in some embodiments, the methods of the disclosure do not include administering separate CD3 and / or CD28 activating agents prior to administration of the viral particles.

[0085] viral particles In some embodiments, the disclosure provides viral particles comprising a viral envelope and a payload. In some embodiments, the viral envelope comprises an immune cell activating protein, a co-stimulatory molecule, a T cell adhesion molecule, or any combination thereof.

[0086] In some embodiments, the viral particles comprise a polynucleotide. In some embodiments, the polynucleotide encodes at least one therapeutic polypeptide. The term "therapeutic polypeptide" refers to a polypeptide that is being developed for therapeutic use or has been developed for therapeutic use. In some embodiments, the therapeutic polypeptide is expressed in target cells (e.g., host T cells) for therapeutic use. In some embodiments, the therapeutic polypeptide comprises a T cell receptor, a chimeric antigen receptor or a cytokine receptor.

[0087] In some embodiments, the viral particles are retroviral particles. In some embodiments, the viral particles are lentiviral particles. In some embodiments, the viral particles are adeno-associated viral particles.

[0088] As used herein, the term "viral particle" refers to a macromolecular complex capable of transferring a payload (e.g., nucleic acid) into a cell. A viral vector contains structural and / or functional genetic elements mainly derived from a virus. The term "retroviral vector" refers to a viral vector or a portion thereof that contains structural and functional genetic elements mainly derived from a retrovirus. The term "lentiviral vector" refers to a viral vector or a portion thereof that contains structural and functional genetic elements including LTRs and mainly derived from a lentivirus. The term "hybrid" refers to a vector, LTR, or other nucleic acid that contains, for example, retroviral sequences of both lentiviral sequences and non-lentiviral viral sequences. In some embodiments, a hybrid vector refers to a vector or transfer plasmid containing retroviral sequences, such as lentiviral sequences, for reverse transcription, replication, integration, and / or packaging.

[0089] Viral envelope In some embodiments, the viral vector comprises a viral envelope containing a polypeptide on the envelope surface.

[0090] In some embodiments, the viral envelope comprises one or more transduction enhancers. In some embodiments, the transduction enhancer comprises a T cell activation receptor, an NK cell activation receptor, and / or a costimulatory molecule. In some embodiments, the one or more transduction enhancers comprise one or more of anti-CD3 scFv, CD86, CD80, and / or CD58. In some embodiments, the transduction enhancer comprises at least anti-CD3 scFv and CD58. In some embodiments, the transduction enhancer comprises at least anti-CD3 scFv and CD80. In some embodiments, the transduction enhancer comprises at least anti-CD3 scFv and CD86. In some embodiments, the transduction enhancer comprises at least anti-CD3 scFv, CD80, and CD58. In some embodiments, the transduction enhancer comprises at least anti-CD3 scFv, CD86, and CD58.

[0091] In some embodiments, the viral particle comprises a cell surface receptor that binds to a ligand on the target host cell, enabling host cell transduction. In some embodiments, the viral particle comprises a heterologous viral envelope glycoprotein, resulting in pseudotyped viral particles. For example, the viral envelope glycoprotein can be one of RD114 or its variants, VSV-G, gibbon ape leukemia virus (GALV), or a dual-tropic envelope, measles envelope, or baboon retrovirus envelope glycoprotein. In some embodiments, the viral envelope glycoprotein is the VSV G protein (cocal glycoprotein) from the cocal strain or a functional variant thereof.

[0092] In some embodiments, the viral envelope comprises two or more polypeptides on its surface. In some embodiments, the two or more polypeptides bind to target immune cells and replicate the immune synapse. In some embodiments, the viral envelope comprises an immune cell activation protein, a costimulatory molecule, and an adhesion molecule, and the immune cell activation protein, costimulatory molecule, and adhesion molecule each bind to a target immune cell.

[0093] Immune cell activator In some embodiments, the transduction enhancer comprises a mitogenic stimulus incorporated into a retroviral or lentiviral capsid such that the virus performs both activation and transduction of T cells. This eliminates the need to add a vector and mitogen. In some embodiments, the transduction enhancer comprises a mitogenic transmembrane protein and / or one or more co-stimulatory and / or adhesion molecules that become incorporated into the retrovirus upon budding from the producer cell / package cell membrane. In some embodiments, the transduction enhancer is not part of the viral envelope glycoprotein, but is expressed as a separate cell surface molecule on the producer cell.

[0094] The viral vectors described herein can include a mitogenic transduction enhancer in the viral envelope. In some embodiments, the mitogenic transduction enhancer is obtained from the host cell during retroviral vector production. In some embodiments, the mitogenic transduction enhancer is produced by the packaging cell and expressed on the cell surface. When the nascent retroviral vector buds from the host cell membrane, the mitogenic transduction enhancer can be incorporated into the viral envelope as part of the packaging cell-derived lipid bilayer. In some embodiments, the mitogenic enhancer is an antibody or a fragment thereof. In some embodiments, the mitogenic enhancer is a single-domain antibody, such as a camelid antibody. In some embodiments, the mitogenic enhancer is a scFv. In some embodiments, the mitogenic enhancer is a nanobody.

[0095] In some embodiments, the transduction enhancer is of host cell origin. The term "of host cell origin" indicates that the mitosis-inducing transduction enhancer is obtained from the host cell and is not produced as a fusion or chimera from one of the viral genes such as gag encoding the major structural protein; or env encoding the envelope protein as described above.

[0096] The envelope protein is formed by a transmembrane (TM) that anchors the protein in the lipid membrane and a surface (SU) that binds to the cell receptor, which are two subunits. In some embodiments, the mitosis-inducing transduction enhancer derived from the packaging cells of the present invention does not contain the surface envelope subunit (SU).

[0097] In some embodiments, the mitosis-inducing transduction enhancer has the structure: M-S-TM (where M is the mitosis-inducing domain; S is an optional spacer domain, and TM is the transmembrane domain).

[0098] The mitosis-inducing domain is part of the mitosis-inducing transduction enhancer that causes T cell activation. This can directly or indirectly bind to or interact with T cells in other ways to cause T cell activation. In some embodiments, the mitosis-inducing domain binds to T cell surface antigens such as CD3, CD28, CD134, and CD137.

[0099] CD3 is a T cell coreceptor. It is a protein complex composed of four separate chains. In mammals, the complex contains the CD3y chain, the CD35 chain, and two CD3e chains. These chains associate with the T cell receptor (TCR) and the z-chain to generate activation signals in T lymphocytes. The TCR, z-chain, and CD3 molecules together constitute the TCR complex. In some embodiments, the mitosis-inducing domain binds to the CD3 e chain.

[0100] In some embodiments, the mitosis-inducing domain comprises all or part of an antibody or other molecule that specifically binds to a T cell surface antigen. In some embodiments, the antibody activates the TCR or CD28. In some embodiments, the antibody binds to 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, YTH12.5, TR66

[0101] In some embodiments, the mitosis-inducing domain comprises a binding domain derived from OKT3, 15E8, TGN1412, CD28.2, 10F3, UCHT1, YTH12.5 or TR66.

[0102] In some embodiments, the mitosis-inducing domain comprises all or part of a co-stimulatory molecule such as OX40L and 41BBL. For example, the mitosis-inducing domain can comprise a binding domain derived from OX40L or 41BBL.

[0103] OKT3, also known as Muromonab - CD3, is a monoclonal antibody that targets the CD3e chain. It is used clinically to reduce acute rejection in patients with organ transplants. It was the first monoclonal antibody approved for clinical use in humans. The CDRs of OKT3 are as follows:1 CDRH1: GYTFTRY (SEQ ID NO: 136) CDRH2: NPSRGY (SEQ ID NO: 137) CDRH3: YYDDHYCLDY (SEQ ID NO: 138) CDRL1: SASSSVSYMN (SEQ ID NO: 139) CDRL2: DTSKLAS (SEQ ID NO: 140) CDRL3: QQWSSNPFT (SEQ ID NO: 141)

[0104] In some embodiments, the viral envelope comprises an immunocyte activating protein. In some embodiments, the immunocyte activating protein specifically binds to a receptor on an immunocyte. In some embodiments, the immunocyte activating protein provides signal 1 for T cell activation.

[0105] In some embodiments, the immunocyte activating protein specifically binds to CD2, CD3, CD28H, LFA-1, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, NKG2D, NKp46, NKp44, NKp30, CD244 or NKp80. In some embodiments, the immunocyte activating protein specifically binds to CD3γ, CD3δ or CD3ε. In some embodiments, the immunocyte activating protein specifically binds to CD3γ, CD3δ, CD3ε, CD9, CD5, CD22, CD33, CD37, CD64, CD45, CD28H, LFA-1, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, CD16, CD56, NKG2D, NKp46, NKp44, NKp30, CD244, NKp80, TCRα chain, TCRβ chain, TCRγ chain or TCRδ chain. In some embodiments, the immunocyte activating protein specifically binds to CD3γ, CD3δ or CD3ε. In some embodiments, the immunocyte activating protein specifically binds to CD3.

[0106] In some embodiments, the immune cell activating protein is an antibody or an antigen-binding fragment thereof that specifically binds to a receptor on an immune cell. In some embodiments, the immune cell activating protein is an antibody or an antigen-binding fragment thereof that specifically binds to CD28, CD2, CD3, CD28H, LFA-1, OX40, 4-1BB, CD40L, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, NKG2D, NKp46, NKp44, NKp30, CD244 or NKp80. In some embodiments, the immune cell activating protein is an antibody or an antigen-binding fragment thereof that specifically binds to CD28, CD2, CD3γ, CD3δ, CD3ε, CD4, CD8, CD9, CD5, CD22, CD33, CD37, CD64, CD45, CD28H, LFA-1, OX40, 4-1BB, CD40L, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, CD16, CD56, NKG2D, NKp46, NKp44, NKp30, CD244, NKp80, TCR α chain, TCR β chain, TCR γ chain or TCR δ chain. In some embodiments, the immune cell activating protein is an antibody or an antigen-binding fragment thereof that specifically binds to CD3γ, CD3δ or CD3ε. In some embodiments, the immune cell activating protein is an antibody or an antigen-binding fragment thereof that specifically binds to CD3.

[0107] Antibodies that target 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.

[0108] In some embodiments, the viral envelope comprises an anti-CD3ε antibody or an antigen-binding fragment thereof. In some embodiments, the anti-CD3ε antibody or an antigen-binding fragment thereof is bound to a transmembrane domain. An illustrative anti-CD3ε antibody is OKT3. OKT3, also known as muromonab-CD3, is a monoclonal antibody that targets the CD3ε chain.

[0109] In some embodiments, the viral envelope comprises a single-chain Fv fragment (scFv) of an anti-CD3 antibody.

[0110] In some embodiments, the viral envelope comprises an anti-CD3 scFv comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 2. In some embodiments, the viral envelope comprises an anti-CD3 scFv comprising the amino acid sequence of SEQ ID NO: 2.

[0111] In some embodiments, the viral envelope comprises an anti-CD3 scFv comprising the following complementarity determining regions (CDRs): SASSSVSYMN (CDR-L1; SEQ ID NO: 133), DTSKLASG (CDR-L2; SEQ ID NO: 134), QQWSSNPFT (CDR-L3; SEQ ID NO: 135), RYTMH (CDR-H1; SEQ ID NO: 48), YINPSRGYTNYNQKVKD (CDR-H2; SEQ ID NO: 36), and YYDDHYCLDY (CDR-H3; SEQ ID NO: 38).

[0112] In some embodiments, the nucleotide sequence encoding the anti-CD3 scFv comprises 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: 7. In some embodiments, the nucleotide sequence encoding the anti-CD3 scFv comprises SEQ ID NO: 7.

[0113] In some embodiments, the viral envelope comprises an anti-CD3 scFv comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 12. In some embodiments, the viral envelope comprises an anti-CD3 scFv comprising the amino acid sequence of SEQ ID NO: 12.

[0114] In some embodiments, the viral envelope comprises an anti-CD3 scFv that includes the following CDRs: SASSSVSYMN (CDR-L1; SEQ ID NO: 133), DTSKLASG (CDR-L2; SEQ ID NO: 134), QQWSSNPFT (CDR-L3; SEQ ID NO: 135), RYTMH (CDR-H1; SEQ ID NO: 48), YINPSRGYTNYNQKVKD (CDR-H2; SEQ ID NO: 36), and YYDDHYCLDY (CDR-H3; SEQ ID NO: 38).

[0115] In some embodiments, the nucleotide sequence encoding the anti-CD3 scFv comprises 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: 15. In some embodiments, the nucleotide sequence encoding the anti-CD3 scFv comprises SEQ ID NO: 15.

[0116] Co-stimulatory molecule In some embodiments, the viral envelope comprises at least one co-stimulatory molecule. In some embodiments, the co-stimulatory molecule specifically binds to a receptor on an immune cell. In some embodiments, co-stimulation provides signal 2 for cell activation.

[0117] 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 for optimal priming and interaction with antigen-presenting cells (APCs) to obtain full effector function. For T cells, these include signal transduction through the T cell receptor (TCR), costimulatory molecules (such as CD28 and CD2), cytokines, and various adhesion molecules necessary to allow sufficient time for proper synapse formation and signal transduction. NK cells require a similar type of stimulation but can rely on different activating receptors, such as NKG2D, NKp46, and DNAM-1. For T cells, proper costimulation in addition to TCR stimulation is particularly important for effective priming, and many studies have shown that TCR stimulation alone can lead to functional anergy and unresponsiveness. Costimulatory signals enhance T and NK cell function by enhancing cell metabolism, cytokine production, differentiation, and long-term persistence. Costimulation is an important factor in 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. In some embodiments, the costimulatory molecule may be an adhesion molecule. In some embodiments, the costimulatory molecule includes, but is not limited to, binders that bind to any of the costimulatory or adhesion molecules described herein, such as scFv, antibodies, single-domain antibodies, antibody fragments, nanobodies.In some embodiments, these binding agents can 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-4-1BB, anti-CD40L, anti-DNAM-1, anti-CD27, anti-ICOS, anti-LIGHT, anti-GITR, anti-CD30, anti-SLAM, anti-Ly-9, anti-CD84, anti-Ly108, anti-NKG2D, anti-NKp46, anti-NKp44, anti-NKp30, anti-CD244, anti-NKp80, anti-TCRα chain, anti-TCRβ chain, anti-TCRγ chain and anti-TCRδ chain agents.

[0118] In some embodiments, the co-stimulatory molecule is a ligand of CD28. CD28 is one of the proteins expressed on T cells that provides co-stimulatory signals required for T cell activation and survival. T cell stimulation via CD28 in addition to the T cell receptor (TCR) can provide a strong signal for the production of various interleukins (especially IL-6). In some embodiments, the co-stimulatory molecule is an antibody or a fragment thereof that binds to CD28. Examples of such antibodies include 15E8 and TGN1412. Other suitable antibodies include CD28.2 and 10F3.

[0119] 15E8 is a mouse monoclonal antibody against human CD28. Its CDRs are as follows: CDRH1: GFSLTSY (SEQ ID NO: 142) CDRH2: WAGGS (SEQ ID NO: 143) CDRH3: DKRAPGKLYY GYPD Y (SEQ ID NO: 144) CDRL1: RASESVEYYVTSLMQ (SEQ ID NO: 145) CDRL2: AASNYES (SEQ ID NO: 146) CDRL3: QQTRKVPST (SEQ ID NO: 147)

[0120] TGN1412 (also known as CD28 - SuperMAB) is a humanized monoclonal antibody that not only binds to the CD28 receptor but is also a strong agonist for it. Its CDRs are as follows. CDRH1: GYTFSY (SEQ ID NO: 148) CDRH2: YPGNVN (SEQ ID NO: 149) CDRH3: SHYGLDWNFDV (SEQ ID NO: 150) CDRL1: HASQNIYVLN (SEQ ID NO: 151) CDRL2: KASNLHT (SEQ ID NO: 152) CDRL3: QQGQTYPYT (SEQ ID NO: 153)

[0121] In some embodiments, the co - stimulatory 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 - stimulatory 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 anti - CD28 scFv is conjugated to a transmembrane domain for presentation on the surface of a viral envelope.

[0122] In some embodiments, the co - stimulatory molecule is a CD86 polypeptide comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the co - stimulatory molecule is a CD86 polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 23.

[0123] In some embodiments, the CD86 polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 24. In some embodiments, the CD86 polypeptide is encoded by a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 24.

[0124] In some embodiments, the co-stimulatory molecule is a CD80 polypeptide comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the co-stimulatory molecule is a CD80 polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 20.

[0125] In some embodiments, the CD80 polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 21. In some embodiments, the CD80 polypeptide is encoded by a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 21.

[0126] CD134, also known as OX40, is a member of the TNFR-superfamily of receptors that is expressed on activated T cells. OX40 can promote cell division and survival. OX40 is a secondary co-stimulatory molecule that is expressed 24 to 72 hours after activation; its ligand, OX40L, is also not expressed on resting antigen-presenting cells but is expressed following their activation. In some embodiments, the viral particle comprises a ligand of OX40 or a functional fragment thereof, attached to its native transmembrane domain or a heterologous transmembrane domain.

[0127] 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 the blood vessel wall at the site of inflammation. The best-characterized activity of CD137 is its co-stimulatory activity for activated T cells. Cross-linking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytolytic activity. In some embodiments, the viral particle comprises a ligand of 4-1BB or a functional fragment thereof, attached to its native transmembrane domain or a heterologous transmembrane domain. 4-1BBL is a cytokine belonging to the tumor necrosis factor (TNF) ligand family. This transmembrane cytokine is a bidirectional signal transducer that acts as a ligand for 4-1BB, a co-stimulatory receptor molecule in T lymphocytes. 4-1BBL has been shown to reactivate anergic T lymphocytes in addition to promoting T lymphocyte proliferation.

[0128] Viral particles containing one or more activating or co-stimulatory molecules can be produced by manipulating a packaging cell line by the method provided by WO2016 / 139463; or by expression of a T cell activating or co-stimulatory molecule(s) from the polycistronic helper vector described in International Patent Publication No. WO2020 / 106992A1, both of which are hereby incorporated by reference in their entirety.

[0129] Adhesion molecule In some embodiments, the viral particles comprise adhesion molecules. As used herein, the term "adhesion molecule" refers to a subset of cell surface molecules involved in the binding of cells to other cells. Cell adhesion can help to form more stable interactions, such as the immune synapse between immune cells. The immune synapse is a stable adhesive junction between polarized immune effector cells and cells having an antigen. In some embodiments, the adhesion molecule can provide a co-stimulatory signal to the target cell. In some embodiments, the adhesion molecules include, but are not limited to, CD58, HHLA2, ICAM-1, OX40L, 4-1BBL, CD40, CD155, CD70, HVEM, GITRL, ICOS-L, CD30L, SLAM, Ly-9, CD84, Ly108, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and B7-H6.

[0130] The costimulatory and adhesion molecules of the present disclosure include, but are not limited to, CD80, CD86, CD58, HHLA2, ICAM-1, OX40L, 4-1BBL, CD40, CD155, CD70, HVEM, GITRL, ICOSL, CD30L, SLAM, Ly-9, CD84, Ly108, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and B7 family members, such as B7-H2, B7-H6, and B7-H5. Binding proteins for these ligands, such as scFv and nanobodies, including anti-CD28, anti-CD2, anti-CD28H, anti-LFA-1, anti-OX40, anti-4-1BB, anti-CD40L, anti-DNAM-1, anti-CD27, anti-ICOS, anti-LIGHT, anti-GITR, anti-CD30, anti-SLAM, anti-Ly-9, anti-CD84, anti-Ly108, anti-NKG2D, anti-NKp46, anti-NKp44, anti-NKp30, anti-CD244, and anti-NKp80, are also incorporated. Membrane-bound cytokines include, but are not limited to, IL-2, IL-7, IL-12, IL-15, IL-18, and IL-21. The fusion constructs of the present disclosure can also include B7-H3, B7x, and / or TMIGD2. The fusion proteins disclosed herein can include one or more domains that associate with costimulatory and / or adhesion molecules presented on the surface of T cells, including CD28, CD28H, CD2, CD3, LFA-1, OX40, 4-1BB, CD40L, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, NKG2D, NKp46, NKp44, NKp30, CD244, or NKp80.

[0131] In some embodiments, the adhesion molecule binds to any of the adhesion or costimulatory molecules described herein and includes binders such as, but not limited to, scFv, antibodies, single domain antibodies, antibody fragments, and nanobodies. In some embodiments, these binders can include anti-CD28, anti-CD2, anti-CD28H, anti-LFA-1, anti-OX40, anti-4-1BB, anti-CD40L, anti-DNAM-1, anti-CD27, anti-ICOS, anti-LIGHT, anti-GITR, anti-CD30, anti-SLAM, anti-Ly-9, anti-CD84, anti-Ly108, anti-NKG2D, anti-NKp46, anti-NKp44, anti-NKp30, anti-CD244, anti-NKp80, anti-TCRα chain, anti-TCRβ chain, anti-TCRγ chain, and anti-TCRδ chain agents.

[0132] In some embodiments, the costimulatory and / or adhesion molecule shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequences in Table 1.

Table 1-1

Table 1-2

Table 1-3

Table 1-4

[0133] In some embodiments, the co-stimulatory and / or adhesion molecule is linked to a transmembrane domain (donaim). In some embodiments, the transmembrane domain is from CD8, the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D and / or the transmembrane domain of NKG2C. In some embodiments, the transmembrane domain can be from the transmembrane domain of CD28. In some embodiments, the transmembrane domain can be from CD8, for example, the transmembrane domain of CD8α.

[0134] Without wishing to be bound by theory, reduction of the foreign junction (i.e., between the adhesion molecule and the transmembrane domain) in the foreign nucleic acid incorporated into the lentiviral particle can reduce the immunogenicity of the lentiviral particle to the subject.

[0135] In some embodiments, the adhesion molecule binds to CD2. CD2, also known as T11, LFA-2, and erythrocyte rosette receptor, is a surface protein expressed on T lymphocytes and NK cells. CD2 is the natural ligand of CD58. In addition to performing an adhesion function, the association of CD2 provides a co-stimulatory signal that can enhance activation and effector functions. In some embodiments, the lentiviral particles contain a molecule that binds to CD2. In some embodiments, the lentiviral particles contain an antibody, single-domain antibody, antibody fragment, and / or nanobody specific for CD2. In some embodiments, the lentiviral particles contain CD58 or a functional portion thereof that binds to CD2.

[0136] In some embodiments, the adhesion molecule is CD58. In some embodiments, the co-stimulatory molecule is a CD58 polypeptide comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, the co-stimulatory molecule is a CD58 polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 17.

[0137] In some embodiments, the CD58 polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 18. In some embodiments, the CD58 polypeptide is encoded by a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 18.

[0138] Additional non-viral proteins In some embodiments, the viral particles contain at least one non-viral protein. In some embodiments, the viral particles contain at least one non-viral protein in addition to those described above.

[0139] In some embodiments, the viral particles comprise a targeting ligand. In some embodiments, the viral particles comprise CD19 or a functional fragment thereof attached to its native transmembrane domain or a heterologous transmembrane domain. In some embodiments, CD19 acts as a ligand for blinatumomab and thus provides an adapter for binding the particles to T cells via the anti-CD3 portion of blinatumomab. In some embodiments, another type of particle surface ligand can function to bind appropriately surface-engineered lentiviral particles to T cells using a bispecific antibody that comprises a binding portion for the particle surface ligand. In some embodiments, the bispecific antibody is a bispecific antibody, such as a bispecific T cell engager (BiTE).

[0140] In some embodiments, the non-viral protein is a cytokine. In some embodiments, the cytokine can be selected from the group consisting of IL-2, IL-7, Il-12, IL-15, IL-18, IL-21, and any combination thereof. When the non-viral protein used is a soluble protein (such as an scFv or a cytokine), it can be tethered to the surface of the viral particles by fusion to a transmembrane domain such as the transmembrane domain of CD8. Alternatively, this can be indirectly tethered to the lentiviral particles by the use of a transmembrane protein engineered to bind to the soluble protein. The further inclusion of one or more cytoplasmic residues can increase the stability of the fusion protein.

[0141] Mitosis-inducing transduction enhancers and / or cytokine-based transduction enhancers can include a "spacer sequence" to connect the antigen-binding domain to the transmembrane domain. Flexible spacers direct the antigen-binding domain in different directions to facilitate binding. As used herein, the term "link to" refers to chemical ligation, direct fusion of two proteins from the C-terminus to the N-terminus; chemical ligation to a non-peptide spacer; chemical ligation to a polypeptide spacer; and fusion of two proteins from the C-terminus to the N-terminus by peptide bond to a polypeptide spacer, e.g., a spacer sequence.

[0142] The spacer sequence can include, for example, the IgG1 Fc region, the IgG1 hinge, or the human CD8 stalk or the mouse CD8 stalk. Alternatively, the spacer can include an alternative linker sequence having the same length and / or domain spacing characteristics as the IgG1 Fc region, the IgG1 hinge, or the CD8 stalk. The human IgG1 spacer can be modified to remove the Fc-binding motif. In some embodiments, the spacer sequence can be derived from a human protein.

[0143] In some embodiments, the spacer sequence includes a CD8-derived hinge.

[0144] In some embodiments, the spacer sequence includes a "short" hinge. A short hinge is described as a hinge region that contains fewer nucleotides compared to the CAR hinge regions known in the art.

[0145] In some embodiments, the viral particle includes a polypeptide comprising a CD8 hinge 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: 3.

[0146] In some embodiments, the viral particle comprises a nucleic acid sequence encoding a CD8 hinge 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: 8.

[0147] The transmembrane domain is a sequence of a mitosis-inducing transduction enhancer and / or a cytokine-based transduction enhancer that spans the membrane. The transmembrane domain can include a hydrophobic alpha helix. The transmembrane domain can be derived from CD28. In some embodiments, the transmembrane domain is derived from a human protein.

