Viral particles with surface stimulating molecules

Abstract

Virus particles for activating and transducing immune cells in vitro or in vivo, as well as compositions and methods of using the same, are provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 339,332 filed on May 6, 2022, U.S. Provisional Application No. 63 / 348,180 filed on June 2, 2022, U.S. Provisional Application No. 63 / 351,064 filed on June 10, 2022, and U.S. Provisional Application No. 63 / 487,734 filed on March 1, 2023, the contents of each of which are incorporated herein by reference in their entirety.

[0003] Incorporation by Reference into the Sequence Listing

[0004] This application contains a sequence listing submitted via EFS-WEB in .XML ST26 format, and is hereby incorporated by reference in its entirety. The .XML copy was created on May 4, 2023, named 061479-505001WO_SeqList_ST26.xml, and is 288 kilobytes in size. Background Art

[0005] Cell therapy often uses in vitro transduction of immune cells to generate a population of therapeutic cells to be introduced into a patient. For example, autologous or allogeneic T cells can be transduced in vitro with a vector encoding a chimeric antigen receptor. The resulting CAR T cells are then infused into the patient.

[0006] Although it is desirable to generate therapeutic cells in vivo by delivering vectors to patients, current methods for transducing immune cells in vivo face challenges in terms of technology, logistics, consistency, cost, and efficacy. Because of the technical challenges of in vivo methods, they have not been widely used, with the main obstacles being the need to activate T cells in vivo to effectively engineer them, and the need to "control" the expansion of these engineered cells after transduction.

[0007] The in vitro manufacturing of cell therapies requires a complex series of steps, starting with the collection of patients’ peripheral blood mononuclear cells through a leukapheresis procedure, followed by genetic modification of the patient’s T cells in a cGMP facility, which introduces delays, risks, and complex logistics to patient care. Subsequently, lymphodepleting chemotherapy is administered prior to infusion of the final drug product. Therefore, there remains a need for improved cell therapies. Summary of the invention

[0008] The present disclosure is at least partially based on the following discovery: virus particles can be made to express cell surface proteins on the viral envelope, thereby simultaneously initiating and transducing immune cells. Specifically, as demonstrated herein, compared with virus particles expressing only TCR targeting molecules, virus particles expressing TCR targeting molecules and co-stimulatory molecules enhance the cell activation and transduction of nucleotides encoding target polypeptides in vivo. In addition, it has been shown that adhesion molecules can further enhance cell activation and transduction in vivo. Without wishing to be bound by theory, virus particles engineered to express TCR targeting molecules (such as CD3 binding proteins) and co-stimulatory molecules (such as CD80 or CD86) activate signals one and two necessary for T cell activation. In addition, it is believed that adhesion molecules stabilize the interaction between immune cells and virus particles, thereby rebuilding immune synapses to allow sufficient nucleotides for cell activation and transduction. The present disclosure also shows the in vivo activation and amplification of non-transduced T cells. Without wishing to be bound by theory, the particles described herein can drive the activation and amplification of tumor-infiltrating lymphocytes and tumor-reactive T cells present in tumor drainage or metastatic lymph nodes, indicating that such particles can be effective at low doses.

[0009] Therefore, in some aspects, the present disclosure provides a kind of virus particle, it comprises virus envelope, and the virus envelope comprises at least one T cell adhesion molecule, at least one co-stimulatory protein or its combination, and immune cell activation protein on the surface of the virus envelope.In some embodiments, T cell adhesion molecule, co-stimulatory protein and immune cell activation protein are each recombinant proteins.

[0010] 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, ICOSL, 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.

[0011] In some or any of the foregoing or related aspects, at least one 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. In some aspects, at least one co-stimulatory molecule is CD80, CD86, or CD80 and CD86.

[0012] In some or any of the foregoing or related aspects, the immune cell 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 alpha chain, TCR beta chain, TCR zeta chain, TCR gamma chain, TCR delta chain, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit or NKp80. In some aspects, the immune cell activation protein is a protein that specifically binds to CD3.

[0013] In some or any of the foregoing or related aspects, the immune cell activation protein is an antibody or 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 activation protein is an antibody or 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.

[0014] In some or any of the foregoing or related aspects, the T cell adhesion molecule is CD58 and the costimulatory molecule is CD80. In other aspects, the T cell adhesion molecule is CD58 and the costimulatory molecule is CD86.

[0015] In some or any of the foregoing or related aspects, the T cell adhesion molecule is CD58, the immune cell activation 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 activation protein is an anti-CD3 antibody or an antigen binding fragment thereof, and the co-stimulatory molecule is CD86.

[0016] In some or any of the foregoing or related aspects, the viral particle comprises a payload. In some aspects, the payload is a nucleic acid. In some aspects, the nucleic acid is a non-coding nucleic acid, optionally wherein the non-coding nucleic acid is a siRNA, miRNA or shRNA. In some aspects, the nucleic acid comprises a nucleotide sequence encoding a polypeptide of interest.

[0017] In some or any of the foregoing or related aspects, the viral particle comprises a vector genome comprising at least one nucleotide sequence encoding a polypeptide of interest.

[0018] In some aspects, the present disclosure provides a virus particle comprising (i) a virus envelope, the virus envelope comprising (a) an immune cell activation protein on the surface of the virus envelope, wherein the immune cell activation protein binds to a T cell receptor, (b) a co-stimulatory molecule and (c) a T cell adhesion molecule, and (ii) a vector genome, the vector genome comprising at least one nucleotide sequence encoding a polypeptide of interest. In some aspects, (a) the immune cell activation protein is a protein that specifically binds to the following: 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) 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 aspects, (a) immune cell activation protein is an antibody or antigen binding fragment thereof that specifically binds to CD3, (b) costimulatory molecule is CD80 or CD86, and (c) T cell adhesion molecule is CD58.

[0019] In some or any of the foregoing or related aspects, the viral envelope comprises a membrane-bound cytokine. In some aspects, the membrane-bound cytokine is selected from: IL-2, IL-7, IL-12, IL-15, IL-18 or IL-21.

[0020] 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 Cocal virus G protein. In some aspects, the viral envelope comprises a Cocal glycoprotein or a functional variant thereof. In some aspects, the Cocal glycoprotein comprises an R354Q mutation compared to SEQ ID NO:5. In some aspects, the Cocal glycoprotein comprises a K47Q mutation compared to SEQ ID NO:5. In some aspects, 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 NO:5, 13, and 19. In some aspects, the Cocal glycoprotein comprises an amino acid sequence selected from SEQ ID NO:5, 13, and 19.

[0021] 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.

[0022] 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 a FKBP-rapamycin complex binding domain (FRB domain) and a FK506 binding protein domain (FKBP). In some aspects, the multipartite cell surface receptor is a rapamycin activated cell surface receptor.

[0023] In some or any of the foregoing or related aspects, the viral particle includes a nucleotide sequence encoding a chimeric antigen receptor (CAR). In some aspects, the viral particle includes a nucleotide sequence encoding a cell surface receptor activated by rapamycin and a nucleotide sequence encoding CAR. In some aspects, the viral particle includes a vector genome, and the vector genome includes from 5' to 3': a nucleotide sequence encoding CAR and a nucleotide sequence encoding a multi-part cell surface receptor. In some aspects, the nucleotide sequence is operably connected. In some aspects, CAR includes an antigen binding domain specific for a cancer-associated antigen, and wherein the multi-part cell surface receptor is a cell surface receptor activated by rapamycin.

[0024] In some or any of the foregoing or related aspects, CAR comprises an antigen binding domain specific for a cancer-associated antigen. In some aspects, the cancer-associated antigen is CD19, BCMA, GPRC5D, ROR1, FcRL5 / FcRH5, alpha-fetoprotein or Her2. In other aspects, CAR is a universal CAR. In some aspects, CAR comprises a hapten binding domain.

[0025] In some aspects, the present disclosure provides a viral particle comprising (i) a viral envelope, the viral envelope comprising on the surface of the viral envelope (a) an immune cell activating protein that specifically binds to CD3, (b) a co-stimulatory molecule, wherein the co-stimulatory molecule binds to CD28, and (c) a T cell adhesion molecule, and (ii) a vector genome, the vector genome comprising (a) a nucleotide sequence encoding a cell surface receptor activated by rapamycin, and (b) a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen binding domain specific for a cancer associated antigen, optionally wherein the nucleotide sequence is operably linked.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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 77 and 78. In some aspects, the multipartite cell surface receptor comprises the amino acid sequence of SEQ ID NO: 77, 78, or 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 83 and 84. In some aspects, the multipartite cell surface receptor is encoded by a nucleotide sequence of SEQ ID NO: 83, 84, or 83 and 84.

[0030] In some or any of the foregoing or related aspects, the vector genome comprises a promoter. In some aspects, the promoter is a MND promoter, a CAG promoter, a SV40 promoter, a SV40 / CD43 promoter, or an EF-1α promoter. In some aspects, the promoter is an inducible promoter.

[0031] In some or any of the foregoing or related aspects, the viral particle is a lentiviral particle.

[0032] 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 in vitro. 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.

[0033] In some aspects, the present disclosure provides a pharmaceutical composition comprising the viral particles described herein and a pharmaceutically acceptable carrier.

[0034] In some aspects, the present disclosure provides a method for transducing a T cell population in a subject, comprising administering a viral particle or a pharmaceutical composition thereof to the subject, wherein the viral particle comprises a nucleotide sequence encoding a polypeptide of interest, wherein the polypeptide of interest is expressed in the T cell population after administration. In some aspects, at least three days after administration of the lentiviral particle, the T cell population 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)-(iii).

[0035] In other aspects, the present disclosure provides methods of generating immune cells expressing a chimeric antigen receptor in a subject in need thereof, comprising administering to the subject a viral particle or pharmaceutical composition described herein, wherein the viral particle comprises a nucleotide sequence encoding a chimeric antigen receptor.

[0036] In a further aspect, the present disclosure provides a method of treating a disease or condition in a subject in need thereof, comprising administering to the subject a viral particle or pharmaceutical composition described herein, wherein the viral particle comprises a nucleotide sequence encoding a therapeutic polypeptide.

[0037] In some or any of the foregoing or related aspects, the viral particles are administered by intraperitoneal, subcutaneous or intralymph node injection. In some aspects, the viral particles are administered by intralymph node injection via the inguinal lymph nodes.

[0038] In some or any of the above or related aspects, the subject in need thereof suffers from a disease or disorder, wherein the disease or disorder comprises a B-cell malignancy, a relapsed / refractory CD19-expressing malignancy, 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), a hematological malignancy, 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.

[0039] In some aspects, the disclosure provides a kit comprising a container comprising a viral particle as described herein and an optional pharmaceutically acceptable carrier, and instructions for transducing T cells in a subject, the instructions comprising administering the viral particle to the subject. In some aspects, the subject suffers from a disease or condition. In some aspects, the instructions comprise administering the viral particle by intraperitoneal, subcutaneous, or intralymph node injection.

[0040] In other aspects, the disclosure provides a kit comprising a container comprising a viral particle as described herein and an optional pharmaceutically acceptable carrier, and instructions for treating a subject in need thereof, the instructions comprising administering the viral particle to the subject. In some aspects, the subject suffers from a disease or condition. In some aspects, the instructions comprise administering the viral particle by intraperitoneal, subcutaneous, or intralymph node injection.

[0041] In some aspects, the disclosure provides a method for using the viral particles described herein to transduce T cells in a subject, comprising administering the viral particles to the subject. In other aspects, the disclosure provides a method for using the viral particles described herein to treat a subject suffering from a disease or condition, comprising administering the viral particles to the subject.

[0042] In some aspects, the disclosure provides the use of the viral particles described herein in the preparation of a medicament for transducing T cells in a subject, comprising administering the viral particles to the subject. In other aspects, the disclosure provides the use of the viral particles described herein in the preparation of a medicament for treating a subject with a disease or condition, comprising administering the viral particles to the subject.

[0043] In some aspects, the present disclosure provides a method for transducing a T cell population of a subject in vitro, comprising contacting the T cell population with a viral particle or a pharmaceutical composition thereof, wherein the viral particle comprises a nucleotide sequence encoding a polypeptide of interest, wherein the polypeptide of interest is expressed in the T cell population after administration, wherein the contact is performed in vitro. In some aspects, the contact is performed during a closed-loop manufacturing process. In some aspects, the T cells have not been previously contacted with an exogenous activator during the manufacturing process. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 is a schematic diagram of T cell activation using exemplary viral particles with an immune cell activating protein (e.g., CD3 scFv), a viral envelope protein (e.g., Cocal), and one or two co-stimulatory molecules.

[0045] Figure 2A Shown is the activation of CD8+ T cells, measured as % CD25+ cells, where viral particles displayed CD3scfv or CD3scfv+CD80.

[0046] Figure 2B Shown is the activation of CD8+ T cells, measured as % CD25+ cells, where viral particles displayed CD3scfv alone, CD3scfv+CD80, or CD3scfv+CD58.

[0047] Figure 2C-2D The CAR expression levels in CD8+ T cells are shown by using viral particles with either CD3scFv alone or CD3scFv+CD80 as the % CAR expression ( Figure 2C ) or total CAR+CD8+T cells ( Figure 2D ) is determined.

[0048] Figure 2E-2FThe CAR expression levels in CD3+ T cells are shown by using viral particles with CD3scfv alone, CD3scfv+CD80, or CD3scfv+CD58 as the % CAR expression ( Figure 2E ) or total CAR+CD3+T cells ( Figure 2F ) is determined.

[0049] Figure 2G-2H Stimulation with IL-2 using viral particles with CD3scfv alone or CD3scfv+CD80 is shown ( Figure 2G ) or rapamycin stimulation ( Figure 2H )'s generated CAR+CD8+T cells.

[0050] Figure 3A Shown are the percentages of CD25(+)CD8 T cells after incubation with viral particles displaying CD3scfv alone, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58.

[0051] Figure 3B Shown are the geometric mean fluorescence intensities (gMFI) of CD25(+)CD8 T cells after incubation with viral particles displaying CD3scfv alone, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58.

[0052] Figure 3C-3E Cytokine production is shown after 3 days of incubation with particles displaying CD3scfv alone, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58. IFN-γ ( Figure 3C )、IL-2( Figure 3D ) and TNF-α( Figure 3E )level.

[0053] Figure 3F-3G CAR expression in CD3+ T cells generated with viral particles expressing CD3scfv alone, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58 (mixed particles) is shown. % CAR expression ( Figure 3F ) and total CAR+T cells ( Figure 3G ).

[0054] Figures 3H-3ICAR expression in CD8+ T cells generated with viral particles expressing CD3scfv alone, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58 (same particles) is shown. % CAR expression ( Figure 3H ) and total CAR+T cells ( Fig. 3I ).

[0055] Figure 3J-3L Cocal staining on CD8+ T cells incubated with viral particles displaying CD3scFv alone, CD3scFv+CD80, CD3scFv+CD58, or CD3scA+CD80+CD58 is shown ( Figure 3J ), CD80 staining ( Figure 3K ) or CD58 staining ( Figure 3L ).

[0056] Figure 3M Principal component analysis of 3 major differentiation clusters based on particulate co-stimulatory molecules using CCR7, CD45RO, CD45RA, CD27, CD25, CAR+, total cells, CD4, and CD8 markers is shown.

[0057] Figure 3N showed that the production of effector T cells (T eff Compared with CD3scFv alone, CD3scFv+CD80 particles produced a population of predominantly central memory (T cm ) phenotype of CAR+T cells.

[0058] Fig.3O showed that the production of effector T cells (T eff Central memory T cells (T cm Compared with CD3scfv alone, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58 particles generated particles with a predominantly central memory (T cm ) phenotype of CAR+T cells.

[0059] Figure 4A Shown are the number of K562.CD19 cells over several days after incubation with anti-CD19 CAR+ T cells generated with viral particles encoding the anti-CD19 CAR and expressing CD3scFv alone, CD3scFv+CD80, CD3scFv+CD58, or CD3scFv+CD80+CD58. Particles were added to PBMCs at an MOI of 10 and tumor cells were added at a PBMC:tumor ratio of 5:1 and then directly plated on the incucyte. CD3scFv+CD80+CD58 CAR T cells were generated using a mixture of individual particles.

[0060] Figure 4B The number of Raji cells over the days following incubation with anti-CD19CAR+ T cells generated with viral particles encoding anti-CD19 CAR and showing CD3scFv alone, CD3scFv+CD80, CD3scFv+CD58, or CD3scFv+CD80+CD58 particles is shown. Particles were added to PBMCs at an MOI of 10 and tumor cells to PBMCs at a 5:1 ratio and then directly plated on the incucyte. CD3scFv+CD80+CD58 CAR T cells were generated using a mixture of individual particles.

[0061] Figure 4C The number of K562.CD19 cells is shown in the days after incubation with anti-CD19CAR+ T cells generated with viral particles encoding anti-CD19 CAR and showing CD3scFv alone, CD3scFv+CD80, CD3scFv+CD58, or CD3scFv+CD80+CD58 particles. Seven days after transduction at an MOI of 10, total CAR+ cells were counted and incubated with K562.CD19 at E:T ratios of 0.5 and 1, respectively. CD3scFv+CD80+CD58 CAR T cells were generated using a mixture of individual particles.

[0062] Figure 4D The number of Raji cells is shown in the days after incubation with anti-CD19CAR+ T cells generated with viral particles encoding anti-CD19 CAR and showing CD3scfv alone, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58 particles. Seven days after transduction at an MOI of 10, total CAR+ cells were counted and incubated with Raji at E:T ratios of 0.5 and 1, respectively. CD3scfv+CD80+CD58 CAR T cells were generated using a mixture of individual particles.

[0063] Figure 4E The number of K562.CD19 cells is shown within days after incubation with anti-CD19CAR+T cells generated with viral particles encoding anti-CD19 CAR and showing CD3scFv alone, CD3scFv+CD80, CD3scFv+CD58, or CD3scFv+CD80+CD58 particles. Seven days after transduction at an MOI of 10, total CAR+ cells were counted and incubated with K562.CD19 cells at a 1:1 E:T ratio, respectively. CD3scFv+CD80+CD58CAR T cells were generated using a single particle with co-stimulatory and adhesion molecules.

[0064] Figure 4FThe number of Nalm6 cells in the days after incubation with anti-CD19 CAR+ T cells generated with viral particles encoding anti-CD19 CAR and displaying CD3scfv alone, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58 is shown. Seven days after transduction at an MOI of 10, total CAR+ cells were counted and incubated with Nalm6 cells at an E:T ratio of 1:1. CD3scfv+CD80+CD58 CAR T cells were generated using a single particle with co-stimulatory molecules and adhesion molecules.

[0065] Figure 5A Shown are the numbers of CAR T cells in blood samples of NSG MHCI / II KO mice 11 days after injection of PMBCs and viral particles displaying either CD3scFv only or CD3scFv+CD80 particles.

[0066] Figure 5B-5C It was shown that only CD3 scFv ( Figure 5B ) or CD3scfv+CD80( Figure 5C ) viral particles.

[0067] Figure 6A-6B Shown are PBMCs from three healthy donors 3 days after transduction with viral particles displaying either CD3scfv alone or CD3scfv+CD80+CD58 particles ( Fig. 6A ) or 7 days ( Figure 7B ), the number of cells expressing CAR.

[0068] Figures 7A-7C The expression of CAR in cells transduced with viral particles pseudotyped with mutant VSVG envelope protein is shown. SupT1 cells ( Fig. 7A ) or PBMC( Figure 7B-7C ) were cultured with viral particles carrying anti-CD19 CAR payload and showing mutant VSVG envelope protein with or without CD3scfv+CD80+CD58. After transduction of PBMCs, CD4+ T cells ( Figure 7B ) and CD8+ T cells ( Figure 7C CAR expression was assessed in .

[0069] Figure 8 Shown are the numbers of CAR-negative T cells in the blood of mice after administration of the indicated doses of particles encoding the anti-CD19 CAR and displaying CD3scFv alone or CD3scFv+CD80+CD58.

[0070] Figures 9A-9CIn vitro particle-T cell binding ( Fig.9A ), T cell activation ( Fig. 9B ) and transduction ( Fig. 9C ).

[0071] Figures 9D-9H Cytokine production after incubation with particles expressing CD3scfv alone, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58 is shown. IFN-γ ( Fig.9D and 9E )、IL-2( Fig.9F and 9G ) and TNF-α( Figure 9H )level.

[0072] Fig.10 It was shown that CD3scfv+CD80+CD58 particles produced CAR+T cells with a less differentiated cell phenotype (measured by expression of CCR7+CD28+CD27+ cell markers) compared to CD3scfv particles alone, while CD3scfv particles produced CAR+T cells with a more differentiated cell phenotype (measured by expression of CD57+).

[0073] Fig.11 The number of Nalm6 cells after continuous stimulation of anti-CD19 CAR+ T cells with Nalm6 tumor cells every 2-3 days is shown. Anti-CD19 CAR+ T cells were generated with viral particles encoding the anti-CD19 CAR transgene and showing CD3scfv alone or CD3scfv+CD80+CD58 particles. Arrows indicate stimulation with Nalm6 tumor cells.

[0074] Fig. 12A The study design and timeline are shown. Fig. 12B Shown are the numbers of cells expressing the activation markers CD25 or CD71 four days after transduction with viral particles expressing CD3scfv alone or CD3scfv+CD80+CD58+. Fig. 12C Shown are the numbers of T cells expressing anti-CD19 CAR in the blood 11 days after transduction with viral particles expressing CD3scFv alone or CD3scFv+CD80+CD58+.

[0075] Figures 13A-13B It was shown that only CD3 scFv ( Fig.13A ) or CD3scfv+CD80+CD58( Fig. 13B ) of viral particles, tumor burden in NSG MHCI / II KO mice.

[0076] Fig.14A The study design and timeline are shown.

[0077] Fig. 14B is a graph showing Cocal staining on CD3+ T cells incubated with engineered particles displaying CD58, CD80 and anti-CD3 scFv triple fusion polypeptides.

[0078] Fig. 14C Figure 2 is a graph showing Cocal staining on engineered particles bound to T cells. The left peak shows CD3- T cells and the right peak shows CD3+ T cells. The engineered particles show CD58, CD80 and anti-CD3 scFv triple fusion polypeptides.

[0079] Fig.14D Shown is the expression of CD25 in CD8+ T cells at day 3 after transduction with viral particles displaying CD58, CD80 and anti-CD3 scFv triple fusion polypeptides.

[0080] Fig.14E Shown is CAR expression in CD8+ T cells at day 7 after transduction with viral particles displaying CD58, CD80 and anti-CD3 scFv triple fusion polypeptides.

[0081] Fig.15 The number of Nalm6 cells after continuous stimulation of anti-CD19 CAR+T cells with Nalm6 tumor cells every 2-3 days is shown. Anti-CD19 CAR+T cells were generated with viral particles encoding the anti-CD19 CAR transgene and displaying the CD3scfv+CD80+CD58 tri-fusion polypeptide. Arrows indicate stimulation with Nalm6 tumor cells. Error bars represent mean ± SEM.

[0082] Fig.16A The study design and timeline are shown. Fig. 16B Shown are the numbers of cells expressing the activation marker CD25 four days after transduction with viral particles displaying CD3scfv+CD80+CD58+triple fusion polypeptides. Fig. 16C Shown are the numbers of cells expressing the activation marker CD71 four days after transduction with viral particles displaying the CD3scfv+CD80+CD58+tri-fusion polypeptide. Fig.16D Shown is the production of IFN-γ after 4 days of incubation with particles displaying the CD3scfv+CD80+CD58 triple fusion polypeptide. Fig.16E Shown are the numbers of T cells expressing anti-CD19 CAR in the blood 11 days after transduction with viral particles displaying CD3scfv+CD80+CD58+ triple fusion polypeptides at a viral dose of 10 or 50 million transducing units (TU). Fig.16FShown are tumor burdens in NSG MHCI / II KO mice following administration of viral particles displaying CD3scfv+CD80+CD58+tri-fusion polypeptides at viral doses of 10 or 50 million transducing units (TU).