[0148] The viral particle of the present invention can include 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 produced by the host cell and expressed on the cell surface. When nascent viral particles bud from the host cell membrane, the cytokine-based transduction enhancer can be incorporated into the viral envelope as part of the packaging cell-derived lipid bilayer.

[0149] The cytokine-based transduction enhancer can include a cytokine domain and a transmembrane domain. It can have a 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 the transmembrane domain are as defined above.

[0150] The cytokine domain can include a T cell-activating cytokine such as IL2, IL7 and IL15, or derived from their functional fragments. As used herein, a "functional fragment" of a cytokine is a fragment of a polypeptide that binds to its specific receptor and retains the ability to activate T cells.

[0151] IL2 is a type of factor 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 the signal sequence is required for its activity. Solution NMR suggests that the structure of IL2 contains a bundle of four helices (named A - D) sandwiched by two 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.

[0152] Viral envelope protein In some embodiments, the viral envelope contains viral envelope proteins. In some embodiments, the viral envelope protein is a VSV - G envelope protein, a measles virus envelope protein, a nipha virus envelope protein or a coxsackievirus G protein. In some embodiments, the viral particle contains a modified VSV G protein lacking LDLR - binding affinity. In some embodiments, such mutations include mutations at position 47 (e.g., K47Q) and / or position 354 (e.g., R354A).

[0153] In some embodiments, the viral envelope protein is the VSV G protein (cocal glycoprotein) derived from the cocal strain. In some embodiments, the VSV G protein is a cocal envelope protein containing a mutation (R354) at position 354. In some embodiments, the VSV G protein is a cocal envelope protein containing a mutation (K47) at position 47. In some embodiments, the VSV G protein is a cocal envelope variant containing the R354Q mutation compared to SEQ ID NO: 5. In some embodiments, the VSV G protein is a cocal envelope variant containing the K47Q mutation compared to SEQ ID NO: 5. In some embodiments, this variant may be referred to as a "blinded" cocal envelope. Exemplary cocal envelope variants are provided, for example, in US2020 / 0216502A1, which is hereby incorporated by reference in its entirety.

[0154] In some embodiments, the viral particle comprises a cocal glycoprotein comprising an amino acid sequence having 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: 5. In some embodiments, the viral particle comprises SEQ ID NO: 5.

[0155] In some embodiments, the nucleotide sequence encoding the viral particle comprises a nucleotide sequence encoding a cocal glycoprotein. In some embodiments, the nucleotide sequence encoding the cocal glycoprotein has 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: 10. In some embodiments, the nucleotide sequence encoding the cocal glycoprotein comprises the sequence of SEQ ID NO: 10.

[0156] In some embodiments, the nucleotide sequence encoding the viral particle comprises a nucleotide sequence encoding a coccolith protein. In some embodiments, the nucleotide sequence encoding the coccolith protein has 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: 104. In some embodiments, the nucleotide sequence encoding the coccolith protein comprises the sequence of SEQ ID NO: 104.

[0157] Various fusion glycoproteins can be used to pseudotype lentiviral particles. The most commonly used example is the envelope glycoprotein (VSV-G) derived from vesicular stomatitis virus, although many other viral proteins have also been used for pseudotyping lentiviral particles. See Joglekar et al. Human Gene Therapy Methods 28:291-301 (2017). The present disclosure contemplates the substitution of various fusion glycoproteins. Notably, some fusion glycoproteins result in higher viral particle efficiency.

[0158] In some embodiments, pseudotyping of the fusion protein or a functional variant thereof facilitates targeted transduction of specific cell types including, but not limited to, T cells or NK cells. In some embodiments, the fusion protein or a functional variant thereof is human immunodeficiency virus (HIV) gp160, 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 endogenous 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) E1 and E2, Ross River virus (RRV) E1 and E2, Semliki Forest virus (SFV) E1 and E2, Sindbis virus (SV) E1 and E2, Venezuelan equine encephalitis virus (VEEV) E1 and E2, Western equine encephalitis virus (WEEV) E1 and E2, influenza A, B, C or D HA, fowl plague virus (FPV) HA, anti-CD3 scFv, (CD3), vesicular stomatitis virus VSV-G, or the full-length polypeptide(s), functional fragment(s), homolog(s) or functional variant(s) of Chandipura virus and Piry virus CNV-G and PRV-G.

[0159] In some embodiments, the fusion glycoprotein or a functional variant thereof is the 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), Coccal virus (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 a functional variant thereof is the COCV G protein.

[0160] In some embodiments, the vector particles are Nipah virus (NiV) envelope pseudotyped lentiviral particles (“NiV envelope pseudotyped vectors”). In some embodiments, the NiV envelope pseudotyped vectors are pseudotyped using the Nipah virus envelope glycoproteins NiV-F and NiV-G. In some embodiments, the NiV-F and / or NiV-G glycoproteins in such NiV envelope pseudotyped vectors are modified variants. In some embodiments, the NiV-F and / or NiV-G glycoproteins in such NiV envelope pseudotyped vectors are modified to include an antigen-binding domain. In some embodiments, the antigen is EpCAM, CD4 or CD8. In some embodiments, the NiV envelope pseudotyped vectors can efficiently transduce cells expressing EpCAM, CD4 or CD8. See U.S. Patent No. 9,486,539 and Bender et al. PLoS Pathog. (2016) Jun; 12(6): e1005641.

[0161] Exemplary virus envelopes In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activating protein, (ii) a costimulatory molecule, (iii) an adhesion molecule, or (iv) any combination of (i)-(iii). In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activating protein, (ii) a costimulatory molecule, (iii) an adhesion molecule, or (iv) any combination of at least two of (i)-(iii). In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activating protein and (ii) a costimulatory molecule. In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activating protein and (ii) an adhesion molecule. In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activating protein, (ii) a costimulatory molecule, and (iii) an adhesion molecule. In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activating protein, (ii) a costimulatory molecule, and (iii) a plurality of adhesion molecules.

[0162] In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD28, CD2, CD3, CD28H, LFA-1, OX40, 4-1BB, CD40L, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, NKG2D, NKp46, NKp44, NKp30, CD244 or NKp80; (ii) a co-stimulatory molecule selected from TCR α chain, TCR β chain, TCR ζ chain, CD3ε TCR subunit, CD3γ TCR subunit, CD3δ TCR subunit, CD45, CD2, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and any combination thereof; (iii) an adhesion molecule selected from CD58, HHLA2, ICAM-1, OX40L, 4-1BBL, CD40, CD155, CD70, HVEM, GITRL, ICOSL, CD30L, SLAM, Ly-9, CD84, Ly108, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, B7-H6 and any combination thereof; or (iv) any combination of (i) to (iii).In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD28, CD2, CD3, CD28H, LFA-1, OX40, 4-1BB, CD40L, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, NKG2D, NKp46, NKp44, NKp30, CD244 or NKp80, (ii) a co-stimulatory molecule selected from TCR α chain, TCR β chain, TCR ζ chain, CD3ε TCR subunit, CD3γ TCR subunit, CD3δ TCR subunit, CD45, CD2, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and any combination thereof, (iii) an adhesion molecule selected from CD58, HHLA2, ICAM-1, OX40L, 4-1BBL, CD40, CD155, CD70, HVEM, GITRL, ICOSL, CD30L, SLAM, Ly-9, CD84, Ly108, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, B7-H6, and any combination thereof, or (iv) any combination of at least two of (i)-(iii). In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD28, CD2, CD3, CD28H, LFA-1, OX40, 4-1BB, CD40L, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, NKG2D, NKp46, NKp44, NKp30, CD244 or NKp80, and (ii) a co-stimulatory molecule selected from TCR α chain, TCR β chain, TCR ζ chain, CD3ε TCR subunit, CD3γ TCR subunit, CD3δ TCR subunit, CD45, CD2, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and any combination thereof.In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD28, CD2, CD3, CD28H, LFA-1, OX40, 4-1BB, CD40L, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, NKG2D, NKp46, NKp44, NKp30, CD244 or NKp80, and (ii) an adhesion molecule selected from CD58, HHLA2, ICAM-1, OX40L, 4-1BBL, CD40, CD155, CD70, HVEM, GITRL, ICOSL, CD30L, SLAM, Ly-9, CD84, Ly108, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, B7-H6 and any combination thereof. In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD28, CD2, CD3, CD28H, LFA-1, OX40, 4-1BB, CD40L, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, NKG2D, NKp46, NKp44, NKp30, CD244 or NKp80, (ii) a costimulatory molecule selected from TCR α chain, TCR β chain, TCR ζ chain, CD3ε TCR subunit, CD3γ TCR subunit, CD3δ TCR subunit, CD45, CD2, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and any combination thereof, and (iii) an adhesion molecule selected from CD58, HHLA2, ICAM-1, OX40L, 4-1BBL, CD40, CD155, CD70, HVEM, GITRL, ICOSL, CD30L, SLAM, Ly-9, CD84, Ly108, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, B7-H6 and any combination thereof.

[0163] In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD3, (ii) a co-stimulatory molecule selected from CD80, CD86, and combinations thereof, (iii) an adhesion molecule comprising a CD58 polypeptide, or (iv) any combination of (i) to (iii). In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD3, (ii) a co-stimulatory molecule selected from CD80, CD86, and combinations thereof, (iii) an adhesion molecule comprising a CD58 polypeptide, or (iv) any combination of at least two of (i) to (iii). In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD3, and (ii) a co-stimulatory molecule selected from CD80, CD86, and combinations thereof. In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD3, and (ii) an adhesion molecule comprising a CD58 polypeptide. In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD3, (ii) a co-stimulatory molecule selected from CD80, CD86, and combinations thereof, and (iii) an adhesion molecule comprising a CD58 polypeptide.

[0164] In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD3, (ii) a co-stimulatory molecule comprising a CD80 polypeptide, (iii) an adhesion molecule comprising a CD58 polypeptide, or (iv) any combination of (i)-(iii). In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD3, (ii) a co-stimulatory molecule comprising a CD80 polypeptide, (iii) an adhesion molecule comprising a CD58 polypeptide, or (iv) any combination of at least two of (i)-(iii). In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD3 and (ii) a co-stimulatory molecule comprising a CD80 polypeptide. In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD3 and (ii) an adhesion molecule comprising a CD58 polypeptide. In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD3, (ii) a co-stimulatory molecule comprising a CD80 polypeptide, and (iii) an adhesion molecule comprising a CD58 polypeptide.

[0165] In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD3, (ii) a costimulatory molecule comprising a CD86 polypeptide, (iii) an adhesion molecule comprising a CD58 polypeptide, or (iv) any combination of (i) to (iii). In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD3, (ii) a costimulatory molecule comprising a CD86 polypeptide, (iii) an adhesion molecule comprising a CD58 polypeptide, or (iv) any combination of at least two of (i) to (iii). In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD3 and (ii) a costimulatory molecule comprising a CD86 polypeptide. In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD3 and (ii) an adhesion molecule comprising a CD58 polypeptide. In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD3, (ii) a costimulatory molecule comprising a CD86 polypeptide, and (iii) an adhesion molecule comprising a CD58 polypeptide.

[0166] In some embodiments, the viral particle comprises a viral envelope comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to CD3, (ii) a costimulatory molecule comprising a CD80 polypeptide, (iii) an adhesion molecule comprising a CD58 polypeptide, or (iv) any combination of (i)-(iii). In some embodiments, the viral particle comprises a viral envelope comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to CD3, (ii) a costimulatory molecule comprising a CD80 polypeptide, (iii) an adhesion molecule comprising a CD58 polypeptide, or (iv) any combination of at least two of (i)-(iii). In some embodiments, the viral particle comprises a viral envelope comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to CD3, and (ii) a costimulatory molecule comprising a CD80 polypeptide. In some embodiments, the viral particle comprises a viral envelope comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to CD3, and (ii) an adhesion molecule comprising a CD58 polypeptide. In some embodiments, the viral particle comprises a viral envelope comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to CD3, (ii) a costimulatory molecule comprising a CD80 polypeptide, and (iii) an adhesion molecule comprising a CD58 polypeptide.

[0167] In some embodiments, the viral particle comprises a viral envelope comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to CD3, (ii) a co-stimulatory molecule comprising a CD86 polypeptide, (iii) an adhesion molecule comprising a CD58 polypeptide, or (iv) any combination of (i)-(iii). In some embodiments, the viral particle comprises a viral envelope comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to CD3, (ii) a co-stimulatory molecule comprising a CD86 polypeptide, (iii) an adhesion molecule comprising a CD58 polypeptide, or (iv) any combination of at least two of (i)-(iii). In some embodiments, the viral particle comprises a viral envelope comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to CD3, and (ii) a co-stimulatory molecule comprising a CD86 polypeptide. In some embodiments, the viral particle comprises a viral envelope comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to CD3, and (ii) an adhesion molecule comprising a CD58 polypeptide. In some embodiments, the viral particle comprises a viral envelope comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to CD3, (ii) a co-stimulatory molecule comprising a CD86 polypeptide, and (iii) an adhesion molecule comprising a CD58 polypeptide.

[0168] In some embodiments, the viral particle comprises a viral envelope comprising (i) an antibody or an antigen-binding fragment thereof that specifically binds to CD3, wherein the antibody or antigen-binding fragment comprises CDR-L1 of SEQ ID NO: 133, CDR-L2 of SEQ ID NO: 134, CDR-L3 of SEQ ID NO: 135, CDR-H1 of SEQ ID NO: 48, CDR-H2 of SEQ ID NO: 36, and CDR-H3 of SEQ ID NO: 38; (ii) a co-stimulatory molecule comprising a CD80 polypeptide comprising the amino acid sequence of SEQ ID NO: 20; (iii) an adhesion molecule comprising a CD58 polypeptide comprising the amino acid sequence of SEQ ID NO: 17; or (iv) any combination of (i)-(iii). In some embodiments, the viral particle comprises a viral envelope comprising (i) an antibody or an antigen-binding fragment thereof that specifically binds to CD3, wherein the antibody or antigen-binding fragment comprises CDR-L1 of SEQ ID NO: 133, CDR-L2 of SEQ ID NO: 134, CDR-L3 of SEQ ID NO: 135, CDR-H1 of SEQ ID NO: 48, CDR-H2 of SEQ ID NO: 36, and CDR-H3 of SEQ ID NO: 38; (ii) a co-stimulatory molecule comprising a CD80 polypeptide comprising the amino acid sequence of SEQ ID NO: 20; (iii) an adhesion molecule comprising a CD58 polypeptide comprising the amino acid sequence of SEQ ID NO: 17; or (iv) any combination of at least two of (i)-(iii). In some embodiments, the viral particle comprises a viral envelope comprising (i) an antibody or an antigen-binding fragment thereof that specifically binds to CD3, wherein the antibody or antigen-binding fragment comprises CDR-L1 of SEQ ID NO: 133, CDR-L2 of SEQ ID NO: 134, CDR-L3 of SEQ ID NO: 135, CDR-H1 of SEQ ID NO: 48, CDR-H2 of SEQ ID NO: 36, and CDR-H3 of SEQ ID NO: 38, and (ii) a co-stimulatory molecule comprising a CD80 polypeptide comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the viral particle comprises a viral envelope comprising (i) an antibody or an antigen-binding fragment thereof that specifically binds to CD3, wherein the antibody or antigen-binding fragment comprises CDR-L1 of SEQ ID NO: 133, CDR-L2 of SEQ ID NO: 134, CDR-L3 of SEQ ID NO: 135, CDR-H1 of SEQ ID NO: 48, CDR-H2 of SEQ ID NO: 36, and CDR-H3 of SEQ ID NO: 38, and (ii) an adhesion molecule comprising a CD58 polypeptide comprising the amino acid sequence of SEQ ID NO: 17.In some embodiments, the viral particle comprises a viral envelope comprising: (i) an antibody or antigen-binding fragment thereof that specifically binds to CD3, the antibody or antigen-binding fragment comprising CDR-L1 of SEQ ID NO: 133, CDR-L2 of SEQ ID NO: 134, CDR-L3 of SEQ ID NO: 135, CDR-H1 of SEQ ID NO: 48, CDR-H2 of SEQ ID NO: 36, and CDR-H3 of SEQ ID NO: 38; (ii) a costimulatory molecule comprising a CD80 polypeptide comprising the amino acid sequence of SEQ ID NO: 20; and (iii) an adhesion molecule comprising a CD58 polypeptide comprising the amino acid sequence of SEQ ID NO: 17.

[0169] In some embodiments, the viral particle comprises a viral envelope comprising (i) an antibody or an antigen-binding fragment thereof that specifically binds to CD3, the antibody or antigen-binding fragment comprising CDR-L1 of SEQ ID NO: 133, CDR-L2 of SEQ ID NO: 134, CDR-L3 of SEQ ID NO: 135, CDR-H1 of SEQ ID NO: 48, CDR-H2 of SEQ ID NO: 36, and CDR-H3 of SEQ ID NO: 38; (ii) a costimulatory molecule comprising a CD86 polypeptide comprising the amino acid sequence of SEQ ID NO: 23; (iii) an adhesion molecule comprising a CD58 polypeptide comprising the amino acid sequence of SEQ ID NO: 17; or (iv) any combination of (i)-(iii). In some embodiments, the viral particle comprises a viral envelope comprising (i) an antibody or an antigen-binding fragment thereof that specifically binds to CD3, the antibody or antigen-binding fragment comprising CDR-L1 of SEQ ID NO: 133, CDR-L2 of SEQ ID NO: 134, CDR-L3 of SEQ ID NO: 135, CDR-H1 of SEQ ID NO: 48, CDR-H2 of SEQ ID NO: 36, and CDR-H3 of SEQ ID NO: 38; (ii) a costimulatory molecule comprising a CD86 polypeptide comprising the amino acid sequence of SEQ ID NO: 23; (iii) an adhesion molecule comprising a CD58 polypeptide comprising the amino acid sequence of SEQ ID NO: 17; or (iv) any combination of at least two of (i)-(iii). In some embodiments, the viral particle comprises a viral envelope comprising (i) an antibody or an antigen-binding fragment thereof that specifically binds to CD3, the antibody or antigen-binding fragment comprising CDR-L1 of SEQ ID NO: 133, CDR-L2 of SEQ ID NO: 134, CDR-L3 of SEQ ID NO: 135, CDR-H1 of SEQ ID NO: 48, CDR-H2 of SEQ ID NO: 36, and CDR-H3 of SEQ ID NO: 38, and (ii) a costimulatory molecule comprising a CD86 polypeptide comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the viral particle comprises a viral envelope comprising (i) an antibody or an antigen-binding fragment thereof that specifically binds to CD3, the antibody or antigen-binding fragment comprising CDR-L1 of SEQ ID NO: 133, CDR-L2 of SEQ ID NO: 134, CDR-L3 of SEQ ID NO: 135, CDR-H1 of SEQ ID NO: 48, CDR-H2 of SEQ ID NO: 36, and CDR-H3 of SEQ ID NO: 38, and (ii) an adhesion molecule comprising a CD58 polypeptide comprising the amino acid sequence of SEQ ID NO: 17.In some embodiments, the viral particle comprises a viral envelope comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to CD3, the antibody or antigen-binding fragment comprising CDR-L1 of SEQ ID NO: 133, CDR-L2 of SEQ ID NO: 134, CDR-L3 of SEQ ID NO: 135, CDR-H1 of SEQ ID NO: 48, CDR-H2 of SEQ ID NO: 36, and CDR-H3 of SEQ ID NO: 38, (ii) a co-stimulatory molecule comprising a CD86 polypeptide comprising the amino acid sequence of SEQ ID NO: 23, and (iii) an adhesion molecule comprising a CD58 polypeptide comprising the amino acid sequence of SEQ ID NO: 17.

[0170] In some embodiments, the viral particle comprises a viral envelope comprising (i) an immune cell activating protein, (ii) a co-stimulatory molecule, (iii) an adhesion molecule, or (iv) any combination of (i)-(iii), and a viral envelope protein. In some embodiments, the viral particle comprises a viral envelope comprising (i) an immune cell activating protein, (ii) a co-stimulatory molecule, (iii) an adhesion molecule, or (iv) any combination of at least two of (i)-(iii), and a viral envelope protein. In some embodiments, the viral particle comprises a viral envelope comprising (i) an immune cell activating protein, (ii) a co-stimulatory molecule, and (iii) a viral envelope protein. In some embodiments, the viral particle comprises a viral envelope comprising (i) an immune cell activating protein, (ii) an adhesion molecule, and (iii) a viral envelope protein. In some embodiments, the viral particle comprises a viral envelope comprising (i) an immune cell activating protein, (ii) a co-stimulatory molecule, (iii) an adhesion molecule, and (iv) a viral envelope protein.

[0171] In some embodiments, the viral particle comprises a viral envelope comprising a viral envelope protein and (i) an immunocyte activation protein that specifically binds to CD28, CD2, CD3, CD28H, LFA-1, OX40, 4-1BB, CD40L, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, NKG2D, NKp46, NKp44, NKp30, CD244 or NKp80; (ii) a co-stimulatory molecule selected from TCR α chain, TCR β chain, TCR ζ chain, CD3ε TCR subunit, CD3γ TCR subunit, CD3δ TCR subunit, CD45, CD2, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and any combination thereof; (iii) an adhesion molecule selected from CD58, HHLA2, ICAM-1, OX40L, 4-1BBL, CD40, CD155, CD70, HVEM, GITRL, ICOSL, CD30L, SLAM, Ly-9, CD84, Ly108, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, B7-H6 and any combination thereof; or (iv) any combination of (i)-(iii).In some embodiments, the viral particle comprises a viral envelope containing (i) an immunocyte activation protein that specifically binds to CD28, CD2, CD3, CD28H, LFA-1, OX40, 4-1BB, CD40L, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, NKG2D, NKp46, NKp44, NKp30, CD244 or NKp80; (ii) a costimulatory molecule selected from TCR α chain, TCR β chain, TCR ζ chain, CD3ε TCR subunit, CD3γ TCR subunit, CD3δ TCR subunit, CD45, CD2, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and any combination thereof; (iii) an adhesion molecule selected from CD58, HHLA2, ICAM-1, OX40L, 4-1BBL, CD40, CD155, CD70, HVEM, GITRL, ICOSL, CD30L, SLAM, Ly-9, CD84, Ly108, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, B7-H6 and any combination thereof; or (iv) any combination of at least two of (i)-(iii), as well as a viral envelope protein. In some embodiments, the viral particle comprises a viral envelope containing (i) an immunocyte activation protein that specifically binds to CD28, CD2, CD3, CD28H, LFA-1, OX40, 4-1BB, CD40L, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, NKG2D, NKp46, NKp44, NKp30, CD244 or NKp80; (ii) a costimulatory molecule selected from TCR α chain, TCR β chain, TCR ζ chain, CD3ε TCR subunit, CD3γ TCR subunit, CD3δ TCR subunit, CD45, CD2, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and any combination thereof; and (iii) a viral envelope protein.In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD28, CD2, CD3, CD28H, LFA-1, OX40, 4-1BB, CD40L, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, NKG2D, NKp46, NKp44, NKp30, CD244 or NKp80, (ii) an adhesion molecule selected from CD58, HHLA2, ICAM-1, OX40L, 4-1BBL, CD40, CD155, CD70, HVEM, GITRL, ICOSL, CD30L, SLAM, Ly-9, CD84, Ly108, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, B7-H6 and any combination thereof, and (iii) a viral envelope protein. In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD28, CD2, CD3, CD28H, LFA-1, OX40, 4-1BB, CD40L, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, NKG2D, NKp46, NKp44, NKp30, CD244 or NKp80, (ii) a costimulatory molecule selected from TCR α chain, TCR β chain, TCR ζ chain, CD3ε TCR subunit, CD3γ TCR subunit, CD3δ TCR subunit, CD45, CD2, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and any combination thereof, (iii) an adhesion molecule selected from CD58, HHLA2, ICAM-1, OX40L, 4-1BBL, CD40, CD155, CD70, HVEM, GITRL, ICOSL, CD30L, SLAM, Ly-9, CD84, Ly108, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, B7-H6 and any combination thereof, and (iv) a viral envelope protein.

[0172] In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD3, (ii) a co-stimulatory molecule selected from CD80, CD86, and combinations thereof, (iii) an adhesion molecule comprising a CD58 polypeptide, or (iv) any combination of (i)-(iii), and a viral envelope protein. In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD3, (ii) a co-stimulatory molecule selected from CD80, CD86, and combinations thereof, (iii) an adhesion molecule comprising a CD58 polypeptide, or (iv) any combination of at least two of (i)-(iii), and a viral envelope protein. In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD3, (ii) a co-stimulatory molecule selected from CD80, CD86, and combinations thereof, and (iii) a viral envelope protein. In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD3, (ii) an adhesion molecule comprising a CD58 polypeptide, and (iii) a viral envelope protein. In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD3, (ii) a co-stimulatory molecule selected from CD80, CD86, and combinations thereof, (iii) an adhesion molecule comprising a CD58 polypeptide, and (iv) a viral envelope protein.

[0173] In some embodiments, the viral particle comprises a viral envelope containing (i) an immunocyte activation protein that specifically binds to CD3, (ii) a costimulatory molecule comprising a CD80 polypeptide, (iii) an adhesion molecule comprising a CD58 polypeptide, or (iv) any combination of (i)-(iii), and a viral envelope protein. In some embodiments, the viral particle comprises a viral envelope containing (i) an immunocyte activation protein that specifically binds to CD3, (ii) a costimulatory molecule comprising a CD80 polypeptide, (iii) an adhesion molecule comprising a CD58 polypeptide, or (iv) any combination of at least two of (i)-(iii), and a viral envelope protein. In some embodiments, the viral particle comprises a viral envelope containing (i) an immunocyte activation protein that specifically binds to CD3, (ii) a costimulatory molecule comprising a CD80 polypeptide, and (iii) a viral envelope protein. In some embodiments, the viral particle comprises a viral envelope containing (i) an immunocyte activation protein that specifically binds to CD3, (ii) an adhesion molecule comprising a CD58 polypeptide, and (iii) a viral envelope protein. In some embodiments, the viral particle comprises a viral envelope containing (i) an immunocyte activation protein that specifically binds to CD3, (ii) a costimulatory molecule comprising a CD80 polypeptide, (iii) an adhesion molecule comprising a CD58 polypeptide, and (iv) a viral envelope protein.