[0083] Fig.17A The study design and timeline are shown. Fig. 17B Shown are the numbers of T cells expressing anti-CD19 CAR from donor 1 and donor 2 in the blood after 14 days of incubation with viral particles in vitro. Fig. 17C Shown are tumor burdens in donor 1 and donor 2 NSG MHCI / II KO mice after administration of T cells generated by in vitro incubation with viral particles. N=7 animals, error bars represent mean±SEM. Fig.17D The study design and timeline of the rechallenge study are shown. Fig.17E Shown are tumor burdens in donor 1 and donor 2 NSG MHCI / II KO mice following administration of T cells generated by in vitro incubation with viral particles and tumor cell re-challenge at day 49. Error bars represent mean ± SEM. Fig.17F is a schematic diagram showing an exemplary fusion protein comprising the extracellular region of CD58 and α-CD3 scFv fused to the N-terminus of CD80 via a linker. DETAILED DESCRIPTION

[0084] In some embodiments, the disclosure provides a viral particle comprising a viral envelope, wherein the viral envelope comprises an immune cell activation protein, a co-stimulatory molecule, a T cell adhesion molecule, and any combination thereof. In some embodiments, the viral particle is a delivery agent for a target molecule. In some embodiments, the molecule is a nucleotide sequence encoding a target polypeptide. In some embodiments, the molecule is a non-coding nucleic acid. In some embodiments, the non-coding nucleic acid is a cDNA, shRNA, microRNA, or siRNA.

[0085] In some embodiments, the viral particles described herein activate and transduce immune cells in vivo. In some embodiments, the viral particles described herein activate and transduce immune cells in vitro. In some embodiments, the viral particles simultaneously activate and transduce immune cells.

[0086] In some embodiments, the viral particles and methods provided herein eliminate the need for pre-activating immune cells before administering the viral particles. In some embodiments, the method includes not pre-activating the immune cells of the subject before administering the viral particles (e.g., no pre-activation within about 1, 2, 3, 4, 5, 6, or 7 days, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks before administering the viral particles). In some embodiments, the pre-activation of immune cells includes activating CD3 and / or CD28 signal transduction in immune cells (e.g., T cells), optionally activated by administering anti-CD3 and / or anti-CD28 antibodies, respectively. Therefore, in some embodiments, the methods of the present disclosure do not include administering a separate CD3 activator and / or CD28 activator before administering the viral particles.

[0087] Virus particles

[0088] In some embodiments, the present disclosure provides a viral particle comprising a viral envelope and a payload. In some embodiments, the viral envelope comprises an immune cell activation protein, a co-stimulatory molecule, a T cell adhesion molecule, or any combination thereof.

[0089] In some embodiments, the viral particle comprises 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 a target cell (e.g., a host T cell) for therapeutic use. In some embodiments, the therapeutic polypeptide comprises a T cell receptor, a chimeric antigen receptor, or a cytokine receptor.

[0090] In some embodiments, the viral particle is a retroviral particle. In some embodiments, the viral particle is a lentiviral particle. In some embodiments, the viral particle is an adeno-associated viral particle.

[0091] 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 primarily derived from a virus. The term "retroviral vector" refers to a viral vector containing structural and functional genetic elements primarily derived from a retrovirus, or a portion thereof. The term "lentiviral vector" refers to a viral vector containing structural and functional genetic elements primarily derived from a lentivirus, or a portion thereof (including LTR). The term "hybrid" refers to a vector, LTR, or other nucleic acid containing a retroviral (e.g., lentiviral) sequence and a non-lentiviral sequence. In some embodiments, a hybrid vector refers to a vector or transfer plasmid containing a retroviral (e.g., lentiviral) sequence for reverse transcription, replication, integration, and / or packaging.

[0092] Virus envelope

[0093] In some embodiments, the viral vector comprises a viral envelope comprising a polypeptide on the surface of the envelope.

[0094] In some embodiments, the viral envelope comprises one or more transduction enhancers. In some embodiments, the transduction enhancer comprises a T cell activating receptor, a NK cell activating receptor and / or a co-stimulatory molecule. In some embodiments, 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.

[0095] In some embodiments, the viral particles comprise cell surface receptors that bind to ligands on target host cells, thereby allowing host cell transduction. In some embodiments, the viral particles comprise heterologous viral envelope glycoproteins, thereby producing pseudotyped viral particles. For example, the viral envelope glycoprotein can be derived from RD114 or one of its variants, VSV-G, gibbon ape leukemia virus (GALV), or an amphoteric envelope, measles envelope, or baboon retrovirus envelope glycoprotein. In some embodiments, the viral envelope glycoprotein is a VSV G protein (Cokar glycoprotein) from a Cokar strain or a functional variant thereof.

[0096] In some embodiments, the viral envelope comprises more than one polypeptide on the surface. In some embodiments, more than one polypeptide binds to a target immune cell and replicates an immune synapse. In some embodiments, the viral envelope comprises an immune cell activation protein, a co-stimulatory molecule, and an adhesion molecule, wherein the immune cell activation protein, the co-stimulatory molecule, and the adhesion molecule each bind to a target immune cell.

[0097] Immune cell activators

[0098] In some embodiments, the transduction enhancer comprises a mitogenic stimulator that is incorporated into a retroviral or lentiviral capsid so that the virus activates and transduces T cells. This eliminates the need to add vectors and mitogens. In some embodiments, the transduction enhancer comprises a mitogenic transmembrane protein and / or one or more co-stimulatory molecules and / or adhesion molecules that are incorporated into the retrovirus when the retrovirus buds from the producer / packaging cell membrane. In some embodiments, the transduction enhancer is expressed as a separate cell surface molecule on the production cell, rather than as part of the viral envelope glycoprotein.

[0099] The viral vectors described herein may include a mitogenic transduction enhancer in the viral envelope. In some embodiments, the mitogenic transduction enhancer is derived from a host cell during the production of a retroviral vector. In some embodiments, the mitogenic transduction enhancer is made by a packaging cell and expressed on the cell surface. When the nascent retroviral vector buds from the host cell membrane, the mitogenic transduction enhancer may be incorporated into the viral envelope as part of a lipid bilayer derived from the packaging cell. 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 camel antibody. In some embodiments, the mitogenic enhancer is a scFv. In some embodiments, the mitogenic enhancer is a nanobody.

[0100] In some embodiments, the transduction enhancer is host cell derived. The term "host cell derived" means that the mitogenic transduction enhancer originates from the above host cells and is not produced as a fusion or chimera from one of the viral genes, such as gag encoding a major structural protein; or env encoding an envelope protein.

[0101] The envelope protein is formed by two subunits: a transmembrane (TM) subunit that anchors the protein to the lipid membrane, and a surface (SU) subunit that binds to cell receptors. In some embodiments, the packaging cell-derived mitogenic transduction enhancer of the present invention does not contain a surface envelope subunit (SU).

[0102] In some embodiments, the mitogenic transduction enhancer has the following structure: MS-TM, wherein M is a mitogenic domain; S is an optional spacer domain, and TM is a transmembrane domain.

[0103] The mitogenic domain is a part of a mitogenic transduction enhancer that causes T cell activation. It can bind to or otherwise interact with T cells directly or indirectly, thereby causing T cell activation. In some embodiments, the mitogenic domain binds to T cell surface antigens such as CD3, CD28, CD134, and CD137.

[0104] CD3 is a T cell co-receptor. It is a protein complex composed of four different chains. In mammals, the complex contains CD3y chain, CD35 chain and two CD3e chains. These chains bind to the T cell receptor (TCR) and z chain to generate activation signals in T lymphocytes. TCR, z chain and CD3 molecules together constitute the TCR complex. In some embodiments, the mitogenic domain binds to the CD3 e chain.

[0105] In some embodiments, the mitogenic 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 TCR or CD28. In some embodiments, the antibody binds to TCR, CD3, or CD28. Examples of such antibodies include: OKT3, 15E8, and TGN1412. Other suitable antibodies include:

[0106] Anti-CD28: CD28.2, 10F3

[0107] Anti-CD3 / TCR: UCHT1, YTH12.5, TR66

[0108] In some embodiments, the mitogenic domain comprises a binding domain from OKT3, 15E8, TGN1412, CD28.2, 10F3, UCHT1, YTH12.5, or TR66.

[0109] In some embodiments, the mitogenic domain comprises all or part of a co-stimulatory molecule, such as OX40L and 41BBL. For example, the mitogenic domain can comprise a binding domain from OX40L or 41BBL.

[0110] OKT3, also known as muromonab-CD3, is a monoclonal antibody targeting the CD3e chain. It is clinically used to reduce acute rejection in organ transplant patients. It is the first monoclonal antibody approved for human clinical use. The CDRs of OKT3 are as follows:

[0111] CDRH1:GYTFTRY(SEQ ID NO:136)

[0112] CDRH2:NPSRGY (SEQ ID NO:137)

[0113] CDRH3:YYDDHYCLDY(SEQ ID NO:138)

[0114] CDRL1:SASSSVSYMN(SEQ ID NO:139)

[0115] CDRL2:DTSKLAS(SEQ ID NO:140)

[0116] CDRL3: QQWSSNPFT(SEQ ID NO:141)

[0117] In some embodiments, the viral envelope comprises an immune cell activation protein. In some embodiments, the immune cell activation protein specifically binds to a receptor on an immune cell. In some embodiments, the immune cell activation protein provides a signal for T cell activation.

[0118] In some embodiments, the immune cell activation 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 immune cell activation protein specifically binds to CD3γ, CD3δ or CD3ε. In some embodiments, the immune cell activation 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 immune cell activation protein specifically binds to CD3γ, CD3δ or CD3ε. In some embodiments, the immune cell activation protein specifically binds to CD3.

[0119] In some embodiments, the immune cell activation protein is an antibody or antigen binding fragment thereof that specifically binds to a receptor on an immune cell. In some embodiments, the immune cell activation protein is an antibody or antigen binding fragment thereof that specifically binds to the following: 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 activation protein is an antibody or antigen binding fragment thereof that specifically binds to the following: 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, TCRα chain, TCRβ chain, TCRγ chain or TCRδ chain. In some embodiments, the immune cell activation protein is an antibody or antigen binding fragment thereof that specifically binds to CD3γ, CD3δ or CD3ε. In some embodiments, the immune cell activation protein is an antibody or antigen binding fragment thereof that specifically binds to CD3.

[0120] Antibodies targeting the polypeptides described herein are known to those skilled in the art. Methods of generating antibodies are known to those skilled in the art.

[0121] 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 coupled to a transmembrane domain. An exemplary anti-CD3ε antibody is OKT3. OKT3, also known as muromonab-CD3, is a monoclonal antibody targeting the CD3ε chain.

[0122] In some embodiments, the viral envelope comprises a single-chain Fv fragment (scFv) of an anti-CD3 antibody.

[0123] 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.

[0124] In some embodiments, the viral envelope comprises an anti-CD3 scFv comprising the following complementarity determining regions (CDRs): SASSVSYMN (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).

[0125] In some embodiments, the nucleotide sequence encoding the anti-CD3 scFv 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: 7. In some embodiments, the nucleotide sequence encoding the anti-CD3 scFv comprises SEQ ID NO: 7.

[0126] 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.

[0127] In some embodiments, the viral envelope comprises an anti-CD3 scFv comprising the following CDRs: SASSVSYMN (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).

[0128] In some embodiments, the nucleotide sequence encoding the anti-CD3 scFv comprises a 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: 15. In some embodiments, the nucleotide sequence encoding the anti-CD3 scFv comprises SEQ ID NO: 15.

[0129] Co-stimulatory molecules

[0130] 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 a signal for cell activation.

[0131] As used herein, the term "co-stimulatory molecule" refers to a molecule that can produce a co-stimulatory signal to a T cell. Lymphocytes such as T cells and natural killer (NK) cells usually require multiple signals and interactions with antigen presenting cells (APC) to achieve the best priming effect, thereby obtaining complete effector functions. T cells include these signal transductions produced by T cell receptors (TCR), co-stimulatory molecules (such as CD28 and CD2), cytokines, and various adhesion molecules required for proper synapse formation and signal transduction by allowing enough time. NK cells require similar types of stimulation, but can rely on different activation receptors, such as NKG2D, NKp46, and DNAM-1. For T cells, in addition to TCR stimulation, appropriate co-stimulation is particularly important for effective priming, and many studies have shown that TCR stimulation alone can lead to functional ineffectiveness and anergy. Co-stimulatory signals enhance the function of T cells and NK cells by enhancing cell metabolism, cytokine production, differentiation, and long-term persistence. Co-stimulation is an important factor in cell proliferation, differentiation, and survival. In some embodiments, co-stimulatory 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, co-stimulatory molecules can also be adhesion molecules. In some embodiments, co-stimulatory molecules include but are not limited to binding agents, such as scFv, antibodies, single domain antibodies, antibody fragments, nanobodies that bind to any co-stimulatory molecules or adhesion molecules described herein. In some embodiments, these binding agents may include anti-CD28 agents, anti-CD2 agents, anti-CD45 agents, anti-CD4 agents, anti-CD5 agents, anti-CD8 agents, anti-CD9 agents, anti-CD16 agents, anti-CD22 agents, anti-CD33 agents, anti-CD37 agents, anti-CD64 agents, anti-CD80 agents, anti-CD86 agents, anti-CD137 agents, anti-CD154 agents, anti-CD28H agents, anti-LFA-1 agents, anti-OX40 agents, anti-4-1BB agents, anti-CD40L agents, anti-DNAM-1 agents, anti-CD27 agents, anti-ICOS agents, anti-LIGHT agents, anti-GITR agents, anti-CD30 agents, anti-SLAM agents, anti-Ly-9 agents, anti-CD84 agents, anti-Ly 108 agents, anti-NKG2D agents, anti-NKp46 agents, anti-NKp44 agents, anti-NKp30 agents, anti-CD244 agents, anti-NKp80 agents, anti-TCRα chain agents, anti-TCRβ chain agents, anti-TCRγ chain agents and anti-TCRδ chain agents.

[0132] In some embodiments, the costimulatory molecule is a ligand of CD28. CD28 is one of the proteins expressed on T cells, which provides the costimulatory signals required for T cell activation and survival. In addition to the T cell receptor (TCR), T cell stimulation by CD28 can provide a powerful signal for the production of various interleukins (especially IL-6). In some embodiments, the costimulatory molecule is an antibody or fragment thereof that binds to CD28. Examples of such antibodies include: 15E8 and TGN1412. Other suitable antibodies include: CD28.2 and 10F3.

[0133] 15E8 is a mouse monoclonal antibody against human CD28. Its CDR is as follows:

[0134] CDRH1:GFSLTSY(SEQ ID NO:142)

[0135] CDRH2: WAGGS (SEQ ID NO: 143)

[0136] CDRH3:DKRAPGKLYYGYPDY(SEQ ID NO:144)

[0137] CDRL1:RASESVEYYVTSLMQ(SEQ ID NO:145)

[0138] CDRL2:AASNYES(SEQ ID NO:146)

[0139] CDRL3:QQTRKVPST(SEQ ID NO:147)

[0140] TGN1412 (also known as CD28-super MAB) is a humanized monoclonal antibody that not only binds to the CD28 receptor but is also a potent agonist. Its CDRs are as follows:

[0141] CDRH1: GYTFSY (SEQ ID NO: 148)

[0142] CDRH2: YPGNVN (SEQ ID NO: 149)

[0143] CDRH3:SHYGLDWNFDV(SEQ ID NO:150)

[0144] CDRL1:HASQNIYVLN(SEQ ID NO:151)

[0145] CDRL2:KASNLHT(SEQ ID NO:152)

[0146] CDRL3:QQGQTYPYT(SEQ ID NO:153)

[0147] 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 anti-CD28 antibody or anti-CD28 scFv. In some embodiments, the anti-CD28 antibody or anti-CD28 scFv is coupled to a transmembrane domain to be displayed on the surface of the viral envelope.

[0148] In some embodiments, the costimulatory molecule is a CD86 polypeptide comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the costimulatory 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.

[0149] 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.

[0150] In some embodiments, the costimulatory molecule is a CD80 polypeptide comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the costimulatory 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.

[0151] 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.

[0152] CD134, also known as OX40, is a member of the TNFR superfamily of receptors and is expressed on activated T cells. OX40 can promote cell division and survival. OX40 is a secondary co-stimulatory molecule expressed 24 to 72 hours after activation; its ligand OX40L is also not expressed on resting antigen-presenting cells, but is expressed after their activation. In some embodiments, the viral particle comprises a ligand of OX40 or a functional fragment thereof coupled to its native transmembrane domain or a heterologous transmembrane domain.

[0153] 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 present in dendritic cells, follicular dendritic cells, natural killer cells, granulocytes and vascular wall cells at the site of inflammation. The most significant activity of CD137 is its co-stimulatory activity on activated T cells. The cross-linking of CD137 enhances T cell proliferation, IL-2 secretion survival and cytolytic activity. In some embodiments, the viral particle comprises a ligand or a functional fragment of 4-1BB coupled to its native transmembrane domain or 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 can serve as a ligand for 4-1BB, which is a co-stimulatory receptor molecule in T lymphocytes. It has been shown that 4-1BBL can reactivate inactive T lymphocytes in addition to promoting T lymphocyte proliferation.

[0154] Viral particles comprising one or more activation molecules or co-stimulatory molecules can be prepared by engineering the packaging cell line by the method provided in WO 2016 / 139463; or by expressing T cell activation molecules or co-stimulatory molecules from a polycistronic helper vector as described in International Patent Publication No. WO 2020 / 106992Al, the entire contents of which are incorporated herein by reference in their entirety.

[0155] Adhesion molecules

[0156] In some embodiments, the viral particles include adhesion molecules. As used herein, the term "adhesion molecules" refers to a subset of cell surface molecules that participate in the binding of cells to other cells. Adhesion cells can contribute to the formation of more stable interactions between immune cells, such as immune synapses. Immune synapses are stable adhesive connections between polarized immune effector cells and antigen-bearing cells. In some embodiments, adhesion molecules can provide costimulatory signals to target cells. In some embodiments, adhesion molecules include but are not limited to CD58, HHLA2, ICAM-1, OX40L, 4-1BBL, CD40, CD155, CD70, HVEM, GITRL, ICOSL, CD30L, SLAM, Ly-9, CD84, Ly108, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6 and B7-H6.

[0157] The co-stimulatory and adhesion molecules disclosed herein 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. Also added are binding proteins such as scfv and nano antibodies of these ligands, 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. Membrane-bound cytokines include but are not limited to IL-2, IL-7, IL-12, IL-15, IL-18 and IL-21. The fusion construct of the present disclosure may also include B7-H3, B7x and / or TMIGD2. The fusion proteins disclosed herein may comprise one or more domains that engage with co-stimulatory molecules and / or adhesion molecules present 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.

[0158] In some embodiments, adhesion molecules include but are not limited to binding agents, such as scFv, antibodies, single domain antibodies, antibody fragments and nano antibodies bound to any adhesion molecules or co-stimulatory molecules described herein. In some embodiments, these binding agents can include anti-CD28 agents, anti-CD2 agents, anti-CD28H agents, anti-LFA-1 agents, anti-OX40 agents, anti-4-1BB agents, anti-CD40L agents, anti-DNAM-1 agents, anti-CD27 agents, anti-ICOS agents, anti-LIGHT agents, anti-GITR agents, anti-CD30 agents, anti-SLAM agents, anti-Ly-9 agents, anti-CD84 agents, anti-Ly108 agents, anti-NKG2D agents, anti-NKp46 agents, anti-NKp44 agents, anti-NKp30 agents, anti-CD244 agents, anti-NKp80 agents, anti-TCR alpha chain agents, anti-TCR beta chain agents, anti-TCR gamma chain agents and anti-TCR delta chain agents.

[0159] In some embodiments, the co-stimulatory molecule and / or adhesion molecule 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 a sequence in Table 1.

[0160] Table 1

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167] In some embodiments, the co-stimulatory molecule and / or adhesion molecule is connected to a transmembrane cell. In some embodiments, the transmembrane domain can be derived from the following transmembrane domains: CD8, α, β or ζ chain of T cell receptor, CD28, CD3ε, 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), 4-1B B(CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80(KLRFI), CD160, CD19, IL2Rβ, IL2Rγ, IL7 Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LF A-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, 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 may be derived from the transmembrane domain of CD28. In some embodiments, the transmembrane domain may be derived from the transmembrane domain of CD8, such as CD8α.

[0168] Without wishing to be bound by theory, reducing the foreign linkages (ie, between the adhesion molecule and the transmembrane domain) in the foreign nucleic acid incorporated into the lentiviral particle may reduce the immunogenicity of the lentiviral particle to a subject.

[0169] 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 a natural ligand for CD58. In addition to performing adhesion functions, the participation of CD2 provides co-stimulatory signals that can enhance activation and effector functions. In some embodiments, the lentiviral particles comprise molecules that bind to CD2. In some embodiments, the lentiviral particles comprise antibodies, single domain antibodies, antibody fragments, and / or nanobodies specific for CD2. In some embodiments, the lentiviral particles comprise CD58 or a functional portion thereof that binds to CD2.

[0170] 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.

[0171] 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.

[0172] Additional non-viral proteins

[0173] In some embodiments, the viral particle comprises at least one non-viral protein. In some embodiments, the viral particle comprises at least one non-viral protein in addition to the above proteins.

[0174] In some embodiments, the viral particle comprises a targeting ligand. In some embodiments, the viral particle comprises CD19 or a functional fragment thereof coupled to its native transmembrane domain or a heterologous transmembrane domain. In some embodiments, CD19 serves as a ligand for blinatumomab, thereby providing a linker for coupling the particle to a T cell via the anti-CD3 portion of blinatumomab. In some embodiments, another type of particle surface ligand can be used to couple appropriately surface engineered lentiviral particles to T cells using a multispecific antibody comprising a binding portion of a particle surface ligand. In some embodiments, the multispecific antibody is a bispecific antibody, such as a bispecific T cell adapter (BiTE).

[0175] 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 scEv or a cytokine), it can be tethered to the surface of the viral particle by fusion to a transmembrane domain (such as the transmembrane domain of CD8). Alternatively, a transmembrane protein engineered to bind to a soluble protein can be used to indirectly connect it to a lentiviral particle. Further including one or more cytoplasmic residues can increase the stability of the fusion protein.

[0176] Mitogenic transduction enhancers and / or cytokine-based transduction enhancers may include a "spacer sequence" to connect the antigen binding domain to the transmembrane domain. Flexible spacers allow the antigen binding domain to be oriented in different directions to facilitate binding. As used herein, the term "coupling" refers to chemical connection, direct fusion of the C-terminus and N-terminus of two proteins; chemical connection with non-peptide space; chemical connection with polypeptide space; and the C-terminus and N-terminus of two proteins are fused to a polypeptide spacer, such as a spacer sequence, by a peptide bond.

[0177] The spacer sequence may, for example, comprise an IgG1 Fc region, an IgG1 hinge, or a human CD8 stem or a mouse CD8 stem. The spacer may alternatively comprise an alternative linker sequence having a length and / or domain spacing properties similar to an IgG1 Fc region, an IgG1 hinge, or a CD8 stem. The human IgG1 spacer may be altered to remove an Fc binding motif. In some embodiments, the spacer sequence may be derived from a human protein.

[0178] In some embodiments, the spacer sequence comprises a CD8-derived hinge.

[0179] In some embodiments, the spacer sequence comprises a "short" hinge. A short hinge is described as a hinge region that comprises fewer nucleotides relative to the CAR hinge region known in the art.

[0180] In some embodiments, the viral particle comprises a polypeptide comprising a CD8 hinge 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:3.

[0181] In some embodiments, the viral particle comprises a nucleic acid sequence encoding a CD8 hinge 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:8.

[0182] The transmembrane domain is a sequence of a mitogenic transduction enhancer and / or a cytokine-based transduction enhancer that spans the membrane. The transmembrane domain may comprise a hydrophobic alpha helix. The transmembrane domain may be derived from CD28. In some embodiments, the transmembrane domain is derived from a human protein.

[0183] The viral particles of the present invention may 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 made by the host cell and expressed on the cell surface. When the nascent viral particle buds from the host cell membrane, the cytokine-based transduction enhancer can be incorporated into the viral envelope as part of the lipid bilayer of the packaging cell source.

[0184] The transduction enhancer based on cytokine can include a cytokine domain and a transmembrane domain. It can have a CS-TM structure, wherein C is a cytokine domain, S is an optional spacer domain (e.g., a spacer sequence), and TM is a transmembrane domain. The spacer domain and the transmembrane domain are as defined above.

[0185] The cytokine domain may comprise a T cell activating cytokine, such as from IL2, IL7 and IL15, or a functional fragment thereof. As used herein, a "functional fragment" of a cytokine is a polypeptide fragment that retains the ability to bind to its specific receptor and activate T cells.

[0186] IL2 is one of the factors secreted by T cells to regulate the growth and differentiation of T cells and some B cells. IL2 is a lymphokine that induces proliferation of reactive T cells. It is secreted as a single glycosylated polypeptide and requires cleavage of a signal sequence for its activity. Solution NMR showed that the structure of IL2 contains a bundle of 4 helices (called AD) flanked by 2 shorter helices and several poorly defined loops. Residues in helix A and in the loop region between helices A and B are important for receptor binding.

[0187] Viral envelope protein

[0188] In some embodiments, the viral envelope comprises a viral envelope protein. In some embodiments, the viral envelope protein is a VSV-G envelope protein, a measles virus envelope protein, a Nipah virus envelope protein, or a Kokar virus G protein. In some embodiments, the viral particles comprise a modified VSV G protein lacking LDLR binding affinity. In some embodiments, these mutations comprise mutations at position 47 (e.g., K47Q) and / or 354 (e.g., R354A).

[0189] In some embodiments, the viral envelope protein is a VSV G protein (Cocal glycoprotein) from a Cocal strain. In some embodiments, the VSV G protein is a Cocal envelope protein containing a mutation at position 354 (R354). In some embodiments, the VSV G protein is a Cocal envelope protein containing a mutation at position 47 (K47). In some embodiments, the VSV G protein is a Cocal envelope variant containing an R354Q mutation relative to SEQ ID NO:5. In some embodiments, the VSV G protein is a Cocal envelope variant containing a K47Q mutation relative to SEQ ID NO:5. In some embodiments, the variant may be referred to as a "blind" Cocal envelope. Exemplary Cocal envelope variants are provided, for example, in US2020 / 0216502A1, which is incorporated herein by reference in its entirety.

[0190] In some embodiments, the viral particle comprises a Cocal glycoprotein 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: 5. In some embodiments, the viral particle comprises SEQ ID NO: 5.

[0191] In some embodiments, the nucleotide sequence encoding the viral particle comprises a nucleotide sequence encoding Cocal glycoprotein. In some embodiments, the nucleotide sequence encoding Cocal glycoprotein 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: 10. In some embodiments, the nucleotide sequence encoding Cocal glycoprotein comprises the sequence of SEQ ID NO: 10.

[0192] In some embodiments, the nucleotide sequence encoding the viral particle comprises a nucleotide sequence encoding Cocal glycoprotein. In some embodiments, the nucleotide sequence encoding Cocal glycoprotein 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: 104. In some embodiments, the nucleotide sequence encoding Cocal glycoprotein comprises the sequence of SEQ ID NO: 104.

[0193] Various fusion glycoproteins can be used for pseudotyped lentiviral particles. Although the most commonly used example is the envelope glycoprotein of vesicular stomatitis virus (VSV-G), many other viral proteins have also been used for pseudotyping of lentiviral particles. See Joglekare et al. Human Gene Therapy Methods 28: 291-301 (2017). The present disclosure contemplates the replacement of various fusion glycoproteins. It is worth noting that some fusion glycoproteins cause higher efficiency of viral particles.

[0194] In some embodiments, pseudotyping of the fusion glycoprotein or its functional variant facilitates targeted transduction of specific cell types (including but not limited to T cells or NK cells). In some embodiments, the fusion glycoprotein or its functional variant 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 simian leukemia virus (BaEV) gp70, Measles virus (MV) H and F, Nipah virus (NiV) H and F, Rabies virus (RabV) G, Mokola virus (MOKV) G, Ebola Zaire virus (EboZ) G, Lymphocytic choriomeningitis virus (LCMV) GP1 and GP2, Baculovirus GP64, Chikungunya virus (CHIKV) 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 Chandipura virus and PRV-G.

[0195] In some embodiments, the fusion glycoprotein or its functional variant is a full-length polypeptide, functional fragment, homologue or functional variant of the G protein of Alagoas vesicular stomatitis virus (VSAV), Carajas vesicular stomatitis virus (CJSV), Chandipura vesicular stomatitis virus (CHPV), Cocal vesicular stomatitis virus (COCV), Indiana vesicular stomatitis virus (VSIV), Isfahan vesicular stomatitis virus (ISFV), Marburg vesicular stomatitis virus (MARAV), New Jersey vesicular stomatitis virus (VSNJV), Bas-Congo virus (BASV). In some embodiments, the fusion glycoprotein or its functional variant is Cocal virus G protein.

[0196] In some embodiments, the viral particle is a Nipah virus (NiV) envelope pseudotyped lentiviral particle ("Nipah envelope pseudotyped vector"). In some embodiments, the Nipah envelope pseudotyped vector is pseudotyped using Nipah virus envelope glycoproteins NiV-F and NiV-G. In some embodiments, the NiV-F and / or NiV-G glycoproteins on this Nipah envelope pseudotyped vector are modified variants. In some embodiments, the NiV-F and / or NiV-G glycoproteins on this Nipah envelope pseudotyped vector are modified to include an antigen binding domain. In some embodiments, the antigen is EpCAM, CD4, or CD8. In some embodiments, the Nipah envelope pseudotyped vector can effectively 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.

[0197] Exemplary Viral Envelopes

[0198] In some embodiments, the viral particles include a viral envelope, and the viral envelope includes (i) immune cell activation proteins, (ii) costimulatory molecules, (iii) adhesion molecules, or (iv) (i)-(iii) any combination. In some embodiments, the viral particles include a viral envelope, and the viral envelope includes (i) immune cell activation proteins, (ii) costimulatory molecules, (iii) adhesion molecules, or (iv) (i)-(iii) any combination of at least two. In some embodiments, the viral particles include a viral envelope, and the viral envelope includes (i) immune cell activation proteins and (ii) costimulatory molecules. In some embodiments, the viral particles include a viral envelope, and the viral envelope includes (i) immune cell activation proteins and (ii) costimulatory molecules. In some embodiments, the viral particles include a viral envelope, and the viral envelope includes (i) immune cell activation proteins and (ii) adhesion molecules. In some embodiments, the viral particles include a viral envelope, and the viral envelope includes (i) immune cell activation proteins, (ii) costimulatory molecules, and (iii) adhesion molecules. In some embodiments, the viral particles include a viral envelope, and the viral envelope includes (i) immune cell activation proteins, (ii) costimulatory molecules, and (iii) multiple adhesion molecules.

[0199] In some embodiments, the viral particle comprises a viral envelope, wherein the viral envelope comprises (i) an immune cell 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, C D5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and any combination thereof, (iii) an adhesion molecule selected from the following: 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, wherein the viral envelope comprises (i) an immune cell 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, (iii) an adhesion molecule selected from the group consisting of 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) to (iii). In some embodiments, the viral particle comprises a viral envelope comprising (i) an immune cell activating 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, NKp3 0, CD244 or NKp80, and (ii) a co-stimulatory molecule selected from the following: TCRα chain, TCRβ chain, TCRζ chain, CD3εTCR subunit, CD3γTCR subunit, CD3δTCR 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, wherein the viral envelope comprises (i) an immune cell activating 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, CD24 4 or NKp80, and (ii) an adhesion molecule selected from the group consisting of 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, wherein the viral envelope comprises (i) an immune cell 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, C D2, 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 the following: 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.

[0200] In some embodiments, the viral particles include a viral envelope, and the viral envelope includes (i) an immune cell activation protein that specifically binds CD3, (ii) a co-stimulatory molecule selected from CD80, CD86, and a combination thereof, (iii) an adhesion molecule comprising a CD58 polypeptide, or (iv) (i)-(iii) any combination. In some embodiments, the viral particles include a viral envelope, and the viral envelope includes (i) an immune cell activation protein that specifically binds CD3, (ii) a co-stimulatory molecule selected from CD80, CD86, and a combination thereof, (iii) an adhesion molecule comprising a CD58 polypeptide, or (iv) (i)-(iii) any combination of at least two. In some embodiments, the viral particles include a viral envelope, and the viral envelope includes (i) an immune cell activation protein that specifically binds CD3, and (ii) a co-stimulatory molecule selected from CD80, CD86, and a combination thereof. In some embodiments, the viral particles include a viral envelope, and the viral envelope includes (i) an immune cell activation protein that specifically binds CD3, and (ii) an adhesion molecule comprising a CD58 polypeptide. In some embodiments, the viral particle comprises a viral envelope comprising (i) an immune cell activating protein that specifically binds to CD3, (ii) a co-stimulatory molecule selected from CD80, CD86, and a combination thereof, and (iii) an adhesion molecule comprising a CD58 polypeptide.

[0201] In some embodiments, the viral particles include a viral envelope, the viral envelope comprising (i) an immune cell 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) (i)-(iii) any combination. In some embodiments, the viral particles include a viral envelope, the viral envelope comprising (i) an immune cell 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) (i)-(iii) any combination of at least two. In some embodiments, the viral particles include a viral envelope, the viral envelope comprising (i) an immune cell activation protein that specifically binds to CD3, and (ii) a co-stimulatory molecule comprising a CD80 polypeptide. In some embodiments, the viral particles include a viral envelope, the viral envelope comprising (i) an immune cell 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) a cell activation protein that immunospecifically binds to CD3, (ii) a co-stimulatory molecule comprising a CD80 polypeptide, and (iii) an adhesion molecule comprising a CD58 polypeptide.

[0202] In some embodiments, the viral particles include a viral envelope, which includes (i) an immune cell 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) (i)-(iii) any combination. In some embodiments, the viral particles include a viral envelope, which includes (i) an immune cell 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) (i)-(iii) any combination of at least two. In some embodiments, the viral particles include a viral envelope, which includes (i) an immune cell activation protein that specifically binds to CD3, and (ii) a co-stimulatory molecule comprising a CD86 polypeptide. In some embodiments, the viral particles include a viral envelope, which includes (i) an immune cell 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 immune cell activating protein that specifically binds to CD3, (ii) a co-stimulatory molecule comprising a CD86 polypeptide, and (iii) an adhesion molecule comprising a CD58 polypeptide.

[0203] In some embodiments, the viral particle comprises a viral envelope, the viral envelope comprising (i) an antibody or 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, or (iv) any combination of (i)-(iii). In some embodiments, the viral particle comprises a viral envelope, the viral envelope comprising (i) an antibody or 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, or (iv) any combination of at least two of (i)-(iii). In some embodiments, the viral particle comprises a viral envelope, the viral envelope comprising (i) an antibody or antigen binding fragment thereof 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, the 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 CD80 polypeptide, and (iii) an adhesion molecule comprising a CD58 polypeptide.

[0204] In some embodiments, the viral particle comprises a viral envelope, the 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) (i)-(iii) any combination. In some embodiments, the viral particle comprises a viral envelope, the 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) (i)-(iii) any combination of at least two. In some embodiments, the viral particle comprises a viral envelope, the 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, the 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.

[0205] In some embodiments, the viral particle comprises a viral envelope comprising (i) an antibody or 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, which 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 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, which 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, 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 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, 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, and (iii) an adhesion molecule comprising a CD58 polypeptide comprising the amino acid sequence of SEQ ID NO:17.

[0206] In some embodiments, the viral particle comprises a viral envelope, which 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 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, which 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 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, which 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, and (ii) a co-stimulatory 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 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, which 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 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.

[0207] In some embodiments, the virus particles include a virus envelope and a virus envelope protein, and the virus envelope includes (i) immune cell activation protein, (ii) costimulatory molecules, (iii) adhesion molecules or (iv) (i)-(iii) any combination. In some embodiments, the virus particles include a virus envelope and a virus envelope protein, and the virus envelope includes (i) immune cell activation protein, (ii) costimulatory molecules, (iii) adhesion molecules or (iv) (i)-(iii) any combination of at least two. In some embodiments, the virus particles include a virus envelope, and the virus envelope includes (i) immune cell activation protein, (ii) costimulatory molecules and (iii) virus envelope proteins. In some embodiments, the virus particles include a virus envelope, and the virus envelope includes (i) immune cell activation protein, (ii) costimulatory molecules, (iii) adhesion molecules and (iii) virus envelope proteins. In some embodiments, the virus particles include a virus envelope, and the virus envelope includes (i) immune cell activation protein, (ii) costimulatory molecules, (iii) adhesion molecules and (iv) virus envelope proteins.

[0208] In some embodiments, the viral particle comprises a viral envelope and a viral envelope protein, wherein the viral envelope comprises (i) an immune cell 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, CD 4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and any combination thereof, (iii) an adhesion molecule selected from the following: 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 and a viral envelope protein, wherein the viral envelope comprises (i) an immune cell 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, C (iii) an adhesion molecule selected from the group consisting of 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, wherein the viral envelope comprises (i) an immune cell activating 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, CD24 4 or NKp80, (ii) a co-stimulatory molecule selected from the following: TCRα chain, TCRβ chain, TCRζ chain, CD3εTCR subunit, CD3γTCR subunit, CD3δTCR 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 immune cell activating 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 NKp8 0, (ii) an adhesion molecule selected from the group consisting of 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, wherein the viral envelope comprises (i) an immune cell 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, CD 4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and any combination thereof, (iii) an adhesion molecule selected from the group consisting of 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.

[0209] In some embodiments, the viral particles include a viral envelope and a viral envelope protein, wherein the viral envelope includes (i) an immune cell activation protein that specifically binds to CD3, (ii) a co-stimulatory molecule selected from CD80, CD86, and a combination thereof, (iii) an adhesion molecule comprising a CD58 polypeptide, or (iv) (i)-(iii) any combination. In some embodiments, the viral particles include a viral envelope and a viral envelope protein, wherein the viral envelope includes (i) an immune cell activation protein that specifically binds to CD3, (ii) a co-stimulatory molecule selected from CD80, CD86, and a combination thereof, (iii) an adhesion molecule comprising a CD58 polypeptide, or (iv) (i)-(iii) any combination of at least two. In some embodiments, the viral particles include a viral envelope, wherein the viral envelope includes (i) an immune cell activation protein that specifically binds to CD3, (ii) a co-stimulatory molecule selected from CD80, CD86, and a combination thereof, and (iii) a viral envelope protein. In some embodiments, the viral particle comprises a viral envelope, the viral envelope comprising (i) an immune cell activating 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, the viral envelope comprising (i) an immune cell activating protein that specifically binds to CD3, (ii) a co-stimulatory molecule selected from CD80, CD86, and a combination thereof, (iii) an adhesion molecule comprising a CD58 polypeptide, and (iv) a viral envelope protein.

[0210] In some embodiments, the viral particles include a viral envelope and a viral envelope protein, wherein the viral envelope includes (i) an immune cell 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) (i)-(iii) any combination. In some embodiments, the viral particles include a viral envelope and a viral envelope protein, wherein the viral envelope includes (i) an immune cell 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) (i)-(iii) any combination of at least two of the above. In some embodiments, the viral particles include a viral envelope, wherein the viral envelope includes (i) an immune cell activation protein that specifically binds to CD3, (ii) a co-stimulatory molecule comprising a CD80 polypeptide, and (iii) a viral envelope protein. In some embodiments, the viral particle comprises a viral envelope, which comprises (i) an immune cell activating 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, which comprises (i) an immune cell activating protein 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 protein.

[0211] In some embodiments, the viral particles include a viral envelope and a viral envelope protein, the viral envelope comprising (i) an immune cell 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) (i)-(iii) any combination. In some embodiments, the viral particles include a viral envelope and a viral envelope protein, the viral envelope comprising (i) an immune cell 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) (i)-(iii) any combination of at least two. In some embodiments, the viral particles include a viral envelope, the viral envelope comprising (i) an immune cell 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 particles include a viral envelope, the viral envelope comprising (i) an immune cell 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 immune cell activating 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.

[0212] In some embodiments, the viral particle comprises a viral envelope and a viral envelope protein, wherein the viral envelope 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, or (iv) any combination of (i)-(iii). In some embodiments, the viral particle comprises a viral envelope and a viral envelope protein, wherein the viral envelope 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, or (iv) any combination of at least two of (i)-(iii). In some embodiments, the viral particle comprises a viral envelope, wherein the viral envelope comprises (i) an antibody or an antigen binding fragment thereof that specifically binds to CD3, (ii) a co-stimulatory molecule comprising a CD80 polypeptide, and (iii) a viral envelope protein. 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) an adhesion molecule comprising a CD58 polypeptide, and (iii) a viral envelope protein.

[0213] In some embodiments, the viral particles comprise a viral envelope, the viral envelope comprising (i) an antibody or 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 protein. In some embodiments, the viral particles comprise a viral envelope and a viral envelope protein, the 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 particles comprise a viral envelope and a viral envelope protein, the 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, (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 antibody or antigen binding fragment thereof 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 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, and (iv) a viral envelope protein.

[0214] In some embodiments, the viral particle comprises a viral envelope and a viral envelope protein, wherein the viral envelope 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 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 and a viral envelope protein, wherein the viral envelope 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 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, which 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 co-stimulatory molecule comprising a CD80 polypeptide comprising the amino acid sequence of SEQ ID NO: 20, and (iii) a viral envelope protein.In some embodiments, the viral particle comprises a viral envelope, which 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) an adhesion molecule comprising a CD58 polypeptide comprising the amino acid sequence of SEQ ID NO: 17, and (iii) a viral envelope protein. In some embodiments, the viral particle comprises a viral envelope, which 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 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, and (iv) a viral envelope protein.

[0215] In some embodiments, the viral particle comprises a viral envelope and a viral envelope protein, wherein the viral envelope 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 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 and a viral envelope protein, wherein the viral envelope 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 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, which 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 co-stimulatory molecule comprising a CD86 polypeptide comprising the amino acid sequence of SEQ ID NO: 23, and (iii) a viral envelope protein.In some embodiments, the viral particle comprises a viral envelope, which 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) an adhesion molecule comprising a CD58 polypeptide comprising the amino acid sequence of SEQ ID NO: 17, and (iii) a viral envelope protein. In some embodiments, the viral particle comprises a viral envelope, which 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 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, and (iv) a viral envelope protein.

[0216] In some embodiments, the viral particle comprises a polypeptide comprising a Gaussia luciferase signal peptide, the signal peptide being operably linked to an anti-CD3 scFv, the anti-CD3 scFv being operably linked to a hinge domain, the hinge domain being operably linked to a transmembrane domain and a cytoplasmic tail derived from a Cocal envelope, the polypeptide being 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:107.

[0217] In some embodiments, the viral particle comprises a polypeptide comprising a Gaussia luciferase signal peptide, the signal peptide being operably linked to an anti-CD3 scFv, the anti-CD3 scFv being operably linked to a hinge domain, the hinge domain being operably linked to a transmembrane domain and a cytoplasmic tail derived from a Cocal envelope, the polypeptide being 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:109.

[0218] In some embodiments, the viral particle comprises a polypeptide comprising a Gaussia luciferase signal peptide, the signal peptide being operably linked to an anti-CD3 scFv, the anti-CD3 scFv being operably linked to a linker, the linker being operably linked to a transmembrane domain derived from glycoprotein A and a cytoplasmic tail derived from the HIV envelope, the polypeptide being 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:111.

[0219] In some embodiments, the viral particle comprises a polypeptide comprising a Gaussia luciferase signal peptide, the signal peptide being operably linked to an anti-CD3 scFv, the anti-CD3 scFv being operably linked to a linker, the linker being operably linked to a transmembrane domain and a cytoplasmic tail derived from the HIV envelope, the polypeptide being 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:113.

[0220] In some embodiments, the viral particle comprises a polypeptide comprising a Gaussia luciferase signal peptide, the signal peptide being operably linked to an anti-CD3 scFv, the anti-CD3 scFv being operably linked to a triple G4S linker, the linker being operably linked to a transmembrane domain and a cytoplasmic tail derived from the HIV envelope, the polypeptide being 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:115.

[0221] In some embodiments, the viral particle comprises a polypeptide comprising a Gaussia luciferase signal peptide, the signal peptide being operably linked to an anti-CD3 scFv, the anti-CD3 scFv being operably linked to a hinge domain, the hinge domain being operably linked to a transmembrane domain derived from a Cocal envelope, a cytoplasmic tail, and a T2A self-cleaving peptide, wherein the polypeptide 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:117.

[0222] In some embodiments, the viral particle comprises a polypeptide comprising a Gaussia luciferase signal peptide, the signal peptide being operably linked to an anti-CD3 scFv, the anti-CD3 scFv being operably linked to a linker, the linker being operably linked to a hinge, a transmembrane domain, and a cytoplasmic tail derived from glycoprotein A, the polypeptide being 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:119.

[0223] In some embodiments, the viral particle comprises a polypeptide comprising a short hinge operably linked to a transmembrane domain, the transmembrane domain operably linked to a cytoplasmic tail derived from Cocal glycoprotein, the polypeptide 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:13.

[0224] In some embodiments, the viral particle comprises a polypeptide comprising a long hinge operably linked to a transmembrane domain, the transmembrane domain operably linked to a cytoplasmic tail derived from Cocal glycoprotein, the polypeptide 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:19.

[0225] In some embodiments, the viral particle comprises a polypeptide comprising a 218 linker, the 218 linker being operably linked to a transmembrane domain of the extracellular domain of human glycoprotein A, the transmembrane domain of the extracellular domain of human glycoprotein A being operably linked to a cytoplasmic tail derived from the HIV viral envelope, the polypeptide being 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:25.

[0226] In some embodiments, the viral particle comprises a polypeptide comprising a 218 linker operably linked to an HIV viral envelope transmembrane domain and a cytoplasmic tail, wherein the polypeptide 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:31.

[0227] In some embodiments, the viral particle comprises a polypeptide comprising a triple G4S linker operably linked to an HIV viral envelope transmembrane domain and a cytoplasmic tail, wherein the polypeptide 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:37.

[0228] In some embodiments, the viral particle comprises a polypeptide comprising a Ser-Gly peptide operably linked to a small extracellular domain, a transmembrane, and a cytoplasmic tail sequence derived from human glycoprotein A, wherein the polypeptide 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:97.

[0229] In some embodiments, the viral particle comprises a polypeptide comprising a transmembrane domain and a cytoplasmic tail sequence derived from human glycoprotein A, wherein the polypeptide 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:105.

[0230] In some embodiments, the viral particle comprises a polypeptide comprising a short hinge operably linked to a Cocal glycoprotein transmembrane domain and a cytoplasmic tail, the polypeptide being 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:43.

[0231] In some embodiments, the viral particle comprises a polypeptide comprising a transmembrane domain and a cytoplasmic tail derived from CD4, wherein the cytoplasmic tail is operably linked to a T2A linker, wherein the polypeptide 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:4.

[0232] In some embodiments, the viral particle comprises a polypeptide comprising a signal peptide sequence derived from Gaussia luciferase, wherein the polypeptide 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:11.

[0233] Virus particle envelope expression cassette

[0234] In some embodiments, a viral envelope expression cassette encoding at least one polypeptide described herein is used to produce the viral particles described herein.

[0235] In some embodiments, viral particles of the present disclosure are produced using a viral envelope expression cassette encoding, from 5' to 3':

[0236] (a) Signal peptide from CD8

[0237] (b) Anti-CD3 scFV

[0238] (c) CD8-derived hinge domain

[0239] (d) CD4 transmembrane domain and cytoplasmic tail

[0240] (e) T2A connector

[0241] (f) Cocalin

[0242] In some embodiments, viral particles of the present disclosure are produced using a viral envelope expression cassette encoding, from 5' to 3':

[0243] (a) Signal peptide derived from Gaussia luciferase

[0244] (b) Anti-CD3 scFV

[0245] (c) Short hinge domain

[0246] (d) Transmembrane domain and cytoplasmic tail derived from Cocalin

[0247] In some embodiments, viral particles of the present disclosure are produced using a viral envelope expression cassette encoding, from 5' to 3':

[0248] (a) Signal peptide derived from Gaussia luciferase

[0249] (b) Anti-CD3 scFV

[0250] (c) Long hinge domain

[0251] (d) Transmembrane domain and cytoplasmic tail derived from Cocalin

[0252] In some embodiments, viral particles of the present disclosure are produced using a viral envelope expression cassette encoding, from 5' to 3':

[0253] (a) Signal peptide derived from Gaussia luciferase

[0254] (b) Anti-CD3 scFV

[0255] (c)218 connector

[0256] (d) Transmembrane domain derived from the extracellular domain of human glycoprotein A

[0257] (e) HIV viral envelope-derived cytoplasmic tail

[0258] In some embodiments, the viral particles of the present disclosure are produced using a viral envelope expression cassette encoding, from 5' to 3':

[0259] (a) Signal peptide derived from Gaussia luciferase

[0260] (b) Anti-CD3 scFV

[0261] (c)218 connector

[0262] (d) Transmembrane domain and cytoplasmic tail derived from HIV viral envelope

[0263] In some embodiments, viral particles of the present disclosure are produced using a viral envelope expression cassette that encodes, from 5' to 3':

[0264] (a) Signal peptide derived from Gaussia luciferase

[0265] (b) Anti-CD3 scFV

[0266] (c) Triple G4S connector

[0267] (d) Transmembrane domain and cytoplasmic tail derived from HIV viral envelope

[0268] In some embodiments, viral particles of the present disclosure are produced using a viral envelope expression cassette encoding, from 5' to 3':

[0269] (a) Signal peptide derived from Gaussia luciferase

[0270] (b) Anti-CD3 scFV

[0271] (c) Short hinge domain

[0272] (d) Transmembrane domain and cytoplasmic tail derived from Cocalin

[0273] (e) T2A connector

[0274] (f) Cocalin

[0275] In some embodiments, the viral expression cassettes described herein comprise nucleotide sequences encoding co-stimulatory molecules and / or adhesion molecules. In some embodiments, the viral expression cassettes described herein comprise nucleotide sequences encoding CD86 polypeptides. In some embodiments, the viral expression cassettes described herein comprise nucleotide sequences encoding CD80 polypeptides. In some embodiments, the viral expression cassettes described herein comprise nucleotide sequences encoding CD58 polypeptides.