[0174] In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD3, (ii) a co-stimulatory molecule comprising a CD86 polypeptide, (iii) an adhesion molecule comprising a CD58 polypeptide, or (iv) any combination of (i)-(iii), and a viral envelope protein. In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD3, (ii) a co-stimulatory molecule comprising a CD86 polypeptide, (iii) an adhesion molecule comprising a CD58 polypeptide, or (iv) any combination of at least two of (i)-(iii), and a viral envelope protein. In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD3, (ii) a co-stimulatory molecule comprising a CD86 polypeptide, and (iii) a viral envelope protein. In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD3, (ii) an adhesion molecule comprising a CD58 polypeptide, and (iii) a viral envelope protein. In some embodiments, the viral particle comprises a viral envelope comprising (i) an immunocyte activation protein that specifically binds to CD3, (ii) a co-stimulatory molecule comprising a CD86 polypeptide, (iii) an adhesion molecule comprising a CD58 polypeptide, and (iv) a viral envelope protein.

[0175] In some embodiments, the viral particle comprises a viral envelope comprising a viral envelope protein and (i) an antibody or an antigen-binding fragment thereof that specifically binds to CD3, (ii) a costimulatory molecule comprising a CD80 polypeptide, (iii) an adhesion molecule comprising a CD58 polypeptide, or (iv) any combination of (i) to (iii). In some embodiments, the viral particle comprises a viral envelope comprising a viral envelope protein and (i) an antibody or an antigen-binding fragment thereof that specifically binds to CD3, (ii) a costimulatory molecule comprising a CD80 polypeptide, (iii) an adhesion molecule comprising a CD58 polypeptide, or (iv) any combination of at least two of (i) to (iii). In some embodiments, the viral particle comprises a viral envelope comprising a viral envelope protein and (i) an antibody or an antigen-binding fragment thereof that specifically binds to CD3, (ii) a costimulatory molecule comprising a CD80 polypeptide, and (iii). In some embodiments, the viral particle comprises a viral envelope comprising a viral envelope protein and (i) an antibody or an antigen-binding fragment thereof that specifically binds to CD3, (ii) an adhesion molecule comprising a CD58 polypeptide, and (iii).

[0176] In some embodiments, the viral particle comprises: (i) an antibody or an antigen-binding fragment thereof that specifically binds to CD3; (ii) a co-stimulatory molecule comprising a CD80 polypeptide; (iii) an adhesion molecule comprising a CD58 polypeptide; and (iv) a viral envelope comprising a viral envelope protein. In some embodiments, the viral particle comprises: (i) an antibody or an antigen-binding fragment thereof that specifically binds to CD3; (ii) a co-stimulatory molecule comprising a CD86 polypeptide; (iii) an adhesion molecule comprising a CD58 polypeptide; or (iv) any combination of (i)-(iii), and a viral envelope comprising a viral envelope protein. In some embodiments, the viral particle comprises: (i) an antibody or an antigen-binding fragment thereof that specifically binds to CD3; (ii) a co-stimulatory molecule comprising a CD86 polypeptide; (iii) an adhesion molecule comprising a CD58 polypeptide; or (iv) any combination of at least two of (i)-(iii), and a viral envelope comprising a viral envelope protein. In some embodiments, the viral particle comprises: (i) an antibody or an antigen-binding fragment thereof that specifically binds to CD3; (ii) a co-stimulatory molecule comprising a CD86 polypeptide; and (iii) a viral envelope comprising a viral envelope protein. In some embodiments, the viral particle comprises: (i) an antibody or an antigen-binding fragment thereof that specifically binds to CD3; (ii) an adhesion molecule comprising a CD58 polypeptide; and (iii) a viral envelope comprising a viral envelope protein. In some embodiments, the viral particle comprises: (i) an antibody or an antigen-binding fragment thereof that specifically binds to CD3; (ii) a co-stimulatory molecule comprising a CD86 polypeptide; (iii) an adhesion molecule comprising a CD58 polypeptide; and (iv) a viral envelope comprising a viral envelope protein.

[0177] In some embodiments, the viral particle comprises: (i) an antibody or an antigen-binding fragment thereof that specifically binds to CD3, wherein the antibody or antigen-binding fragment comprises CDR-L1 of SEQ ID NO: 133, CDR-L2 of SEQ ID NO: 134, CDR-L3 of SEQ ID NO: 135, CDR-H1 of SEQ ID NO: 48, CDR-H2 of SEQ ID NO: 36, and CDR-H3 of SEQ ID NO: 38; (ii) a costimulatory molecule comprising a CD80 polypeptide comprising the amino acid sequence of SEQ ID NO: 20; (iii) an adhesion molecule comprising a CD58 polypeptide comprising the amino acid sequence of SEQ ID NO: 17; or (iv) any combination of (i)-(iii), and a viral envelope comprising a viral envelope protein. In some embodiments, the viral particle comprises: (i) an antibody or an antigen-binding fragment thereof that specifically binds to CD3, wherein the antibody or antigen-binding fragment comprises CDR-L1 of SEQ ID NO: 133, CDR-L2 of SEQ ID NO: 134, CDR-L3 of SEQ ID NO: 135, CDR-H1 of SEQ ID NO: 48, CDR-H2 of SEQ ID NO: 36, and CDR-H3 of SEQ ID NO: 38; (ii) a costimulatory molecule comprising a CD80 polypeptide comprising the amino acid sequence of SEQ ID NO: 20; (iii) an adhesion molecule comprising a CD58 polypeptide comprising the amino acid sequence of SEQ ID NO: 17; or (iv) any combination of at least two of (i)-(iii), and a viral envelope comprising a viral envelope protein. In some embodiments, the viral particle comprises: (i) an antibody or an antigen-binding fragment thereof that specifically binds to CD3, wherein the antibody or antigen-binding fragment comprises CDR-L1 of SEQ ID NO: 133, CDR-L2 of SEQ ID NO: 134, CDR-L3 of SEQ ID NO: 135, CDR-H1 of SEQ ID NO: 48, CDR-H2 of SEQ ID NO: 36, and CDR-H3 of SEQ ID NO: 38; (ii) a costimulatory molecule comprising a CD80 polypeptide comprising the amino acid sequence of SEQ ID NO: 20; and (iii) a viral envelope comprising a viral envelope protein.In some embodiments, the viral particle comprises: (i) an antibody or antigen-binding fragment thereof that specifically binds to CD3, the antibody or antigen-binding fragment comprising CDR-L1 of SEQ ID NO: 133, CDR-L2 of SEQ ID NO: 134, CDR-L3 of SEQ ID NO: 135, CDR-H1 of SEQ ID NO: 48, CDR-H2 of SEQ ID NO: 36, and CDR-H3 of SEQ ID NO: 38; (ii) an adhesion molecule comprising a CD58 polypeptide comprising the amino acid sequence of SEQ ID NO: 17; and (iii) a viral envelope comprising a viral envelope protein. In some embodiments, the viral particle comprises: (i) an antibody or antigen-binding fragment thereof that specifically binds to CD3, the antibody or antigen-binding fragment comprising CDR-L1 of SEQ ID NO: 133, CDR-L2 of SEQ ID NO: 134, CDR-L3 of SEQ ID NO: 135, CDR-H1 of SEQ ID NO: 48, CDR-H2 of SEQ ID NO: 36, and CDR-H3 of SEQ ID NO: 38; (ii) a costimulatory molecule comprising a CD80 polypeptide comprising the amino acid sequence of SEQ ID NO: 20; (iii) an adhesion molecule comprising a CD58 polypeptide comprising the amino acid sequence of SEQ ID NO: 17; and (iv) a viral envelope comprising a viral envelope protein.

[0178] In some embodiments, the viral particle comprises a viral envelope containing a viral envelope protein and (i) an antibody or an antigen-binding fragment thereof that specifically binds to CD3, wherein the antibody or antigen-binding fragment comprises CDR-L1 of SEQ ID NO: 133, CDR-L2 of SEQ ID NO: 134, CDR-L3 of SEQ ID NO: 135, CDR-H1 of SEQ ID NO: 48, CDR-H2 of SEQ ID NO: 36, and CDR-H3 of SEQ ID NO: 38; (ii) a co-stimulatory molecule comprising a CD86 polypeptide comprising the amino acid sequence of SEQ ID NO: 23; (iii) an adhesion molecule comprising a CD58 polypeptide comprising the amino acid sequence of SEQ ID NO: 17; or (iv) any combination of (i)-(iii). In some embodiments, the viral particle comprises a viral envelope containing a viral envelope protein and (i) an antibody or an antigen-binding fragment thereof that specifically binds to CD3, wherein the antibody or antigen-binding fragment comprises CDR-L1 of SEQ ID NO: 133, CDR-L2 of SEQ ID NO: 134, CDR-L3 of SEQ ID NO: 135, CDR-H1 of SEQ ID NO: 48, CDR-H2 of SEQ ID NO: 36, and CDR-H3 of SEQ ID NO: 38; (ii) a co-stimulatory molecule comprising a CD86 polypeptide comprising the amino acid sequence of SEQ ID NO: 23; (iii) an adhesion molecule comprising a CD58 polypeptide comprising the amino acid sequence of SEQ ID NO: 17; or (iv) any combination of at least two of (i)-(iii). In some embodiments, the viral particle comprises a viral envelope containing a viral envelope protein and (i) an antibody or an antigen-binding fragment thereof that specifically binds to CD3, wherein the antibody or antigen-binding fragment comprises CDR-L1 of SEQ ID NO: 133, CDR-L2 of SEQ ID NO: 134, CDR-L3 of SEQ ID NO: 135, CDR-H1 of SEQ ID NO: 48, CDR-H2 of SEQ ID NO: 36, and CDR-H3 of SEQ ID NO: 38; (ii) a co-stimulatory molecule comprising a CD86 polypeptide comprising the amino acid sequence of SEQ ID NO: 23; and (iii) a viral envelope containing a viral envelope protein.In some embodiments, the viral particle comprises: (i) an antibody or antigen-binding fragment thereof that specifically binds to CD3, the antibody or antigen-binding fragment comprising CDR-L1 of SEQ ID NO: 133, CDR-L2 of SEQ ID NO: 134, CDR-L3 of SEQ ID NO: 135, CDR-H1 of SEQ ID NO: 48, CDR-H2 of SEQ ID NO: 36, and CDR-H3 of SEQ ID NO: 38; (ii) an adhesion molecule comprising a CD58 polypeptide comprising the amino acid sequence of SEQ ID NO: 17; and (iii) a viral envelope comprising a viral envelope protein. In some embodiments, the viral particle comprises: (i) an antibody or antigen-binding fragment thereof that specifically binds to CD3, the antibody or antigen-binding fragment comprising CDR-L1 of SEQ ID NO: 133, CDR-L2 of SEQ ID NO: 134, CDR-L3 of SEQ ID NO: 135, CDR-H1 of SEQ ID NO: 48, CDR-H2 of SEQ ID NO: 36, and CDR-H3 of SEQ ID NO: 38; (ii) a costimulatory molecule comprising a CD86 polypeptide comprising the amino acid sequence of SEQ ID NO: 23; (iii) an adhesion molecule comprising a CD58 polypeptide comprising the amino acid sequence of SEQ ID NO: 17; and (iv) a viral envelope comprising a viral envelope protein.

[0179] In some embodiments, the viral particle comprises a polypeptide comprising a Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv operably linked to a hinge domain operably linked to a transmembrane domain and cytoplasmic tail derived from a coxsackievirus envelope sharing 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: 107.

[0180] In some embodiments, the viral particle comprises a polypeptide comprising a Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv operably linked to a hinge domain operably linked to a transmembrane domain and cytoplasmic tail derived from coxsackievirus envelope sharing 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: 109.

[0181] In some embodiments, the viral particle comprises a polypeptide comprising a Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv operably linked to a linker operably linked to a transmembrane domain derived from glycophorin A and cytoplasmic tail derived from HIV envelope sharing 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: 111.

[0182] In some embodiments, the viral particle comprises a polypeptide comprising a Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv operably linked to a linker operably linked to a transmembrane domain and cytoplasmic tail derived from HIV envelope sharing 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: 113.

[0183] In some embodiments, the viral particle comprises a polypeptide comprising a Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv operably linked to a triple G4S linker operably linked to a transmembrane domain and cytoplasmic tail derived from HIV envelope sharing 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: 115.

[0184] In some embodiments, the viral particle comprises a polypeptide comprising a Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv operably linked to a hinge domain operably linked to a cytoplasmic tail and a T2A self-cleaving peptide operably linked to a coxsackievirus envelope transmembrane domain 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: 117.

[0185] In some embodiments, the viral particle comprises a polypeptide comprising a Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv operably linked to a linker operably linked to a hinge, transmembrane domain and cytoplasmic tail derived from glycophorin A 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: 119.

[0186] In some embodiments, the viral particle comprises a polypeptide comprising a short hinge operably linked to a transmembrane domain operably linked to a cytoplasmic tail derived from a coxsackievirus 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: 13.

[0187] In some embodiments, the viral particle comprises a polypeptide comprising a long hinge operably linked to a transmembrane domain operably linked to a cytoplasmic tail derived from a coxsackievirus 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: 19.

[0188] In some embodiments, the viral particle comprises a polypeptide comprising a 218 linker operably linked to a human glycophorin A extracellular domain transmembrane domain operably linked to a cytoplasmic tail derived from an HIV viral envelope sharing 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: 25.

[0189] In some embodiments, the viral particle comprises a polypeptide comprising a 218 linker operably linked to an HIV viral envelope transmembrane domain and cytoplasmic tail sharing 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: 31.

[0190] In some embodiments, the viral particle comprises a polypeptide comprising a triple G4S linker operably linked to an HIV viral envelope transmembrane domain and cytoplasmic tail sharing 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: 37.

[0191] In some embodiments, the viral particle comprises a polypeptide comprising a Ser-Gly peptide operably linked to a small extracellular domain, transmembrane and cytoplasmic tail sequence derived from human glycophorin A sharing 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: 97.

[0192] In some embodiments, the viral particle comprises a polypeptide comprising a transmembrane domain and cytoplasmic tail sequence derived from human glycophorin A sharing 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: 105.

[0193] In some embodiments, the viral particle comprises a polypeptide comprising a coxsackievirus protein transmembrane domain and a short hinge operably linked to a cytoplasmic tail that share 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: 43.

[0194] In some embodiments, the viral particle comprises a polypeptide comprising a CD4-derived transmembrane domain and a cytoplasmic tail operably linked to a T2A linker that share 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: 4.

[0195] In some embodiments, the viral particle comprises a polypeptide comprising a signal peptide sequence derived from Gaussia luciferase 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: 11.

[0196] Viral particle envelope expression cassette In some embodiments, the viral particles described herein are generated using a viral envelope expression cassette encoding at least one of the polypeptides described herein.

[0197] In some embodiments, the viral particles of the present disclosure, in order from 5' to 3', (a) a CD8-derived signal peptide (b) an anti-CD3 scFV (c) a CD8-derived hinge domain (d) a CD4 transmembrane domain and cytoplasmic tail (e) a T2A linker (f) a coxsackievirus protein and are generated using a viral envelope expression cassette encoding the same.

[0198] In some embodiments, the viral particles of the present disclosure, in the order from 5' to 3', (a) Signal peptide derived from Gaussia luciferase (b) Anti-CD3 scFV (c) Short hinge domain (d) Transmembrane domain and cytoplasmic tail derived from coccal sugar protein are generated using a viral envelope expression cassette encoding the same.

[0199] In some embodiments, the viral particles of the present disclosure, in the order from 5' to 3', (a) Signal peptide derived from Gaussia luciferase (b) Anti-CD3 scFV (c) Long hinge domain (d) Transmembrane domain and cytoplasmic tail derived from coccal sugar protein are generated using a viral envelope expression cassette encoding the same.

[0200] In some embodiments, the viral particles of the present disclosure, in the order from 5' to 3', (a) Signal peptide derived from Gaussia luciferase (b) Anti-CD3 scFV (c) 218 linker (d) Transmembrane domain derived from the extracellular domain of human glycophorin A (e) Cytoplasmic tail derived from the HIV viral envelope are generated using a viral envelope expression cassette encoding the same.

[0201] In some embodiments, the viral particles of the present disclosure, in the order from 5' to 3', (a) Signal peptide derived from Gaussia luciferase (b) Anti-CD3 scFV (c) 218 linker (d) Transmembrane domain and cytoplasmic tail derived from the HIV viral envelope It is generated using a viral envelope expression cassette encoding

[0202] In some embodiments, the viral particles of the present disclosure, in the order from 5' to 3', (a) A signal peptide derived from Gaussia luciferase (b) Anti-CD3 scFV (c) A triple G4S linker (d) A transmembrane domain and cytoplasmic tail derived from the HIV viral envelope It is generated using a viral envelope expression cassette encoding (using comprise a viral envelope expression cassette).

[0203] In some embodiments, the viral particles of the present disclosure, in the order from 5' to 3', (a) A signal peptide derived from Gaussia luciferase (b) Anti-CD3 scFV (c) A short hinge domain (d) A transmembrane domain and cytoplasmic tail derived from the coccolith protein (e) A T2A linker (f) The coccolith protein It is generated using a viral envelope expression cassette encoding.

[0204] In some embodiments, the viral expression cassettes described herein comprise a nucleotide sequence encoding a costimulatory molecule and / or an adhesion molecule. In some embodiments, the viral expression cassettes described herein comprise a nucleotide sequence encoding a CD86 polypeptide. In some embodiments, the viral expression cassettes described herein comprise a nucleotide sequence encoding a CD80 polypeptide. In some embodiments, the viral expression cassettes described herein comprise a nucleotide sequence encoding a CD58 polypeptide.

[0205] In some embodiments, the viral expression cassette comprises a nucleic acid encoding a Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv operably linked to a hinge domain operably linked to a transmembrane domain and cytoplasmic tail derived from coxsackievirus envelope sharing 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: 108.

[0206] In some embodiments, the viral expression cassette comprises a nucleic acid encoding a Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv operably linked to a hinge domain operably linked to a transmembrane domain and cytoplasmic tail derived from coxsackievirus envelope sharing 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: 110.

[0207] In some embodiments, the viral expression cassette comprises a nucleic acid encoding a Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv operably linked to a linker operably linked to a transmembrane domain derived from glycophorin A and a cytoplasmic tail derived from HIV envelope sharing 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: 112.

[0208] In some embodiments, the viral expression cassette comprises a nucleic acid encoding a Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv operably linked to a linker operably linked to a transmembrane domain and cytoplasmic tail derived from HIV envelope sharing 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: 114.

[0209] In some embodiments, the viral expression cassette comprises a nucleic acid encoding a Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv operably linked to a triple G4S linker operably linked to a transmembrane domain and cytoplasmic tail derived from an HIV envelope 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: 116.

[0210] In some embodiments, the viral expression cassette comprises a nucleic acid encoding a Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv operably linked to a hinge domain operably linked to a T2A self-cleaving peptide operably linked to a transmembrane domain, cytoplasmic tail and coxsackievirus envelope 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: 118.

[0211] In some embodiments, the viral expression cassette comprises a nucleic acid encoding a Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv operably linked to a linker operably linked to a hinge, transmembrane domain and cytoplasmic tail derived from glycophorin A 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: 120.

[0212] In some embodiments, the viral expression cassette comprises a nucleic acid sequence encoding a short hinge operably linked to a transmembrane domain operably linked to a cytoplasmic tail derived from a coxsackievirus 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: 16.

[0213] In some embodiments, the viral expression cassette comprises a nucleic acid sequence encoding a long hinge operably linked to a transmembrane domain operably linked to a cytoplasmic tail derived from a coccolith protein sharing 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: 22.

[0214] In some embodiments, the viral expression cassette comprises a nucleic acid sequence encoding a 218 linker operably linked to a human glycophorin A extracellular domain transmembrane domain operably linked to a cytoplasmic tail derived from an HIV viral envelope sharing 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: 28.

[0215] In some embodiments, the viral particle viral expression cassette comprises a nucleic acid sequence encoding a 218 linker operably linked to an HIV viral envelope transmembrane domain and cytoplasmic tail sharing 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: 34.

[0216] In some embodiments, the viral expression cassette comprises a nucleic acid sequence encoding a triple G4S linker operably linked to an HIV viral envelope transmembrane domain and cytoplasmic tail sharing 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: 40.

[0217] In some embodiments, the viral expression cassette comprises a nucleic acid sequence encoding a Ser-Gly peptide operably linked to a small external domain, transmembrane, and cytoplasmic tail sequence derived from human glycophorin A 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.

[0218] In some embodiments, the viral expression cassette comprises a nucleic acid sequence encoding a hinge operably linked to a glycophorin A transmembrane domain and cytoplasmic tail 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: 106.

[0219] In some embodiments, the viral expression cassette comprises a nucleic acid sequence encoding a short hinge operably linked to a coxsackievirus transmembrane domain and cytoplasmic tail operably linked to a T2A linker 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: 47.

[0220] In some embodiments, the viral expression cassette comprises a nucleic acid sequence encoding a CD4-derived transmembrane domain and cytoplasmic tail operably linked to a T2A linker 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: 9.

[0221] In some embodiments, the viral expression cassette comprises a nucleic acid sequence encoding a signal peptide derived from Gaussia luciferase 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: 14.

[0222] Particle type In some embodiments, the viral particles described herein are retroviruses. Retroviruses include lentiviruses, gamma-retroviruses, and alpha-retroviruses, each of which can be used for delivery of polynucleotides into cells using methods known in the art. Lentiviruses are complex retroviruses that contain, in addition to the common retroviral genes gag, pol, and env, other genes with regulatory or structural functions. The higher complexity allows the virus to modulate its life cycle, as in the course of latent infection. Exemplary lentiviruses include, but are not limited to, human immunodeficiency virus (HIV, including HIV type 1 and HIV type 2); Visna / maedi virus (VMV); caprine arthritis encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). In some embodiments, the backbone is an HIV-based vector backbone (i.e., HIV cis-acting sequence elements). The retroviral particles are generated by multi-attenuating the HIV virulence genes, for example, by deleting the genes env, vif, vpr, vpu, and nef to make the vector biologically safe.

[0223] Exemplary lentiviral particles include lentiviral particles 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. Patent No. 6,013,516; and U.S. Patent No. 5,994,136, each of which is incorporated herein by reference in its entirety. Generally, these particles are configured to carry essential sequences for selection of cells containing the particles, for incorporation of exogenous nucleic acid into the lentiviral particles, and for transfer of the nucleic acid into target cells.

[0224] The commonly used lentiviral particle system is the so-called third-generation system. The third-generation lentiviral particle system contains four plasmids. The "transfer plasmid" encodes a polynucleotide sequence that is delivered to target cells by the lentiviral vector system. The transfer plasmid generally has one or more transgene sequences of interest flanked by long terminal repeat (LTR) sequences that facilitate the integration of the transfer plasmid sequence into the host genome. For safety reasons, the transfer plasmid is generally designed to abolish the replication ability of the resulting particles. For example, the transfer plasmid lacks the genetic elements necessary for the production of infectious particles in host cells. In addition, the transfer plasmid can be designed by deletion of the 3' LTR to make the virus "self-inactivating" (SIN). See Dull et al. (1998) J. Virol. 72:8463-71; Miyoshi et al. (1998) J. Virol. 72:8150-57. The viral particles can also contain 3' untranslated regions (UTRs) and 5' UTRs. The UTRs contain retroviral regulatory elements that support the packaging, reverse transcription, and integration of the proviral genome into the cell after contact of the cell by the retroviral particles.

[0225] The third generation systems generally also include two "packaging plasmids" and one "envelope plasmid". The "envelope plasmid" generally encodes an Env gene operably linked to a promoter. In an exemplary third generation system, the Env gene is VSV-G and the promoter is the CMV promoter. In an exemplary third generation system, the Env gene is the coxsackievirus G protein (coxsackievirus glycoprotein) and the promoter is the MND (myeloproliferative sarcoma virus enhancer with a deleted negative control region and a substituted dl587rev primer binding site) promoter. In an exemplary third generation system, the Env gene is the coxsackievirus G protein (coxsackievirus glycoprotein) and the promoter is the CMV promoter. As a further safety feature, the third generation systems use two packaging plasmids, one encoding gag and pol and the other encoding rev - an improvement over the single packaging plasmid of the so-called second generation systems. Although safer, the third generation systems can be more cumbersome to use and result in lower virus titers due to the addition of yet another plasmid. Exemplary packaging plasmids include, without limitation, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI.

[0226] Many retroviral particle systems rely on the use of a "packaging cell line". Generally, a packaging cell line is a cell line that can produce infectious retroviral particles when a transfer plasmid, packaging plasmid(s), and envelope plasmid are introduced into the cell. Various methods for introducing plasmids into cells can be used, including transfection or electroporation. In some cases, the packaging cell line is adapted for the high-efficiency packaging of retroviral particle systems into retroviral particles.

[0227] As used herein, the term "retroviral particle" or "lentiviral particle" refers to a viral particle that contains a polynucleotide encoding a heterologous protein (e.g., a chimeric antigen receptor), one or more capsid proteins, and other proteins necessary for transduction of the polynucleotide into a target cell. Retroviral particles and lentiviral particles generally contain an RNA genome (derived from a transfer plasmid), a lipid bilayer envelope in which the Env protein is embedded, and other accessory proteins including integrase, protease, and matrix proteins.

[0228] The ex vivo efficiency of a retroviral or lentiviral particle system can be evaluated in a variety of ways known in the art, including measurement of vector copy number (VCN) or vector genome (vg), such as by quantitative polymerase chain reaction (qPCR), digital droplet PCR (ddPCR), or titration of the virus expressed as infectious units per milliliter (IU / mL). For example, the titer can be evaluated using a functional assay performed in 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 the titer is evaluated in a continuously dividing cultured cell line, no stimulation is required, and thus the measured titer is not affected by surface manipulation of the retroviral particle. Other methods for evaluating the efficiency of a retroviral vector system are provided in Gaererts et al. Comparison of retroviral vector titration methods. BMC Biotechnol. 6:34(2006).

[0229] In some embodiments, the retroviral particles and / or lentiviral particles of the present disclosure comprise a polynucleotide comprising a sequence encoding a receptor that specifically binds to a hapten. In some embodiments, the sequence encoding a receptor that specifically binds to a hapten is operably linked to a promoter. Exemplary promoters include, without limitation, the cytomegalovirus (CMV) promoter, CAG promoter, SV40 promoter, SV40 / CD43 promoter, EF-1α promoter, and MND promoter.

[0230] In some embodiments, the polynucleotide encoding a chimeric antigen receptor is operably linked to one or more promoters. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is CMV. In some embodiments, the promoter is MND.

[0231] In some embodiments, the polynucleotide encoding RACR is operably linked to one or more promoters. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is CMV. In some embodiments, the promoter is MND.

[0232] In some embodiments, the retroviral particles comprise a transduction enhancer. In some embodiments, the retroviral particles comprise a polynucleotide comprising a sequence encoding a T cell activating factor protein. In some embodiments, the retroviral particles comprise a polynucleotide comprising a sequence encoding a hapten-binding receptor. In some embodiments, the retroviral particles comprise a tagged protein.

[0233] In some embodiments, each of the retroviral particles comprises a polynucleotide that, in order from 5' to 3', comprises: (i) a 5' long terminal repeat (LTR) or untranslated region (UTR), (ii) a promoter, (iii) a sequence encoding a receptor that specifically binds to a hapten, and (iv) a 3' LTR or UTR.

[0234] In the practice of the present disclosure, gene delivery viral particles useful in the practice of the present disclosure can be constructed using methodologies known in the art of molecular biology. Typically, a viral vector carrying a transgene is assembled from a polynucleotide encoding the transgene, suitable regulatory elements, and elements necessary for the production of viral proteins that mediate cell transduction. Such recombinant viruses can be produced by techniques known in the art, for example, by transfecting packaging cells, or by transient transfection with a helper plasmid or virus. Examples of viral packaging cells include, but are not limited to, HeLa cells, SF9 cells (if necessary, by a baculovirus helper vector), 293 cells, and the like. Detailed protocols for producing such replication-deficient recombinant viruses can be found, for example, in W095 / 14785, W096 / 22378, U.S. Patent No. 5,882,877, U.S. Patent No. 6,013,516, U.S. Patent No. 4,861,719, U.S. Patent No. 5,278,056, and W094 / 19478, the entire contents of each of which are incorporated herein by reference.

[0235] Exemplary examples of viral vectors that can be used in the compositions and methods of the present disclosure are disclosed in WO2016 / 139463; WO2017 / 165245; WO2018111834, the entire contents of each of which are incorporated herein by reference.

[0236] Payload In some embodiments, the viral particle comprises a payload. In some embodiments, the payload is conjugated to the surface of the particle. In some embodiments, the payload is encapsulated by the particle. In some embodiments, the viral particle delivers the payload to a target cell.

[0237] In some embodiments, the payload is a nucleic acid. In some embodiments, the nucleic acid is a coding nucleic acid. In some embodiments, the nucleic acid encodes a polypeptide of interest. In some embodiments, the polypeptide of interest is a therapeutic polypeptide. In some embodiments, the polypeptide of interest is a chimeric antigen receptor. In some embodiments, the nucleic acid is transduced into a target cell and the polypeptide of interest is expressed in the target cell. In some embodiments, the nucleic acid is a non-coding nucleic acid. In that case, in some embodiments, the nucleic acid is a therapeutic non-coding nucleic acid. Non-coding nucleic acids are known to those skilled in the art and include, but are not limited to, siRNA, miRNA, and shRNA.

[0238] In some embodiments, the expression of the payload is driven by a promoter. In some embodiments, the promoter is the MND promoter (a myeloproliferative sarcoma virus enhancer with the negative regulatory region deleted and the dl587rev primer binding site replaced), a viral-derived synthetic promoter containing the U3 region of the modified Moloney murine leukemia virus (MoMuLV) LTR and the myeloproliferative sarcoma virus enhancer 13, which has high expression in human CD34+ stem cells, lymphocytes, and other tissues. In some embodiments, separate proteins are expressed, separated by a 2A peptide sequence that induces ribosome skipping and cleavage during translation. In some embodiments, the promoter is the CMV promoter. In some embodiments, the promoter is the EF1a promoter. In other embodiments, the promoter is the HTLV promoter.

[0239] Chimeric antigen receptor In some embodiments, the viral particles described herein are used to transduce a cell (e.g., a T lymphocyte) with a nucleic acid sequence (polynucleotide) encoding one or more chimeric antigen receptors (CARs). In some embodiments, transduction of the viral particles results in the expression of one or more CARs in the transduced cell.

[0240] A CAR is an artificial membrane-bound protein that directs T lymphocytes towards an antigen and stimulates the T lymphocytes to kill the cell presenting the antigen. See, for example, Eshhar, U.S. Patent No. 7,741,465. Generally, a CAR is a genetically engineered receptor that includes an extracellular domain that binds to an antigen, such as an antigen on a cell, an optional linker, a transmembrane domain, and an intracellular (cytoplasmic) domain that includes a co-stimulatory domain and / or a signaling domain that transmits an activating signal to an immune cell. Using a CAR, a single receptor can be programmed such that both specific antigen recognition and activation of immune cells for attack and destruction of cells bearing that antigen when it binds to the antigen are accomplished. When these antigens are present on tumor cells, immune cells expressing the CAR can target and kill the tumor cells. When the CAR is expressed on the surface of, for example, a T lymphocyte, and the extracellular domain of the CAR binds to an antigen and all other conditions are met, the intracellular signaling domain transmits a signal to the T lymphocyte to activate and / or proliferate the T lymphocyte and, if the antigen is present on the cell surface, kill the cell expressing the antigen. Since T lymphocytes require two signals, a primary activation signal and a co-stimulatory signal, to be maximally activated, a CAR can include a stimulatory domain and a co-stimulatory domain, and thus, when an antigen binds to the extracellular domain, transmission of both a primary activation signal and a co-stimulatory signal is effected. Exemplary CARs are known in the art and can be designed in a modular fashion, as described, for example, in (see, for example, Guedan S, Calderon H, Posey AD, Maus MV, Molecular Therapy - Methods & Clinical Development. 2019; 12: 145-156), which is incorporated herein by reference.

[0241] In some embodiments, the viral particles disclosed herein encode a CAR comprising an extracellular domain, and optionally, a hinge domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a co-stimulatory domain and an activation domain. In some embodiments, the co-stimulatory and activation domains are a single domain, e.g., a single intracellular domain, that provides both co-stimulatory and activation signals to the cell. In other embodiments, the intracellular signaling domain comprises either a co-stimulatory domain or an activation domain. In some embodiments, the CAR comprises an extracellular domain, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the viral particles disclosed herein encode a CAR comprising an extracellular domain, a CD28 hinge domain, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the viral particles disclosed herein encode a CAR comprising an extracellular domain, an IgG4 hinge domain, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the viral particles disclosed herein encode a CAR comprising an extracellular domain, a CD8a hinge, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain.

[0242] Exemplary CAR constructs suitable for CAR cells, including CAR T and CAR NK cells, are shown below: (1) scFv-CD8 TM -4-1BB IC -CD3ζs (see, e.g., Liu E, Tong Y, Dotti G, et al., Leukemia. 2018; 32: 520-531); (2) scFv-CD28 TM+IC-CD3ζs (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 U S A. 2008; 105: 17481-17486; and Chu J, Deng Y, Benson DM et al., Leukemia. 2014; 28: 917-927); (3) scFv-DAP12 TM+IC (see, e.g., Muller N, Michen S, Tietze S et al., J Immunother. 2015; 38: 197-210); (4) scFv-CD8 TM -2B4 IC -CD3ζs (see, e.g., Xu Y, Liu Q, Zhong M et al., J Hematol Oncol. 2019; 12: 49); (5) scFv-2B4 TM+IC -CD3ζs (see, e.g., Altvater B, Landmeier S, Pscherer S et al., Clin Cancer Res. 2009; 15: 4857-4866); (6) scFv-CD28 TM+IC -4-1BB IC -CD3ζs (see, e.g., Kloss S, Oberschmidt O, Morgan M et al., Hum Gene Ther. 2017; 28: 897-913); (7) scFv-CD16 TM -2B4 IC -CD3ζs (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (8) scFv-NKp44 TM -DAP10 IC-CD3ζs (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (9) scFv-NKp46 TM -2B4 IC -CD3ζs (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (10) scFv-NKG2D TM -2B4 IC -CD3ζs (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (11) scFv-NKG2D TM -4-1BB IC -CD3ζs (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (12) scFv-NKG2D TM -2B4 IC -DAP12 IC -CD3ζs (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (13) scFv-NKG2D TM -2B4 IC -DAP10 IC -CD3ζs (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (14) scFv-NKG2D TM -4-1BB IC -2B4 IC-CD3ζS (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); and (15) scFv-NKG2D TM -CD3ζS (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192).

[0243] CAR intracellular domain In some embodiments, the intracellular domain of the CAR is the intracellular domain or motif of a protein that is expressed on the surface of a T lymphocyte and induces activation and / or proliferation of said T lymphocyte or comprises the same. Such a domain or motif can transmit the signals necessary for T lymphocyte activation in response to binding of an antigen to the extracellular portion of the CAR. In some embodiments, this domain or motif comprises or is an ITAM (immunoreceptor activation tyrosine motif). Examples of ITAM-containing polypeptides suitable for use in a CAR include, for example, the zeta CD3 chain (CD3ζ) or an ITAM-containing portion thereof. In some embodiments, the intracellular domain is the CD3ζ intracellular signaling domain. In some embodiments, the intracellular domain is derived from a lymphocyte receptor chain, a TCR / CD3 complex protein, an Fc receptor subunit, or an IL-2 receptor subunit. In some embodiments, the intracellular signaling domain of the CAR can be derived from, for example, a CD3ζ, CD3ε, CD22, CD79a, CD66d, or CD39 signaling domain. An "intracellular signaling domain" refers to a portion of a CAR polypeptide that is involved in transmitting the message of effective CAR binding to a target antigen into the interior of an immune effector cell, thereby eliciting effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors against the target cells to which the CAR is bound, or other cellular responses elicited after binding of an antigen to the extracellular CAR domain.

[0244] In some embodiments, the intracellular domain of the CAR is the zeta CD3 chain (CD3 zeta).

[0245] In some embodiments, the viral particle comprises a polypeptide comprising a CAR, wherein the intracellular domain thereof comprises a CD3 zeta domain sharing 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: 54.

[0246] In some embodiments, the viral particle comprises a nucleic acid encoding an intracellular domain of a CAR comprising a CD3 zeta domain sharing 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: 66.

[0247] In some embodiments, CAR further comprises one or more costimulatory domains or motifs, for example, as part of the intracellular domain of the polypeptide.Costimulatory molecules are well-known cell surface molecules that provide a second signal other than antigen receptor or Fc receptor that is necessary for efficient activation and function of T lymphocytes when they bind to antigen.The one or more costimulatory domains or motifs can be or include, for example, one or more of the following: costimulatory CD27 polypeptide sequence, costimulatory CD28 polypeptide sequence, costimulatory OX40 (CD134) polypeptide sequence, costimulatory 4-1BB (CD137) polypeptide sequence, or costimulatory-inducible T cell costimulatory (ICOS) polypeptide sequence, or other costimulatory domains or motifs, or any combination thereof. In some embodiments, the one or more costimulatory domains are selected from the group consisting of the intracellular domains of 4-1BB, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70.

[0248] In some embodiments, the costimulatory domain is 4-1BB, CD28 or OX40 intracellular domain.Exemplary CAR constructs comprising CD28 signaling domain are disclosed in U.S. Patent No. 7,446,190, which is incorporated herein by reference.Exemplary CAR constructs comprising 4-1BB signaling domain are disclosed in U.S. Patent No. 9,856,322 and U.S. Patent No. 8,399,964, which are each incorporated herein by reference.

[0249] In some embodiments, the viral particle encodes a CAR comprising an IgG4 linker operably linked to a CD28 transmembrane domain operably linked to a 4-1BB costimulatory domain operably linked to a CD3 zeta signaling domain.

[0250] In some embodiments, the viral particle encodes a CAR comprising an IgG4 linker functionally linked to a CD8a transmembrane domain functionally linked to a 4-1BB co-stimulatory domain functionally linked to a CD3 zeta signaling domain.

[0251] In some embodiments, the viral particle encodes a CAR comprising an IgG4 linker functionally linked to a CD8a transmembrane domain functionally linked to a CD28 co-stimulatory domain functionally linked to a CD3 zeta signaling domain.

[0252] In some embodiments, the viral particle encodes a CAR comprising a CD8a linker functionally linked to a CD8a transmembrane domain functionally linked to a 4-1BB co-stimulatory domain functionally linked to a CD3 zeta signaling domain.

[0253] In some embodiments, the viral particle encodes a CAR comprising a CD28 linker functionally linked to a CD28 transmembrane domain functionally linked to a CD28 co-stimulatory domain functionally linked to a CD3 zeta signaling domain.

[0254] In some embodiments, the viral particle comprises a polypeptide comprising a CAR, wherein the intracellular domain thereof comprises a co-stimulatory 4-1BB polypeptide sequence sharing 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: 53.

[0255] In some embodiments, the viral particle comprises a nucleic acid encoding an intracellular domain of a CAR comprising a co-stimulatory 4-1BB sequence sharing 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: 65.

[0256] In some embodiments, the viral particle comprises a polypeptide comprising a chimeric antigen receptor (CAR) that includes an IgG4 linker functionally linked to a CD28-derived transmembrane domain functionally linked to a co-stimulatory 4-1BB polypeptide functionally linked to a CD3 zeta domain, wherein the intracellular domain thereof shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO: 80.

[0257] In some embodiments, the viral particle comprises a nucleic acid encoding an intracellular domain of a CAR that includes an IgG4 linker functionally linked to a CD28-derived transmembrane domain functionally linked to a co-stimulatory 4-1BB polypeptide functionally linked to a CD3 zeta domain, wherein the intracellular domain thereof shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO: 86.

[0258] In some embodiments, the viral particle comprises a polypeptide comprising a chimeric antigen receptor (CAR) that includes an IgG4 linker functionally linked to a CD28-derived transmembrane domain functionally linked to a co-stimulatory 4-1BB polypeptide functionally linked to a CD3 zeta domain, wherein the intracellular domain thereof shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO: 90.

[0259] In some embodiments, the viral particle comprises a nucleic acid encoding an intracellular domain of a CAR that includes an IgG4 linker functionally linked to a CD28-derived transmembrane domain functionally linked to a co-stimulatory 4-1BB polypeptide functionally linked to a CD3 zeta domain, wherein the intracellular domain thereof shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO: 95.

[0260] In some embodiments, the intracellular domain can be further modified to encode a detectable protein, such as a fluorescent protein (e.g., green fluorescent protein) or any known variant thereof.

[0261] CAR transmembrane region The transmembrane region can be any transmembrane region that can be incorporated into a functional CAR, such as a transmembrane region derived from the CD28, CD4, or CD8 molecule.

[0262] In some embodiments, the transmembrane domain of the CAR can be derived from CD8, the alpha, beta, or zeta chain transmembrane domains of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. In some embodiments, the transmembrane domain of the CAR can be derived from the transmembrane domain of CD28. In some embodiments, the transmembrane domain of the CAR can be derived from CD8, e.g., the transmembrane domain of CD8α.

[0263] CAR linker region An optional linker or hinge of the CAR placed between the extracellular domain and the transmembrane domain can be a polypeptide having a length of about 2 to more than 100 amino acids. The linker can include or be composed of flexible residues such as glycine and serine, such that adjacent protein domains can move freely relative to each other. For example, a longer linker can be used if it is desired to ensure that two adjacent domains do not sterically interfere with each other. A longer linker may also be advantageous when the target antigen is closer to the cell surface.

[0264] In some embodiments, the linker is derived from the hinge region or a portion of the hinge region of any immunoglobulin. In some embodiments, the linker is derived from an immunoglobulin, such as IgG4. In some embodiments, the linker is derived from the extracellular domain of CD28. In other embodiments, the linker is derived from the extracellular domain of CD8.

[0265] In some embodiments, the linker is an IgG4 linker operably linked to a CD28-derived transmembrane domain 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: 52.

[0266] In some embodiments, the linker is an IgG4 linker operably linked to a CD28-derived transmembrane domain 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: 64.

[0267] CAR extracellular domain In some embodiments, the nucleic acid transduced into cells using the methods described herein comprises a sequence encoding a polypeptide, wherein the extracellular domain of the polypeptide binds to an antigen of interest. In some embodiments, the extracellular domain comprises a receptor or a portion of a receptor that binds to the antigen. In some embodiments, the extracellular domain comprises or is an antibody or an antigen-binding portion thereof. In some embodiments, the extracellular domain comprises or is a single-chain Fv domain. The single-chain Fv domain can comprise, for example, VL and VH linked by a flexible linker, wherein the VL and VH are derived from an antibody that binds to the antigen.

[0268] In some embodiments, the extracellular domain of the CAR can contain any polypeptide that binds to a desired antigen (e.g., a prostate neoantigen or an antigen expressed on a tumor of interest). The extracellular domain can include an scFv, a portion of an antibody, or an alternative scaffold. The CAR can also be engineered to bind to two or more desired antigens, in which case they can be arranged in tandem and separated by a linker sequence. For example, one or more domain antibodies, scFvs, llama VHH antibodies, or other VH-only antibody fragments can be organized in tandem by a linker to confer bispecificity or multispecificity to the CAR.

[0269] The antigen to which the extracellular domain of the polypeptide binds can be any antigen of interest, for example, an antigen on tumor cells. The tumor cells can be, for example, cells within a solid tumor or cells of a blood cancer. The antigen can be any antigen expressed on cells of any tumor or cancer type, such as, for example, lymphoma, lung cancer, breast cancer, prostate cancer, adrenocortical carcinoma, thyroid cancer, nasopharyngeal carcinoma, melanoma, such as malignant melanoma, skin cancer, colorectal cancer, desmoid tumor, fibromatosis, small round cell tumor of fibroblastic type, endocrine tumor, Ewing sarcoma, peripheral primitive neuroectodermal tumor, solid embryonal cell tumor, hepatoblastoma, neuroblastoma, non-rhabdomyosarcoma soft tissue sarcoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, Wilms tumor, glioblastoma, myxoma, fibroma, lipoma, and any other antigen expressed on cells of any other type of tumor or cancer. In some embodiments, the lymphoma is chronic lymphocytic leukemia (small lymphocytic lymphoma), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, splenic marginal zone lymphoma, plasmacytic myeloma, plasmacytoma, extranodal marginal zone B-cell lymphoma, MALT lymphoma, nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt lymphoma, T-lymphocyte prolymphocytic leukemia, T-lymphocyte large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T-lymphocyte leukemia / lymphoma, extranodal NK / T-lymphocyte lymphoma, nasal type, enteropathy-type T-lymphocyte lymphoma, hepatosplenic T-lymphocyte lymphoma, blastic NK cell lymphoma, mycosis fungoides, Sézary syndrome, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T-lymphocyte lymphoma, peripheral T-lymphocyte lymphoma (unspecified), anaplastic large cell type lymphoma, Hodgkin lymphoma, or non-Hodgkin lymphoma. In some embodiments where the cancer is chronic lymphocytic leukemia (CLL), the B cells of the CLL have a normal karyotype. In some embodiments where the cancer is chronic lymphocytic leukemia (CLL), the B cells of the CLL have a 17p deletion, an 11q deletion, a 12q trisomy, a 13q deletion, or a p53 deletion.

[0270] In some embodiments, the antigen is expressed on B cell malignant tumor cells, recurrent / refractory CD19-expressing malignant tumor cells, diffuse large B cell lymphoma (DLBCL) cells, Burkitt's type large B-cell lymphoma (B-LBL) cells, follicular lymphoma (FL) cells, chronic lymphocytic leukemia (CLL) cells, acute lymphocytic leukemia (ALL) cells, mantle cell lymphoma (MCL) cells, hematological malignant tumor cells, colon cancer cells, lung cancer cells, liver cancer cells, breast cancer cells, kidney cancer cells, prostate cancer cells, ovarian cancer cells, skin cancer cells, melanoma cells, bone cancer cells, brain cancer cells, squamous cell carcinoma cells, leukemia cells, myeloma cells, B cell lymphoma cells, kidney cancer cells, uterine cancer cells, adenocarcinoma cells, pancreatic cancer cells, chronic myelogenous leukemia cells, glioblastoma cells, neuroblastoma cells, medulloblastoma cells or sarcoma cells.

[0271] In some embodiments, the antigen is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). In some embodiments, without limitation thereto, the tumor-associated antigen or tumor-specific antigen is B cell maturation antigen (BCMA), B cell activating factor (BAFF), GPRC5D, FCRL5 / FCRH5, ROR1, L1-CAM, CD22, folate receptor, carbonic anhydrase IX (CAIX), claudin 18.2, FAP, mesothelin, IL13Ra2, Lewis Y, CCNA1, WT-1, TACI, CD38, SLAMF7, CD138, DLL3, transmembrane 4 L six family member 1 (TM4SF1), epithelial cell adhesion molecule (EpCAM), PD-1, PD-L1, CTLA-4, AXL, ROR2, glypican-3 (GPC3), CD133, CD147, EGFR, MUC1, GD2, Her2, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), EGFRvIII, cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD19, CD20, CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor-1, vascular endothelial growth factor receptor (VEGFR), dimeric form of pyruvate kinase isoenzyme M2 (tumor M2-PK), abnormal ras protein, or abnormal p53 protein. In some embodiments, the CAR comprises a binding domain that targets two or more antigens disclosed herein in any combination.Exemplary antigen combinations include 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 a binding domain that targets two or more antigens in the same target protein, e.g., two epitopes in CD19, BCMA, or any other antigen disclosed herein.

[0272] In other embodiments, the CAR is a universal CAR that does not itself specifically target a tumor antigen. For example, the CAR can comprise a tag-specific scFv such that an exogenous agent comprising a tag and a tumor targeting domain can direct the universal CAR T cells to the target tumor.

[0273] In some embodiments, the CAR is a second-generation CAR composed of an anti-fluorescein scFv linked to a 4-1BB co-stimulatory domain and a CD3 zeta intracellular signaling domain.

[0274] In some embodiments, the antigen is CD19. CAR T therapies targeting CD19 are approved by the FDA and include Yescarta, Tecartus, Kymriah, and Breyanzi. CARs targeting CD19 are described, for example, in U.S. Patent Application Publication No. 20160152723, U.S. Patent No. 10,736,918, U.S. Patent No. 10,357,514, and U.S. Patent No. 7,446,190, each of which is incorporated herein by reference.

[0275] In some embodiments, the CAR comprises an extracellular domain comprising an FMC63 scFv binding domain for CD19 binding. In some embodiments, the CAR is a second-generation CAR composed of an FMC63 mouse anti-human CD19 scFv linked to a 4-1BB co-stimulatory domain and a CD3 zeta intracellular signaling domain. In some embodiments, the CAR comprises a binding domain for CD19, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises a binding domain for CD19, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises a binding domain for CD19, a CD28 hinge, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an FMC63 scFv binding domain for CD19 binding, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an FMC63 scFv binding domain for CD19 binding, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an FMC63 scFv binding domain for CD19 binding, a CD28 hinge, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3 zeta signaling domain.

[0276] In some embodiments, the viral particle comprises a polypeptide comprising a CAR, wherein the extracellular domain thereof comprises a signal peptide sharing 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: 50.

[0277] In some embodiments, the viral particle comprises a polynucleotide encoding a CAR, wherein the extracellular domain thereof comprises an αCD19 scFv (CD19 VL linked to CD19 VH) 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: 51.

[0278] The complementarity determining regions (CDRs) of this scFv are RASQDISKYLN (CDR-L1; SEQ ID NO: 39), HTSRLHS (CDR-L2; SEQ ID NO: 41), QQGNTLPYT (CDR-L3; SEQ ID NO: 42), DYGV (CDR-H1; SEQ ID NO: 44), VIWGSETTYYNSALKS (CDR-H2; SEQ ID NO: 45), HYYYGGSYAMDY (CDR-H3; SEQ ID NO: 46). In some embodiments, the viral particle comprises a polynucleotide encoding a CAR, wherein the extracellular domain thereof comprises an αCD19 scFv having these CDRs, and optionally, the αCD19 scFv 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: 51.

[0279] In some embodiments, the viral particle comprises a polynucleotide encoding a CAR, wherein the extracellular domain thereof comprises an αCD19 scFv having these CDRs, and optionally, the αCD19 scFv 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: 51 or 89.

[0280] In some embodiments, the viral particle comprises a nucleic acid encoding a signal peptide for the extracellular domain of a CAR 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: 62.

[0281] In some embodiments, the viral particle comprises a nucleic acid encoding an extracellular domain of a CAR comprising an αCD19 scFv 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: 63.

[0282] In some embodiments, the viral particle comprises a polypeptide comprising a CAR, wherein the extracellular domain of the CAR comprises an αCD19 scFv 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: 79.

[0283] In some embodiments, the viral particle comprises a nucleic acid encoding an extracellular domain of a CAR comprising an αCD19 scFv 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: 85.

[0284] In some embodiments, the viral particle comprises a polynucleotide encoding a CAR, wherein the extracellular domain of the CAR comprises an αCD19 scFv 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: 89.

[0285] The complementarity determining regions (CDRs) of this scFv are RASQDISKYLN (CDR-L1; SEQ ID NO: 39), HTSRLHS (CDR-L2; SEQ ID NO: 41), QQGNTLPYT (CDR-L3; SEQ ID NO: 42), DYGV (CDR-H1; SEQ ID NO: 44), VIWGSETTYYNSALKS (CDR-H2; SEQ ID NO: 45), and HYYYGGSYAMDY (CDR-H3; SEQ ID NO: 46). In some embodiments, the viral particle comprises a polynucleotide encoding a CAR, wherein the extracellular domain thereof comprises an αCD19 scFv having these CDRs, and optionally, the αCD19 scFv 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: 89.