[0276] In some embodiments, the viral expression cassette comprises a nucleic acid encoding a Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv, the anti-CD3 scFv operably linked to a hinge domain, the hinge domain operably linked to a Cocal envelope-derived transmembrane domain and a cytoplasmic tail, wherein the nucleic acid 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:108.

[0277] In some embodiments, the viral expression cassette comprises a nucleic acid encoding a Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv, the anti-CD3 scFv operably linked to a hinge domain, the hinge domain operably linked to a Cocal envelope-derived transmembrane domain and a cytoplasmic tail, wherein the nucleic acid 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:110.

[0278] In some embodiments, the viral expression cassette comprises a nucleic acid encoding a Gaussia luciferase signal peptide, which is operably linked to an anti-CD3 scFv, which is operably linked to a linker, which is operably linked to a transmembrane domain derived from glycoprotein A and a cytoplasmic tail derived from the HIV envelope, and the nucleic acid 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:112.

[0279] In some embodiments, the viral expression cassette comprises a nucleic acid encoding a Gaussia luciferase signal peptide, which is operably linked to an anti-CD3 scFv, which is operably linked to a linker, which is operably linked to a transmembrane domain and a cytoplasmic tail derived from the HIV envelope, and the nucleic acid 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:114.

[0280] In some embodiments, the viral expression cassette comprises a nucleic acid encoding a Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv, the anti-CD3 scFv operably linked to a triple G4S linker, the linker operably linked to a transmembrane domain and a cytoplasmic tail derived from the HIV envelope, and the nucleic acid 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:116.

[0281] In some embodiments, the viral expression cassette comprises a nucleic acid encoding a Gaussia luciferase signal peptide operably linked to an anti-CD3 scFv, the anti-CD3 scFv operably linked to a hinge domain, the hinge domain operably linked to a transmembrane domain derived from a Cokal envelope, a cytoplasmic tail, and a T2A self-cleaving peptide operably linked to the Cokal envelope, wherein the nucleic acid 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: 118.

[0282] In some embodiments, the viral expression cassette comprises a nucleic acid encoding a Gaussia luciferase signal peptide, which is operably linked to an anti-CD3 scFv, which is operably linked to a linker, which is operably linked to a hinge, a transmembrane domain, and a cytoplasmic tail derived from glycoprotein A, and the nucleic acid 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:120.

[0283] 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 Cocal glycoprotein, wherein the nucleic acid sequence 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:16.

[0284] 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 Cocal glycoprotein, wherein the nucleic acid sequence 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:22.

[0285] In some embodiments, the viral expression cassette comprises a nucleic acid sequence encoding a 218 linker, which is operably linked to the transmembrane domain of the extracellular domain of human glycoprotein A, and the transmembrane domain of the extracellular domain of human glycoprotein A is operably linked to a cytoplasmic tail derived from the HIV viral envelope, and the nucleic acid sequence 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:28.

[0286] In some embodiments, the viral portion viral expression cassette comprises a nucleic acid sequence encoding a 218 linker operably linked to an HIV viral envelope transmembrane domain and a cytoplasmic tail, wherein the nucleic acid sequence 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:34.

[0287] 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 a cytoplasmic tail, wherein the nucleic acid sequence 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:40.

[0288] In some embodiments, the viral expression cassette comprises a nucleic acid sequence encoding a Ser-Gly peptide operably linked to a small extracellular domain, transmembrane, and cytoplasmic tail sequence derived from human glycoprotein A, wherein the nucleic acid sequence 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:98.

[0289] In some embodiments, the viral expression cassette comprises a nucleic acid sequence encoding a hinge operably linked to the glycoprotein A transmembrane domain and the cytoplasmic tail, wherein the nucleic acid sequence 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:106.

[0290] In some embodiments, the viral expression cassette comprises a nucleic acid sequence encoding a short hinge operably linked to a Cokar glycoprotein transmembrane domain and a cytoplasmic tail, which is operably linked to a T2A linker, wherein the nucleic acid sequence 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:47.

[0291] In some embodiments, the viral expression cassette comprises a nucleic acid sequence encoding a CD4-derived transmembrane domain and a cytoplasmic tail, which is operably linked to a T2A linker, and the nucleic acid sequence 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:9.

[0292] In some embodiments, the viral expression cassette comprises a nucleic acid sequence encoding a signal peptide derived from Gaussia luciferase, wherein the nucleic acid sequence 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:14.

[0293] Particle Type

[0294] In some embodiments, the viral particles described herein are retroviruses. Retroviruses include lentiviruses, γ-retroviruses, and α-retroviruses, and each virus can be used to deliver polynucleotides to cells using methods known in the art. Lentivirus is a complex retrovirus that contains other genes with regulatory or structural functions in addition to the common retroviral genes gag, pol, and env. Higher complexity enables the virus to regulate its life cycle, just as in the process of latent infection. Exemplary lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); Vesna-Medi 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 includes a vector backbone based on HIV (e.g., HIV cis-acting sequence elements). Retroviral particles are generated by multiple attenuation of HIV virulence genes (e.g., deletion of the genes env, vif, vpr, vpu, and nef) to render the vector biologically safe.

[0295] Exemplary lentiviral particles include those described in Naldini et al. (1996) Science 272:263-7; Zufferey et al. (1998) J. Virol. 72:9873-9880; Dull et al. (1998) J. Virol. 72:8463-8471; U.S. Pat. No. 6,013,516; and U.S. Pat. No. 5,994,136, the entire contents of which are incorporated herein by reference. In general, these particles are configured to carry the necessary sequences for selecting cells containing the particles, incorporating foreign nucleic acids into the lentiviral particles, and transferring the nucleic acids into target cells.

[0296] Commonly used lentiviral particle systems are so-called third generation systems. The third generation lentiviral particle system includes four plasmids. "Transfer plasmid" encodes a polynucleotide sequence delivered to the target cell by the lentiviral vector system. The transfer plasmid usually has one or more target transgenic sequences, which are flanked by long terminal repeat (LTR) sequences, which help integrate the transfer plasmid sequence into the host genome. For safety reasons, transfer plasmids are usually designed to make the produced particles unable to replicate. For example, the transfer plasmid lacks the genetic elements necessary to produce infectious particles in the host cell. In addition, the transfer plasmid can be designed to lack 3'LTR, making the virus "self-inactivated" (SIN). See Ull et al. (1998) J.Virol.72:8463-71; Miyoshi et al. (1998) J.Virol.72:8150-57. Viral particles may also include 3' untranslated regions (UTR) and 5'UTR. The UTR contains retroviral regulatory elements that support packaging, reverse transcription, and integration of the proviral genome into cells following contact of retroviral particles with cells.

[0297] The third generation system also generally includes two "packaging plasmids" and "envelope plasmids". 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 a CMV promoter. In an exemplary third generation system, the Env gene is Cokar G protein (Cokar glycoprotein) and the promoter is a MND (myeloproliferative sarcoma virus enhancer, negative control region deletion, dl587rev primer binding site substitution) promoter. In an exemplary third generation system, the Env gene is Cokar G protein (Cokar glycoprotein) and the promoter is a CMV promoter. The third generation system uses two packaging plasmids, one encoding gag and pol and the other encoding rev as a further safety feature - an improvement over the single packaging plasmid of the so-called second generation system. Although the third generation system is safer, it is more cumbersome to use and the virus titer is reduced due to the addition of additional plasmids. Exemplary packaging plasmids include, but are not limited to, pMD2.G, pRSV-rev, pMDLG-pRRE and pRRL-GOI.

[0298] Many retroviral particle systems rely on the use of a "packaging cell line". Typically, a packaging cell line is a cell line whose cells are capable of producing infectious retroviral particles when a transfer plasmid, packaging plasmid (one or more) and envelope plasmid are introduced into the cell. Various methods of introducing plasmids into cells can be used, including transfection or electroporation. In some cases, a packaging cell line is suitable for efficiently packaging a retroviral particle system into retroviral particles.

[0299] As used herein, the term "retroviral particle" or "lentiviral particle" refers to a virus particle comprising a polynucleotide encoding a heterologous protein (e.g., a chimeric antigen receptor), one or more capsid proteins, and other proteins required for transduction of the polynucleotide into a target cell. Retroviral particles and lentiviral particles typically include an RNA genome (derived from a transfer plasmid), a lipid bilayer envelope in which the Env protein is embedded, and other auxiliary proteins, including integrase, protease, and matrix protein.

[0300] The in vitro efficiency of retroviral or lentiviral particle systems can be evaluated by various methods known in the art, including measuring vector copy number (VCN) or vector genome (vg), such as by quantitative polymerase chain reaction (qPCR), digital droplet PCR (ddPCR) or viral titer in per milliliter infection unit (lU / mL). For example, titer can be evaluated using a functional assay performed on a cultured tumor cell line HT1080 as described in Humbert et al. Development of Third-generation Cocal Envelope Producer Cell Lines for Robust Retroviral Gene Transfer into Hematopoietic Stem Cells and T-cells. Molecular Therapy 24: 1237–1246 (2016). When titer is evaluated on a cultured cell line that continues to divide, stimulation is not required, so the titer measured is not affected by retroviral particle surface engineering. Other methods for assessing the efficiency of retroviral vector systems are provided in Gaererts et al. Comparison of retroviral vector titration methods. BMC Biotechnol. 6:34 (2006).

[0301] 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 the receptor that specifically binds to a hapten is operably linked to a promoter. Exemplary promoters include, but are not limited to, a cytomegalovirus (CMV) promoter, a CAG promoter, a SV40 promoter, a SV40 / CD43 promoter, an EF-1α promoter, and a MND promoter.

[0302] In some embodiments, the polynucleotide encoding the 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.

[0303] 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.

[0304] In some embodiments, the retroviral particle comprises a transduction enhancer. In some embodiments, the retroviral particle comprises a polynucleotide comprising a sequence encoding a T cell activation protein. In some embodiments, the retroviral particle comprises a polynucleotide comprising a sequence encoding a hapten binding receptor. In some embodiments, the retroviral particle comprises a tag protein.

[0305] In some embodiments, each retroviral particle comprises a polynucleotide comprising, 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 a hapten, and (iv) a 3' LTR or UTR.

[0306] Gene delivery virus particles for the practice of the present disclosure can be constructed using methods known in the field of molecular biology. Typically, the viral vector carrying a transgenic gene is assembled from polynucleotides encoding transgenic genes, suitable regulatory elements, and elements necessary for producing viral proteins that mediate cell transduction. Such recombinant viruses can be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with auxiliary plasmids or viruses. Examples of viral packaging cells include, but are not limited to, HeLa cells, SF9 cells (optionally with baculovirus auxiliary vectors), 293 cells, etc. The detailed scheme for producing such replication-defective recombinant viruses can be found in, for example, WO95 / 14785, WO96 / 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 WO94 / 19478, the complete contents of each of which are incorporated herein by reference.

[0307] Illustrative 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; each of which is incorporated herein by reference in its entirety.

[0308] Payload

[0309] In some embodiments, the viral particle comprises a payload. In some embodiments, the payload is bound 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.

[0310] 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 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.

[0311] In some embodiments, the expression of payload is driven by a promoter. In some embodiments, promoter is MND promoter (myeloproliferative sarcoma virus enhancer, negative control region deletion, d1587rev primer binding site replacement), which is a synthetic promoter of viral origin, containing the U3 region of the modified Moloney leukemia virus (MoMuLV) LTR with myeloproliferative sarcoma virus enhancer 13, and has high expression in human CD34+ stem cells, lymphocytes and other tissues. In some embodiments, a single protein is expressed, separated by the 2A peptide sequence that induces ribosome jumping and cutting during translation. In some embodiments, promoter is a CMV promoter. In some embodiments, promoter is an EF1a promoter. In other embodiments, promoter is an HTLV promoter.

[0312] Chimeric Antigen Receptor

[0313] In some embodiments, the viral particles described herein are used to transduce nucleic acid sequences (polynucleotides) encoding one or more chimeric antigen receptors (CAR) into cells (e.g., T lymphocytes). In some embodiments, the transduction of viral particles results in the expression of one or more CARs in the transduced cells.

[0314] CAR is an artificial membrane-bound protein that guides T lymphocytes to antigens and stimulates T lymphocytes to kill cells that display antigens. For example, see Eshhar, U.S. Patent No. 7,741,465. Typically, CAR is a genetically engineered receptor, comprising an extracellular domain bound to an antigen (e.g., an antigen on a cell), an optional linker, a transmembrane domain, and an intracellular (cytoplasmic) domain comprising a co-stimulatory domain and / or a signal transduction domain that transmits an activation signal to an immune cell. Using CAR, a single receptor can be programmed so that it can both recognize a specific antigen and activate immune cells to attack and destroy cells carrying the antigen when bound to the antigen. When these antigens are present on tumor cells, immune cells expressing CAR can target and kill tumor cells. When all other conditions are met, when CAR is expressed on the surface of, for example, T lymphocytes, and the extracellular domain of CAR binds to the antigen, the intracellular signal transduction domain transmits signals to T lymphocytes to activate and / or proliferate, and if the antigen is present on the cell surface, cells expressing the antigen are killed. Since T lymphocytes require two signals, namely primary activation signals and costimulatory signals, in order to maximize activation, CAR can include a stimulatory domain and a costimulatory domain, so that the antigen is combined with the extracellular domain, causing the transmission of primary activation signals and costimulatory signals. Exemplary CAR is known in the art and can be designed in a modular manner, for example, as described in Guedan S, Calderon H, Posey AD, Maus MV, Molecular Therapy-Methods & Clinical Development.2019; 12: 145-156, which is incorporated by reference.

[0315] In some embodiments, the viral particles disclosed herein encode CARs comprising an extracellular domain, an optional hinge domain, a transmembrane domain, and an intracellular signal transduction domain. In some embodiments, the intracellular signal transduction domain comprises a costimulatory domain and an activation domain. In some embodiments, the costimulatory domain and the activation domain are single domains, such as a single intracellular domain that provides a costimulatory signal and an activation signal to a cell. In other embodiments, the intracellular signal transduction domain comprises a costimulatory domain or an activation domain. In some embodiments, CAR comprises an extracellular domain, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signal transduction domain. In some embodiments, the viral particles disclosed herein encode an extracellular domain, a CD28 hinge domain, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3ζ signal transduction domain. In some embodiments, the viral particles disclosed herein encode extracellular domain, IgG4 hinge domain, CD28 transmembrane domain, 4-1BB costimulatory domain and CD3 ζ signal transduction domain. In some embodiments, the viral particles disclosed herein encode CAR comprising extracellular domain, CD8a hinge, CD28 transmembrane domain, 4-1BB costimulatory domain and CD3 ζ signal transduction domain.

[0316] Exemplary CAR constructs suitable for use in CAR cells, including CAR T cells and CAR NK cells, are provided below:

[0317] (1) scFv-CD8 TM -4-1BB IC -CD3ζ (see, e.g., Liu E, Tong Y, Dotti G, et al., Leukemia. 2018; 32: 520-531);

[0318] (2) scFv-CD28 TM+IC -CD3ζ (see e.g., Han J, Chu J, Keung CW et al., SciRep. 2015;5:11483; Kruschinski A, Moosmann A, Poschke I et al., Proc Natl Acad SciU S A. 2008;105:17481-17486; and Chu J, Deng Y, Benson DM et al. al.,Leukemia.2014;28:917-927);

[0319] (3) scFv-DAP12 TM+IC(See, e.g., Muller N, Michen S, Tietze S et al., J Immunother. 2015; 38: 197-210);

[0320] (4) scFv-CD8 TM -2B4 IC -CD3ζ (see, e.g., Xu Y, Liu Q, Zhong M et al., J Hematol Oncol. 2019; 12:49);

[0321] (5) scFv-2B4 TM+IC -CD3ζ (see, e.g., Altvater B, Landmeier S, Pscherer S et al., Clin Cancer Res. 2009; 15: 4857-4866);

[0322] (6) scFv-CD28 TM+IC -4-1BB IC -CD3ζ (see, e.g., Kloss S, Oberschmidt O, Morgan Met al., Hum Gene Ther. 2017; 28: 897-913);

[0323] (7) scFv-CD16 TM -2B4 IC -CD3ζ (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192);

[0324] (8)scFv-NKp44 TM -DAP10 IC -CD3ζ (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192);

[0325] (9)scFv-NKp46 TM -2B4 IC -CD3ζ (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192);

[0326] (10)scFv-NKG2D TM -2B4IC -CD3ζ (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192);

[0327] (11)scFv-NKG2D TM -4-1BB IC -CD3ζ (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192);

[0328] (12)scFv-NKG2D TM -2B4 IC -DAP12 IC -CD3ζ (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192);

[0329] (13)scFv-NKG2D TM -2B4 IC -DAP10 IC -CD3ζ (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192);

[0330] (14)scFv-NKG2D TM -4-1BB IC -2B4 IC -CD3ζ (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); and

[0331] (15)scFv-NKG2D TM -CD3ζ (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192).

[0332] CAR intracellular domain

[0333] In some embodiments, the intracellular domain of CAR is or is included in the intracellular domain or motif of a protein expressed on the surface of T lymphocytes and triggering the activation and / or proliferation of the T lymphocytes. Such a domain or motif can transmit a signal, which is necessary for activating T lymphocytes in response to antigen binding to the extracellular part of CAR. In some embodiments, the domain or motif includes or is ITAM (immunoreceptor tyrosine activation motif). Suitable ITAM-containing polypeptides for CAR include, for example, ζCD3 chains (CD3ζ) or parts thereof containing ITAM. In some embodiments, the intracellular domain is a CD3ζ intracellular signal transduction domain. In some embodiments, the intracellular domain is from a lymphocyte receptor chain, a TCR / CD3 complex protein, an Fc receptor subunit, or an IL-2 receptor subunit. In some embodiments, the intracellular signal transduction domain of CAR can be derived from, for example, a signal transduction domain of CD3ζ, CD3ε, CD22, CD79a, CD66d, or CD39. "Intracellular signal transduction domain" refers to a part of the CAR polypeptide that is involved in transmitting information about effective CAR binding to the target antigen to the interior of the immune effector cell to trigger effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors to CAR-bound target cells, or other cellular responses triggered by antigen binding to the extracellular CAR domain.

[0334] In some embodiments, the intracellular domain of the CAR is the zeta CD3 chain (CD3ζ).

[0335] In some embodiments, the viral particle comprises a polypeptide comprising a CAR whose intracellular domain comprises a CD3 zeta domain 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:54.

[0336] In some embodiments, the viral particle comprises a nucleic acid encoding an intracellular domain of a CAR comprising a CD3 zeta domain 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:66.

[0337] In some embodiments, CAR additionally includes one or more costimulatory domains or motifs, for example, as part of the intracellular domain of a polypeptide.Costimulatory molecules are well-known cell surface molecules other than antigen receptors or Fc receptors, which provide a second signal required for effective activation and function of T lymphocytes after binding to an antigen. The one or more costimulatory domains or motifs may, for example, be or include one or more of the following: costimulatory CD27 polypeptide sequences, costimulatory CD28 polypeptide sequences, costimulatory OX40 (CD134) polypeptide sequences, costimulatory 4-1BB (CD137) polypeptide sequences, or costimulatory inducible T cell costimulation (ICOS) polypeptide sequences, or other costimulatory domains or motifs, or any combination thereof. In some embodiments, the one or more co-stimulatory domains are selected from the group consisting of the following domains: 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.

[0338] In some embodiments, the costimulatory domain is an intracellular domain of 4-1BB, CD28 or OX40. An exemplary CAR construct comprising a CD28 signal transduction domain is disclosed in U.S. Patent No. 7,446,190, which is incorporated by reference. The construct of an exemplary CAR comprising a 4-1BB signal transduction domain is disclosed in U.S. Patent No. 9,856,322 and U.S. Patent No. 8,399,964, each of which is incorporated by reference.

[0339] In some embodiments, the viral particle encodes a CAR comprising an IgG4 linker operably linked to a CD28 transmembrane domain, which is operably linked to a 4-1BB co-stimulatory domain, which is operably linked to a CD3ζ signaling domain.

[0340] In some embodiments, the viral particle encodes a CAR comprising an IgG4 linker operably linked to a CD8a transmembrane domain, which is operably linked to a 4-1BB co-stimulatory domain, which is operably linked to a CD3ζ signaling domain.

[0341] In some embodiments, the viral particle encodes a CAR comprising an IgG4 linker operably linked to a CD8a transmembrane domain, which is operably linked to a CD28 co-stimulatory domain, which is operably linked to a CD3ζ signaling domain.

[0342] In some embodiments, the viral particle encodes a CAR comprising a CD8a linker, which is operably linked to a CD8a transmembrane domain, which is operably linked to a 4-1BB co-stimulatory domain, and which is operably linked to a CD3ζ signaling domain.

[0343] In some embodiments, the viral particle encodes a CAR comprising a CD28 linker operably linked to a CD28 transmembrane domain, which is operably linked to a CD28 co-stimulatory domain, which is operably linked to a CD3ζ signaling domain.

[0344] In some embodiments, the viral particle comprises a polypeptide comprising a CAR, the intracellular domain of which comprises a co-stimulatory 4-1BB polypeptide 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:53.

[0345] In some embodiments, the viral particle comprises a nucleic acid encoding an intracellular domain of a CAR comprising a co-stimulatory 4-1BB 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:65.

[0346] In some embodiments, the viral particle comprises a polypeptide comprising a CAR, the intracellular domain of the CAR comprising an IgG4 linker, the IgG4 linker being operably linked to a transmembrane domain derived from CD28, the transmembrane domain derived from CD28 being operably linked to a co-stimulatory 4-1BB polypeptide, the co-stimulatory 4-1BB polypeptide being operably linked to a CD3 ζ domain, and the polypeptide being 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: 80.

[0347] In some embodiments, the viral particle comprises a nucleic acid encoding an intracellular domain of a CAR, the intracellular domain of the CAR comprising an IgG4 linker, the IgG4 linker being operably linked to a transmembrane domain derived from CD28, the transmembrane domain derived from CD28 being operably linked to a co-stimulatory 4-1BB polypeptide, the co-stimulatory 4-1BB polypeptide being operably linked to a CD3 zeta domain, and the nucleic acid being 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: 86.

[0348] In some embodiments, the viral particle comprises a polypeptide comprising a CAR, the intracellular domain of the CAR comprising an IgG4 linker, the IgG4 linker being operably linked to a transmembrane domain derived from CD28, the transmembrane domain derived from CD28 being operably linked to a co-stimulatory 4-1BB polypeptide, the co-stimulatory 4-1BB polypeptide being operably linked to a CD3 ζ domain, and the polypeptide being 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: 90.

[0349] In some embodiments, the viral particle comprises a nucleic acid encoding an intracellular domain of a CAR, the intracellular domain of the CAR comprising an IgG4 linker, the IgG4 linker being operably linked to a transmembrane domain derived from CD28, the transmembrane domain derived from CD28 being operably linked to a co-stimulatory 4-1BB polypeptide, the co-stimulatory 4-1BB polypeptide being operably linked to a CD3 zeta domain, and the nucleic acid being 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: 95.

[0350] In some embodiments, the intracellular domain can be further modified to encode, for example, a detectable fluorescent protein (eg, green fluorescent protein) or any known variant thereof.

[0351] CAR transmembrane region

[0352] The transmembrane region can be any transmembrane region that can be incorporated into a functional CAR, such as a transmembrane region from a CD28, CD4 or CD8 molecule.

[0353] In some embodiments, the transmembrane domain of the CAR can be derived from the transmembrane domain of CD8, α, β or ζ chain of the T cell receptor, CD28, CD3ε, 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, IL2Rβ, IL2Rγ, IL 7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LF A-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, 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 CAR can be derived from the transmembrane domain of CD28. In some embodiments, the transmembrane domain of the CAR can be derived from the transmembrane domain of CD8, such as CD8α.

[0354] CAR linker region

[0355] The optional linker or hinge of the CAR between the extracellular domain and the transmembrane domain can be a polypeptide of about 2 to more than 100 amino acids in length. The linker can contain or consist of flexible residues such as glycine and serine, so that adjacent protein domains can move freely with each other. For example, when it is necessary to ensure that two adjacent domains do not interfere with each other in space, a longer linker can be used. When the target antigen is closer to the cell surface, a longer linker can also have an advantage.