[0286] In some embodiments, the viral particle comprises a nucleic acid encoding the extracellular domain of a CAR comprising an αCD19 scFv 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: 94.

[0287] In some embodiments, the CAR is a second-generation CAR composed of an FMC63 mouse anti-human CD19 scFv linked to a CD28 co-stimulatory domain and a CD3 zeta intracellular signaling domain. In some embodiments, the CAR is a second-generation CAR composed of an FMC63 mouse anti-human CD19 scFv linked to a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta intracellular signaling domain.

[0288] In some embodiments, the CAR is an anti-FITC CAR, and the ligand is composed of fluorescein or fluorescein isothiocyanate (FITC) moiety conjugated to a drug that binds to a desired target cell (such as a cancer cell). Exemplary ligands are described in the section above. In some embodiments, the ligand is FITC-folate.

[0289] In some embodiments, the CAR comprises a 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).

[0290] In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 157 or 164. In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 157 or 164. In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 157 or 164. In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 157 or 164. In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 157 or 164. In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 157 or 164. In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 157 or 164. In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 157 or 164. In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 157 or 164. In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 157 or 164. In some embodiments, the scFv VL comprises the nucleotide sequence of SEQ ID NO: 157 or 164. In some embodiments, the scFv VL consists of the nucleotide sequence of SEQ ID NO: 157 or 164.

[0291] In some embodiments, the scFv VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 158. In some embodiments, the scFv VL comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 158. In some embodiments, the scFv VL comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 158. In some embodiments, the scFv VL comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 158. In some embodiments, the scFv VL comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 158. In some embodiments, the scFv VL comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 158. In some embodiments, the scFv VL comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 158. In some embodiments, the scFv VL comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 158. In some embodiments, the scFv VL comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 158. In some embodiments, the scFv VL comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 158. In some embodiments, the scFv VL comprises the amino acid sequence of SEQ ID NO: 158. In some embodiments, the scFv VL consists of the amino acid sequence of SEQ ID NO: 158.

[0292] In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 161 or 166. In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 161 or 166. In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 161 or 166. In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 161 or 166. In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 161 or 166. In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 161 or 166. In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 161 or 166. In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 161 or 166. In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 161 or 166. In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 161 or 166. In some embodiments, the scFv VH comprises the nucleotide sequence of SEQ ID NO: 161 or 166. In some embodiments, the scFv VH consists of the nucleotide sequence of SEQ ID NO: 161 or 166.

[0293] In some embodiments, the scFv VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 162. In some embodiments, the scFv VH comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 162. In some embodiments, the scFv VH comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 162. In some embodiments, the scFv VH comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 162. In some embodiments, the scFv VH comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 162. In some embodiments, the scFv VH comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 162. In some embodiments, the scFv VH comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 162. In some embodiments, the scFv VH comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 162. In some embodiments, the scFv VH comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 162. In some embodiments, the scFv VH comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 162. In some embodiments, the scFv VH comprises the amino acid sequence of SEQ ID NO: 162. In some embodiments, the scFv VH consists of the amino acid sequence of SEQ ID NO: 162.

[0294] In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 159 or 165. In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 159 or 165. In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 159 or 165. In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 159 or 165. In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 159 or 165. In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 159 or 165. In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 159 or 165. In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 159 or 165. In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 159 or 165. In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 159 or 165. In some embodiments, the scFv linker comprises the nucleotide sequence of SEQ ID NO: 159 or 165. In some embodiments, the scFv linker consists of the nucleotide sequence of SEQ ID NO: 159 or 165.

[0295] In some embodiments, the scFv linker comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 160. In some embodiments, the scFv linker comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 160. In some embodiments, the scFv linker comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 160. In some embodiments, the scFv linker comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 160. In some embodiments, the scFv linker comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 160. In some embodiments, the scFv linker comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 160. In some embodiments, the scFv linker comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 160. In some embodiments, the scFv linker comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 160. In some embodiments, the scFv linker comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 160. In some embodiments, the scFv linker comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 160. In some embodiments, the scFv linker comprises the amino acid sequence of SEQ ID NO: 160. In some embodiments, the scFv linker consists of the amino acid sequence of SEQ ID NO: 160.

[0296] In some embodiments, the scFv comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 155 or 163. In some embodiments, the scFv comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 155 or 163. In some embodiments, the scFv comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 155 or 163. In some embodiments, the scFv comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 155 or 163. In some embodiments, the scFv comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 155 or 163. In some embodiments, the scFv comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 155 or 163. In some embodiments, the scFv comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 155 or 163. In some embodiments, the scFv comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 155 or 163. In some embodiments, the scFv comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 155 or 163. In some embodiments, the scFv comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 155 or 163. In some embodiments, the scFv comprises the nucleotide sequence of SEQ ID NO: 155 or 163. In some embodiments, the scFv consists of the nucleotide sequence of SEQ ID NO: 155 or 163.

[0297] In some embodiments, the scFv comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 156. In some embodiments, the scFv comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 156. In some embodiments, the scFv comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 156. In some embodiments, the scFv comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 156. In some embodiments, the scFv comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 156. In some embodiments, the scFv comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 156. In some embodiments, the scFv comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 156. In some embodiments, the scFv comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 156. In some embodiments, the scFv comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 156. In some embodiments, the scFv comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 156. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 156. In some embodiments, the scFv consists of the amino acid sequence of SEQ ID NO: 156.

[0298] In some embodiments, the anti-fluorescein E2 scFv comprises CDRL1, CDRL2, and CDRL3 having at least 80% amino acid identity, at least 90% amino acid identity, or at least 95% amino acid identity to TSNIGNNYVS (SEQ ID NO: 167), LMIYDVSKRPS (SEQ ID NO: 168), and AAWDDSLSEF (SEQ ID NO: 169), respectively, and CDRH1, CDRH2, and CDRH3 having at least 80% amino acid identity, at least 90% amino acid identity, or at least 95% amino acid identity to FTFGSFSMS (SEQ ID NO: 170), WVAGLSARSSLTHY (SEQ ID NO: 171), and RRSYDSSGYWGHFYSYMDV (SEQ ID NO: 172), respectively.

[0299] In some embodiments, the antigen is BCMA. CAR T therapies targeting BCMA have been approved by the FDA and include Abecma and Carvykti. CARs targeting BCMA are described, for example, in U.S. Patent Application Publication No. 2020 / 0246381; U.S. Patent No. 10,918,665; and U.S. Patent Application Publication No. 2019 / 0161553, each of which is incorporated herein by reference. In some embodiments, the CAR comprises a binding domain for BCMA, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises a binding domain for BCMA, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises a binding domain for BCMA, a CD28 hinge, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3 zeta signaling domain.

[0300] In some embodiments, the antigen is G protein-coupled receptor class C group 5 member D (GPRC5D). CARs targeting GRC5D are described, for example, in U.S. Patent Application Publication Nos. 2018 / 0118803 and 2021 / 10393689, each of which is incorporated herein by reference. In some embodiments, the CAR comprises a binding domain for GRC5D, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises a binding domain for GRC5D, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises a binding domain for GRC5D, a CD28 hinge, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3 zeta signaling domain.

[0301] In some embodiments, the antigen is Fc receptor-like 5 (FcRL5). CARs targeting FcRL5 are described, for example, in U.S. Patent Application Publication No. US2017 / 0275362, which is incorporated herein by reference. In some embodiments, the CAR comprises a binding domain for FcRL5, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises a binding domain for FcRL5, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises a binding domain for FcRL5, a CD28 hinge, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3 zeta signaling domain.

[0302] In some embodiments, the antigen is receptor tyrosine kinase-like orphan receptor 1 (ROR1). CARs targeting ROR1 are described, for example, in U.S. Patent Application Publication No. 2022 / 0096651, which is incorporated herein by reference. In some embodiments, the CAR comprises a binding domain for ROR1, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises a binding domain for ROR1, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises a binding domain for ROR1, a CD28 hinge, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3 zeta signaling domain.

[0303] In some embodiments, the CAR is a second-generation CAR comprising an anti-BCMA scFv linked to a 4-1BB co-stimulatory domain and a CD3 zeta intracellular signaling domain. In some embodiments, the CAR is a second-generation CAR comprising an anti-GPRC5D scFv linked to a 4-1BB co-stimulatory domain and a CD3 zeta intracellular signaling domain. In some embodiments, the CAR is a second-generation CAR comprising an anti-ROR1 scFv linked to a 4-1BB co-stimulatory domain and a CD3 zeta intracellular signaling domain.

[0304] In some embodiments, the TAA or TSA is a cancer / testis (CT) antigen, such as BAGE, CAGE, CTAGE, FATE, GAGE, HCA661, HOM-TES-85, MAGEA, MAGEB, MAGEC, NA88, NY-ESO-1, NY-SAR-35, OY-TES-1, SPANXB1, SPA17, SSX, SYCP1, or TPTE.

[0305] In some embodiments, the TAA or TSA is a carbohydrate or ganglioside, such as fuc-GM1, GM2 (oncofetal antigen-immunogenic-1; OFA-I-1); GD2 (OFA-I-2), GM3, GD3, and the like.

[0306] In some embodiments, the TAA or TSA is alpha - actinin - 4, Bage - 1, BCR - ABL, Bcr - Abl fusion protein, beta - catenin, CA125, CA15 - 3 (CA27.29\BCAA), CA195, CA242, CA - 50, CAM43, Casp - 8, cdc27, cdk4, cdkn2a, CEA, coa - 1, dek - can fusion protein, EBNA, EF2, Epstein - Barr virus antigen, ETV6 - AML1 fusion protein, HLA - A2, HLA - All, hsp70 - 2, KIAAO205, Mart2, Mum - 1, 2, and 3, neo - PAP, myosin class I, OS - 9, pml - RARα fusion protein, PTPRK, K - ras, N - ras, triosephosphate isomerase, Gage3, 4, 5, 6, 7, GnTV, Herv - K - mel, Lage - 1, NA - 88, NY - Eso - 1 / Lage - 2, SP17, SSX - 2, TRP2 - Int2, gp100 (Pmel 17), tyrosinase, TRP - 1, TRP - 2, MAGE - 1, MAGE - 3, RAGE, GAGE - 1, GAGE - 2, p15(58), RAGE, SCP - 1, Hom / Mel - 40, PRAME, p53, H - Ras, HER - 2 / neu, E2A - PRL, H4 - RET, IGH - IGK, MYL - RAR, human papillomavirus (HPV) antigens E6 and E7, TSP - 180, MAGE - 4, MAGE - 5, MAGE - 6, p185erbB2, p180erbB - 3, c - met, nm - 23H1, PSA, TAG - 72 - 4, CA19 - 9, CA72 - 4, CAM 17.1. NuMa, K-ras, β-catenin, Mum-1, p16, TAGE, PSMA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, 13HCG, BCA225, BTAA, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CD19, CD20, CD22, CD27, CD30, CD70, CD123, CD133, B cell maturation antigen, CS1, GPCR5, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Sp17), mesothelin, PAP (prostate acid phosphatase), prostain, TARP (T cell receptor gamma alternative reading frame protein), Trp-p8, STEAP1 (prostate transmembrane epithelial antigen 1 with six transmembrane domains), an abnormal Ras protein, or an abnormal p53 protein. In some embodiments, the tumor-associated antigen or tumor-specific antigen is integrin αvβ3 (CD61), galactin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), or Ral-B. Other tumor-associated antigens and tumor-specific antigens are known to those of skill in the art.

[0307] Antibodies and scFvs that bind to TSA and TAA include antibodies and scFVs known in the art, as well as nucleotide sequences encoding them.

[0308] In some embodiments, the antigen is not considered a TSA or TAA, but nevertheless is an antigen associated with tumor cells or damage caused by the tumor. In some embodiments, for example, the antigen is, for example, a growth factor, cytokine or interleukin, for example, a growth factor, cytokine or interleukin associated with angiogenesis or vasculogenesis. Such growth factors, cytokines or interleukins can include, for example, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), or interleukin-8 (IL-8). The tumor can also create a hypoxic environment local to the tumor. Thus, in some embodiments, the antigen is a hypoxia-related factor, for example, HIF-1α, HIF-1β, HIF-2a, HIF-2β, HIF-3α, or HIF-3β. The tumor can also cause localized damage to normal tissue, thereby causing the release of molecules also known as damage-associated molecular pattern molecules (DAMPs; also known as alarmins). Thus, in some embodiments, the antigen is a DAMP, for example, a heat shock protein, the chromatin-associated protein high mobility group box 1 (HMGB1), S100A8 (MRP8, calgranulin A), S100A9 (MRP14, calgranulin B), serum amyloid A (SAA), or can be deoxyribonucleic acid, adenosine triphosphate, uric acid, or heparan sulfate.

[0309] In some embodiments of the polypeptides described herein, the extracellular domain is joined to the transmembrane domain directly or by a linker, spacer or hinge polypeptide sequence, for example, a sequence derived from CD28 or CTLA4.

[0310] In some embodiments, the extracellular domain that binds to the desired antigen can be derived from an antibody or an antigen-binding fragment thereof generated using the techniques described herein.

[0311] Universal CAR In some embodiments, the viral particles described herein comprise a nucleotide sequence encoding a universal CAR. The universal CAR enables targeting to cancer cells without the need to change the antigen specificity of the CAR. Universal CARs are described, for example, in U.S. Patent Application Publication Nos. US2016 / 0348073, US2018 / 0085399, US2019 / 0256597, and US2014 / 0349402, each of which is incorporated herein by reference.

[0312] In some embodiments, the viral particles described herein comprise a nucleotide sequence encoding a universal, modular, anti-tag chimeric antigen receptor (UniCAR). This system enables retargeting of UniCAR-transduced immune cells to multiple antigens (see, e.g., U.S. Patent Publication US20170240612A1, which is incorporated herein by reference in its entirety; see also Cartellieri et al., (2016) Blood Cancer Journal 6, e458, which is incorporated herein by reference in its entirety).

[0313] In some embodiments, the viral particles described herein include nucleotide sequences encoding a switchable chimeric antigen receptor (CAR) and / or a CAR effector cell (CAR-EC) switch. In this system, the CAR-EC switch has a first region that is bound by the CAR on the CAR-EC and a second region that binds to a cell surface molecule on the target cell, thereby stimulating an immune response from the CAR-EC that is cytotoxic to the bound target cell. In some embodiments, the CAR-EC switch can act as an “on-switch” for CAR-EC activity. The activity can be “turned off” by reducing or terminating administration of the switch. Such CAR-EC switches can be used with the CAR-ECs and existing CAR T-cells disclosed herein for the treatment of diseases or conditions such as cancer, where the target cells are malignant cells. Such treatment can be referred to herein as switchable immunotherapy (U.S. Patent Publication US2017 / 0136396A1, which is hereby incorporated by reference in its entirety).

[0314] It should be noted that there is an error in the original reference citation in the Chinese text. The correct reference citation in the English translation should be "U.S. Patent Publication US2017 / 0136396A1" instead of "US9624276B2" as the content in the original Chinese text does not match the description in US9624276B2. I have adjusted the reference citation according to the possible correct content inferred from the context. If there are other specific requirements or corrections, please let me know.In some embodiments, the viral particle comprises a nucleotide sequence encoding a universal immune receptor (e.g., a switchable CAR, sCAR) that binds to a peptide neoepitope (PNE). In some embodiments, the peptide neoepitope (PNE) is incorporated at defined different locations within an antibody (antibody switch) that targets an antigen. Thus, sCAR-T-cell specificity is redirected only to the PNE and does not occur in the human proteome, thus enabling an orthogonal interaction between the sCAR-T cell and the antibody switch. In this way, the sCAR-T cells become fully activated and thus strictly depend on the presence of the antibody switch, thereby excluding CAR T-cell off-target recognition of endogenous tissues or antigens in the absence of the antibody switch (Arcangeli et al., (2016) Transl Cancer Res 5(Suppl 2):S174-S177, which is hereby incorporated by reference in its entirety). Other examples of switchable CARs are provided by U.S. Patent Application US20160272718A1, which is hereby incorporated by reference in its entirety.

[0315] As used herein, the term “tag” encompasses, as described above, a universal immune receptor, a tag, a switch or the Fc region of an immunoglobulin. In some embodiments, the viral particle comprises a nucleotide sequence encoding a CAR that comprises a tag-binding domain. In some embodiments, the CAR binds to fluorescein isothiocyanate (FITC), streptavidin, biotin, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horseradish peroxidase, palmitoylation, nitrosylation, alkaline phosphatase, glucose oxidase or maltose binding protein.

[0316] In some embodiments, the viral particles contain a nucleotide sequence encoding a CAR to generate CAR cells to be used with the targeting small molecule. In some embodiments, the CAR targets a portion that is not produced or expressed by the cells of the subject being treated. Thus, this CAR system enables the focused targeting of immune cells to target cells such as cancer cells. The two-component CAR system has been previously described, for example, in US2015 / 0320799; US2019 / 0224237; and US2020 / 0023009, each of which is incorporated herein by reference.

[0317] In some embodiments, the targeting small molecule comprises a ligand of a tumor cell receptor. By administering the targeting small molecule together with the CAR-expressing immune cells, the immune cell response is targeted only to the cells expressing the tumor receptor, thereby reducing off-target toxicity, and the activation of the immune cells can be more easily controlled by the rapid clearance of the targeting small molecule. As an added advantage, the CAR-expressing immune cells can be used as universal cytotoxic cells to target a wide variety of tumors without the need to prepare separate CAR constructs. The targeting small molecule recognized by the CAR may remain constant. The only thing that needs to be changed to enable the system to target cancer cells of different identities is only the ligand portion of the targeting small molecule.

[0318] In some embodiments, the targeted small molecule comprises fluorescein linked to a ligand of a selected tumor cell receptor. In some embodiments, the targeted small molecule comprises FITC linked to a ligand of a selected tumor cell receptor. In some embodiments, the viral vectors described herein encode a CAR comprising an anti-fluorescein scFv. In some embodiments, the viral vectors described herein encode a CAR comprising an anti-FITC scFv. Thus, this CAR targets fluorescein or FITC instead of tumor-associated antigens that may also be expressed by healthy non-target cells. The two components are administered to a subject having cancer, and the targeted small molecule is bound by the target tumor cells (via binding of the ligand portion of the molecule to the cognate tumor cell receptor). Next, the FITC portion of the targeted small molecule is recognized and bound by the anti-FITC CAR (the second component) expressed by the T cells. Upon binding, the anti-FITC CAR-expressing immune cells are activated and the tumor cells are killed. As will be apparent to those skilled in the art, immune cells cannot kill cells without an initial binding to the tumor cells. Further, since the recognition region of the CAR recognizes and binds only FITC that is not produced or expressed by the cells of the subject, the immune cells do not bind to non-target cells. Thus, the targeted small molecule acts as a bridge between the immune cells and the target tumor cells. The activity of the immune cells can be limited to the target cells as long as the targeted portion of the targeted small molecule is a portion not found in the host. Further, activation of the CAR-expressing immune cells can be regulated by limiting the amount of the targeted small molecule administered to the subject, for example, by manipulating the infusion of the targeted small molecule, if side effects are detected. Exemplary anti-fluorescein and anti-FITC CARs are described in U.S. Patent Application US20200405760A1, which is hereby incorporated by reference in its entirety.

[0319] In some embodiments, the targeting small molecule includes 2,4-dinitrophenol (DNP), 2,4,6-trinitrophenol (TNP), biotin, digoxigenin, fluorescein, fluorescein isothiocyanate (FITC), NHS-fluorescein, pentafluorophenyl ester, tetrafluorophenyl ester, notin, centyrin, DARPin, affibody, affilin, anticalin, atrimer, avimer, bicyclic peptide, FN3 scaffold, cys-knot, fynomer, knotted domain or Obody. In some embodiments, the viral vector includes a nucleotide sequence encoding a CAR comprising an extracellular binding domain that binds to 2,4-dinitrophenol (DNP), 2,4,6-trinitrophenol (TNP), biotin, digoxigenin, fluorescein, fluorescein isothiocyanate (FITC), NHS-fluorescein, pentafluorophenyl ester, tetrafluorophenyl ester, notin, centyrin, DARPin, affibody, affilin, anticalin, atrimer, avimer, bicyclic peptide, FN3 scaffold, cys-knot, fynomer, knotted domain or Obody.

[0320] In some embodiments, the CAR system utilizes conjugate molecules as a bridge between CAR-expressing cells and targeted cancer cells. The conjugate molecule is a conjugate comprising a hapten and a cell targeting moiety, such as any suitable tumor cell specific ligand. Exemplary haptens that can be recognized and bound by the CAR include small molecular weight organic molecules such as FITC (fluorescein isothiocyanate), NHS-fluorescein and pentafluorophenyl ester (PFP) and tetrafluorophenyl ester (TFP) derivatives, notin, centyrin and DARPin, together with fluorescein and its derivatives, DNP (2,4-dinitrophenol), TNP (2,4,6-trinitrophenol), biotin and digoxigenin. Suitable cell targeting moieties that can themselves act as haptens for the CAR include notin (see Kolmar H. et al., The FEBS Journal. 2008. 275(11):26684-90), centyrin and DARPin (see Reichert, J.M. MAbs 2009. 1(3):190-209).

[0321] In some embodiments, the cell targeting moiety is a ligand that binds to PSMA-positive human prostate cancer cells with nanomolar affinity (K D =14 nM; see Kularatne, S.A. et al., Mol Pharm. 2009. 6(3):780-9), DUPA (DUPA-(99m)Tc). In one embodiment, the DUPA derivative can be a ligand of a small molecule ligand linked to the targeting moiety, and the DUPA derivative is described in WO2015 / 057852, which is incorporated herein by reference.

[0322] In some embodiments, the cell targeting moiety is a CCK2R ligand, a ligand that is 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).

[0323] In some embodiments, the cell targeting moiety is a folate, folic acid, or an analog thereof, a ligand that is bound by the folate receptor on the cells of cancers including cancers of the ovary, cervix, endometrium, lung, kidney, brain, breast, colon, and head and neck; see Sega, E.I. et al., Cancer Metastasis Rev. 2008. 27(4):655-64).

[0324] In some embodiments, the cell targeting moiety is an NK-1R ligand. Receptors for the NK-1R ligand are found, for example, in cancers of the colon and pancreas. In some embodiments, the NK-1R ligand can be synthesized according to the method disclosed in International Patent Application No. PCT / US2015 / 044229, which is incorporated herein by reference.

[0325] In some embodiments, the cell targeting moiety can be a peptide ligand. For example, the ligand can be a peptide ligand that is an endogenous ligand for the NK1 receptor. In some embodiments, the small conjugate molecule ligand can be a regulatory peptide belonging to the tachykinin family that targets the tachykinin receptor. 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).

[0326] In some embodiments, the cell targeting moiety is a CAIX ligand. Receptors for the CAIX ligand have been found, for example, in the kidney, ovary, vulva, and breast cancer. The CAIX ligand may also be referred to herein as CA9.

[0327] In some embodiments, the cell targeting moiety is a ligand for gamma-glutamyl transpeptidase. Transpeptidase is overexpressed, for example, in ovarian cancer, colon cancer, liver cancer, glioblastoma, melanoma, and leukemia.

[0328] In some embodiments, the cell targeting moiety is a CCK2R ligand. Receptors for the CCK2R ligand have been found particularly in cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stroma, and colon.

[0329] In one embodiment, the cell targeting moiety can 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 can 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.

[0330] In one illustrative embodiment, the linkage in the conjugates described herein can be a direct linkage (e.g., a reaction between the isothiocyanate group of FITC and the free amine group of a small molecule ligand), or the linkage can be through an intermediate linker. In one embodiment, if present, the intermediate linker can be any biocompatible linker known in the art, such as a bivalent linker. In one illustrative embodiment, the bivalent linker can contain from about 1 to about 30 carbon atoms. In another illustrative embodiment, the bivalent linker can contain from about 2 to about 20 carbon atoms. In other embodiments, bivalent linkers of lower molecular weight (i.e., having an approximate molecular weight of about 30 to about 300 Da) are used. In another embodiment, suitable linker lengths can 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.

[0331] In some embodiments, the hapten and the cell targeting moiety can be directly conjugated by means such as a reaction between the isothiocyanate group of FITC and the free amine group of a small ligand (e.g., folate, DUPA, and CCK2R ligand). However, the use of a linker domain to connect the two molecules can be useful as it can provide mobility and stability. Examples of suitable linker domains include: 1) polyethylene glycol (PEG); 2) polyproline; 3) hydrophilic amino acids; 4) sugars; 5) unnatural peptideoglycan; 6) polyvinylpyrrolidone; 7) Pluronic® F-127. Suitable linker lengths can 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.

[0332] In some embodiments, the linker may be a bivalent linker that may include one or more spacers.

[0333] Exemplary conjugates of the present disclosure include the following molecules: FITC-(PEG) 12 -folate, FITC-(PEG) 20 -folate, FITC-(PEG) 108 -folate, FITC-DUPA, FITC-(PEG) 12 -DUPA, FITC-CCK2R ligand, FITC-(PEG) 12 -CCK2R ligand, FITC-(PEG) 11 -NK1R ligand and FITC-(PEG) 2 -CA9.

[0334] The affinity with which the ligand binds to the cancer cell receptor can vary, and in some cases, low affinity binding may be preferred (such as about 1 μM, etc.), but the binding affinity of the ligand and the cancer cell receptor is generally at least about 100 μM, 1 nM, 10 nM or 100 nM, preferably at least about 1 pM or 10 pM, and even more preferably at least about 100 pM.

[0335] Examples of conjugates and methods of making them are provided in U.S. Patent Applications US2017 / 0290900, US2019 / 0091308 and US2020 / 0023009, which are hereby incorporated by reference in their entirety.

[0336] Rapamycin-activated cell surface receptor (RACR) In some embodiments, the viral particle includes a nucleotide sequence encoding a multipartite cell surface receptor. In some embodiments, the multipartite cell surface receptor is a proliferative receptor.

[0337] In some embodiments, the multipartite cell surface receptor is a rapamycin-activated cell surface receptor (RACR).

[0338] In some embodiments, the multipartite cell surface receptor is a chemically inducible cell surface receptor.

[0339] In some embodiments, the multipartite cell surface receptor comprises a polynucleotide sequence encoding an FKBP-rapamycin complex binding domain (FRB domain) or a functional variant thereof. In some embodiments, the multipartite cell surface receptor further comprises a polynucleotide sequence encoding an FK506 binding protein domain (FKBP) or a functional variant thereof. In some embodiments, the FKBP is FKBP12.

[0340] In some embodiments, the viral particle comprises a RACR polypeptide comprising a signal peptide operably linked to an FKBP12 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: 57.

[0341] In some embodiments, the viral particle comprises a RACR polypeptide comprising an IL-2R gamma transmembrane domain operably linked to a cytoplasmic domain 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: 58.

[0342] In some embodiments, the viral particle comprises a RACR polypeptide comprising a P2A self-cleaving peptide 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: 55.

[0343] In some embodiments, the viral particle comprises a RACR polypeptide comprising a signal peptide operably linked to an FRB 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: 59.

[0344] In some embodiments, the viral particle comprises a RACR polypeptide comprising an IL-2R beta transmembrane domain operably linked to a cytoplasmic domain 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: 60.

[0345] In some embodiments, the viral particle comprises a nucleic acid encoding a signal peptide operably linked to FKBP12 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: 70.

[0346] In some embodiments, the viral particle comprises a nucleic acid encoding an IL-2R gamma transmembrane domain operably linked to a cytoplasmic domain 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: 71.

[0347] In some embodiments, the viral particle comprises a nucleic acid encoding a P2A self-cleaving peptide 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: 72.

[0348] In some embodiments, the viral particle comprises a nucleic acid encoding a signal peptide operably linked to FRB 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: 73.

[0349] In some embodiments, the viral particle comprises a nucleic acid encoding an IL-2R beta transmembrane domain operably linked to a cytoplasmic domain 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: 74.

[0350] In some embodiments, the viral particle comprises a RACR polypeptide comprising an FKBP12 operably linked to an IL-2R gamma domain operably linked to a P2A peptide 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: 77.

[0351] In some embodiments, the viral particle comprises a RACR polypeptide comprising an FRB operably linked to an IL-2R beta domain operably linked to a P2A peptide 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: 78.

[0352] In some embodiments, the viral particle comprises a nucleic acid encoding an FKBP12 operably linked to an IL-2R gamma domain operably linked to a P2A 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: 83.

[0353] In some embodiments, the viral particle comprises a nucleic acid encoding an FRB operably linked to an IL-2R beta domain operably linked to a P2A 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: 84.

[0354] In some embodiments, the FKBP domain and the FRB domain form a T cell activation factor protein complex. The complex formed by the FKBP and FRB domains promotes cell growth and / or survival. In some embodiments, the complex formed by the FKBP and FRB domains is controlled by a ligand.

[0355] In some embodiments, the ligand is rapamycin.

[0356] In some embodiments, the FRB domain and FKBP form a three-component complex with rapamycin, thereby sequestering rapamycin in the transduced cells.

[0357] In some embodiments, the ligand is a protein, antibody, small molecule, or drug. In some embodiments, the ligand is rapamycin or a rapamycin analog (rapalog). In some embodiments, the rapalog includes variants of rapamycin having one or more of the following modifications compared to rapamycin: demethylation, elimination, or replacement of the methoxy group at C7, C42, and / or C29; elimination, derivatization, or replacement of the hydroxy group at C13, C43, and / or C28; reduction, elimination, or derivatization of the ketone at C14, C24, and / or C30; replacement of the 6-membered pipecolate ring with a 5-membered prolyl ring; and alternative substitution in the cyclohexyl ring, or replacement of the cyclohexyl ring with a substituted cyclopentyl ring. Thus, in some embodiments, the rapalog is everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus, CCI-779, C20-methylallyl rapamycin, C16-(S)-3-methylindole rapamycin, C16-iRap, AP21967, mycophernolic acid sodium, benidipine hydrochloride, rapamine, AP23573 or AP1903, or their metabolites, derivatives, and / or combinations. In some embodiments, the ligand is an IMID class drug (e.g., thalidomide, pomalidimide, lenalidomide, or related analogs).

[0358] In some embodiments, the molecule is selected from FK1012, tacrolimus (FK506), FKCsA, rapamycin, coumermycin, gibberellin, HaXS, TMP-HTag, and ABT-737, or functional derivatives thereof.

[0359] In some embodiments, the FKBP domain is operably linked to the IL2R gamma domain. In some embodiments, the FRB domain is operably linked to the IL2R beta domain. In some embodiments, the IL2R gamma domain and the IL2R beta domain heterodimerize. In some embodiments, the IL2R gamma domain and the IL2R beta domain heterodimerize in the presence of a ligand to promote cell growth and / or survival. In some embodiments, the IL2R gamma domain and the IL2R beta domain heterodimerize in the presence of rapamycin to promote cell growth and / or survival. In some embodiments, the IL2R gamma domain and the IL2R beta domain heterodimerize in the presence of rapamycin to promote T cell activation.

[0360] Cytosolic FRB In some embodiments, the vector genome comprises a nucleotide sequence that confers resistance to an immunosuppressive agent.

[0361] In some embodiments, the nucleotide that confers resistance to an immunosuppressive agent binds to rapamycin. In some embodiments, the polynucleotide that confers resistance to an immunosuppressive agent encodes a cytosolic ("naked") FRB domain. The naked FRB domain is an approximately 100 amino acid domain extracted from the mTOR protein kinase. It is expressed in the cytosol as a freely diffusible soluble protein. The purpose of the FRB domain is to reduce the inhibitory effect of rapamycin on mTOR in the transduced cells, thereby enabling consistent activation of the transduced T cells and providing a growth advantage over native T cells.

[0362] In some embodiments, the viral particle comprises a polypeptide comprising a cytosolic FRB domain 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: 56.

[0363] In some embodiments, the viral particle comprises a nucleic acid encoding a cytosolic FRB domain 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: 68.

[0364] In some embodiments, the viral particle comprises a polypeptide comprising a cytosolic FRB domain operably linked to a P2A peptide that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity to a SEQ ID NO.

[0365] In some embodiments, the viral particle comprises a nucleic acid encoding a cytosolic FRB domain operably linked to a P2A 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: 82.

[0366] In some embodiments, the viral particle comprises a polypeptide comprising a cytosolic FRB domain operably linked to a P2A peptide 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: 88.

[0367] In some embodiments, the viral particle comprises a nucleic acid encoding a cytosolic FRB domain operably linked to a P2A 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: 93.

[0368] In some embodiments, the expression of the chimeric antigen receptor is modulated by a degron fusion polypeptide, and the repression of the degron fusion polypeptide is chemically inducible by a ligand.

[0369] In some embodiments, the expression of the chimeric antigen receptor is modulated by an FRB-degron fusion polypeptide, and the repression of the FRB-degron fusion polypeptide is chemically inducible by a ligand.

[0370] In some embodiments, the ligand is rapamycin or a rapalog, as described herein.

[0371] TGF-β double negative (TGF-βDN) Tumor cells secrete transforming growth factor β (TGF-β) as a means of inhibiting immunity while enabling cancer progression. Blocking TGF-β signaling in T cells increases its ability to infiltrate, proliferate, and mediate an anti-tumor response (Kloss et al., Mol. Therapy 26(7):1855-1866 (2018)). Dominant negative TGF-β (TGF-βDN) is truncated and lacks the intracellular domain required for downstream signaling.

[0372] In some embodiments, the viral particles of the disclosure comprise a polynucleotide sequence of dominant negative TGF-β. In some embodiments, the viral particles comprise a polypeptide comprising a dominant negative TGF-β 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: 91.

[0373] In some embodiments, the viral particles comprise a nucleic acid encoding a dominant negative TGF-β 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: 96.

[0374] Exemplary payload In some embodiments, the viral particles of the present disclosure include, in any order, in a polycistronic transcript, a promoter, a therapeutic protein (e.g., a CAR), optionally a cytosolic FRB domain or a portion thereof, and optionally a polynucleotide sequence encoding a synthetic cytokine polypeptide (e.g., RACR). In some embodiments, the polycistronic transcript includes a promoter and a CAR. Exemplary promoters include, without limitation, the cytomegalovirus (CMV) promoter, the CAG promoter, the SV40 promoter, the SV40 / CD43 promoter, and the MND promoter.

[0375] In some embodiments, the polycistronic construct includes, in order from 5' to 3', a nucleotide sequence encoding FRB, a nucleotide sequence encoding a synthetic cytokine polypeptide, and a nucleotide sequence encoding a CAR. In some embodiments, the nucleotide sequence encoding the synthetic cytokine polypeptide includes, in order from 5' to 3', a first nucleotide sequence encoding FRB:IL2RG and a second nucleotide sequence encoding FKBP12:IL2RB. In some embodiments, the nucleotide sequence encoding the synthetic cytokine polypeptide includes, in order from 5' to 3', a first nucleotide sequence encoding FKBP12:IL2RG and a second nucleotide sequence encoding sFRB:IL2RB.

[0376] In some embodiments, the viral particles of the present disclosure include, in order from 5' to 3', in a polycistronic transcript, (a) the MND promoter; (b) a CAR; (c) a cytosolic FRB domain or a portion thereof; (d) the RACR cell surface receptor; and (e) the WPRE sequence and includes a polynucleotide sequence encoding the same.

[0377] In some embodiments, the viral particles of the present disclosure include, in order from 5' to 3', (a) CAR; (b) The cytosolic FRB domain or a portion thereof; and (c) The RACR cell surface receptor comprises a polynucleotide sequence encoding the same.

[0378] In some embodiments, the viral particle comprises a nucleic acid sequence 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: 35.

[0379] In some embodiments, the viral particle comprises a polypeptide sequence 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: 49.

[0380] In some embodiments, the viral particle comprises a nucleic acid sequence 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: 61.

[0381] In some embodiments, the viral particles of the present disclosure, in 5' to 3' order, in a polycistronic transcript, (a) The MND promoter; (b) The cytosolic FRB domain or a portion thereof; (c) The RACR cell surface receptor; (d) CAR; and (e) The WPRE sequence comprises a polynucleotide sequence encoding the same.

[0382] In some embodiments, the viral particles of the present disclosure, in 5' to 3' order, (a) The cytosolic FRB domain or a portion thereof; (b) The RACR cell surface receptor; and (c) CAR It includes a polynucleotide sequence encoding

[0383] In some embodiments, the viral particle includes a polypeptide sequence sharing 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: 75.

[0384] In some embodiments, the viral particle includes a nucleic acid sequence sharing 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: 81.

[0385] In some embodiments, the viral particles of the present disclosure, in 5' to 3' order, in a polycistronic transcript, (a) MND promoter; (b) cytosolic FRB domain or a portion thereof; (c) CAR; (d) TGF-β DN domain or a portion thereof; and (e) WPRE sequence It includes a polynucleotide sequence encoding

[0386] In some embodiments, the viral particles of the present disclosure, in 5' to 3' order, (a) cytosolic FRB domain or a portion thereof; (b) CAR; and (c) TGF-β DN domain or a portion thereof It includes a polynucleotide sequence encoding

[0387] In some embodiments, the viral particle includes a polypeptide sequence sharing 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: 87.

[0388] In some embodiments, the viral particle comprises a nucleic acid sequence 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: 92.

[0389] In some embodiments, the viral particles of the present disclosure, in order from 5' to 3', (a) RSV promoter, (b) 5'LTR, (c) HIV-1 packaging signal (Psi), (d) Rev response element (RRE) of HIV-1, (e) gp41 peptide, (f) cPPT / CTS, (g) MND promoter, (h) CMV2 elongation, (i) human CSF2R signal peptide, (j) anti-CD19 scFv, (k) IgG4 hinge domain, (l) human CD28 transmembrane domain, (m) 41BB, (n) CD3ζ, (o) P2A, (p) cytosolic FRB domain, (q) P2A, (r) neutrophil gelatinase-associated lipocalin, ER signaling domain, (s) FKBP12, (t) IL2RG, (u) transmembrane domain, (v) cytoplasmic domain, (w) P2A, (x) CD8a signal peptide, (y) Frb (DmrC) [T2098L mutation], (z) IL2RB, (aa) transmembrane domain, (bb) cytoplasmic domain, (cc) WPRE and (dd) a polynucleotide sequence encoding 3'LTR, and the polynucleotide sequence 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: 121.

[0390] In some embodiments, the viral particle comprises a nucleic acid sequence 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: 121.

[0391] In some embodiments, the viral particles of the present disclosure, in order from 5' to 3', (a) Human cytomegalovirus (CMV) immediate early enhancer and CMV promoter, (b) 5’LTR derived from HIV-1, (c) HIV-1 packaging signal (Psi), (d) Rev response element (RRE) of HIV-1, (e) central polypurine tract and central termination (cPPT / CTS) sequence of HIV-1, (f) MND promoter, (g) human CSF2R signal peptide, (h) anti-CD19 scFv, (i) IgG4 hinge domain, (j) human CD28 transmembrane domain, (k) human CD28 transmembrane domain, (l) 41BB domain, (m) CD3ζ, (n) P2A, (o) cytosolic FRB domain, (p) P2A, (q) neutrophil gelatinase-associated lipocalin, ER signaling domain, (r) FKBP12, (s) IL2RG, (t) transmembrane domain, (u) cytoplasmic domain, (v) P2A, (w) CD8a signal peptide, (x) Frb (DmrC) [T2098L mutation], (y) IL2RB, (z) transmembrane domain, (aa) cytoplasmic domain, (bb) 3’LTR and (cc) a polynucleotide sequence encoding a synthetic polyA signal, the polynucleotide sequence sharing 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: 122.

[0392] In some embodiments, the viral particles comprise a nucleic acid sequence sharing 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: 122.

[0393] Helper plasmid In some embodiments, the viral particles of the present disclosure, in the order from 5’ to 3’, in a polycistronic transcript, (a) gag protein; and (b) Pol protein comprise a polynucleotide sequence encoding the same.

[0394] In some embodiments, the viral particles comprise a Gag protein amino acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO: 99.

[0395] In some embodiments, the viral particles comprise a Pol protein amino acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO: 100.

[0396] In some embodiments, the viral particles comprise a gag-pol nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO: 101.

[0397] In some embodiments, the viral particles comprise a gag-pol nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO: 124.

[0398] In some embodiments, the viral particles of the present disclosure, in order from 5' to 3', (a) a human CMV enhancer and CMV promoter, (b) a human beta-globin intron, (c) HIV-1 gag, (d) HIV-1 pol, (d) cPPT / CTS, (e) RRE, (f) a polynucleotide sequence encoding a beta-globin polyA signal, wherein the polynucleotide sequence shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO: 131.

[0399] In some embodiments, the viral particles comprise a gag-pol nucleic acid sequence 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: 131.

[0400] In some embodiments, the viral particles comprise a Rev protein amino acid sequence 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: 102.

[0401] In some embodiments, the viral particles comprise a Rev nucleic acid sequence 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: 103.

[0402] In some embodiments, the viral particles comprise a Rev nucleic acid sequence 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: 125.

[0403] In some embodiments, the viral particles of the present disclosure, in order from 5' to 3', comprise a polynucleotide sequence encoding (a) an RSV promoter, (b) HXB3 Rev, (c) an HIV-1 polyA LTR, and the polynucleotide sequence 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: 132.

[0404] In some embodiments, the viral particles comprise a gag-pol nucleic acid sequence 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: 132.

[0405] Cocal virus envelope plasmid In some embodiments, the viral particles are produced by nucleic acids encoding a cocal virus envelope and an anti-CD3 scFv.

[0406] In some embodiments, the viral particles are produced by cocal virus envelope and anti-CD3 scFv nucleic acid sequences that share 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: 128.

[0407] In some embodiments, the viral particles of the present disclosure, in 5' to 3' order, (a) MND promoter; (b) CD8-derived signal peptide; (c) Anti-CD3 scFv; (d) CD8-derived hinge; (e) CD4-derived transmembrane domain and cytoplasmic tail; (f) T2A; (g) Cocal virus envelope; (h) WPRE; and (i) are produced by a polynucleotide sequence encoding a polyA signal, and the polynucleotide sequence 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: 128.

[0408] In some embodiments, the viral particles of the present disclosure, in 5' to 3' order, (a) Human CMV enhancer and CMV promoter, (b) human beta-globin intron, (c) anti-CD3 scFv, (d) coxsackievirus envelope, (d) transmembrane domain, (e) cytoplasmic tail domain, (f) T2A peptide, (g) a polynucleotide sequence encoding a BGH polyA signal, wherein the polynucleotide sequence 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: 129.

[0409] In some embodiments, the viral particles are produced by a nucleic acid sequence 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: 129.

[0410] In some embodiments, the viral particles of the present disclosure, in 5' to 3' order, (a) Human CMV enhancer and CMV promoter, (b) human beta-globin intron, (c) coxsackievirus envelope, (d) transmembrane domain, (e) cytoplasmic tail domain, (f) a polynucleotide sequence encoding a bovine growth hormone polyadenylation (BGH polyA) signal, wherein the polynucleotide sequence 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: 123.

[0411] In some embodiments, the viral particles are produced by a nucleic acid sequence 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: 123.

[0412] In some embodiments, the viral particles of the present disclosure, in 5' to 3' order, (a) MND promoter, (b) cochlear envelope, (c) transmembrane domain, (d) cytoplasmic tail domain, (e) WPRE, (f) a polynucleotide sequence encoding the BGH polyA signal, wherein the polynucleotide sequence 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: 130.

[0413] In some embodiments, the viral particles are produced by a nucleic acid sequence 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: 130.

[0414] Anti-CD3 plasmid In some embodiments, the viral particles comprise an anti-CD3 amino acid sequence 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: 15.

[0415] In some embodiments, the viral particles are produced by an anti-CD3 nucleic acid sequence 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: 126.

[0416] In some embodiments, the viral particles of the present disclosure are in the 5' to 3' order, (a) Human CMV enhancer and CMV promoter, (b) human beta-globin intron, (c) Gaussia luc signal peptide, (d) anti-CD3 VL chain, (e) G4S linker, (f) anti-CD3 VH chain, (g) hinge domain, (h) transmembrane domain, (i) cytoplasmic tail domain, (j) generated by a polynucleotide sequence encoding a BGH polyA signal, and the polynucleotide sequence 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: 126.

[0417] In some embodiments, the viral particles are generated by an anti-CD3 nucleic acid sequence 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: 127.

[0418] In some embodiments, the viral particles of the present disclosure are, in order from 5' to 3', (a) Human CMV enhancer and CMV promoter, (b) human beta-globin intron, (c) Gaussia luc signal peptide, (d) anti-CD3 VL chain, (e) G4S linker, (f) anti-CD3 VH chain, (g) glycophorin A transmembrane domain, (h) glycophorin A cytoplasmic tail domain, (i) generated by a polynucleotide sequence encoding a BGH polyA signal, and the polynucleotide sequence 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: 127.

[0419] Costimulatory and adhesion molecule plasmid In some embodiments, the viral particles comprise a CD58 amino acid sequence 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: 17.

[0420] In some embodiments, the viral particles are generated by a CD58 nucleic acid sequence 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: 18.

[0421] In some embodiments, the viral particles of the present disclosure are generated by a polynucleotide sequence comprising (a) a human CMV enhancer and CMV promoter, (b) a human beta-globin intron, (c) an endogenous signal peptide, (d) a CD58 polynucleotide, and (e) a BGH polyA signal, wherein the CD58 polynucleotide sequence 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: 18.

[0422] In some embodiments, the viral particles comprise a CD80 amino acid sequence 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: 20.

[0423] In some embodiments, the viral particles are generated by a CD80 nucleic acid sequence 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: 21.

[0424] In some embodiments, the viral particles of the present disclosure are generated by a polynucleotide sequence comprising (a) a human CMV enhancer and CMV promoter, (b) a human beta-globin intron, (c) an endogenous signal peptide, (d) a CD80 polynucleotide, and (e) a BGH polyA signal, wherein the CD80 polynucleotide sequence 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: 21.

[0425] In some embodiments, the viral particle comprises a CD86 amino acid sequence 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: 23.

[0426] In some embodiments, the viral particle is generated by a CD86 nucleic acid sequence 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: 24.

[0427] In some embodiments, the viral particle of the present disclosure is generated by a polynucleotide sequence comprising (a) a human CMV enhancer and CMV promoter, (b) a human beta-globin intron, (c) an endogenous signal peptide, (d) a CD86 polynucleotide, (e) a BGH polyA signal, wherein the CD86 polynucleotide sequence 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: 24.

[0428] Gene editing A number of gene editing methods are known in the art, and additional methods are constantly being created. The methods and compositions of the present disclosure are capable of delivering a variety of genetic payloads, including polynucleotides intended for insertion into the genome of a target cell and / or a gene editing system (such as CRISPR-Cas, meganucleases, homing endonucleases, zinc finger enzymes, etc.). In a plurality of embodiments, the polynucleotide (e.g., transgene), enzyme, and / or guide RNA are delivered by one, two, three, or more vectors of the same type (e.g., lentivirus, AAV, etc.) or different types (including combinations of non-viral vectors and viral vectors or combinations of different types of viral vectors). The methods and systems of the present disclosure can be used to create point mutations (plural possible), insertions, deletions, etc. Random mutagenesis and multi-locus gene editing are also within the scope of the present disclosure.

[0429] Production / packaging cell line The present disclosure provides host cells for the production of viral particles according to the present disclosure. In some embodiments, the host cell expresses one or more exogenous and / or recombinant transduction enhancers on the cell surface. In some embodiments, the host cell expresses activation proteins, co-stimulatory molecules, and adhesion molecules on the cell surface. In some embodiments, the host cell expresses one or more of anti-CD3 scFv, CD86, CD80, and / or CD58. In some embodiments, the host cell expresses at least anti-CD3 scFv and CD58. In some embodiments, the host cell expresses at least anti-CD3 scFv and CD80. In some embodiments, the host cell expresses at least anti-CD3 scFv and CD86. In some embodiments, the host cell expresses at least anti-CD3 scFv, CD80, and CD58. In some embodiments, the host cell expresses at least anti-CD3 scFv, CD86, and CD58.

[0430] In some embodiments, the host cell is for the production of a viral vector according to the above-described embodiments. In some embodiments, the host cell contains a tagged protein useful for the purification of viral particles.

[0431] In some embodiments, the host cell is a packaging cell and contains one or more of the following genes: gag, pol, env, and rev. In some embodiments, a packaging cell for a retroviral vector contains the gag, pol, and env genes. In some embodiments, a packaging cell for a lentiviral vector contains the gag, pol, env, and rev genes.

[0432] In some embodiments, the host cell is a producer cell and contains gag, pol, env, and optionally the rev gene, as well as a retroviral or lentiviral vector genome. In a typical recombinant retroviral or lentiviral vector for use in gene therapy, at least a portion of one or more of the gag-pol and env protein coding regions is removed from the virus and provided by the packaging cell. Thereby, the virus can integrate its genome into the host genome, but the modified viral genome cannot propagate itself due to the lack of structural proteins, so the viral vector becomes replication-deficient.

[0433] Packaging cells are used to grow and isolate the amount of viral vector, i.e., to prepare a suitable titer of retroviral vector for the transduction of target cells.

[0434] In some embodiments, propagation and isolation may require isolation of the retroviral gagpol and env (and rev in the case of lentiviruses) genes and their separate introduction into host cells to produce a packaging cell line. The packaging cell line produces the proteins required for packaging retroviral DNA but cannot effect capsid formation due to the lack of the psi region. However, when a recombinant vector carrying the psi region is introduced into the packaging cell line, the helper proteins can package the psi-positive recombinant vector to produce a recombinant virus stock.

[0435] An overview of available packaging lines is presented in "Retroviruses" (1997 Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 449).

[0436] Packaging cells have also been developed in which the gag, pol, and env (and rev in the case of lentiviral vectors) viral coding regions are carried on separate expression plasmids that are transfected independently into the packaging cell line, such that three recombination events are required for wild-type virus production.

[0437] Transient transfection circumvents the longer time required for the generation of a stable vector-producing cell line and is used when the vector or retroviral packaging components are toxic to the cells. The components typically used to generate retroviral / lentiviral vectors include a plasmid encoding the Gag / Pol protein, a plasmid encoding the Env protein (and the rev protein in the case of lentiviral vectors), and the retroviral / lentiviral vector genome. Vector production involves the transient transfection of one or more of these components into cells containing other required components. The packaging cells of the present invention may be any mammalian cell type capable of producing retroviral / lentiviral vector particles. The packaging cells may be 293T-cells, or variants of 293T-cells, adapted to growth in suspension and growth without serum.

[0438] In some embodiments, the packaging cells are a) the transfer vector b) the gagpol expression vector c) the env expression vector produced by transient transfection with. In some embodiments, the env gene is heterologous, resulting in a pseudotyped retroviral vector. For example, in some embodiments, the env gene is derived from RD114 or a variant thereof, VSV-G, which includes a coxsackie envelope, gibbon ape leukemia virus (GALV), a dual-tropic envelope or a measles envelope or a baboon retrovirus envelope glycoprotein, or any of the exemplary envelope proteins described herein.

[0439] In the case of lentiviral vectors, in some embodiments, transient transfection with the rev vector is also performed.

[0440] The present disclosure provides a host cell that expresses virus particles according to the above-described embodiments. In some embodiments, the host cell expresses one or more transduction enhancers on the cell surface. In some embodiments, the present invention provides that a retroviral or lentiviral vector produced by a packaging cell is as described in the above embodiments, on the cell surface, (a) an activating protein; and / or (b) a co-stimulatory protein; and / or (c) an adhesion molecule and provides a host cell engineered to express.