[0356] 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.

[0357] In some embodiments, the linker is an IgG4 linker that is operably linked to the CD28-derived transmembrane domain, and the IgG4 linker 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:52.

[0358] In some embodiments, the linker is an IgG4 linker that is operably linked to a CD28-derived transmembrane domain, and the IgG4 linker 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:64.

[0359] CAR extracellular domain

[0360] In some embodiments, the nucleic acid transduced into the cell 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 may comprise, for example, a VL connected to a VH by a flexible linker, wherein the VL and VH are from an antibody that binds to the antigen.

[0361] In some embodiments, the extracellular domain of CAR may include any polypeptide that binds to the desired antigen (e.g., a prostate neoantigen or an antigen expressed on a target tumor). The extracellular domain may include a portion of an scFv, an antibody, or an alternative scaffold. CAR can also be engineered to bind to two or more desired antigens, which can be arranged in series and separated by a linker sequence. For example, one or more domain antibodies, scFv, camel VHH antibodies, or other VH-only antibody fragments can be organized in series by a linker to provide bispecific or multispecific CAR.

[0362] The antigen bound by the extracellular domain of the polypeptide can be any target antigen, for example, it can be an antigen on a tumor cell. The tumor cell can be, for example, a cell in a solid tumor or a blood cancer cell. The antigen can be any antigen expressed on a cell of any tumor or cancer type, for example, a cell of the following tumor or cancer: lymphoma, lung cancer, breast cancer, prostate cancer, adrenocortical carcinoma, thyroid cancer, nasopharyngeal carcinoma, melanoma (e.g., malignant melanoma), skin cancer, colorectal cancer, desmoid tumor, desmoplastic small round cell tumor, endocrine tumor, Ewing's sarcoma, peripheral primitive neuroectodermal tumor, solid germ cell tumor, hepatoblastoma, neuroblastoma, non-rhabdomyosarcoma soft tissue sarcoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, Wilms' tumor, glioblastoma, myxoma, fibroma, lipoma, etc. In some embodiments, the lymphoma can be chronic lymphocytic leukemia (small lymphocytic lymphoma), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, Waldenstrom's macroglobulinemia, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal zone B-cell lymphoma, MALT lymphoma, intranodal 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's lymphoma, T-lymphocyte prolymphocytes, The invention relates to a method for treating a patient with a cancer of the present invention, wherein the cancer is a chronic lymphocytic leukemia (CLL), wherein the B cells of the CLL have a normal karyotype. In some embodiments, wherein the cancer is chronic lymphocytic leukemia (CLL), the B cells of the CLL carry a 17p deletion, a 11q deletion, a 12q trisomy, a 13q deletion, or a p53 deletion.

[0363] In some embodiments, the antigen is expressed on the following cells: B cell malignancy cells, relapsed / refractory CD19 expressing malignancy cells, diffuse large B cell lymphoma (DLBCL) cells, Burkitt's 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 malignancy 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 cancer cells, leukemia cells, myeloma cells, B cell lymphoma cells, kidney cancer cells, uterine cancer cells, adenocarcinoma cells, pancreatic cancer cells, chronic myeloid leukemia cells, glioblastoma cells, neuroblastoma cells, medulloblastoma cells or sarcoma cells.

[0364] In some embodiments, the antigen is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). In some embodiments, without limitation, 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, carboxyanhydrase IX (CAIX), Claudin 18.2, FAP, mesothelin, IL13Ra2, Lewis Y, CCNA1, WT-1, TACI, CD38, SLAMF7, CD138, DLL3, transmembrane 4L6 family member 1 (TM4SF1), epithelial cell adhesion molecule (EpCAM), PD-1, PD-L1, CTLA-4, AXL, ROR2, phosphatidylinositol proteoglycan-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, calcium-binding protein, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, black The invention relates to melanoma-associated antigen (MAGE), CD19, CD20, CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acid protein (GFAP), fluid protein of giant cystic disease (GCDFP-15), HMB-45 antigen, protein melanin-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-1, vascular endothelial growth factor receptor (VEGFR), dimer of M2-type pyruvate kinase isozyme (tumor M2-PK), abnormal ras protein or abnormal p53 protein. In some embodiments, CAR comprises a binding domain targeting 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, CAR comprises binding domains targeting two or more antigens on the same target protein, such as two epitopes in CD19, BCMA or any other antigen disclosed herein.

[0365] In other embodiments, the CAR is a universal CAR and does not specifically target a tumor antigen itself. For example, the CAR may comprise a tag-specific scFv such that an exogenous agent comprising a tag and a tumor targeting domain can guide the universal CAR T cell to the target tumor.

[0366] 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 CD3ζ intracellular signaling domain.

[0367] In some embodiments, the antigen is CD19. CAR T therapy targeting CD19 has been approved by the FDA, including Yescarta, Tecartus, Kymriah and Breyanzi. CAR targeting CD19 is described in, for example, U.S. 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 by reference.

[0368] In some embodiments, CAR comprises an extracellular domain comprising an FMC63 scFv binding domain for CD19 binding. In some embodiments, CAR is a second generation CAR, consisting of FMC63 mouse anti-human CD19 scFv connected to a 4-1BB costimulatory domain and a CD3 ζ intracellular signal transduction domain. In some embodiments, CAR comprises a binding domain of CD19, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB costimulatory domain, and a CD3 ζ signal transduction domain. In some embodiments, CAR comprises a binding domain of CD19, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 ζ signal transduction domain. In some embodiments, CAR comprises a binding domain of CD19, a CD28 hinge, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3 ζ signal transduction domain. In some embodiments, CAR comprises an extracellular domain containing an FMC63 scFv binding domain for CD19 binding, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 ζ signal transduction domain. In some embodiments, CAR comprises an extracellular domain containing an FMC63 scFv binding domain for CD19 binding, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 ζ signal transduction domain. In some embodiments, CAR comprises an extracellular domain containing an FMC63 scFv binding domain for CD19 binding, a CD28 hinge, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3 ζ signal transduction domain.

[0369] In some embodiments, the viral particle comprises a polypeptide comprising a CAR, the extracellular domain of which comprises a signal peptide 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:50.

[0370] In some embodiments, the viral particle comprises a polynucleotide encoding a CAR whose extracellular domain comprises an αCD19 scFv (CD19 VL linked to CD19 VH) 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:51.

[0371] The complementarity determining regions (CDRs) of the 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), VIWGSETTYYN SALKS (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, the extracellular domain of the CAR comprising an αCD19 scFv having these CDRs, wherein optionally the αCD19 scFv 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: 51.

[0372] In some embodiments, the viral particle comprises a polynucleotide encoding a CAR whose extracellular domain comprises an αCD19 scFv having these CDRs, wherein optionally the αCD19 scFv 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:51 or 89.

[0373] In some embodiments, the viral particle comprises a nucleic acid encoding a signal peptide of the extracellular domain of CAR, wherein the nucleic acid 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:62.

[0374] In some embodiments, the viral particle comprises a nucleic acid encoding an extracellular domain of a CAR comprising an αCD19 scFv 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:63.

[0375] In some embodiments, the viral particle comprises a polypeptide comprising a CAR whose extracellular domain comprises an αCD19 scFv 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:79.

[0376] In some embodiments, the viral particle comprises a nucleic acid encoding an extracellular domain of a CAR comprising an αCD19 scFv 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:85.

[0377] In some embodiments, the viral particle comprises a polynucleotide encoding a CAR whose extracellular domain comprises an αCD19 scFv 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:89.

[0378] The complementarity determining regions (CDRs) of the 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), VIWGSETTYYN SALKS (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, the extracellular domain of the CAR comprising an αCD19 scFv having these CDRs, wherein optionally the αCD19 scFv 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: 89.

[0379] In some embodiments, the viral particle comprises a nucleic acid encoding an extracellular domain of a CAR comprising an αCD19 scFv 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:94.

[0380] In some embodiments, CAR is a second generation CAR, consisting of FMC63 mouse anti-human CD19 scFv connected to a CD28 costimulatory domain and a CD3 ζ intracellular signal transduction domain. In some embodiments, CAR is a second generation CAR, consisting of FMC63 mouse anti-human CD19 scFv connected to a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 ζ intracellular signal transduction domain.

[0381] In some embodiments, CAR is an anti-FITC CAR, and the ligand is composed of a fluorescein or fluorescein isothiocyanate (FITC) portion bound to an agent that binds to a desired target cell (e.g., a cancer cell). The above section describes an exemplary ligand. In some embodiments, the ligand is FITC-folic acid.

[0382] In some embodiments, 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 joint and a heavy chain variable domain (VH).

[0383] 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.

[0384] 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.

[0385] 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.

[0386] 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.

[0387] 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.

[0388] 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.

[0389] 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.

[0390] 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.

[0391] In some embodiments, the anti-fluorescein E2 scFv comprises CDRL1, CDRL2, and CDRL3 that have at least 80% amino acid identity, at least 90% amino acid identity, or at least 95% amino acid identity with TSNIGNNYVS (SEQ ID NO: 167), LMIYDVSKRPS (SEQ ID NO: 168), and AAWDDSLSEF (SEQ ID NO: 169), respectively, and CDRH1, CDRH2, and CDRH3 that have at least 80% amino acid identity, at least 90% amino acid identity, or at least 95% amino acid identity with FTFGSFSMS (SEQ ID NO: 170), WVAGLSARSSLTHY (SEQ ID NO: 171), and RRSYDSSGYWGHFYSYMDV (SEQ ID NO: 172), respectively.

[0392] In some embodiments, antigen is BCMA.CAR-T therapy targeting BCMA has been approved by the FDA, including Abecma and Carvykti.CAR targeting BCMA is described in, for example, U.S. Publication No. 2020 / 0246381, U.S. Patent No. 10,918,665, and U.S. Publication No. 2019 / 0161553, all of which are incorporated herein by reference.In some embodiments, CAR includes a binding domain, CD8a hinge, CD8a transmembrane domain, 4-1BB costimulatory domain, and CD3ζ signal transduction domain of BCMA.In some embodiments, CAR includes a binding domain, IgG4 hinge, CD28 transmembrane domain, 4-1BB costimulatory domain, and CD3ζ signal transduction domain of BCMA.In some embodiments, CAR includes a binding domain, CD28 hinge, CD28 transmembrane domain, CD28 costimulatory domain, and CD3ζ signal transduction domain of BCMA.

[0393] In some embodiments, the antigen is G protein coupled receptor C class group 5 member D (GPRC5D). The CAR targeting GRC5D is described in, for example, U.S. Publication Nos. 2018 / 0118803 and 2021 / 10393689, both of which are incorporated herein by reference. In some embodiments, CAR includes a binding domain, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB costimulatory domain, and a CD3 ζ signal transduction domain of GRC5D. In some embodiments, CAR includes a binding domain, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 ζ signal transduction domain of GRC5D. In some embodiments, CAR includes a binding domain, a CD28 hinge, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3 ζ signal transduction domain of GRC5D.

[0394] In some embodiments, the antigen is Fc receptor-like 5 (FcRL5). CAR targeting FcRL5 is described in, for example, U.S. Publication No. US2017 / 0275362, which is incorporated herein by reference. In some embodiments, CAR includes a binding domain, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 ζ signal transduction domain of FcRL5. In some embodiments, CAR includes a binding domain, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 ζ signal transduction domain of FcRL5. In some embodiments, CAR includes a binding domain, a CD28 hinge, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3 ζ signal transduction domain of FcRL5.

[0395] In some embodiments, the antigen is a receptor tyrosine kinase-like orphan receptor 1 (ROR1). The CAR of target ROR1 is described in, for example, U.S. Publication No. 2022 / 0096651, which is incorporated herein by reference. In some embodiments, CAR comprises a binding domain, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signal transduction domain of ROR1. In some embodiments, CAR comprises a binding domain, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signal transduction domain of ROR1. In some embodiments, CAR comprises a binding domain, a CD28 hinge, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3ζ signal transduction domain of ROR1.

[0396] In some embodiments, CAR is a second generation CAR, consisting of an anti-BCMA scFv connected to a 4-1BB costimulatory domain and a CD3 ζ intracellular signaling domain. In some embodiments, CAR is a second generation CAR, consisting of an anti-GPRC5D scFv connected to a 4-1BB costimulatory domain and a CD3 ζ intracellular signaling domain. In some embodiments, CAR is a second generation CAR, consisting of an anti-ROR1 scFv connected to a 4-1BB costimulatory domain and a CD3 ζ intracellular signaling domain.

[0397] 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.

[0398] In some embodiments, the TAA or TSA is a carbohydrate or ganglioside, such as fuc-GM1, GM2 (carcinoembryonic antigen-immunogenic-1; OFA-I-1); GD2 (OFA-I-2), GM3, GD3, etc.

[0399] In some embodiments, the TAA or TSA is alpha-actinin-4, Bage-1, BCR-ABL, Bcr-Abl fusion protein, beta-catenin, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, 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-A11, 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, Gage 3, 4, 5, 6, 7, GnTV, Herv-K-mel, Lage-1, NA-88, NY-Eso-1 / Lage-2, SP17, SSX-2, TRP2-Int2, gpl00 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, RAGE, GAGE-1, GAGE-2, pl5(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, pl85erbB2, pl80erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1. NuMa, K-ras, 13-Catenin, Mum-1, pl6, 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, TAG7 2, 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 (prostatic acid phosphatase), prostein, TARP (T cell receptor gamma variable reading frame protein), Trp-p8, STEAP1 (prostatic six transmembrane epithelial antigen 1), abnormal ras protein or abnormal p53 protein. In some embodiments, the tumor-associated antigen or tumor-specific antigen is integrin αvβ3 (CD61), galactosylprotein, K-Ras (V-Ki-ras2 Kirsten rat sarcoma virus oncogene) or Ral-B. Other tumor-associated and tumor-specific antigens are known to those skilled in the art. .

[0400] Antibodies and scFvs that bind to TSA and TAA include antibodies and scFvs known in the art, and nucleotide sequences encoding them.

[0401] In some embodiments, the antigen is an antigen that is not considered to be a TSA or TAA, but it is still associated with damage caused by tumor cells or tumors. In some embodiments, for example, the antigen is, for example, a growth factor, a cytokine, or an interleukin, such as a growth factor, a cytokine, or an interleukin associated with angiogenesis or vasculogenesis. Such growth factors, cytokines, or interleukins may 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). Tumors can also produce a hypoxic environment locally in the tumor. Therefore, in some embodiments, the antigen is a hypoxia-related factor, such as HIF-1α, HIF-1β, HIF-2α, HIF-2β, HIF-3α, or HIF-3β. Tumors can also cause local damage to normal tissues, resulting in the release of molecules known as damage-associated molecular pattern molecules (DAMPs; also known as alarmins). Thus, in some embodiments, the antigen is a DAMP, such as a heat shock protein, the chromatin-associated protein high mobility group protein B1 (HMGB1), S100A8 (MRP8, calgranulin A), S100A9 (MRP14, calgranulin B), serum amyloid A (SAA), or may be deoxyribonucleic acid, adenosine triphosphate, uric acid, or heparin sulfate.

[0402] In some embodiments of the polypeptides described herein, the extracellular domain is directly linked to the transmembrane domain or is linked to the transmembrane domain via a linker, spacer, or hinge polypeptide sequence (e.g., a sequence from CD28 or a sequence from CTLA4).

[0403] In some embodiments, the extracellular domain that binds a desired antigen can be derived from an antibody or antigen-binding fragment thereof generated using the techniques described herein.

[0404] Universal CAR

[0405] In some embodiments, the viral particles described herein include a nucleotide sequence encoding a universal CAR. Universal CAR allows targeting cancer cells without changing the antigen specificity of CAR. For example, universal CAR is described in U.S. Publication Nos. US2016 / 0348073, US2.018 / 0085399, US 2019 / 0256597, and US2014 / 0349402, all of which are incorporated herein by reference.

[0406] In some embodiments, the viral particles described herein include nucleotide sequences encoding universal, modular, anti-tag chimeric antigen receptors (UniCAR). The system allows UniCAR-implanted immune cells to be retargeted to multiple antigens (see, e.g., U.S. Patent Publication US20170240612 A1, which is incorporated herein by reference in its entirety; Cartellieri et al., (2016) Blood Cancer Journal 6, e458, which is incorporated herein by reference in its entirety).

[0407] In some embodiments, the viral particles described herein include nucleotide sequences encoding convertible CAR and / or CAR effector cell (CAR-EC) switches. In this system, the CAR-EC switch has a first region bound to the CAR on the CAR-EC and a second region bound to the cell surface molecule on the target cell, thereby stimulating the immune response of the CAR-EC with cytotoxicity 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 stopping the application of the switch. These CAR-EC switches can be used with CAR-EC disclosed herein and existing CAR T cells for the treatment of diseases or disorders, such as cancer, wherein the target cell is a malignant cell. Such treatments may be referred to herein as convertible immunotherapy (US Patent Publication US9624276 B2, which is incorporated herein by reference in its entirety).

[0408] In some embodiments, the viral particle comprises a nucleotide sequence encoding a universal immune receptor (e.g., convertible CAR, sCAR) bound to a peptide neo-epitope (PNE). In some embodiments, the peptide neo-epitope (PNE) has been incorporated into different positions defined in the antibody targeting the antigen (antibody switch). Therefore, sCAR-T cells are specific only to PNE and do not occur in the human proteome, thereby allowing orthogonal interactions between sCAR-T cells and antibody switches. In this way, sCAR-T cells strictly rely on the presence of an antibody switch to be fully activated, thereby excluding the off-target recognition of endogenous tissues or antigens by CAR-T cells in the absence of an antibody switch (Arcangeli et al., (2016) Transl Cancer Res 5 (Suppl 2): ​​S174-S177 (which is incorporated herein by reference in its entirety). U.S. Patent Application US20160272718A1 provides other examples of convertible CARs, which are incorporated herein by reference in their entirety.

[0409] As used herein, the term "tag" encompasses the Fc region of a universal immune receptor, tag, switch or immunoglobulin as described above. In some embodiments, the viral particle comprises a nucleotide sequence encoding a CAR comprising a tag binding domain. In some embodiments, CAR binds fluorescein isothiocyanate (FITC), streptavidin, biotin, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horseradish peroxidase, palmitoylation, nitrosylation, alkaline alanine phosphatase, glucose oxidase or maltose binding protein.

[0410] In some embodiments, the viral particle comprises a nucleotide sequence encoding a CAR to produce a CAR cell for use with a targeted small molecule. In some embodiments, CAR targets a portion of the cells of the treated subject that is not produced or expressed. Therefore, this CAR system can focus immune cells on target cells, such as cancer cells. The two-component CAR system has been previously described in, for example, US2015 / 0320799, US 2019 / 0224237, and US2020 / 0023009, all of which are incorporated herein by reference.

[0411] In some embodiments, the targeting small molecule comprises a ligand of a tumor cell receptor. By administering the targeting small molecule together with the immune cells expressing CAR, the immune cell response can be targeted only to cells expressing tumor receptors, thereby reducing off-target toxicity, and the activation of immune cells can be more easily controlled due to the rapid clearance of the targeting small molecule. Another advantage is that CAR-expressing immune cells can be used as universal cytotoxic cells to target a variety of tumors without the need to prepare separate CAR constructs. The targeting small molecules recognized by CAR can also remain unchanged. Only the ligand portion of the targeting small molecule needs to be changed to enable the system to target cancer cells of different identities.

[0412] In some embodiments, the targeting small molecule comprises a fluorescein connected to a ligand of a selected tumor cell receptor. In some embodiments, the targeting small molecule comprises FITC connected to a ligand of a selected tumor cell receptor. In some embodiments, the viral vector described herein encodes a CAR comprising an anti-fluorescein scFv. In some embodiments, the viral vector described herein encodes a CAR comprising an anti-FITC scFv. Therefore, the CAR targets fluorescein or FITC, rather than tumor-associated antigens that may also be expressed by healthy non-target cells. These two components are applied to a subject with cancer, and the targeting small molecule binds to the target tumor cell (by binding to the homologous tumor cell receptor through the ligand portion of the molecule). Then, the FITC portion of the targeting small molecule is recognized and bound by the anti-FITC CAR (second component) expressed by the T cell. After binding, immune cells expressing anti-FITC CAR are activated and tumor cells are killed. It will be clear to those skilled in the art that immune cells cannot kill cells if they do not first bind to tumor cells. It is further obvious that immune cells will not bind to non-target cells because the recognition region of CAR will only recognize and bind to FITC, and FITC is not produced or expressed by the subject's cells. Therefore, the targeted small molecule acts as a bridge between immune cells and target tumor cells. As long as the targeted portion of the targeted small molecule is a portion that does not exist in the host, the activity of the immune cells can be limited to the target cells. In addition, the activation of immune cells expressing CAR can be regulated by limiting the amount of targeted small molecules administered to the subject, for example, by manipulating the infusion of targeted small molecules if side effects are detected. Exemplary anti-fluorescein and anti-FITC CARs are described in U.S. Patent Application US20200405760A1, which is incorporated herein by reference in its entirety.

[0413] In some embodiments, the targeting small molecule comprises 2,4-dinitrophenol (DNP), 2,4,6-trinitrophenol (TNP), biotin, digoxigenin, fluorescein, fluorescein isothiocyanate (FITC), NHS-fluorescein, pentafluorophenyl ester, tetrafluorophenyl ester, kinetochoresin, centyrin, DARPin, affinity body, affilin, anticalin, atrimer, avimer, bicyclic peptide, FN3 scaffold, cys-knot, fynomer, Kunitz domain or Obody. In some embodiments, the viral vector comprises a nucleotide sequence encoding a CAR comprising an extracellular binding domain that binds 2,4-dinitrophenol (DNP), 2,4,6-trinitrophenol (TNP), biotin, digoxigenin, fluorescein, fluorescein isothiocyanate (FITC), NHS-fluorescein, pentafluorophenyl ester, tetrafluorophenyl ester, kinetochoresin, centyrin, DARPin, affinity body, affilin, anticalin, atrimer, avimer, bicyclic peptide, FN3 scaffold, cys-knot, fynomer, Kunitz domain or Obody.

[0414] In some embodiments, the CAR system uses a conjugated molecule as a bridge between CAR-expressing cells and targeted cancer cells. The conjugated molecule is a conjugate comprising a hapten and a cell targeting portion (such as any suitable tumor cell-specific ligand). Exemplary haptens that can be recognized and bound by CAR include small molecular weight organic molecules, such as DNP (2,4-dinitrophenol), TNP (2,4,6-trinitrophenol), biotin and digoxin, as well as fluorescein and its derivatives, including FITC (fluorescein isothiocyanate), NHS-fluorescein, pentafluorophenyl ester (PFP) and tetrafluorophenyl ester (TFP) derivatives, scutellarin, centyrin and DARPin. Suitable cell targeting moieties themselves can serve as haptens of CAR, including scutellarin (see Kolmar H.etal., The FEBS Journal.2008.275 (11): 26684-90), centyrin and DARPin (see Reichert, JMMAbs 2009.1 (3): 190–209).

[0415] In some embodiments, the cell targeting moiety is DUPA (DUPA-(99m)Tc), a ligand that binds to PSMA-positive human prostate cancer cells with nanomolar affinity (K D=14 nM; see Kularatne, SA et al., Mol Pharm. 2009. 6(3):780-9). In one embodiment, the DUPA derivative can be a ligand of a small molecule ligand linked to a targeting moiety, and the DUPA derivative is described in WO 2015 / 057852, which is incorporated herein by reference.

[0416] In some embodiments, the cell targeting moiety is a CCK2R ligand, a ligand that binds to CCK2R-positive cancer cells (e.g., thyroid cancer, lung cancer, pancreatic cancer, ovarian cancer, brain cancer, gastric cancer, gastrointestinal stromal cancer, and colon cancer; see Wayua. C. et al., Molecular Pharmaceutics. 2013. ePublication).

[0417] In some embodiments, the cell targeting moiety is folate, folic acid, or an analog thereof, a ligand that binds to a folate receptor on cancer cells, including ovarian cancer, cervical cancer, endometrial cancer, lung cancer, kidney cancer, brain cancer, breast cancer, colon cancer, and head and neck cancer; see Sega, EI et al., Cancer Metastasis Rev. 2008. 27(4):655-64.

[0418] In some embodiments, the cell targeting moiety is a NK-1R ligand. For example, receptors for NK-1R ligands are found in colon cancer and pancreatic cancer. In some embodiments, the NK-1R ligand can be synthesized according to the methods disclosed in International Patent Application No. PCT / US2015 / 044229. This patent is incorporated herein by reference.

[0419] 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 of the NK1 receptor. In some embodiments, the small conjugated 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 (neuromodulatory peptide K), (see Hennig et al., International Journal of Cancer: 61, 786-792).

[0420] In some embodiments, the cell targeting moiety is a CAIX ligand. For example, receptors for the CAIX ligand are found in renal cancer, ovarian cancer, vulvar cancer, and breast cancer. The CAIX ligand may also be referred to herein as CA9.

[0421] In some embodiments, the cell targeting moiety is a ligand for gamma glutamyl transpeptidase. For example, transpeptidase is overexpressed in ovarian cancer, colon cancer, liver cancer, astrocytoma, melanoma, and leukemia.

[0422] In some embodiments, the cell targeting moiety is a CCK2R ligand. Receptors for CCK2R ligands are found in thyroid cancer, lung cancer, pancreatic cancer, ovarian cancer, brain cancer, gastric cancer, gastrointestinal stromal cancer, and colon cancer, among others.

[0423] In one embodiment, the mass of the cell targeting moiety can be 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 mass of the small molecule ligand can be 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.

[0424] In an exemplary embodiment, the connection in the conjugate described herein can be a direct connection (e.g., a reaction between the isothiocyanate group of FITC and the free amine group of the small molecule ligand) or the connection can be carried out through an intermediate linker. In one embodiment, if present, the intermediate linker can be any biocompatible linker known in the art, such as a divalent linker. In an exemplary embodiment, the divalent linker can contain about 1 to about 30 carbon atoms. In another exemplary embodiment, the divalent linker can contain about 2 to about 20 carbon atoms. In other embodiments, a lower molecular weight divalent linker (i.e., a linker with an approximate molecular weight of about 30 to about 300 Da) is used. In another embodiment, suitable linker lengths include, but are not limited to, 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.

[0425] In some embodiments, the hapten and cell targeting moiety can be directly conjugated by reaction between the isothiocyanate group of FITC and the free amine group of a small ligand (e.g., folate, DUPA, and CCK2R ligand). However, it may be helpful to use a linker domain to connect the two molecules because it can provide flexibility and stability. Examples of suitable linker domains include: 1) polyethylene glycol (PEG); 2) polyproline; 3) hydrophilic amino acids; 4) sugars; 5) non-natural peptidoglycans; 6) polyvinyl pyrrolidone; 7) pluronic F-127. Suitable linker lengths include, but are not limited to, 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.

[0426] In some embodiments, the linker can be a bivalent linker, which can include one or more spacers.

[0427] Exemplary conjugates of the present disclosure include the following molecules: (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.

[0428] Although the affinity with which a ligand binds to a cancer cell receptor can vary, and in some cases low affinity binding may be preferred (e.g., about 1 μM), the binding affinity of a ligand to a cancer cell receptor is typically 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.

[0429] Examples of conjugates and methods for making them are provided in U.S. patent applications US2017 / 0290900, US 2019 / 0091308, and US2020 / 0023009, all of which are incorporated herein by reference.

[0430] Rapamycin-activated cell surface receptor (RACR)

[0431] In some embodiments, the viral particle comprises a nucleotide sequence encoding a multipartite cell surface receptor. In some embodiments, the multipartite cell surface receptor is a proliferation receptor.

[0432] In some embodiments, the multipartite cell surface receptor is a rapamycin-activated cell surface receptor (RACR).

[0433] In some embodiments, the multipartite cell surface receptor is a chemically inducible cell surface receptor.

[0434] In some embodiments, the multipartite cell surface receptor comprises a polynucleotide sequence encoding a 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 a FK506 binding protein domain (FKBP) or a functional variant thereof. In some embodiments, the FKBP is FKBP12.

[0435] In some embodiments, the viral particle comprises a RACR polypeptide comprising a signal peptide operably linked to FKBP 12, wherein the polypeptide 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:57.

[0436] In some embodiments, the viral particle comprises a RACR polypeptide comprising an IL-2Rγ transmembrane domain operably linked to a cytoplasmic domain, the RACR polypeptide being 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:58.

[0437] In some embodiments, the viral particle comprises a RACR polypeptide comprising a P2A self-cleaving peptide, wherein the RACR polypeptide 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:55.

[0438] In some embodiments, the viral particle comprises a RACR polypeptide comprising a signal peptide operably linked to a FRB, wherein the RACR polypeptide 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:59.

[0439] In some embodiments, the viral particle comprises a RACR polypeptide comprising an IL-2Rβ transmembrane domain operably linked to a cytoplasmic domain, the RACR polypeptide being 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:60.

[0440] In some embodiments, the viral particle comprises a nucleic acid encoding a signal peptide operably linked to FKBP12, wherein the nucleic acid 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:70.

[0441] In some embodiments, the viral particle comprises a nucleic acid encoding an IL-2Rγ transmembrane domain operably linked to a cytoplasmic domain, wherein the nucleic acid 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:71.

[0442] In some embodiments, the viral particle comprises a nucleic acid encoding a P2A self-cleaving peptide 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:72.

[0443] In some embodiments, the viral particle comprises a nucleic acid encoding a signal peptide operably linked to an FRB, wherein the nucleic acid 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:73.

[0444] In some embodiments, the viral particle comprises a nucleic acid encoding an IL-2Rβ transmembrane domain operably linked to a cytoplasmic domain, wherein the nucleic acid 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:74.

[0445] In some embodiments, the viral particle comprises a RACR polypeptide comprising FKBP12, FKBP12 is operably linked to an IL-2Rγ domain, the IL-2Rγ domain is operably linked to a P2A peptide, and the RACR polypeptide 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:77.

[0446] In some embodiments, the viral particle comprises a RACR polypeptide comprising a FRB, the FRB being operably linked to an IL-2Rβ domain, the IL-2Rβ domain being operably linked to a P2A peptide, the RACR polypeptide being 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:78.

[0447] In some embodiments, the viral particle comprises a nucleic acid encoding FKBP12, which is operably linked to an IL-2Rγ domain, which is operably linked to P2A, and the nucleic acid 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:83.

[0448] In some embodiments, the viral particle comprises a nucleic acid encoding FRB, which is operably linked to an IL-2Rβ domain, which is operably linked to P2A, and the nucleic acid 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:84.

[0449] In some embodiments, the FKBP domain and the FRB domain form a T cell activation 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.

[0450] In some embodiments, the ligand is rapamycin.

[0451] In some embodiments, the FRB domain and FKBP form a tripartite complex with rapamycin, thereby sequestering rapamycin in the transduced cell.

[0452] In some embodiments, the ligand is a protein, an antibody, a small molecule or a drug. In some embodiments, the ligand is rapamycin or a rapamycin analog (rapalog). In some embodiments, the rapamycin analog comprises a rapamycin variant with one or more modifications relative to rapamycin: demethylation, elimination or replacement of the methoxy group at C7, C42 and / or C29; elimination, derivatization or replacement of the hydroxyl group at C13, C43 and / or C28; reduction, elimination or derivatization of ketones at C14, C24 and / or C30; replacement of the 6-membered pipecolic acid ring with a 5-membered prolyl ring; and substitution substitution on the cyclohexyl ring or replacement of the cyclohexyl ring with a substituted cyclopentyl ring. Therefore, in some embodiments, the rapamycin analog is everolimus, novolimus, pimecrolimus, lirolimus, tacrolimus, temsirolimus, euclid, zotarolimus, CCI-779, C20-methallyl rapamycin, Cl6-(S)-3-methylindorapamycin, C16-iRap, AP21967, mycophenolate sodium, benidipine hydrochloride, rapamine, AP23573 or AP1903, or a metabolite, derivative and / or combination thereof. In some embodiments, the ligand is an IMID drug (e.g., thalidomide, pomalidomide, lenalidomide or related analogs).

[0453] In some embodiments, the molecule is selected from FK1012, tacrolimus (FK506), FKCsA, rapamycin, coumermycin, gibberellin, HaXS, TMP-HTag and ABT-737 or a functional derivative thereof.

[0454] In some embodiments, the FKBP domain is operably connected to the IL2Rγ domain. In some embodiments, the FRB domain is operably connected to the IL2Rβ domain. In some embodiments, the IL2Rγ domain and the IL2Rβ domain are heterodimerized. In some embodiments, the IL2Rγ domain and the IL2Rβ domain are heterodimerized in the presence of a ligand to promote the growth and / or survival of cells. In some embodiments, the IL2Rγ domain and the IL2Rβ domain are heterodimerized in the presence of rapamycin to promote the growth and / or survival of cells. In some embodiments, the IL2Rγ domain and the IL2Rβ domain are heterodimerized in the presence of rapamycin to promote T cell activation.

[0455] Cytoplasmic FRB

[0456] In some embodiments, the vector genome comprises a nucleotide sequence that confers resistance to an immunosuppressant. In some embodiments, the nucleotides that confer resistance to an immunosuppressant are combined with rapamycin. In some embodiments, the polynucleotides that confer resistance to an immunosuppressant encode a cytoplasmic ("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 cytoplasm as a freely diffusible soluble protein. The purpose of the FRB domain is to reduce the inhibitory effect of rapamycin on mTOR in transduced cells, which should allow the transduced T cells to be continuously activated and give them a proliferation advantage over natural T cells.

[0457] In some embodiments, the viral particle comprises a polypeptide comprising a cytoplasmic FRB domain, the polypeptide being 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:56.

[0458] In some embodiments, the viral particle comprises a nucleic acid encoding a cytoplasmic FRB domain 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:68.

[0459] In some embodiments, the viral particle comprises a polypeptide comprising a cytoplasmic FRB domain operably linked to a P2A peptide, the polypeptide being 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:.

[0460] In some embodiments, the viral particle comprises a nucleic acid encoding a cytoplasmic FRB domain operably linked to P2A, wherein the nucleic acid 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:82.

[0461] In some embodiments, the viral particle comprises a polypeptide comprising a cytoplasmic FRB domain operably linked to a P2A peptide, wherein the polypeptide 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:88.

[0462] In some embodiments, the viral particle comprises a nucleic acid encoding a cytoplasmic FRB domain operably linked to P2A, wherein the nucleic acid 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:93.

[0463] In some embodiments, expression of the chimeric antigen receptor is regulated by a degron fusion polypeptide, and wherein inhibition of the degron fusion polypeptide is chemically induced by a ligand.

[0464] In some embodiments, expression of the chimeric antigen receptor is regulated by a FRB-degron fusion polypeptide, and wherein inhibition of the FRB-degron fusion polypeptide is chemically induced by a ligand.

[0465] In some embodiments, the ligand is rapamycin or a rapamycin analog as described herein.

[0466] TGF-β double negative (TGF-βDN)

[0467] Tumor cells secrete transforming growth factor β (TGF-β) as a means of suppressing immunity while allowing cancer to progress. Blocking TGF-β signaling in T cells can enhance their ability to penetrate, proliferate, and mediate anti-tumor responses (Kloss et al., Mol. Therapy 26(7): 1855-1866 (2018)). Dominant negative TGF-β (TGF-βDN) is truncated and lacks the intracellular domain necessary for downstream signaling.

[0468] In some embodiments, the viral particles of the present disclosure comprise a dominant negative TGF-β polynucleotide sequence. In some embodiments, the viral particles comprise a dominant negative TGF-β-containing polypeptide 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: 91.

[0469] In some embodiments, the viral particle comprises a nucleic acid encoding a dominant negative TGF-β 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:96.

[0470] Example Payloads

[0471] In some embodiments, the viral particles of the present disclosure include polynucleotide sequences encoding the following in any order on a polycistronic transcript: a promoter, a therapeutic protein (e.g., CAR), optionally a cytoplasmic FRB domain or a portion thereof, and optionally a synthetic cytokine polypeptide (e.g., RACR). In some embodiments, the polycistronic transcript includes a promoter and a CAR. Exemplary promoters include, but are not limited to, a cytomegalovirus (CMV) promoter, a CAG promoter, a SV40 promoter, a SV40 / CD43 promoter, and a MND promoter.

[0472] In some embodiments, the polycistronic construct comprises a nucleotide sequence encoding FRB, a nucleotide sequence encoding a synthetic cytokine polypeptide, and a nucleotide sequence encoding CAR from 5' to 3'. In some embodiments, the nucleotide sequence encoding the synthetic cytokine polypeptide comprises a first nucleotide sequence encoding FRB:IL2RG and a second nucleotide sequence encoding FKBP12:IL2RB from 5' to 3'. In some embodiments, the nucleotide sequence encoding the synthetic cytokine polypeptide comprises a first nucleotide sequence encoding FKBP12:IL2RG and a second nucleotide sequence encoding sFRB:IL2RB from 5' to 3'.

[0473] In some embodiments, the viral particles of the present disclosure comprise, from 5' to 3' on the polycistronic transcript, a polynucleotide sequence encoding:

[0474] (a) MND promoter;

[0475] (b) CAR;

[0476] (c) a cytoplasmic FRB domain or a portion thereof;

[0477] (d) RACR cell surface receptor; and

[0478] (e) WPRE sequence

[0479] In some embodiments, the viral particles of the present disclosure comprise, from 5' to 3', a polynucleotide sequence encoding:

[0480] (a) CAR;

[0481] (b) a cytoplasmic FRB domain or a portion thereof; and

[0482] (c) RACR cell surface receptor.

[0483] In some embodiments, the viral particle comprises a nucleic 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:35.

[0484] In some embodiments, the viral particle comprises a polypeptide 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:49.

[0485] In some embodiments, the viral particle comprises a nucleic 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:61.

[0486] In some embodiments, the viral particles of the present disclosure comprise, from 5' to 3' on the polycistronic transcript, a polynucleotide sequence encoding:

[0487] (a) MND promoter;

[0488] (b) a cytoplasmic FRB domain or a portion thereof;

[0489] (c) RACR cell surface receptor;

[0490] (d) CAR; and

[0491] (e) WPRE sequence.

[0492] In some embodiments, the viral particles of the present disclosure comprise, from 5' to 3', a polynucleotide sequence encoding:

[0493] (a) a cytoplasmic FRB domain or a portion thereof;

[0494] (b) RACR cell surface receptor; and

[0495] (c)CAR.

[0496] In some embodiments, the viral particle comprises a polypeptide 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:75.

[0497] In some embodiments, the viral particle comprises a nucleic 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:81.

[0498] In some embodiments, the viral particles of the present disclosure comprise, from 5' to 3' on the polycistronic transcript, a polynucleotide sequence encoding:

[0499] (a) MND promoter;

[0500] (b) a cytoplasmic FRB domain or a portion thereof;

[0501] (c) CAR;

[0502] (d) a TGF-β DN domain or a portion thereof; and

[0503] (e) WPRE sequence.

[0504] In some embodiments, the viral particles of the present disclosure comprise, from 5' to 3', a polynucleotide sequence encoding:

[0505] (a) a cytoplasmic FRB domain or a portion thereof;

[0506] (b) CAR; and

[0507] (c) TGF-β DN domain or a portion thereof.

[0508] In some embodiments, the viral particle comprises a polypeptide 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:87.

[0509] In some embodiments, the viral particle comprises a nucleic 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:92.

[0510] In some embodiments, the viral particles of the present disclosure comprise, from 5' to 3', a polynucleotide sequence encoding:

[0511] (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 extension, (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) cytoplasmic 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) 3'LTR, and the polynucleotide sequence 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:121.

[0512] In some embodiments, the viral particle comprises a nucleic 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:121.

[0513] In some embodiments, the viral particles of the present disclosure comprise, from 5' to 3', a polynucleotide sequence encoding:

[0514] (a) Human cytomegalovirus (CMV) immediate early enhancer and CMV promoter, (b) 5'LTR 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) CD3C, (n) P2A, (o) cytoplasmic 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) synthetic polyA signal, and the polynucleotide sequence is the same as SEQ ID NO:122 is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical.

[0515] In some embodiments, the viral particle comprises a nucleic 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:122.

[0516] Helper plasmid

[0517] In some embodiments, the viral particles of the present disclosure comprise, from 5' to 3' on the polycistronic transcript, a polynucleotide sequence encoding:

[0518] (a) gag protein; and

[0519] (b) Pol protein.

[0520] In some embodiments, the viral particle comprises a Gag protein 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:99.

[0521] In some embodiments, the viral particle comprises a Pol protein 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:100.

[0522] In some embodiments, the viral particle comprises a gag-pol nucleic 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:101.

[0523] In some embodiments, the viral particle comprises a gag-pol nucleic 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:124.

[0524] In some embodiments, the viral particles of the present disclosure comprise, from 5' to 3', a polynucleotide sequence encoding:

[0525] (a) human CMV enhancer and CMV promoter, (b) human β-globin intron, (c) HIV-1 gag, (d) HIV-1 pol, (d) cPPT / CTS, (e) RRE, (f) β-globin polyA signal, and the polynucleotide sequence has 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.

[0526] In some embodiments, the viral particle comprises a gag-pol nucleic 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:131.

[0527] In some embodiments, the viral particle comprises a Rev protein 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:102.

[0528] In some embodiments, the viral particle comprises a Rev nucleic 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:103.

[0529] In some embodiments, the viral particle comprises a Rev nucleic 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:125.

[0530] In some embodiments, the viral particles of the present disclosure comprise, from 5' to 3', a polynucleotide sequence encoding:

[0531] (a) RSV promoter, (b) HXB3 Rev, (c) HIV-1 polyALTR, and the polynucleotide sequence 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:132.

[0532] In some embodiments, the viral particle comprises a gag-pol nucleic 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:132.

[0533] Cocal Envelope Plasmid

[0534] In some embodiments, viral particles are produced from nucleic acids encoding the Cocal envelope and anti-CD3 scFv.

[0535] In some embodiments, the viral particles produced have a Cocal envelope and an anti-CD3 scFv nucleic 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:128.

[0536] In some embodiments, the viral particles of the present disclosure are produced from a polynucleotide sequence encoding from 5' to 3':

[0537] (a) MND promoter;

[0538] (b) CD8-derived signal peptide;

[0539] (c) anti-CD3 scFv;

[0540] (d) CD8-derived hinge;

[0541] (e) CD4-derived transmembrane domain and cytoplasmic tail;

[0542] (f) T2A;

[0543] (g) Cocal envelope;

[0544] (h) WPRE; and

[0545] (i) a polyA signal, and the polynucleotide sequence 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: 128.

[0546] In some embodiments, the viral particles of the present disclosure are produced from a polynucleotide sequence encoding from 5' to 3':

[0547] (a) human CMV enhancer and CMV promoter, (b) human β-globin intron, (c) anti-CD3 scFv, (d) Cocal envelope, (d) transmembrane domain, (e) cytoplasmic tail domain, (f) T2A peptide, (g) BGH polyA signal, and the polynucleotide sequence has 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: 129.

[0548] In some embodiments, the viral particles are produced by a nucleic 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:129.

[0549] In some embodiments, the viral particles of the present disclosure are produced from a polynucleotide sequence encoding from 5' to 3':

[0550] (a) human CMV enhancer and CMV promoter, (b) human β-globin intron, (c) Cocal envelope, (d) transmembrane domain, (e) cytoplasmic tail domain, (f) bovine growth hormone polyadenylation (BGH polyA) signal, and the polynucleotide sequence has 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:123.

[0551] In some embodiments, the viral particles are produced by a nucleic 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:123.

[0552] In some embodiments, the viral particles of the present disclosure are produced from a polynucleotide sequence encoding from 5' to 3':

[0553] (a) MND promoter, (b) Cocal envelope, (c) transmembrane domain, (d) cytoplasmic tail domain, (e) WPRE, (f) BGH polyA signal, and the polynucleotide sequence 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:130.

[0554] In some embodiments, the viral particles are produced by a nucleic 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:130.

[0555] Anti-CD3 plasmid

[0556] In some embodiments, the viral particle comprises an anti-CD3 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:15.

[0557] In some embodiments, the viral particles are produced by an anti-CD3 nucleic 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:126.

[0558] In some embodiments, the viral particles of the present disclosure are produced from a polynucleotide sequence encoding from 5' to 3':

[0559] (a) human CMV enhancer and CMV promoter, (b) human β-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) BGH polyA signal, and the polynucleotide sequence 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: 126.

[0560] In some embodiments, the viral particles are produced by an anti-CD3 nucleic 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:127.

[0561] In some embodiments, the viral particles of the present disclosure are produced from a polynucleotide sequence encoding from 5' to 3':

[0562] (a) human CMV enhancer and CMV promoter, (b) human β-globin intron, (c) Gaussia luc signal peptide, (d) anti-CD3 VL chain, (e) G4S linker, (f) anti-CD3 VH chain, (g) glycoprotein A transmembrane domain, (h) glycoprotein A cytoplasmic tail domain, (i) BGH polyA signal, and the polynucleotide sequence 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: 127.

[0563] Co-stimulatory and adhesion molecule plasmids

[0564] In some embodiments, the viral particle comprises a CD58 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:17.

[0565] In some embodiments, the viral particles are produced by a CD58 nucleic 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:18.

[0566] In some embodiments, the viral particles of the present disclosure are produced by a polynucleotide sequence comprising: (a) a human CMV enhancer and a CMV promoter, (b) a human β-globin intron, (c) an endogenous signal peptide, (d) a CD58 polynucleotide, (e) a BGH polyA signal, and the CD58 polynucleotide sequence 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:18.

[0567] In some embodiments, the viral particle comprises a CD80 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:20.

[0568] In some embodiments, the viral particles are produced by a CD80 nucleic 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:21.

[0569] In some embodiments, the viral particles of the present disclosure are produced by a polynucleotide sequence comprising: (a) a human CMV enhancer and a CMV promoter, (b) a human β-globin intron, (c) an endogenous signal peptide, (d) a CD80 polynucleotide, (e) a BGH polyA signal, and the CD80 polynucleotide sequence 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:21.

[0570] In some embodiments, the viral particle comprises a CD86 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:23.

[0571] In some embodiments, the viral particles are produced by a CD86 nucleic 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:24.

[0572] In some embodiments, the viral particles of the present disclosure are produced by a polynucleotide sequence comprising: (a) a human CMV enhancer and a CMV promoter, (b) a human β-globin intron, (c) an endogenous signal peptide, (d) a CD86 polynucleotide, and (e) a BGH polyA signal, and the CD86 polynucleotide sequence 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:24.

[0573] Gene Editing

[0574] A variety of gene editing methods are known in the art, and other methods are constantly being created. The methods and compositions disclosed herein can deliver a variety of genetic payloads, including polynucleotides for inserting into the target cell genome and / or gene editing systems (CRISPR-Cas, giant nucleases, homing endonucleases, zinc finger enzymes, etc.). In an embodiment, polynucleotides (e.g., transgenics), enzymes, and / or guide RNAs are delivered in one, two, three or more of the same type (e.g., lentivirus, AAV, etc.) or different types of vectors (comprising, for example, a combination of non-viral and viral vectors or different types of viral vectors). The methods and systems disclosed herein can be used to produce point mutations, insertions, deletions, etc. Random mutagenesis and multi-site gene editing also fall within the scope of this disclosure.

[0575] Production / packaging cell lines

[0576] The present disclosure provides host cells for producing 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 activating 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.

[0577] In some embodiments, the host cell is used to produce a viral vector according to the preceding embodiments. In some embodiments, the host cell comprises a tag protein that can be used to purify the viral particles.

[0578] In some embodiments, the host cell is a packaging cell and comprises one or more of the following genes: gag, pol, env, and rev. In some embodiments, packaging cells for retroviral vectors comprise gag, pol, and env genes. In some embodiments, packaging cells for lentiviral vectors comprise gag, pol, env, and rev genes.

[0579] In some embodiments, the host cell is a production cell and comprises gag, pol, env and optional rev genes and a retroviral or lentiviral vector genome. In a typical recombinant retroviral or lentiviral vector for gene therapy, at least a portion of one or more gag-pol and env protein coding regions may be removed from the virus and provided by the packaging cell. This renders the viral vector replication-defective because the virus is able to integrate its genome into the host genome, but the modified viral genome is unable to propagate itself due to the lack of structural proteins.

[0580] Packaging cells are used to propagate and isolate large quantities of viral vectors, i.e. to prepare retroviral vectors of suitable titers for transduction of target cells.

[0581] In some embodiments, propagation and isolation may require isolation of retroviral gagpol and env (and rev in the case of lentivirus) genes and their separate introduction into host cells to produce packaging cell lines. The packaging cell line produces the proteins required for packaging retroviral DNA, but it is unable to achieve encapsidation 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 auxiliary proteins can package the psi-positive recombinant vector to produce a recombinant virus stock.

[0582] An overview of available packaging systems is given in "Retroviruses" (1997 Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 449).

[0583] 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 independently transfected into the packaging cell line, thus requiring three recombinant elements for the production of wild-type virus.