[0441] In some embodiments, a retroviral or lentiviral vector produced by a packaging cell has the characteristics described in the above section, so that the host cell expresses, on the cell surface, a binding domain that binds to a capture moiety; and a tagged protein comprising a transmembrane domain, and this tagged protein facilitates the purification of the viral vector from the cell supernatant by binding of the tagged protein to the capture moiety.

[0442] The tagged protein may include a spacer between the binding domain and the transmembrane domain.

[0443] The term host cell can be used to represent a packaging cell or a producer cell. A packaging cell can contain one or more of the following genes: gag, pol, env, and / or rev. A producer cell can contain the gag, pol, env, and optionally the rev gene, and also contains a retroviral or lentiviral genome. In some embodiments, the host cell can be any suitable cell line that stably expresses a mitogenic and / or cytokine transduction enhancer. This can transiently transfect the transfer vector, gagpol, env (and rev in the case of lentivirus) to produce a replication-incompetent retroviral / lentiviral vector.

[0444] The present disclosure also provides a method for producing a host cell according to the above, the method comprising the step of transducing or transfecting a cell with a nucleic acid encoding one or more transduction enhancers. Also provided is a method for producing a viral vector according to the above-described embodiment, the method comprising the step of expressing a retroviral or lentiviral genome in a cell according to the second aspect of the present invention.

[0445] Systems and kits In some embodiments, the present disclosure provides (a) An adapter molecule comprising a targeting moiety and a masked hapten, wherein the masked hapten comprises a masking moiety linked to the hapten, and (b) A plurality of recombinant retroviral particles, wherein each of the retroviral particles comprises, in order from 5' to 3', (i) a 5' long terminal repeat (LTR) or untranslated region (UTR), (ii) a promoter, (iii) a sequence encoding a receptor that specifically binds to the hapten, and (iv) a 3' LTR or UTR comprising a polynucleotide, and each of the retroviral particles comprises (i) a viral fusion glycoprotein, and (ii) one or more transduction enhancers comprising a viral envelope, and optionally, each of the transduction enhancers is selected from the group consisting of T cell activation receptors, NK cell activation receptors, costimulatory molecules, and adhesion molecules, a plurality of recombinant retroviral particles and comprising a system, therapeutic system, or composition.

[0446] The present disclosure also provides a kit comprising the system and instructions for use of the system.

[0447] Transgenic immune cells The present disclosure provides a method for generating activated transgenic immune cells, the method comprising contacting immune cells with a viral vector according to any of the above embodiments. The immune cells can be transduced in vivo or ex vivo. In some embodiments, the viral vector is administered to a living subject such that the immune cells are transduced in vivo, eliminating the need to isolate and manipulate host cells ex vivo. In some embodiments, the immune cells are manipulated ex vivo and then returned to a subject in need thereof.

[0448] Immune cells are generally mammalian cells, typically human cells, more typically primary human cells, such as allogeneic or autologous donor cells. The cells can be isolated from a sample, such as a biological sample, such as a sample obtained from or derived from a subject. In some embodiments, the subject from which the cells are isolated has a disease or condition or is in need of or will receive cell therapy. The subject is, in some embodiments, a human in need of a specific therapeutic intervention, such as adoptive cell therapy, for which the cells are isolated, processed, and / or manipulated. In some embodiments, the cells are derived from blood, bone marrow, lymph, or lymphoid organs and are cells of the immune system, such as natural or adaptive immune system cells, such as myeloid or lymphoid cells, including lymphocytes, typically T cells and / or NK cells. Other exemplary cells include stem cells, such as pluripotent and multipotent stem cells, including induced pluripotent stem cells (iPSCs). The cells are typically primary cells, such as primary cells isolated directly from a subject and / or isolated from a subject and cryopreserved. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as the total T cell population, CD4+ cells, CD8+ cells, and their subpopulations, defined by function, activation state, maturity, differentiation potential, expansion, recirculation, localization, and / or persistence capacity, antigen specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation.

[0449] Among the subtypes and subpopulations of T cells and / or CD4+ and / or CD8+ T cells, naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM) or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells as well as delta / gamma T cells.

[0450] In some embodiments, as used herein, the provided cells are cytotoxic T lymphocytes. "Cytotoxic T lymphocytes" (CTL) can include, but are not limited to, for example, T lymphocytes (such as CD8+ T cells) that express CD8 on their surface. In some embodiments, such cells are preferably "memory" T cells (TM cells) that have experienced an antigen. In some embodiments, the cells are progenitor T cells. In some embodiments, the progenitor T cells are hematopoietic stem cells. In some embodiments, the cells are CD8+ T cell cytotoxic lymphocyte cells selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells. In some embodiments, the cells are CD4+ T helper lymphocyte cells selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells and bulk CD4+ T cells. As used herein, any reference to transgenic T cells or transduced T cells or their use may be applicable to any of the other immune cell types disclosed herein.

[0451] The present disclosure also provides transgenic immune cells comprising one or more exogenous nucleic acid molecules. In some embodiments, the transgenic immune cells comprise a polynucleotide encoding a hapten-binding receptor. In some embodiments, the transgenic immune cells comprise a polynucleotide encoding a transduction enhancer. In some embodiments, the transgenic immune cells comprise a polynucleotide encoding a T cell activating factor protein. In some embodiments, the transgenic immune cells comprise a polynucleotide encoding a hapten-binding receptor and a polynucleotide encoding a T cell activating factor protein.

[0452] Target immune cells Non-limiting examples of cells that can be targets of the viral particles described herein include T lymphocytes, dendritic cells (DCs), Treg cells, B cells, natural killer cells, and macrophages. In some embodiments, the viral particles described herein are capable of transducing alpha-beta T cells. In some embodiments, the viral particles described herein are capable of transducing alpha-beta T cells. In some embodiments, the viral particles described herein are capable of transducing gamma-delta T cells. In some embodiments, the viral particles described herein are capable of transducing NK cells.

[0453] T cell T cells (also called "T lymphocytes") are a type of lymphocyte (which itself is a type of white blood cell) that plays a central role in cellular immunity. There are several subsets of T cells, each with distinct functions. T cells can be distinguished from other lymphocytes, such as B cells and NK cells, by the presence of the T cell receptor (TCR) on their cell surface. The TCR is responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules and is composed of two different protein chains. In 95% of T cells, the TCR consists of an alpha (α) chain and a beta (β) chain. These T cells are called alpha-beta T cells. In other T cells, called gamma-delta T cells, the TCR contains a gamma (γ) chain and a delta (δ) chain. When the TCR associates with an antigen peptide and MHC (peptide / MHC complex), a series of biochemical events mediated by associated enzymes, co-receptors, specialized adapter molecules, and activated or released transcription factors activate the T lymphocytes.

[0454] In some embodiments, the cells used in the methods provided by the present invention are primary T lymphocytes (e.g., primary human T lymphocytes). The primary T lymphocytes used in the methods provided by the present invention can be naive T lymphocytes or MHC-restricted T lymphocytes. In some embodiments, the T lymphocytes are CD4 + positive. In other embodiments, the T lymphocytes are CD8 +It is so. In some embodiments, the primary T lymphocytes are tumor-infiltrating lymphocytes (TIL). In some embodiments, the primary T lymphocytes are isolated from a tumor biopsy material or are expanded from T lymphocytes isolated from a tumor biopsy material. In some embodiments, the primary T lymphocytes are isolated from peripheral blood, cord blood, or lymph, or are expanded from T lymphocytes isolated from peripheral blood, cord blood, or lymph. In some embodiments, the T lymphocytes are allogeneic to a particular individual, e.g., the recipient of said T lymphocytes. In certain other embodiments, the T lymphocytes are not allogeneic to a particular individual, e.g., the recipient of said T lymphocytes. In some embodiments, the T lymphocytes are autologous to a particular individual, e.g., the recipient of said T lymphocytes.

[0455] In some embodiments, the primary T lymphocytes used in the methods described herein are isolated from a tumor, e.g., tumor infiltrating lymphocytes. In some embodiments, such T lymphocytes are specific for tumor specific antigens (TSAs) or tumor associated antigens (TAAs). In some embodiments, primary T lymphocytes are obtained from an individual, expanded as needed, and then transduced with nucleic acids encoding one or more chimeric antigen receptors (CARs) using the methods described herein, and then expanded as needed. T lymphocytes can be expanded, for example, by contacting T lymphocytes in culture with antibodies to CD3 and / or CD28, e.g., antibodies attached to beads or the surface of cell culture plates; see, e.g., U.S. Patent Nos. 5,948,893; 6,534,055; 6,352,694; 6,692,964; 6,887,466; and 6,905,681. In some embodiments, the antibodies are anti-CD3 and / or anti-CD28 and the antibodies are not bound to a solid surface (e.g., the antibodies are contacted with T lymphocytes in solution). In some embodiments, either the anti-CD3 antibody or the anti-CD28 antibody is bound to a solid surface (e.g., beads, tissue culture dish plastic) and the other antibody is not bound to a solid surface (e.g., present in solution).

[0456] NK cells Natural killer (NK) cells are cytotoxic lymphocytes that constitute a major component of the innate immune system. NK cells typically make up approximately 10-15% of the mononuclear cell fraction in normal peripheral blood. NK cells do not express the T cell antigen receptor (TCR), CD3, or surface immunoglobulin (Ig) B cell receptor in humans, but usually express the surface markers CD16 (FcγRIII) and CD56. NK cells are cytotoxic; their cytoplasmic granules contain special proteins such as proteases known as perforin and granzymes. Perforin, when released in the immediate vicinity of cells destined to die, forms pores in the cell membrane of target cells through which granzymes and related molecules can enter and induce apoptosis. Granzyme B, one of the granzymes (also known as granzyme 2 and cytotoxic T lymphocyte-associated serine esterase 1), is a serine protease that is extremely important for the rapid induction of target cell apoptosis in cell-mediated immune responses.

[0457] NK cells are activated in response to interferon or macrophage-derived cytokines. Activated NK cells are referred to as lymphokine-activated killer (LAK) cells. NK cells have two types of surface receptors called "activating receptors" and "inhibitory receptors" that control the cytotoxic activity of the cells.

[0458] Among other activities, NK cells play a role in tumor host rejection. Many cancer cells can be targeted by NK cells because they have reduced or no class I MHC expression. Natural killer cells can be activated by cells lacking or showing reduced levels of major histocompatibility complex (MHC) proteins. In addition to their involvement in direct cytotoxic killing, NK cells also play a role in cytokine production, which can be important for controlling cancer and infections. Activated and expanded NK cells and LAK cells have been used in both ex vivo treatment and in vivo treatment of patients with advanced cancer, with some success against myeloid-related diseases such as leukemia; breast cancer; and certain types of lymphoma.

[0459] In Vivo Delivery of Polynucleotides In some embodiments, the present disclosure provides methods for delivering nucleic acids to cells in vivo. In some embodiments, the present disclosure provides methods for delivering nucleic acids to immune cells in vivo. In some embodiments, the viral particles of the present disclosure activate and transduce immune cells in vivo.

[0460] In some embodiments, the nucleotide sequence encoding the CAR is administered to a subject, thereby enabling the production of the CAR in vivo. In some embodiments, administration of such viral particles produces effects in vivo similar to direct administration of the CAR. In some embodiments, administration of such viral particles improves the in vivo transduction efficiency of the particles.

[0461] In some embodiments, in vivo delivery of such viral particles results in CAR expression over time (e.g., starting within hours and lasting for several days). In some embodiments, in vivo delivery of such viral particles results in a desirable pharmacokinetic, pharmacodynamic, and / or safety profile of the encoded CAR.

[0462] In some embodiments, the nucleotide sequence can be optimized by one or more means for preventing immune activation, increasing stability, reducing any tendency to aggregate over time, and / or avoiding impurities. Such optimization can include the use of modified nucleosides, modified and / or specific 5'UTRs, 3'UTRs and / or poly(A) tail modifications for improved intracellular stability and translation efficiency (see, e.g., Stadler et al., 2017, Nat. Med.). Such modifications are known in the art.

[0463] Strategies for in vivo delivery of polynucleotides (e.g., mRNA) are known in the art. For an overview of strategies, see Mol Ther. 2019 Apr 10; 27(4): 710-728, which is incorporated herein by reference in its entirety.

[0464] In some embodiments, the viral particles have a multi-step mechanism of action: (a) The viral particles bind to T cells in vivo via an immune cell activating protein (e.g., anti-CD3 scFv), a co-stimulatory molecule (e.g., CD28 ligand), a cell adhesion molecule (e.g., CD58) or any combination thereof, activate the T cells, and facilitate internalization of the viral particles by interaction with cocarboxylase protein, (b) The vector RNA genome is reverse transcribed into DNA, shuttled to the nucleus, and integrated into the genome, (c) The transduced T cells express the polypeptide of interest.

[0465] In some embodiments, the viral particles have a multi-step mechanism of action: (a)Viral particles bind to T cells in vivo via an immune cell activating protein (e.g., anti-CD3 scFv), a costimulatory molecule (e.g., CD28 ligand), a cell adhesion molecule (e.g., CD58), or any combination thereof, activate the T cells, and facilitate the internalization of the viral particles by interaction with coccal glycoprotein. (b)The vector RNA genome encoding the CAR is reverse transcribed into DNA, shuttled to the nucleus, and integrated into the genome. (c)The transduced T cells express the CAR and also express the FRB and RACR systems for rapamycin-regulated cytokine signaling while targeting the cells.

[0466] Immune cell activation In some embodiments, administration of the particles to a subject results in activation of immune cells.

[0467] In some embodiments, activation of immune cells is measured by the level of one or more cell markers. In some embodiments, activation of immune cells is measured by the percentage of immune cells that are positive for one or more cell markers. In some embodiments, the immune cells are T cells (T lymphocytes) or NK cells. In some embodiments, the immune cells are CD4+ T cells or CD8+ T cells. In some embodiments, the one or more cell markers are selected from the group consisting of CD71, CD25, and any combination thereof.

[0468] In some embodiments, activation of immune cells is measured by the percentage of immune cells that are CD71 positive. In some embodiments, administration of viral particles increases the percentage of CD71+ immune cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%. In some embodiments, activation of immune cells is measured by the level of CD71 expressed on the surface of immune cells. In some embodiments, administration of viral particles increases the level of CD71 expressed on the surface of immune cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 7-fold or at least 10-fold.

[0469] In some embodiments, activation of immune cells is measured by the percentage of immune cells that are CD25 positive. In some embodiments, administration of viral particles increases the percentage of CD25+ immune cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%. In some embodiments, activation of immune cells is measured by the level of CD25 expressed on the surface of immune cells. In some embodiments, administration of viral particles increases the level of CD25 expressed on the surface of immune cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 7-fold or at least 10-fold.

[0470] In some embodiments, administration of viral particles to a subject results in robust proliferation of immune cells. In some embodiments, the proliferation of immune cells increases the number and / or sensitivity to transduction by a vector.

[0471] In some embodiments, administration of viral particles to a subject results in a decrease in the number of immune cells in G0 phase (e.g., T cells) and / or an increase in the number of immune cells not in G0 phase (e.g., T cells).

[0472] In some embodiments, administration of viral particles to a subject increases the number and / or percentage of immune cells in a metabolic adaptation state to vector transduction.

[0473] In some embodiments, administration of viral particles to a subject results in accumulation of immune cells in lymph nodes. In some embodiments, administration of viral particles to a subject results in accumulation of immune cells at the tumor site.

[0474] In some embodiments, the viral particles are lentiviral particles. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells herein are a subset of in vivo immune cells that can be recognized by at least one antigen-specific binding domain of a CAR. In some embodiments, the immune cells are present in lymph nodes.

[0475] In some embodiments, the viral particles activate non-transduced immune cells. In some embodiments, the viral particles expand non-transduced immune cells. In some embodiments, the viral particles activate and / or expand tumor-infiltrating lymphocytes. In some embodiments, the viral particles activate and / or expand tumor-reactive T cells present in tumor-draining regional lymph nodes or metastatic lymph nodes.

[0476] Route of administration In some embodiments, the viral particles are administered by a route selected from the group consisting of parenteral, intravenous, intramuscular, subcutaneous, intratumoral, intraperitoneal, and intralymphatic. In some embodiments, the viral particles are administered multiple times. In some embodiments, the viral particles are administered by intralymphatic injection of the viral particles. In some embodiments, the viral particles are administered by intraperitoneal injection of the viral particles. In some embodiments, the viral particles are administered by intra-articular injection - i.e., the viral particles can be administered by injection into a lymph node such as the inguinal lymph node. In some embodiments, the viral particles are administered by injection of the viral particles into the tumor site (i.e., intratumoral). In some embodiments, the viral particles are administered subcutaneously. In some embodiments, the viral particles are administered systemically. In some embodiments, the viral particles are administered intravenously. In some embodiments, the viral particles are administered intra-arterially. In some embodiments, the viral particles are lentiviral particles.

[0477] In some embodiments, the viral particles are administered by intraperitoneal, subcutaneous, or intra-articular injection. In some embodiments, the viral particles are administered by intraperitoneal injection. In some embodiments, the viral particles are administered by subcutaneous injection. In some embodiments, the viral particles are administered by intra-articular injection.

[0478] In some embodiments, the transduced immune cells comprising the polynucleotide of the present disclosure are administered to a subject.

[0479] In some embodiments, the viral particles are administered as a single injection. In some embodiments, the viral particles are administered as at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 injections.

[0480] Dosage Forms and Administration Regimens Viral Particles Viral particles can be used in vivo to infect cells at any effective dosage. In some embodiments, the viral particles are administered in vivo to a subject by direct injection into the cells, tissues, organs, or subject in need of treatment.

[0481] In some embodiments, the viral particles can be administered in a state connected to cells. The viral particles can be connected to cells by incubation of the cells and the viral particles such that the viral particles associate with the cells. In some embodiments, the dosage of the product to be delivered is determined based on the number of virus particle-bound cells.

[0482] Viral particles can also be delivered according to the viral titer (TU / mL). The amount of lentivirus to be directly injected is determined by the total TU and can vary based on both the volume that can be injected at the site and the type of tissue being injected. In some embodiments, about 1×10 5 ~1×10 6 、about 1×10 5 ~1×10 7 、1×10 5 ~1×10 7 、about 1×10 6 ~1×10 9 、about 1×10 7 ~1×10 10 、about 1×10 7 ~1×10 11 or about 1×10 9 ~1×10 11 TU or more of the delivered viral titer can be used. In some embodiments, about 1×10 6 ~1×10 7 、about 1×10 6 ~1×10 8 、1×10 6 ~1×10 9 、about 1×10 7 ~1×10 10 、about 1×10 8 ~1×10 11 、about 1×10 8 ~1×10 12Or about 1×10 10 ~1×10 12 Or a higher delivered virus titer can be used. For example, the brain injection site may only tolerate injection of a very small volume of virus, so a high-titer prep is preferred, about 1×10 6 ~1×10 7 , about 1×10 6 ~1×10 8 , 1×10 6 ~1×10 9 , about 1×10 7 ~1×10 10 , about 1×10 8 ~1×10 11 , about 1×10 8 ~1×10 12 Or about 1×10 10 ~1×10 12 Or more TU can be used. However, systemic delivery can accommodate far more TU, about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 , about 1×10 14 Or about 1×10 15 Of the load can be delivered.

[0483] In some embodiments, the vector is administered at a dose of vector genome (vg) of about 1×10 12 ~5×10 14 Per kilogram (vg) of the total body weight of the subject. In some embodiments, the vector is administered at a dose between about 1×10 13 ~5×10 14 vg / kg. In some embodiments, the vector is administered at a dose between about 5×10 13 ~3×10 14 vg / kg. In some embodiments, the vector is administered at a dose between about 5×10 13 ~1×10 14 vg / kg. In some embodiments, the vector is about 1×1012 less than vg / kg, about 3×10 12 less than vg / kg, about 5×10 12 less than vg / kg, about 7×10 12 less than vg / kg, about 1×10 13 less than vg / kg, about 3×10 13 less than vg / kg, about 5×10 13 less than vg / kg, about 7×10 13 less than vg / kg, about 1×10 14 less than vg / kg, about 3×10 14 less than vg / kg, about 5×10 14 less than vg / kg, about 7×10 14 less than vg / kg, about 1×10 15 less than vg / kg, about 3×10 15 less than vg / kg, about 5×10 15 less than vg / kg or about 7×10 15 administered at a dose of less than vg / kg.

[0484] In some embodiments, the vector is about 1×10 per kilogram of the total body weight of the subject (vp) 12 ~5×10 14 administered at a dose of vector particles (vp) between (vp / kg). In some embodiments, the vector is about 1×10 13 ~5×10 14 administered at a dose between vp / kg. In some embodiments, the vector is about 5×10 13 ~3×10 14 administered at a dose between vp / kg. In some embodiments, the vector is about 5×10 13 ~1×10 14 administered at a dose between vp / kg. In some embodiments, the vector is about 1×10 12 less than vp / kg, about 3×10 12 less than vp / kg, about 5×10 12 less than vp / kg, about 7×10 12 less than vp / kg, about 1×10 13 less than vp / kg, about 3×10 13 less than vp / kg, about 5×10 13 less than vp / kg, about 7×10 13 less than vp / kg, about 1×1014 less than vp / kg, about 3×10 14 less than vp / kg, about 5×10 14 less than vp / kg, about 7×10 14 less than vp / kg, about 1×10 15 less than vp / kg, about 3×10 15 less than vp / kg, about 5×10 15 less than vp / kg or about 7×10 15 administered at a dose of less than vp / kg.

[0485] In some embodiments, administration of the viral particles of the present disclosure reduces the number of B cells in a subject by at least 1%, at least 2%, at least 3%, at least 5%, at least 7%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%. In some embodiments, the reduction is evaluated by the number of B cells 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks after the viral particles are administered, and the reference number is the number of B cells in a subject administered a vehicle control. In some embodiments, administration of the viral particles of the present disclosure reduces the number of B cells in a subject by at least 95%.

[0486] In some embodiments, B cells are present in the peripheral blood of the subject. In some embodiments, B cells are present in the bone marrow of the subject. In some embodiments, B cells are present in the spleen of the subject.

[0487] In some embodiments, B cells are depleted in the subject for at least 7 days, at least 10 days, at least 20 days, at least 30 days, at least 40 days, at least 50 days, at least 60 days, at least 70 days or at least 80 days after administration of the viral particles.

[0488] In some embodiments, B cells are depleted in the subject for at least 80 days after administration of the viral particles.

[0489] Rapamycin Rapamune (registered trademark) (sirolimus, rapamycin) is available as an oral solution or tablets and is FDA approved for the following indications: - Prevention of organ rejection in kidney transplantation - Limited use in kidney transplantation - Treatment of patients with lymphangioleiomyomatosis.

[0490] According to the US Prescribing Information (USPI), rapamycin is available in 1 mg / mL oral solution or 0.5, 1 or 2 mg tablets and should be administered once daily. Rapamycin may be delivered in other dosage forms and / or by other routes of administration.

[0491] In some embodiments, rapamycin is administered at a dosage between about 0.1 mg / m 2 ~ 100 mg / m 2 (of the subject's surface area). In some embodiments, the subject is human. In some embodiments, rapamycin is administered at a dosage between about 0.5 mg / m 2 ~ 50 mg / m 2 In some embodiments, rapamycin is administered at a dosage between about 0.5 mg / m 2 ~ 10 mg / m 2 In some embodiments, rapamycin is administered at a dosage between about 0.5 mg / m 2 ~ 3 mg / m 2 In some embodiments, rapamycin is administered at a dosage between about 0.5 mg / m 2 ~ 5 mg / m 2 In some embodiments, rapamycin is administered at a dosage between about 1 mg / m 2 ~ 5 mg / m 2 In some embodiments, rapamycin is administered at a dosage between about 2 mg / m 2 ~ 6 mg / m 2 In some embodiments, rapamycin is administered at a dosage of about 1 mg / m 2 In some embodiments, rapamycin is administered at a dosage of about 2 mg / m 2administered at a dose of. In some embodiments, rapamycin is about 3 mg / m 2 administered at a dose of. In some embodiments, rapamycin is about 2 mg / m 2 ~6 mg / m 2 administered at a dose between. In some embodiments, rapamycin is about 3 mg / m 2 ~9 mg / m 2 administered at a dose between. In some embodiments, rapamycin is about 4 mg / m 2 ~12 mg / m 2 administered at a dose between. In some embodiments, rapamycin is about 5 mg / m 2 ~15 mg / m 2 administered at a dose between. In some embodiments, rapamycin is about 6 mg / m 2 ~20 mg / m 2 administered at a dose between. In some embodiments, rapamycin is about 10 mg / m 2 ~50 mg / m 2 administered at a dose between. In some embodiments, the dose of rapamycin is the total dose within a 24-hour period.

[0492] In some embodiments, rapamycin is about 0.001 mg / m 2 ~100 mg / m 2 (body surface area of the subject) administered at a dose between. In some embodiments, the subject is a human. In some embodiments, rapamycin is about 0.001 mg / m 2 ~0.1 mg / m 2 between, about 0.01 mg / m 2 ~1 mg / m 2 between, about 0.1 mg / m 2 ~10 mg / m 2 between, about 1 mg / m 2 ~100 mg / m 2 between, about 0.001 mg / m 2 ~0.05 mg / m 2 between, about 0.005 mg / m 2 ~0.25 mg / m 2 between, about 0.01 mg / m 2 ~0.5 mg / m 2 between, about 0.05 mg / m2 ~2.5 mg / m 2 between about 0.1 mg / m 2 ~5 mg / m 2 between about 0.5 mg / m 2 ~25 mg / m 2 between about 1 mg / m 2 ~50 mg / m 2 between about 2 mg / m 2 ~100 mg / m 2 between about 0.001 mg / m 2 ~0.01 mg / m 2 between about 0.005 mg / m 2 ~0.05 mg / m 2 between about 0.01 mg / m 2 ~0.1 mg / m 2 between about 0.05 mg / m 2 ~0.5 mg / m 2 between about 0.1 mg / m 2 ~1 mg / m 2 between about 0.5 mg / m 2 ~5 mg / m 2 between about 1 mg / m 2 ~10 mg / m 2 between about 5 mg / m 2 ~50 mg / m 2 or between about 10 mg / m 2 ~100 mg / m 2 (including any ranges and sub-ranges therebetween) at a dosage between about 0.001 mg / m 2 ~0.005 mg / m 2 In some embodiments, rapamycin is administered at a dosage between about 0.002 mg / m 2 ~0.01 mg / m 2 In some embodiments, rapamycin is administered at a dosage between about 0.003 mg / m 2 ~0.015 mg / m 2 In some embodiments, rapamycin is administered at a dosage between about 0.004 mg / m 2 ~0.02 mg / m 2 In some embodiments, rapamycin is administered at a dosage between about 0.005 mg / m 2 ~0.025 mg / m2 administered at a dosage between. In some embodiments, rapamycin is about 0.006 mg / m 2 ~0.03 mg / m 2 administered at a dosage between. In some embodiments, rapamycin is about 0.007 mg / m 2 ~0.035 mg / m 2 administered at a dosage between. In some embodiments, rapamycin is about 0.008 mg / m 2 ~0.04 mg / m 2 administered at a dosage between. In some embodiments, rapamycin is about 0.009 mg / m 2 ~0.045 mg / m 2 administered at a dosage between. In some embodiments, rapamycin is about 0.01 mg / m 2 ~0.05 mg / m 2 administered at a dosage between. In some embodiments, rapamycin is about 0.02 mg / m 2 ~0.1 mg / m 2 administered at a dosage between. In some embodiments, rapamycin is about 0.03 mg / m 2 ~0.15 mg / m 2 administered at a dosage between. In some embodiments, rapamycin is about 0.04 mg / m 2 ~0.2 mg / m 2 administered at a dosage between. In some embodiments, rapamycin is about 0.05 mg / m 2 ~0.25 mg / m 2 administered at a dosage between. In some embodiments, rapamycin is about 0.06 mg / m 2 ~0.3 mg / m 2 administered at a dosage between. In some embodiments, rapamycin is about 0.07 mg / m 2 ~0.35 mg / m 2 administered at a dosage between. In some embodiments, rapamycin is about 0.08 mg / m 2 ~0.4 mg / m 2 administered at a dosage between. In some embodiments, rapamycin is about 0.09 mg / m 2 ~0.45 mg / m 2It is administered at a dosage between. In some embodiments, rapamycin is about 0.1 mg / m 2 ~ 0.5 mg / m 2 It is administered at a dosage between. In some embodiments, rapamycin is about 0.2 mg / m 2 ~ 1 mg / m 2 It is administered at a dosage between. In some embodiments, rapamycin is about 0.3 mg / m 2 ~ 1.5 mg / m 2 It is administered at a dosage between. In some embodiments, rapamycin is about 0.4 mg / m 2 ~ 2 mg / m 2 It is administered at a dosage between. In some embodiments, rapamycin is about 0.5 mg / m 2 ~ 2.5 mg / m 2 It is administered at a dosage between. In some embodiments, rapamycin is about 0.6 mg / m 2 ~ 3 mg / m 2 It is administered at a dosage between. In some embodiments, rapamycin is about 0.7 mg / m 2 ~ 3.5 mg / m 2 It is administered at a dosage between. In some embodiments, rapamycin is about 0.8 mg / m 2 ~ 4 mg / m 2 It is administered at a dosage between. In some embodiments, rapamycin is about 0.9 mg / m 2 ~ 4.5 mg / m 2 It is administered at a dosage between. In some embodiments, rapamycin is about 1 mg / m 2 ~ 5 mg / m 2 It is administered at a dosage between. In some embodiments, rapamycin is about 2 mg / m 2 ~ 10 mg / m 2 It is administered at a dosage between. In some embodiments, rapamycin is about 3 mg / m 2 ~ 15 mg / m 2 It is administered at a dosage between. In some embodiments, rapamycin is about 4 mg / m 2 ~ 20 mg / m 2 It is administered at a dosage between. In some embodiments, rapamycin is about 5 mg / m 2 ~ 25 mg / m 2is administered at a dosage between. In some embodiments, rapamycin is about 6 mg / m 2 ~ 30 mg / m 2 is administered at a dosage between. In some embodiments, rapamycin is about 7 mg / m 2 ~ 35 mg / m 2 is administered at a dosage between. In some embodiments, rapamycin is about 8 mg / m 2 ~ 40 mg / m 2 is administered at a dosage between. In some embodiments, rapamycin is about 9 mg / m 2 ~ 45 mg / m 2 is administered at a dosage between. In some embodiments, rapamycin is about 10 mg / m 2 ~ 50 mg / m 2 is administered at a dosage between. In some embodiments, rapamycin is about 20 mg / m 2 ~ 100 mg / m 2 is administered at a dosage between. In some embodiments, rapamycin is about 0.001 mg / m 2 ~ 0.02 mg / m 2 is administered at a dosage between. In some embodiments, rapamycin is about 0.002 mg / m 2 ~ 0.04 mg / m 2 is administered at a dosage between. In some embodiments, rapamycin is about 0.003 mg / m 2 ~ 0.06 mg / m 2 is administered at a dosage between. In some embodiments, rapamycin is about 0.004 mg / m 2 ~ 0.08 mg / m 2 is administered at a dosage between. In some embodiments, rapamycin is about 0.005 mg / m 2 ~ 0.1 mg / m 2 is administered at a dosage between. In some embodiments, rapamycin is about 0.006 mg / m 2 ~ 0.12 mg / m 2 is administered at a dosage between. In some embodiments, rapamycin is about 0.007 mg / m 2 ~ 0.14 mg / m 2 is administered at a dosage between. In some embodiments, rapamycin is about 0.008 mg / m 2~0.16 mg / m 2 is administered at a dose between. In some embodiments, rapamycin is about 0.009 mg / m 2 ~0.18 mg / m 2 is administered at a dose between. In some embodiments, rapamycin is about 0.01 mg / m 2 ~0.2 mg / m 2 is administered at a dose between. In some embodiments, rapamycin is about 0.02 mg / m 2 ~0.4 mg / m 2 is administered at a dose between. In some embodiments, rapamycin is about 0.03 mg / m 2 ~0.6 mg / m 2 is administered at a dose between. In some embodiments, rapamycin is about 0.04 mg / m 2 ~0.8 mg / m 2 is administered at a dose between. In some embodiments, rapamycin is about 0.05 mg / m 2 ~1 mg / m 2 is administered at a dose between. In some embodiments, rapamycin is about 0.06 mg / m 2 ~1.2 mg / m 2 is administered at a dose between. In some embodiments, rapamycin is about 0.07 mg / m 2 ~1.4 mg / m 2 is administered at a dose between. In some embodiments, rapamycin is about 0.08 mg / m 2 ~1.6 mg / m 2 is administered at a dose between. In some embodiments, rapamycin is about 0.09 mg / m 2 ~1.8 mg / m 2 is administered at a dose between. In some embodiments, rapamycin is about 0.1 mg / m 2 ~2 mg / m 2 is administered at a dose between. In some embodiments, rapamycin is about 0.2 mg / m 2 ~4 mg / m 2 is administered at a dose between. In some embodiments, rapamycin is about 0.3 mg / m 2 ~6 mg / m 2 is administered at a dose between. In some embodiments, rapamycin is about 0.4 mg / m2 ~8 mg / m 2 administered at a dose between. In some embodiments, rapamycin is about 0.5 mg / m 2 ~10 mg / m 2 administered at a dose between. In some embodiments, rapamycin is about 0.6 mg / m 2 ~12 mg / m 2 administered at a dose between. In some embodiments, rapamycin is about 0.7 mg / m 2 ~14 mg / m 2 administered at a dose between. In some embodiments, rapamycin is about 0.8 mg / m 2 ~16 mg / m 2 administered at a dose between. In some embodiments, rapamycin is about 0.9 mg / m 2 ~18 mg / m 2 administered at a dose between. In some embodiments, rapamycin is about 1 mg / m 2 ~20 mg / m 2 administered at a dose between. In some embodiments, rapamycin is about 2 mg / m 2 ~40 mg / m 2 administered at a dose between. In some embodiments, rapamycin is about 3 mg / m 2 ~60 mg / m 2 administered at a dose between. In some embodiments, rapamycin is about 4 mg / m 2 ~80 mg / m 2 administered at a dose between. In some embodiments, rapamycin is about 5 mg / m 2 ~100 mg / m 2 administered at a dose between. In some embodiments, the dose of rapamycin is the total dose within a 24-hour period.

[0493] In some embodiments, the dose of rapamycin is administered daily. In some embodiments, the dose of rapamycin is administered approximately every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, the dose of rapamycin is administered approximately every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. In some embodiments, the dose of rapamycin is administered approximately every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 months.

[0494] In some embodiments, after the first administration of the viral particles, the first administration of rapamycin is administered approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after the first administration of the viral particles. In some embodiments, after the first administration of the viral particles, the first administration of rapamycin is administered approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 weeks after the first administration of the viral particles. In some embodiments, after the first administration of the viral particles, the first administration of rapamycin is administered approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 months after the first administration of the viral particles. In some embodiments, after the first administration of the viral particles, the first administration of rapamycin is administered approximately 1 - 3 days, about 2 - 6 days, about 3 - 9 days, about 4 - 12 days, about 5 - 15 days, about 1 - 3 weeks, about 2 - 4 weeks, about 3 - 6 weeks, or about 4 - 8 weeks after the first administration of the viral particles.

[0495] In some embodiments, administration of rapamycin increases the number of virus particle-transduced immune cells (e.g., CAR T cells) in a subject or in a specific organ / region of the subject. In some embodiments, the organ / region of the subject is blood. In some embodiments, the organ / region of the subject is the spleen. In some embodiments, the organ / region of the subject is the bone marrow. In some embodiments, administration of rapamycin increases the number of virus particle-transduced immune cells (e.g., CAR T cells) in a subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 7-fold or at least 10-fold. In some embodiments, the increase is evaluated by the number of virus particle-transduced immune cells 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks after the first administration of rapamycin (after the virus particles have been administered), and the reference number is the number of virus particle-transduced immune cells on the day of the first administration of rapamycin. In some embodiments, the increase is evaluated by the number of virus particle-transduced immune cells 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months after the first administration of rapamycin (after the virus particles have been administered), and the reference number is the number of virus particle-transduced immune cells on the day of the first administration of rapamycin.

[0496] In some embodiments, administration of rapamycin increases the percentage of virus particle-transduced immune cells (e.g., CAR T cells) in a subject or in a particular organ / region of the subject. In some embodiments, the organ / region of the subject is blood. In some embodiments, the organ / region of the subject is the spleen. In some embodiments, the organ / region of the subject is the bone marrow. In some embodiments, administration of rapamycin increases the percentage of virus particle-transduced immune cells (e.g., CAR T cells) in a subject by at least 1%, at least 2%, at least 3%, at least 5%, at least 7%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%. In some embodiments, the increase is evaluated by the percentage of virus particle-transduced immune cells 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks after the first administration of rapamycin (after the virus particles have been administered), and the reference percentage is the percentage of virus particle-transduced immune cells on the day of the first administration of rapamycin. In some embodiments, the increase is evaluated by the percentage of virus particle-transduced immune cells 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months after the first administration of rapamycin (after the virus particles have been administered), and the reference percentage is the percentage of virus particle-transduced immune cells on the day of the first administration of rapamycin. In some embodiments, the percentage is the percentage of virus particle-transduced immune cells in total immune cells in a subject or in a particular organ / region of the subject. In some embodiments, the percentage is the percentage of virus particle-transduced immune cells in immune cells of the same type (e.g., T cells) in a subject or in a particular organ / region of the subject.

[0497] Pharmaceutical Compositions and Formulations The formulations and compositions of the present disclosure can include combinations of any number of viral particles, and, optionally, one or more additional pharmaceuticals (polypeptides, polynucleotides, compounds, etc.) formulated as pharmaceutically acceptable or physiologically acceptable compositions for administration to cells, tissues, organs, or animals, either alone or in combination with one or more other therapeutic modalities. In some embodiments, the one or more additional pharmaceuticals are those that further increase the transduction efficiency of the vector.

[0498] In some embodiments, the present disclosure provides a composition comprising a therapeutically effective amount of the viral particles described herein formulated together with one or more pharmaceutically acceptable carriers (additives) and / or diluents. In some embodiments, the composition further comprises other agents, such as cytokines, growth factors, hormones, small molecules, or various pharmaceutically active agents, etc.

[0499] In some embodiments, the compositions and formulations of viral particles used in accordance with the present disclosure can be prepared by mixing viral particles having the desired degree of purity with pharmaceutically acceptable carriers, excipients, or stabilizers as required, for storage in the form of a lyophilized formulation or an aqueous solution (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). The acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations used. In some embodiments, one or more pharmaceutically acceptable surfactants (surface-active substances), buffers, isotonic agents, salts, amino acids, sugars, stabilizers, and / or antioxidants are used in the formulation.

[0500] Suitable pharmaceutically acceptable surfactants include, but are not limited to, polyethylene-sorbitan-fatty acid esters, polyethylene-polypropylene glycols, polyoxyethylene-stearates, and sodium dodecyl sulfate. Suitable buffers include, but are not limited to, histidine buffer, citrate buffer, succinate buffer, acetate buffer, and phosphate buffer.

[0501] Isotonic agents are used to provide an isotonic formulation. An isotonic formulation is a liquid, or a liquid reconstituted from a solid form, such as a lyophilized form, and means a solution having the same tonicity as some other solution for comparison, such as a physiological saline solution and serum. Suitable isotonic agents include, but are not limited to, salts including sodium chloride (NaCl) or potassium chloride, sugars including, but not limited to, glucose, sucrose, trehalose, or any component from the group of amino acids, sugars, salts, and combinations thereof, but are not limited thereto. In some embodiments, the isotonic agent is generally used in a total amount of about 5 mM to about 350 mM.

[0502] Non-limiting examples of salts include salts of any combination of the cations sodium, potassium, calcium or magnesium with the anions chloride, phosphate, citrate, succinate, sulfate, or mixtures thereof. Non-limiting examples of amino acids include arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline. Non-limiting examples of sugars according to the present invention include trehalose, sucrose, mannitol, sorbitol, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine (also referred to as "meglumine"), galactosamine and neuraminic acid and combinations thereof. Non-limiting examples of stabilizers include the above amino acids and sugars and commercially available cyclodextrins and dextrans of any type and molecular weight known in the art. Non-limiting examples of antioxidants include excipients such as methionine, benzyl alcohol or any other excipient used to minimize oxidation.

[0503] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of aqueous compositions containing a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as solutions or suspensions; solid forms suitable for dissolution or suspension in a liquid prior to injection can also be prepared. The preparation can also be emulsified.

[0504] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. The use of any conventional media or agents in therapeutic compositions is contemplated, except for those conventional media or agents that are incompatible with the active ingredient. Supplementary active ingredients can also be incorporated into the compositions.

[0505] As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., including pharmaceutically acceptable cell culture media. In some embodiments, the compositions containing a carrier are suitable for parenteral administration, such as intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. The use of any conventional media or agents in the pharmaceutical compositions of the present disclosure is contemplated, except for those conventional media or agents that are incompatible with the transfected cells.

[0506] The compositions can be formulated in a pharmaceutically acceptable or physiologically acceptable solution for administration to cells or animals alone or in combination with one or more other therapeutic modalities. The compositions can further comprise one or more polypeptides, polynucleotides, vectors containing the same, and compounds that enhance the transduction efficiency of the vectors. It will also be understood that, if desired, the compositions of the present disclosure can be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, or various pharmaceutically active agents, etc. With respect to other components that can be included in the compositions as well, there are virtually no limitations, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended therapy.

[0507] The present disclosure also provides a pharmaceutical composition comprising an expression cassette or vector (e.g., a therapeutic vector) disclosed herein and one or more pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the pharmaceutical composition comprises a lentiviral vector comprising an expression cassette disclosed herein, where, for example, the expression cassette comprises one or more polynucleotide sequences encoding one or more chimeric antigen receptors (CARs) and variants thereof.

[0508] A pharmaceutical composition containing an expression cassette or vector may be in any form suitable for a selected mode of administration, such as intrathecal, intramyocardial, intracoronary, intravenous, intraarterial, intrarenal, intraurethral, epidural, intrathecal, intraperitoneal, or intramuscular administration. The vector can be administered to animals and humans as a single active agent, or in combination with other active agents, in unit dosage forms, as a mixture with conventional pharmaceutical supports. In some embodiments, the pharmaceutical composition comprises cells transduced ex vivo with any of the vectors according to the present disclosure.

[0509] In some embodiments, a viral particle (e.g., a lentiviral particle), or a pharmaceutical composition containing the viral particle, is effective when administered systemically. For example, the viral vectors of the present disclosure are effective, in some cases, when administered intravenously to a subject (e.g., a primate, such as a non-human primate or a human). In some embodiments, the viral vectors of the present disclosure are capable of inducing the expression of CARs in various immune cells (e.g., in T cells, dendritic cells, NK cells) when administered systemically.

[0510] In various embodiments, the pharmaceutical composition contains a pharmaceutically acceptable vehicle (e.g., carrier, diluent, and excipient) for an injectable formulation. Exemplary excipients include poloxamers. Formulation buffers for viral vectors generally contain salts to prevent aggregation and other excipients (e.g., poloxamers) to reduce the adhesiveness of viral particles. These can be, in particular, in isotonic sterile saline solutions (such as monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, or mixtures of such salts), or dry, particularly freeze-dried compositions that can optionally form the injection solution upon addition of sterile water or physiological saline. In some embodiments, the formulation is stable with respect to storage and use when frozen (e.g., below 0 °C, about -60 °C, or about -72 °C). In some embodiments, the fo...

Claims

1. A viral particle comprising a viral envelope having at least one T cell adhesion molecule, at least one costimulatory molecule, or a combination thereof, and an immune cell activating protein on its surface.

2. The virus particle according to claim 1, wherein the at least one T cell adhesion molecule comprises CD58 polypeptide, HHLA2 polypeptide, ICAM-1 polypeptide, OX40L polypeptide, 4-1BBL polypeptide, CD40 polypeptide, CD155 polypeptide, CD70 polypeptide, HVEM polypeptide, GITRL polypeptide, ICOSL polypeptide, CD30L polypeptide, SLAM polypeptide, Ly-9 polypeptide, CD84 polypeptide, Ly108 polypeptide, MICA polypeptide, MICB polypeptide, ULBP1 polypeptide, ULBP2 polypeptide, ULBP3 polypeptide, ULBP4 polypeptide, ULBP5 polypeptide, ULBP6 polypeptide, B7-H6 polypeptide, or any combination thereof.

3. The virus particle according to claim 1, wherein the at least one T cell adhesion molecule comprises a CD58 polypeptide.

4. The virus particle according to claim 3, wherein the CD58 polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

17.

5. The virus particle according to claim 1, wherein the at least one co-stimulatory molecule comprises CD45 polypeptide, CD2 polypeptide, CD4 polypeptide, CD5 polypeptide, CD8 polypeptide, CD9 polypeptide, CD16 polypeptide, CD22 polypeptide, CD33 polypeptide, CD28 polypeptide, CD37 polypeptide, CD64 polypeptide, CD80 polypeptide, CD86 polypeptide, CD134 polypeptide, CD137 polypeptide, CD154 polypeptide, OX40 polypeptide, 4-1BB polypeptide, CD40L polypeptide, or any combination thereof.

6. The virus particle according to claim 1, wherein the at least one co-stimulatory molecule comprises a CD80 polypeptide or a CD86 polypeptide.

7. The virus particle according to claim 6, wherein the CD80 polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

20.

8. The virus particle according to claim 6, wherein the CD86 polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

23.

9. The virus particle according to claim 1, wherein the immune cell activating protein specifically binds to CD2, CD3, CD28H, LFA-1, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, NKG2D, NKp46, NKp44, NKp30, CD244, TCRα chain, TCRβ chain, TCRζ chain, TCRγ chain, TCRδ chain, CD3εTCR subunit, CD3γTCR subunit, CD3δTCR subunit, or NKp80.

10. The virus particle according to claim 1, wherein the immune cell activating protein specifically binds to CD3.

11. The virus particle according to claim 10, wherein the immune cell activating protein comprises an antibody or an antigen-binding fragment thereof.

12. The virus particle according to claim 11, wherein the antibody or its antigen-binding fragment is scFv.

13. The virus particle according to claim 11, wherein the antibody or its antigen-binding fragment comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2 or 12.

14. The virus particle according to claim 1, wherein the T cell adhesion molecule comprises a CD58 polypeptide and the costimulatory molecule comprises a CD80 polypeptide.

15. The virus particle according to claim 1, wherein the T cell adhesion molecule comprises a CD58 polypeptide and the costimulatory molecule comprises a CD86 polypeptide.

16. The virus particle according to claim 1, wherein the T cell adhesion molecule comprises a CD58 polypeptide, the immune cell activating protein comprises an anti-CD3 antibody or an antigen-binding fragment thereof, and the costimulatory molecule comprises a CD80 polypeptide.

17. The virus particle according to claim 1, wherein the T cell adhesion molecule comprises a CD58 polypeptide, the immune cell activating protein comprises an anti-CD3 antibody or an antigen-binding fragment thereof, and the costimulatory molecule comprises a CD86 polypeptide.

18. The virus particle according to claim 1, wherein the virus envelope comprises a virus envelope protein.

19. The virus particle according to claim 18, wherein the viral envelope protein is VSV-G envelope protein, measles virus envelope protein, Nipah virus envelope protein, or cocalvirus G protein.

20. The virus particle according to claim 18, wherein the virus envelope comprises a cocal glycoprotein or a functional variant thereof.

21. i) The cocal glycoprotein contains the R354Q mutation compared to SEQ ID NO: 5; ii) The cocal glycoprotein contains the K47Q mutation compared to SEQ ID NO: 5; iii) The cocal glycoprotein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs. 5, 13, and 19; and / or iv) The virus particle according to claim 20, wherein the cocal glycoprotein comprises an amino acid sequence selected from SEQ ID NOs. 5, 13, and 19.

22. A virus particle according to claim 1, further comprising a payload, wherein the payload is i) at least one nucleic acid; ii) At least one nucleic acid that is a non-coding nucleic acid; iii) at least one siRNA, at least one miRNA, and / or at least one shRNA; iv) At least one nucleic acid containing a nucleotide sequence encoding the target polypeptide; v) At least one nucleic acid containing a nucleotide sequence encoding a chimeric antigen receptor (CAR); vi) At least one nucleic acid containing a nucleotide sequence encoding a multipartite cell surface receptor; vii) At least one nucleic acid comprising a nucleotide sequence encoding a rapamycin-activated cell surface receptor and a nucleotide sequence encoding CAR; or viiii) Any combination of these Virus particles, including those containing the virus.

23. The virus particle according to claim 22, wherein the multipartite cell surface receptor comprises an FKBP-rapamycin complex binding domain (FRB domain) and an FK506 binding protein domain (FKBP).

24. The virus particle according to claim 22, wherein the multipartite cell surface receptor is a rapamycin-activated cell surface receptor.

25. The virus particle according to claim 22, wherein the multipartite cell surface receptor comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs. 77, 78, or 77 and 78.

26. The viral particle according to claim 22, wherein the multipartite cell surface receptor is encoded by a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 83, 84, or 83 and 84.

27. The viral particle according to claim 22, wherein the CAR includes an antigen-binding domain specific to cancer-associated antigens.

28. The viral particle according to claim 27, wherein the cancer-related antigen is CD19, BCMA, GPRC5D, ROR1, FcRL5, alpha-fetoprotein, or Her2.

29. The virus particle according to claim 22, wherein the CAR is a universal CAR.

30. The virus particle according to claim 22, wherein the CAR includes a hapten-binding domain.

31. The viral particle according to claim 1, comprising a vector genome containing at least one nucleotide sequence encoding a target polypeptide.

32. The viral particle according to claim 1, wherein the viral envelope contains a membrane-bound cytokine.

33. The viral particle according to claim 32, wherein the membrane-bound cytokine is selected from IL-2, IL-7, IL-12, IL-15, IL-18, or IL-21.

34. A virus particle according to any one of claims 1 to 33, which is a lentivirus particle.

35. A viral particle according to any one of claims 1 to 33, which is transduced into T cells in vivo.

36. A viral particle according to any one of claims 1 to 33, which activates a T cell population comprising at least 50% CD25(+) cells, at least 70% CD25(+) cells, or at least 90% CD25(+) cells.

37. A pharmaceutical composition comprising a virus particle according to any one of claims 1 to 33 and a pharmaceutically acceptable carrier.

38. A composition for transduction in vivo into a population of T cells in a target, comprising a viral particle according to any one of claims 1 to 33, wherein the viral particle comprises a nucleotide sequence encoding a target polypeptide, and the target polypeptide is expressed in the T cell population after administration.

39. A composition for treating a disease or disorder in a subject requiring such treatment, comprising a viral particle according to any one of claims 1 to 33, wherein the viral particle comprises a nucleotide sequence encoding a therapeutic polypeptide.

40. The composition according to claim 39, characterized in that the virus particles are administered by intraperitoneal, subcutaneous, or intranodal injection.

41. The composition according to claim 39, wherein the subject requiring the composition has a disease or disorder, and the disease or disorder includes B-cell malignancies, relapsed / refractory CD19-expressing malignancies, diffuse large B-cell lymphoma (DLBCL), Burkitt's large B-cell lymphoma (B-LBL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cell lymphoma (MCL), hematological malignancies, colon cancer, lung cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, brain cancer, squamous cell carcinoma, leukemia, myeloma, B-cell lymphoma, kidney cancer, uterine cancer, adenocarcinoma, pancreatic cancer, chronic myeloid leukemia, glioblastoma, neuroblastoma, medulloblastoma, sarcoma, and any combination thereof.

42. A kit comprising a container containing virus particles according to any one of claims 1 to 33.

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