[0584] Transient transfection avoids the long time required to produce stable vector cell lines and is used when the vector or retroviral packaging components are toxic to the cells. The components commonly used to produce retroviral / lentiviral vectors include plasmids encoding Gag / Pol proteins, plasmids encoding Env proteins (and rev proteins in the case of lentiviral vectors), and retroviral / lentiviral vector genomes. Vector production involves transient transfection of one or more of these components into cells containing other required components. The packaging cells of the present invention can be any mammalian cell type capable of producing retroviral / lentiviral vector particles. The packaging cells can be 293T cells, or variants of 293T cells suitable for suspension growth and serum-free growth.

[0585] In some embodiments, packaging cells are made by transient transfection of:

[0586] a) Transfer vector

[0587] b) gagpol expression vector

[0588] c) env expression vector. In some embodiments, the env gene is heterologous, thereby generating a pseudotyped retroviral vector. For example, in some embodiments, the env gene is from RD1 14 or one of its variants, VSV-G, including the Cocal envelope, Gibbon Ape Leukemia Virus (GALV), the amphibious envelope or the measles envelope or the baboon retrovirus envelope glycoprotein, or any exemplary envelope protein discussed herein.

[0589] In the case of lentiviral vectors, in some embodiments, transient transfection is also performed using a rev vector.

[0590] The present disclosure provides host cells expressing viral particles according to the aforementioned embodiments. In some embodiments, the host cell expresses one or more transduction enhancers on the cell surface. In some embodiments, the present invention provides host cells that are engineered to express on the cell surface:

[0591] (a) activator protein; and / or

[0592] (b) a co-stimulatory protein; and / or

[0593] (c) Adhesion molecules

[0594] The retroviral or lentiviral vector produced by the packaging cell is as described in the previous embodiment.

[0595] In some embodiments, the host cell also expresses a tag protein on the cell surface, which comprises: a binding domain that binds to a capture portion; and a transmembrane domain, wherein the tag protein facilitates the purification of the viral vector from the cell supernatant through the binding of the tag protein to the capture portion, so that the retrovirus or lentiviral vector produced by the packaging cell has the characteristics described in the preceding section.

[0596] The tag protein may also comprise a spacer between the binding domain and the transmembrane domain.

[0597] The term "host cell" can be used to describe packaging cells or production cells. Packaging cells may contain one or more of the following genes: gag, pol, env and / or rev. Production cells may contain gag, pol, env and optional rev genes, and also contain a retroviral or lentiviral genome. In some embodiments, the host cell can be any suitable cell line that stably expresses mitosis and / or cytokine transduction enhancers. It can be transiently transfected with transfer vectors, gagpol, env (and rev in lentivirus) to produce a retroviral / lentiviral vector with insufficient replication capacity.

[0598] The present disclosure also provides a method for preparing the above-mentioned host cell, comprising the step of transducing or transfecting the cell with a nucleic acid encoding one or more transduction enhancers. A method for producing a viral vector according to the aforementioned embodiment is also provided, comprising the step of expressing a retroviral or lentiviral genome in a cell according to the second aspect of the present invention.

[0599] Systems and Kits

[0600] In some embodiments, the present disclosure provides a system, therapeutic system or composition comprising:

[0601] (a) an adaptor molecule comprising a targeting moiety and a masking hapten, wherein the masking hapten comprises a masking moiety attached to the hapten; and

[0602] (b) a plurality of recombinant retroviral particles,

[0603] Each retroviral particle comprises from 5' to 3' a polynucleotide comprising:

[0604] (i) 5' long terminal repeat (LTR) or untranslated region (UTR),

[0605] (ii) a promoter,

[0606] (iii) a sequence encoding a receptor that specifically binds to the hapten, and

[0607] (iv) 3' LTR or UTR; and

[0608] Each retroviral particle comprises a viral envelope comprising:

[0609] (i) viral fusion glycoprotein, and

[0610] (ii) one or more transduction enhancers

[0611] Optionally, each transduction enhancer is selected from a T cell activating receptor, a NK cell activating receptor, a co-stimulatory molecule and an adhesion molecule.

[0612] The present disclosure also provides a kit comprising the system and instructions for use of the system.

[0613] Genetically modified immune cells

[0614] The present invention provides a method for preparing activated transgenic immune cells, comprising the step of contacting immune cells with a viral vector according to any of the foregoing embodiments. Immune cells can be transduced in vivo or in vitro. In some embodiments, the viral vector is administered to a living subject so that the immune cells are transduced in vivo without the need to separate and manipulate host cells in vitro. In some embodiments, the immune cells are manipulated in vitro and then returned to the subject in need.

[0615] Immune cells are usually mammalian cells, and typically human cells, more typically primary human cells, such as allogeneic or autologous donor cells. Cells can be isolated from samples (such as biological samples, such as samples obtained from subjects or derived from). In some embodiments, the subject of the isolated cell is a subject suffering from a disease or illness or needs cell therapy or will be administered cell therapy. In some embodiments, the subject is a person who needs a specific treatment intervention, such as the adoptive cell therapy of separation, processing and / or engineering cells. In some embodiments, the cell is derived from blood, bone marrow, lymph or lymphoid organs, and is a cell of the immune system, such as a cell of the innate or adaptive immune system, such as myeloid cells or lymphocytes, including lymphocytes, typically T cells and / or NK cells. Other exemplary cells include stem cells, such as pluripotent stem cells (multipotent stem cell) and pluripotent stem cells (pluripotent stem cell), including induced pluripotent stem cells (iPSC). These cells are typically primary cells, such as cells directly separated from subjects and / or cells separated and frozen from subjects. In some embodiments, the cells include one or more T cell subsets or other cell types, such as the entire T cell population, CD4+ cells, CD8+ cells, and subsets thereof, such as cells defined by function, activation state, maturity, differentiation potential, expansion, recirculation, localization and / or persistence ability, antigen specificity, antigen receptor type, presence in a specific organ or compartment, marker or cytokine secretion characteristics, and / or degree of differentiation.

[0616] Subtypes and subpopulations of T cells and / or CD4+ and / or CD8+ T cells include naive T (TN) cells, effector T cells (TEFF), memory T cells and subtypes thereof, 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, mucosal-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH 17 cells, TH9 cells, TH22 cells, follicular helper T cells, α / β T cells and δ / γ T cells.

[0617] In some embodiments, the cell provided is a cytotoxic T lymphocyte." Cytotoxic T lymphocyte" (CTL) may include, but is not limited to, for example, T lymphocytes (such as CD8+T cells) expressing CD8 on its surface. In some embodiments, such cells are preferably "memory" T cells (TM cells) with antigen experience. In some embodiments, the cell is a precursor T cell. In some embodiments, the precursor T cell is a hematopoietic stem cell. In some embodiments, the cell is a CD8+T cytotoxic lymphocyte, which is selected from initial CD8+T cells, central memory CD8+T cells, effector memory CD8+T cells and a large number of CD8+T cells. In some embodiments, the cell is a CD4+T helper lymphocyte, which is selected from initial CD4+T cells, central memory CD4+T cells, effector memory CD4+T cells and a large number of CD4+T cells.

[0618] As used herein, any reference to transgenic T cells or transduced T cells, or uses thereof, may also apply to any other immune cell type disclosed herein.

[0619] 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 activation protein. In some embodiments, the transgenic immune cells comprise a polynucleotide encoding a hapten binding receptor and a polynucleotide encoding a T cell activation protein.

[0620] Target immune cells

[0621] Non-limiting examples of cells that can be targeted by the viral particles described herein include T lymphocytes, dendritic cells (DC), Treg cells, B cells, natural killer cells, and macrophages. In some embodiments, the viral particles described herein can transduce αβT cells. In some embodiments, the viral particles described herein can transduce αβT cells. In some embodiments, the viral particles described herein can transduce γδT cells. In some embodiments, the viral particles described herein can transduce NK cells.

[0622] T cells

[0623] T cells ("T lymphocytes") are a type of lymphocyte (itself a type of white blood cell) that plays a central role in cell-mediated immunity. There are several subpopulations of T cells, each with different functions. T cells can be distinguished from other lymphocytes (such as B cells and NK cells) by the T cell receptor (TCR) on the 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 is composed of an alpha chain and a beta 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 binds to an antigenic peptide and MHC (peptide / MHC complex), the T lymphocyte is activated through a series of biochemical events mediated by associated enzymes, co-receptors, specialized adaptor molecules, and activated or released transcription factors.

[0624] In some embodiments, the cell used in the method provided herein is a primary T lymphocyte (e.g., primary human T lymphocyte). The primary T lymphocyte used in the method provided herein can be an initial T lymphocyte or an MHC restricted T lymphocyte. In some embodiments, the T lymphocyte is CD4+. In other embodiments, the T lymphocyte is CD8+. In some embodiments, the primary T lymphocyte is a tumor infiltrating lymphocyte (TIL). In some embodiments, the primary T lymphocyte has been separated from a tumor biopsy or has been expanded from the T lymphocytes separated from the tumor biopsy. In some embodiments, the primary T lymphocyte has been separated from peripheral blood, cord blood or lymph, or has been expanded from the T lymphocytes separated from peripheral blood, cord blood or lymph. In some embodiments, T lymphocytes are allogeneic for a specific individual (e.g., the recipient of the T lymphocyte). In some other embodiments, T lymphocytes are not allogeneic for a certain individual (e.g., the recipient of the T lymphocyte). In some embodiments, the T lymphocytes are autologous to a particular individual (eg, the recipient of the T lymphocytes).

[0625] In some embodiments, the primary T lymphocytes used in the methods described herein are separated from tumors, such as tumor infiltrating lymphocytes. In some embodiments, such T lymphocytes are specific to tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). In some embodiments, primary T lymphocytes are obtained from individuals, optionally amplified, and then transduced with nucleic acids encoding one or more chimeric antigen receptors (CARs) using the methods described herein, and then optionally amplified. T lymphocytes can be amplified, for example, by contacting T lymphocytes in culture with CD3 and / or CD28 antibodies (e.g., antibodies attached to the surface of beads or cell culture plates) to amplify; see, for example, 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 antibody is anti-CD3 and / or anti-CD28, and the antibody is not bound to a solid surface (e.g., the antibody contacts the T lymphocytes in the solution). In some embodiments, the anti-CD3 antibody or the anti-CD28 antibody is bound to a solid surface (eg, a bead, tissue culture dish plastic), while the other antibody is not bound to a solid surface (eg, present in solution).

[0626] NK cells

[0627] Natural killer (NK) cells are cytotoxic lymphocytes that are a major component of the innate immune system. NK cells normally comprise approximately 10% to 15% of the mononuclear cell fraction in normal peripheral blood. NK cells do not express T-cell antigen receptors (TCRs), CD3, or surface immunoglobulin (Ig) B-cell receptors, but in humans they normally express the surface markers CD16 (FcγRIII) and CD56. NK cells are cytotoxic; small granules in their cytoplasm contain specialized proteins, such as perforin, and proteases called granzymes. When perforin is released into the vicinity of cells to be killed, it forms pores in the cell membrane of the target cell through which granzymes and associated molecules can enter, thereby inducing apoptosis. One of the granzymes, granzyme B (also known as granzyme 2 and cytotoxic T-lymphocyte-associated serine esterase 1), is a serine protease that is essential for the rapid induction of apoptosis in target cells in cell-mediated immune responses.

[0628] NK cells are activated in response to interferon or macrophage-derived cytokines, and the activated NK cells are called lymphokine-activated killer (LAK) cells. NK cells have two types of surface receptors, called "activating receptors" and "inhibitory receptors," which control the cytotoxic activity of the cells.

[0629] Among other activities, NK cells play a role in host rejection of tumors. Since many cancer cells have reduced or absent expression of class I MHC, they can become targets for NK cells. Natural killer cells can be activated by cells that lack or exhibit reduced levels of major histocompatibility complex (MHC) proteins. In addition to participating in direct cytotoxic killing, NK cells also play a role in the production of cytokines, which are important for controlling cancer and infection. Activated and expanded NK cells and LAK cells have been used for in vitro and in vivo treatment of patients with advanced cancer and have achieved some success in the treatment of bone marrow-related diseases such as leukemia, breast cancer, and some types of lymphoma.

[0630] In vivo delivery of polynucleotides

[0631] In some embodiments, the present disclosure provides a method of delivering nucleic acid to cells in vivo. In some embodiments, the present disclosure provides a method of delivering nucleic acid to immune cells in vivo. In some embodiments, the viral particles of the present disclosure activate and transduce immune cells in vivo.

[0632] In some embodiments, the nucleotide sequence encoding CAR is administered to the subject, which allows CAR to be produced in vivo. In some embodiments, the administration of such viral particles produces an effect similar to that of directly administering CAR in vivo. In some embodiments, the administration of such viral particles can increase the transduction efficiency of the particles in vivo.

[0633] In some embodiments, the in vivo delivery of such viral particles can produce CAR expression over time (e.g., starting within a few hours and continuing for several days). In some embodiments, the in vivo delivery of such viral particles causes the encoded CAR to have ideal pharmacokinetic, pharmacodynamic and / or safety characteristics.

[0634] In some embodiments, the nucleotide sequence can be optimized by one or more methods to prevent immune activation, increase stability, reduce any aggregation tendency (such as over time) and / or avoid impurities. Such optimization can include the use of modified nucleosides, modified and / or specific 5'UTR, 3'UTR and / or poly (A) tail modifications to improve intracellular stability and translation efficiency (see, e.g., Stadler et al., 2017, Nat. Med.). Such modifications are known in the art.

[0635] Strategies for in vivo delivery of polynucleotides (e.g., mRNA) are known in the art. For a review of strategies, please refer to Mol. Ther. 2019 Apr 10; 27 (4): 710–728, which is incorporated herein by reference in its entirety.

[0636] In some embodiments, the viral particle has a multi-step mechanism of action:

[0637] (a) the viral particles bind to T cells in vivo via immune cell activation proteins (e.g., anti-CD3 scFv), co-stimulatory molecules (e.g., CD28 ligands), cell adhesion molecules (e.g., CD58), or any combination thereof, activating T cells and promoting internalization of the viral particles by interacting with Cocalin;

[0638] (b) the vector RNA genome is reverse transcribed into DNA, transported to the cell nucleus, and integrated into the genome; and

[0639] (c) The transduced T cells express the polypeptide of interest.

[0640] In some embodiments, the viral particle has a multi-step mechanism of action:

[0641] (a) the viral particles bind to T cells in vivo via immune cell activation proteins (e.g., anti-CD3 scFv), co-stimulatory molecules (e.g., CD28 ligands), cell adhesion molecules (e.g., CD58), or any combination thereof, activating T cells and promoting internalization of the viral particles by interacting with Cocalin;

[0642] (b) The vector RNA genome encoding the CAR is reverse transcribed into DNA, transported to the cell nucleus and integrated into the genome; and

[0643] (c) Transduced T cells express CAR and target cells, and also express the FRB and RACR systems for rapamycin-controlled cytokine signaling.

[0644] Immune cell activation

[0645] In some embodiments, administration of the particles to a subject results in activation of immune cells.

[0646] In some embodiments, the activation of immune cells is measured by the level of one or more cell markers. In some embodiments, the activation of immune cells is measured by the percentage of immune cells that are positive for one or more cell markers. In some embodiments, immune cells are T cells (T lymphocytes) or NK cells. In some embodiments, immune cells are CD4+T cells or CD8+T cells. In some embodiments, one or more cell markers are selected from CD71, CD25 and any combination thereof.

[0647] In some embodiments, the activation of immune cells is measured by the percentage of CD71-positive immune cells. In some embodiments, the 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, the activation of immune cells is measured by the level of CD71 expressed on the surface of immune cells. In some embodiments, the 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 times, at least 2 times, at least 3 times, at least 5 times, at least 7 times or at least 10 times.

[0648] In some embodiments, the activation of immune cells is measured by the percentage of CD25 positive immune cells. In some embodiments, the 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, the activation of immune cells is measured by the level of CD25 expressed on the surface of immune cells. In some embodiments, the 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 times, at least 2 times, at least 3 times, at least 5 times, at least 7 times, or at least 10 times.

[0649] In some embodiments, administration of the viral particles to a subject results in active proliferation of immune cells. In some embodiments, proliferation of immune cells increases number and / or sensitivity to vector transduction.

[0650] In some embodiments, administration of the viral particles to a subject results in a decrease in the number of immune cells (eg, T cells) in the GO stage and / or an increase in the number of immune cells (eg, T cells) in the non-GO stage.

[0651] In some embodiments, administration of viral particles to a subject increases the number and / or percentage of immune cells that are in a metabolic state suitable for transduction by the vector.

[0652] In some embodiments, administration of the viral particles to a subject results in accumulation of immune cells in lymph nodes. In some embodiments, administration of the viral particles to a subject results in accumulation of immune cells at a tumor site.

[0653] In some embodiments, the viral particle is a lentiviral particle. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell here is a subpopulation of immune cells in vivo, which can be recognized by at least one antigen-specific binding domain of CAR. In some embodiments, the immune cell resides in a lymph node.

[0654] In some embodiments, the viral particles activate non-transduced immune cells. In some embodiments, the viral particles amplify non-transduced immune cells. In some embodiments, the viral particles activate and / or amplify tumor-infiltrating lymphocytes. In some embodiments, the viral particles activate and / or amplify tumor-reactive T cells present in tumor-draining or metastatic lymph nodes.

[0655] Route of administration

[0656] In some embodiments, the viral particles are administered by a route selected from the following: 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 viral particles. In some embodiments, the viral particles are administered by intraperitoneal injection of viral particles. In some embodiments, the viral particles are administered by intralymphatic injection—that is, the viral particles can be administered by injection into lymph nodes (e.g., inguinal lymph nodes). In some embodiments, the viral particles are administered by injecting the viral particles into the tumor site (i.e., intratumor). 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 intraarterially. In some embodiments, the viral particles are slow viral particles.

[0657] In some embodiments, the viral particles are administered by intraperitoneal, subcutaneous, or intralymph node 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 intralymph node injection.

[0658] In some embodiments, transduced immune cells comprising a polynucleotide of the disclosure are administered to a subject.

[0659] In some embodiments, the viral particles are administered in a single injection. In some embodiments, the viral particles are administered in 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.

[0660] Dosage form and dosing regimen

[0661] Virus particles

[0662] The viral particles can infect cells in vivo at any effective dose. In some embodiments, the viral particles are administered to a subject by direct injection into a cell, tissue, organ or subject in need of treatment.

[0663] In some embodiments, the viral particles can be administered with cells. The viral particles can be associated with cells by incubating the viral particles with cells so that the viral particles are associated with cells. In some embodiments, the dose of the product to be delivered is determined based on the number of viral particles bound to cells.

[0664] Viral particles can also be delivered based on viral titer (TU / mL). The amount of lentivirus injected directly is determined by the total TU and can vary depending on the amount that can be injected into the site and the type of tissue to be injected. In some embodiments, the viral titer delivered per injection can be about 1×10 5 Up to 1×10 6 , about 1×10 5 Up to 1×10 7 , 1×10 5 Up to 1×10 7 , about 1×10 6 Up to 1×10 9 , about 1×10 7 Up to 1×10 10 , about 1×10 7 Up to 1×10 11 , or about 1×10 9 Up to 1×10 11 TU or greater. In some embodiments, the virus titer delivered per injection can be about 1×10 6 Up to 1×10 7 , about 1×10 6 Up to 1×10 8 , 1×10 6 Up to 1×10 9 , about 1×10 7 Up to 1×10 10 , about 1×10 8 Up to 1×10 11 , about 1×10 8 Up to 1×10 12 , or about 1×10 10 Up to 1×10 12 For example, the brain injection site may only allow very small amounts of virus to be injected, so a high titer preparation is preferred, using approximately 1 × 10 6 Up to 1×10 7 , about 1×10 6 Up to 1×108 , 1×10 6 Up to 1×10 9 , about 1×10 7 Up to 1×10 10 , about 1×10 8 Up to 1×10 11 , about 1×10 8 Up to 1×10 12 , or about 1×10 10 Up to 1×10 12 or larger TU. However, systemic delivery can accommodate larger TUs, delivering approximately 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 load.

[0665] In some embodiments, the carrier is present at about 1×10 12 Up to 5×10 14 The vector genome (vg) is administered at a dose of about 1×10 13 Up to 5×10 14 In some embodiments, the carrier is administered at a dose of about 5×10 13 Up to 3×10 14 In some embodiments, the carrier is administered at a dose of about 5×10 13 Up to 1×10 14 vg / kg. In some embodiments, the vector is administered at a dose of less than about 1×10 12 vg / kg, less than about 3×10 12 vg / kg, less than about 5×10 12 vg / kg, less than about 7×10 12 vg / kg, less than about 1×10 13 vg / kg, less than about 3×10 13 vg / kg, less than about 5×10 13 vg / kg, less than about 7×10 13 vg / kg, less than about 1×10 14 vg / kg, less than about 3×10 14 vg / kg, less than about 5×10 14 vg / kg, less than about 7×10 14vg / kg, less than about 1×10 15 vg / kg, less than about 2×10 15 vg / kg, less than about 5×10 15 vg / kg, or less than about 7×10 15 vg / kg.

[0666] In some embodiments, the carrier is present at about 1×10 12 Up to 5×10 14 The vector particles (vp) are administered at a dose (vp / kg). In some embodiments, the vector is administered at a dose of about 1×10 13 Up to 5×10 14 vp / kg. In some embodiments, the carrier is administered at a dose of about 5×10 13 Up to 3×10 14 vp / kg. In some embodiments, the carrier is administered at a dose of about 5×10 13 Up to 1×10 14 vp / kg. In some embodiments, the vector is administered at a dose of less than about 1×10 12 vp / kg, less than about 3×10 12 vp / kg, less than about 5×10 12 vp / kg, less than about 7×10 12 vp / kg, less than about 1×10 13 vp / kg, less than about 3×10 13 vp / kg, less than about 5×10 13 vp / kg, less than about 7×10 13 vp / kg, less than about 1×10 14 vp / kg, less than about 3×10 14 vp / kg, less than about 5×10 14 vp / kg, less than about 7×10 14 vp / kg, less than about 1×10 15 vp / kg, less than about 2×10 15 vp / kg, less than about 5×10 15 vp / kg, or less than about 7×10 15 vp / kg.

[0667] In some embodiments, administration of the viral particles of the present disclosure reduces the number of B cells in the 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 assessed by the number of B cells at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks after administration of the viral particles, wherein the reference number is the number of B cells in the subject administered with the vector control. In some embodiments, administration of the viral particles of the present disclosure reduces the number of B cells in the subject by at least 95%.

[0668] In some embodiments, the B cells are located in the peripheral blood of the subject. In some embodiments, the B cells are located in the bone marrow of the subject. In some embodiments, the B cells are located in the spleen of the subject.

[0669] In some embodiments, the subject's B cells are depleted 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.

[0670] In some embodiments, the subject's B cells are depleted for at least 80 days after administration of the viral particles.

[0671] Rapamycin

[0672] (sirolimus, sirolimus) is available in oral solution and tablet form and is FDA-approved for the following indications:

[0673] Prevention of kidney transplant rejection

[0674] Restrictions on use in kidney transplantation

[0675] Treatment of patients with lymphangioleiomyostenosis

[0676] According to the United States Prescribing Information (USPI), rapamycin is available as a 1 mg / mL oral solution or as a 0.5, 1 or 2 mg tablet for once daily administration. Rapamycin may also be delivered in other dosage forms and / or by other routes of administration.

[0677] In some embodiments, rapamycin is administered at a dose of about 0.1 mg / m 2 Up to 100 mg / m 2 In some embodiments, the subject is a human. In some embodiments, the rapamycin is administered at a dose of about 0.5 mg / m 2 Up to 50 mg / m 2In some embodiments, rapamycin is administered at a dose of about 0.5 mg / m 2 Up to 10 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.5 mg / m 2 Up to 3 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.5 mg / m 2 Up to 5 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 1 mg / m 2 Up to 5 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 2 mg / m 2 Up to 6 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 2 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 3 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 2 mg / m 2 Up to 6 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 3 mg / m 2 Up to 9 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 4 mg / m 2 Up to 12 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 5 mg / m 2 Up to 15 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 6 mg / m 2 Up to 20 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 10 mg / m 2 Up to 50 mg / m 2 In some embodiments, the dose of rapamycin is the total dose over 24 hours.

[0678] In some embodiments, rapamycin is administered at a dose of about 0.001 mg / m 2 Up to 100 mg / m 2 In some embodiments, the subject is a human. In some embodiments, the administered dose of rapamycin is about 0.001 mg / m 2 Up to 0.1 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.5 mg / m 2 Up to 10 mg / m 2 , 0.01mg / m2 Up to 1 mg / m 2 , 0.1mg / m 2 Up to 10 mg / m 2 , 1mg / m 2 Up to 100 mg / m 2 , 0.001mg / m 2 Up to 0.05 mg / m 2 , 0.005mg / m 2 Up to 0.25 mg / m 2 , 0.01mg / m 2 Up to 0.5 mg / m 2 , 0.05mg / m 2 Up to 2.5 mg / m 2 , 0.1mg / m 2 Up to 5 mg / m 2 , 0.5mg / m 2 Up to 25 mg / m 2 , 1mg / m 2 Up to 50 mg / m 2 , 2mg / m 2 Up to 100 mg / m 2 , 0.001mg / m 2 Up to 0.01 mg / m 2 , 0.005mg / m 2 Up to 0.05 mg / m 2 , 0.01mg / m 2 Up to 0.1 mg / m 2 , 0.05mg / m 2 Up to 0.5 mg / m 2 , 0.1mg / m 2 Up to 1 mg / m 2 , 0.5mg / m 2 Up to 5 mg / m 2 , 1mg / m 2 Up to 10 mg / m 2 , 5mg / m 2 Up to 50 mg / m 2 , 10mg / m 2 Up to 100 mg / m 2 In some embodiments, the dosage of rapamycin administered is about 0.001 mg / m 2 Up to 0.005 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.002 mg / m 2 Up to 0.01 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.003 mg / m2 Up to 0.015 mg / m 2 In some embodiments, the dosage of rapamycin administered is about 0.004 mg / m 2 Up to 0.02 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.005 mg / m 2 Up to 0.025 mg / m 2 In some embodiments, the dosage of rapamycin administered is about 0.006 mg / m 2 Up to 0.03 mg / m 2 In some embodiments, the dosage of rapamycin administered is about 0.007 mg / m 2 Up to 0.035 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.008 mg / m 2 Up to 0.04 mg / m 2 In some embodiments, the dosage of rapamycin administered is about 0.009 mg / m 2 Up to 0.045 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.01 mg / m 2 Up to 0.05 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.02 mg / m 2 Up to 0.1 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.03 mg / m 2 Up to 0.15 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.04 mg / m 2 Up to 0.2 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.05 mg / m 2 Up to 0.25 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.06 mg / m 2 Up to 0.3 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.07 mg / m 2 Up to 0.35 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.08 mg / m 2 Up to 0.4 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.09 mg / m 2 Up to 0.45 mg / m 2In some embodiments, rapamycin is administered at a dose of about 0.1 mg / m 2 Up to 0.5 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.2 mg / m 2 Up to 1 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.3 mg / m 2 Up to 1.5 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.4 mg / m 2 Up to 2 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.5 mg / m 2 Up to 2.5 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.6 mg / m 2 Up to 3 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.7 mg / m 2 Up to 3.5 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.8 mg / m 2 Up to 4 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.9 mg / m 2 Up to 4.5 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 1 mg / m 2 Up to 5 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 2 mg / m 2 Up to 10 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 3 mg / m 2 Up to 15 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 4 mg / m 2 Up to 20 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 5 mg / m 2 Up to 25 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 6 mg / m 2 Up to 30 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 7 mg / m 2 Up to 35 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 8 mg / m 2Up to 40 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 9 mg / m 2 Up to 45 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 10 mg / m 2 Up to 50 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 20 mg / m 2 Up to 100 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.001 mg / m 2 Up to 0.02 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.002 mg / m 2 Up to 0.04 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.003 mg / m 2 Up to 0.06 mg / m 2 In some embodiments, the dosage of rapamycin administered is about 0.004 mg / m 2 Up to 0.08 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.005 mg / m 2 Up to 0.1 mg / m 2 In some embodiments, the dosage of rapamycin administered is about 0.006 mg / m 2 Up to 0.12 mg / m 2 In some embodiments, the dosage of rapamycin administered is about 0.007 mg / m 2 Up to 0.14 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.008 mg / m 2 Up to 0.16 mg / m 2 In some embodiments, the dosage of rapamycin administered is about 0.009 mg / m 2 Up to 0.18 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.01 mg / m 2 Up to 0.2 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.02 mg / m 2 Up to 0.4 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.03 mg / m 2 Up to 0.6 mg / m 2In some embodiments, rapamycin is administered at a dose of about 0.04 mg / m 2 Up to 0.8 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.05 mg / m 2 Up to 1 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.06 mg / m 2 Up to 1.2 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.07 mg / m 2 Up to 1.4 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.08 mg / m 2 Up to 1.6 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.09 mg / m 2 Up to 1.8 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.1 mg / m 2 Up to 2 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.2 mg / m 2 Up to 4 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.3 mg / m 2 Up to 6 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.4 mg / m 2 Up to 8 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.5 mg / m 2 Up to 10 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.6 mg / m 2 Up to 12 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.7 mg / m 2 Up to 14 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.8 mg / m 2 Up to 16 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 0.9 mg / m 2 Up to 18 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 1 mg / m 2 Up to 20 mg / m 2In some embodiments, rapamycin is administered at a dose of about 2 mg / m 2 Up to 40 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 3 mg / m 2 Up to 60 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 4 mg / m 2 Up to 80 mg / m 2 In some embodiments, rapamycin is administered at a dose of about 5 mg / m 2 Up to 100 mg / m 2 In some embodiments, the dose of rapamycin is the total dose over 24 hours.

[0679] In some embodiments, a dose of rapamycin is administered daily. In some embodiments, a 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, a dose of rapamycin is administered approximately every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. In some embodiments, a dose of rapamycin is administered approximately every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 months.

[0680] In some embodiments, after the first administration of the viral particles, the first dose of rapamycin is administered about 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 dose of rapamycin is administered about 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 dose of rapamycin is administered about 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 dose of rapamycin is administered about 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.

[0681] In some embodiments, the administration of rapamycin increases the number of immune cells (e.g., CAR T cells) transduced by viral particles in a specific organ / region of a subject or subject. In some embodiments, the subject's organ / region is blood. In some embodiments, the subject's organ / region is spleen. In some embodiments, the subject's organ / region is bone marrow. In some embodiments, the administration of rapamycin increases the number of immune cells (e.g., CAR-T cells) transduced by viral particles 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 times, at least 2 times, at least 3 times, at least 5 times, at least 7 times, or at least 10 times. In some embodiments, the increase is assessed by the number of immune cells transduced by viral particles 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks after the first dose of rapamycin (once the viral particles are administered), wherein the reference number is the number of immune cells transduced by viral particles on the day of the first dose of rapamycin. In some embodiments, the increase is assessed by the number of immune cells transduced by viral particles 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months after the first dose of rapamycin (once the viral particles are administered), wherein the reference number is the number of immune cells transduced by viral particles on the day of the first dose of rapamycin.

[0682] In some embodiments, the administration of rapamycin increases the percentage of immune cells (e.g., CAR T cells) transduced by viral particles in a specific organ / region of a subject or subject. In some embodiments, the subject's organ / region is blood. In some embodiments, the subject's organ / region is spleen. In some embodiments, the subject's organ / region is bone marrow. In some embodiments, the administration of rapamycin increases the percentage of immune cells (e.g., CAR-T cells) transduced by viral particles of the 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 assessed by the percentage of immune cells transduced by viral particles 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks after the first dose of rapamycin (once viral particles are administered), wherein the reference percentage is the percentage of immune cells transduced by viral particles on the day of the first dose of rapamycin. In some embodiments, the increase is assessed by the percentage of immune cells transduced by viral particles 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months after the first dose of rapamycin (once the viral particles are administered), wherein the reference percentage is the percentage of immune cells transduced by viral particles on the day of the first dose of rapamycin. In some embodiments, the percentage is the percentage of immune cells transduced by viral particles in the subject or a specific organ / region of the subject to the total immune cells. In some embodiments, the percentage is the percentage of immune cells transduced by viral particles in the same type of immune cells (e.g., T cells) of the subject or a specific organ / region of the subject.

[0683] Pharmaceutical compositions and preparations

[0684] The preparations and compositions disclosed herein may comprise any number of combinations of viral particles, and optionally one or more additional agents (polypeptides, polynucleotides, compounds, etc.), which are formulated into pharmaceutically acceptable or physiologically acceptable compositions for administration to cells, tissues, organs or animals alone or in combination with one or more other therapeutic modalities. In some embodiments, one or more additional agents further improve the transduction efficiency of the vector.

[0685] In some embodiments, the present disclosure provides a composition comprising a therapeutically effective amount of a viral particle as described herein, formulated 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.

[0686] In some embodiments, compositions and formulations of viral particles used in accordance with the present disclosure can be prepared for storage in the form of lyophilized preparations or aqueous solutions by mixing viral particles having the desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients or stabilizers are nontoxic to the recipient at the doses and concentrations employed. In some embodiments, one or more pharmaceutically acceptable surfactants, buffers, isotonic agents, salts, amino acids, sugars, stabilizers and / or antioxidants are used in the formulations.

[0687] Suitable pharmaceutically acceptable surfactants include, but are not limited to, polyethylene-sorbitol-fatty acid esters, polyethylene-polypropylene glycol, polyoxyethylene-stearate and sodium lauryl sulfate. Suitable buffers include, but are not limited to, histidine buffer, citrate buffer, succinate buffer, acetate buffer and phosphate buffer.

[0688] Isotonic agents are used to provide isotonic preparations. Isotonic preparations are liquids, or liquids reconstructed by solid forms (e.g., lyophilized forms), representing solutions with the same tension as some other solutions (e.g., physiological saline and serum) compared therewith. Suitable isotonic agents include, but are not limited to, salts, including but not limited to 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. In some embodiments, the consumption of isotonic agents is generally about 5mM to about 350mM.

[0689] The non-limiting examples of salts include salts of cation sodium, potassium, calcium or magnesium and anions chloride, phosphoric acid, citric acid, succinic acid, sulfuric acid or any combination thereof. The 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. According to the present invention, the non-limiting examples of sugars include trehalose, sucrose, mannitol, sorbitol, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine (also referred to as "methylglucamine"), galactosamine and neuraminic acid and combinations thereof. The non-limiting examples of stabilizers include amino acids and sugars as described above and commercially available cyclodextrins and dextran of any type and molecular weight known in the art. The non-limiting examples of antioxidants include excipients such as methionine, benzyl alcohol or any other excipients for minimizing oxidation.

[0690] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to the human body. The formulation of aqueous compositions containing proteins as active ingredients is well known in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for dissolution or suspension in liquids prior to injection may also be prepared. The formulation may also be emulsified.

[0691] 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, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active ingredient, they may be considered for use in the treatment composition. Supplementary active ingredients may also be added to the composition.

[0692] As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, etc., including pharmaceutically acceptable cell culture media. In some embodiments, the composition comprising the carrier is 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 injection solutions or dispersions. The use of such media and medicaments for pharmaceutically active substances is well known in the art. Unless any conventional media or medicaments are incompatible with the transduced cells, it is considered to be used in the pharmaceutical composition of the present disclosure.

[0693] The composition may also include one or more polypeptides, polynucleotides, vectors comprising polynucleotides, compounds that increase vector transduction efficiency, and the composition is formulated into a pharmaceutically acceptable or physiologically acceptable solution for administration to cells or animals alone or in combination with one or more other treatment modalities. It should also be understood that the composition of the present disclosure may also be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, or various pharmaceutically active agents, if desired. There are almost no restrictions on other components that may be included in the composition, as long as these additional agents do not adversely affect the ability of the composition to provide the intended treatment.

[0694] The present disclosure also provides a pharmaceutical composition comprising an expression cassette or vector disclosed herein (e.g., a therapeutic vector) and one or more pharmaceutically acceptable carriers, diluents or excipients. In some embodiments, the pharmaceutical composition comprises a lentiviral vector containing an expression cassette disclosed herein, for example, wherein the expression cassette comprises one or more polynucleotide sequences encoding one or more chimeric antigen receptors (CAR) and variants thereof.

[0695] The pharmaceutical composition containing the expression cassette or vector can be in any form suitable for the selected mode of administration, such as intraventricular, intramyocardial, intracoronary, intravenous, intraarterial, intrarenal, intraurethral, ​​epidural, intrathecal, intraperitoneal or intramuscular administration. The vector can be used as the sole active agent, or in combination with other active agents, in a unit administration form, as a mixture with conventional pharmaceutical carriers, and administered to animals and humans. In some embodiments, the pharmaceutical composition comprises cells transduced in vitro with any vector according to the present disclosure.

[0696] In some embodiments, the viral particles (e.g., lentiviral particles) or pharmaceutical compositions comprising the viral particles are effective when systemically administered. For example, the viral vectors of the present disclosure are administered intravenously to subjects (e.g., primates, such as non-human primates or humans) and have curative effects in some cases. In some embodiments, the viral vectors of the present disclosure can induce the expression of CAR in various immune cells when systemically administered (e.g., in T cells, dendritic cells, NK cells).

[0697] In various embodiments, the pharmaceutical composition contains a pharmaceutically acceptable medium (e.g., carrier, diluent, and excipient) for an injectable preparation. Exemplary excipients include poloxamer. The formulation buffer of the viral vector generally contains salts to prevent aggregation and other excipients (e.g., poloxamer) that reduce the viscosity of the viral particles. These can be particularly isotonic, sterile saline solutions (monosodium phosphate or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc. or mixtures of these salts), or dried, particularly freeze-dried compositions, which, according to the circumstances, can constitute an injectable solution after adding sterile water or saline. In some embodiments, the preparation can be stably stored and used when frozen (e.g., below 0°C, about -60°C, or about -72°C). In some embodiments, the preparation is a cryopreservation solution.

[0698] The formulation of the disclosed pharmaceutical compositions, pharmaceutically acceptable excipients and carrier solutions are well known to those skilled in the art, as are the development of appropriate dosages and treatment regimens using the specific compositions described herein in various treatment regimens, including, for example, oral, parenteral, intravenous, intranasal, intraperitoneal and intramuscular administration and formulation.

[0699] In some cases, it is desirable to deliver the compositions disclosed herein parenterally, intravenously, intramuscularly or intraperitoneally, such as in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 (each of which is incorporated herein by reference in its entirety). Solutions of the active compounds as free bases or pharmacologically acceptable salts can be prepared in water with suitable mixing with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and oils. Under normal conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms.

[0700] The pharmaceutical form suitable for injection includes sterile aqueous solution or dispersion, and sterile powder for temporary preparation of sterile injection solution or dispersion (U.S. Patent No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases, the form should be sterile and should have fluidity that is easy to inject. It should remain stable under production and storage conditions, and should prevent the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium, containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), their appropriate mixtures and / or vegetable oils. Appropriate fluidity can be maintained, for example, by using a coating (such as lecithin), by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. Various antibacterial an...

Claims

1. A virus particle comprising a virus envelope, wherein the virus envelope comprises at least one T cell adhesion molecule, at least one co-stimulatory protein or a combination thereof, and an immune cell activation protein on the surface of the virus envelope.

2. The viral particle according to claim 1, wherein the at least one T cell adhesion molecule is selected from the group consisting of: 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.

3. The viral particle of claim 1 or 2, wherein the at least one T cell adhesion molecule is CD58.

4. The viral particle according to any one of claims 1-3, wherein the at least one co-stimulatory molecule is selected from the group consisting of: 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.

5. The viral particle according to any one of claims 1 to 4, wherein the at least one co-stimulatory molecule is CD80, CD86, or CD80 and CD86.

6. The viral particle according to any one of claims 1 to 5, wherein the immune cell 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, TCRα chain, TCRβ chain, TCRζ chain, TCRγ chain, TCRδ chain, CD3εTCR subunit, CD3γTCR subunit, CD3δTCR subunit or NKp80.

7. The viral particle according to any one of claims 1 to 6, wherein the immune cell activation protein is a protein that specifically binds to CD3.

8. The viral particle according to any one of claims 1 to 7, wherein 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.

9. The viral particle according to any one of claims 1 to 8, wherein the immune cell activating protein is an antibody or an antigen-binding fragment thereof that binds to CD3.

10. The viral particle of claim 9, wherein the antibody or antigen-binding fragment thereof that binds CD3 is an anti-CD3 scFv.

11. The viral particle according to any one of claims 1 to 10, wherein the T cell adhesion molecule is CD58 and the co-stimulatory molecule is CD80.

12. The viral particle according to any one of claims 1 to 10, wherein the T cell adhesion molecule is CD58 and the co-stimulatory molecule is CD86.

13. The viral particle according to any one of claims 1 to 10, wherein the T cell adhesion molecule is CD58, the immune cell activation protein is an anti-CD3 antibody or an antigen-binding fragment thereof, and the co-stimulatory molecule is CD80.

14. The viral particle according to any one of claims 1 to 10, wherein the T cell adhesion molecule is CD58, the immune cell activation protein is an anti-CD3 antibody or an antigen-binding fragment thereof, and the co-stimulatory molecule is CD86.

15. The viral particle according to any one of claims 1 to 14, comprising a payload.

16. The viral particle of claim 15, wherein the payload is a nucleic acid.

17. The viral particle of claim 16, wherein the nucleic acid is a non-coding nucleic acid, optionally wherein the non-coding nucleic acid is a siRNA, miRNA or shRNA.

18. The viral particle of claim 17, wherein the nucleic acid comprises a nucleotide sequence encoding a polypeptide of interest.

19. The viral particle according to any one of claims 1 to 14, comprising a vector genome comprising at least one nucleotide sequence encoding a polypeptide of interest.

20. A viral particle comprising (i) a viral envelope, wherein the viral envelope comprises (a) an immune cell activation protein on the surface of the viral envelope, wherein the immune cell activation protein binds to a T cell receptor, (b) a co-stimulatory molecule and (c) a T cell adhesion molecule, and (ii) a vector genome, wherein the vector genome comprises at least one nucleotide sequence encoding a polypeptide of interest.

21. The viral particle of claim 20, wherein (a) the immune cell 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 or NKp80, and (b) the co-stimulatory molecule is selected from: CD45, CD2, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, (c) the T cell adhesion molecule is selected from the group consisting of: 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.

22. The viral particle according to claim 20 or 21, wherein (a) the immune cell activation 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.

23. The viral particle of any one of claims 1-22, wherein the viral envelope comprises a membrane-bound cytokine.

24. The viral particle of claim 23, wherein the membrane-bound cytokine is selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18, or IL-21.

25. The viral particle of any one of claims 1-24, wherein the viral envelope comprises a viral envelope protein.

26. The viral particle of claim 25, wherein the viral envelope protein is a VSV-G envelope protein, a measles virus envelope protein, a Nipah virus envelope protein, or a Cocal virus G protein.

27. The viral particle according to claim 26, wherein the viral envelope comprises Cocal glycoprotein or a functional variant thereof.

28. The viral particle of claim 27, wherein the Cocal glycoprotein comprises a R354Q mutation compared to SEQ ID NO:

5.

29. The viral particle of claim 27 or 28, wherein 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.

30. The viral particle of claim 27 or 28, wherein the Cocal glycoprotein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 13 and 19.

31. The viral particle of any one of claims 9-19 and 22-30, wherein the anti-CD3 binding antibody or antigen-binding fragment thereof 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.

32. The viral particle of any one of claims 9-19 and 22-30, wherein the anti-CD3 binding antibody or antigen-binding fragment thereof comprises SEQ ID NO:2 or SEQ ID NO:

12.

33. The viral particle of any one of claims 19-32, wherein at least one nucleotide sequence encodes a multipartite cell surface receptor.

34. The viral particle of claim 33, wherein the multipartite cell surface receptor comprises a FKBP-rapamycin complex binding domain (FRB domain) and a FK506 binding protein domain (FKBP).

35. The viral particle of claim 33, wherein the multipartite cell surface receptor is a rapamycin-activated cell surface receptor.

36. The viral particle of any one of claims 19-35, wherein at least one nucleotide sequence encodes a chimeric antigen receptor (CAR).

37. The viral particle according to any one of claims 19-32, comprising a nucleotide sequence encoding a cell surface receptor activated by rapamycin and a nucleotide sequence encoding a CAR.

38. The viral particle of claim 36 or 37, wherein the CAR comprises an antigen binding domain specific for a cancer associated antigen.

39. The viral particle of claim 37, wherein the cancer-associated antigen is CD19, BCMA, GPRC5D, ROR1, FcRL5, alpha-fetoprotein, or Her2.

40. The viral particle of claim 36 or 37, wherein the CAR is a universal CAR.

41. The viral particle of claim 36 or 37, wherein the CAR comprises a hapten binding domain.

42. The viral particle of any one of claims 19-32, wherein the vector genome comprises from 5' to 3': a nucleotide sequence encoding a CAR and a nucleotide sequence encoding a multipartite cell surface receptor.

43. The viral particle of claim 42, wherein the nucleotide sequences are operably linked.

44. The viral particle of claim 42 or 43, wherein the CAR comprises an antigen binding domain specific for a cancer associated antigen, wherein the multipartite cell surface receptor is a rapamycin-activated cell surface receptor.

45. The viral particle of claim 44, wherein the cancer associated antigen is CD19, BCMA, GPRC5D, ROR1, FcRL5, alpha-fetoprotein or Her2.

46. ​​A viral particle comprising (i) a viral envelope, wherein the viral envelope comprises on the surface of the viral envelope (a) an immune cell activating protein that specifically binds to CD3, (b) a co-stimulatory molecule, wherein the co-stimulatory molecule binds to CD28, and (c) a T cell adhesion molecule, and (ii) a vector genome, wherein the vector genome comprises (a) a nucleotide sequence encoding a cell surface receptor activated by rapamycin, and (b) a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen binding domain specific for a cancer-associated antigen, optionally wherein the nucleotide sequence is operably linked.

47. The viral particle of any one of claims 2-19 and 21-46, wherein 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.

48. The viral particle of any one of claims 2-19 and 21-46, wherein CD58 comprises the amino acid sequence of SEQ ID NO:

17.

49. The viral particle of any one of claims 4-11, 13, 15-19, 21-45, 47-48, wherein 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.

50. The viral particle of any one of claims 4-11, 13, 15-19, 21-45, 47-48, wherein CD80 comprises the amino acid sequence of SEQ ID NO:

20.

51. The viral particle of any one of claims 4-10, 12, 14, 15-19, 21-45, 47-48, wherein 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.

52. The viral particle of any one of claims 4-10, 12, 14, 15-19, 21-45, 47-48, wherein CD86 comprises the amino acid sequence of SEQ ID NO:

23.

53. The viral particle of any one of claims 33-45 and 47-52, 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% or at least 99% identical to SEQ ID NO: 77, 78, or 77 and 78.

54. The viral particle of any one of claims 33-45 and 47-52, wherein the multipartite cell surface receptor comprises the amino acid sequence of SEQ ID NO: 77, 78, or 77 and 78.

55. The viral particle of any one of claims 33-45 and 47-54, wherein the multi-part 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 83 and 84.

56. The viral particle of any one of claims 33-45 and 47-54, wherein the multipartite cell surface receptor is encoded by the nucleotide sequence of SEQ ID NO: 83, 84, or 83 and 84.

57. The viral particle of any one of claims 19-56, wherein the vector genome comprises a promoter.

58. The viral particle of claim 57, wherein the promoter is MND promoter, CAG promoter, SV40 promoter, SV40 / CD43 promoter or EF-1α promoter.

59. The viral particle of claim 57, wherein the promoter is an inducible promoter.

60. The viral particle of any one of claims 1-59, wherein the viral particle is a lentiviral particle.

61. The viral particle of any one of claims 1-60, wherein the viral particle transduces T cells in vivo.

62. The viral particle of any one of claims 1-61, wherein the viral particle activates a T cell population comprising at least 50% CD25(+) cells, at least 70% CD25(+) cells, or at least 90% CD25(+) cells.

63. A pharmaceutical composition comprising the viral particle of any one of claims 1-62 and a pharmaceutically acceptable carrier.

64. A method for transducing a T cell population in a subject, comprising administering to the subject the viral particle of any one of claims 1-62 or the pharmaceutical composition of claim 63, wherein the viral particle comprises a nucleotide sequence encoding a polypeptide of interest, wherein the polypeptide of interest is expressed in the T cell population after administration.

65. The method of claim 64, wherein at least three days after administration of the lentiviral particles, the T cell population 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)-(iii).

66. A method for producing immune cells expressing a chimeric antigen receptor in a subject in need thereof, comprising administering to the subject the viral particle of any one of claims 1-62 or the pharmaceutical composition of claim 63, wherein the viral particle comprises a nucleotide sequence encoding the chimeric antigen receptor.

67. A method of treating a disease or condition in a subject in need thereof, comprising administering to the subject the viral particle of any one of claims 1-62 or the pharmaceutical composition of claim 63, wherein the viral particle comprises a nucleotide sequence encoding a therapeutic polypeptide.

68. The method of any one of claims 64-67, wherein the viral particles are administered by intraperitoneal, subcutaneous or intralymph node injection.

69. The method of claim 68, wherein the viral particles are administered by intranodal injection via the inguinal lymph nodes.

70. The method of any one of claims 64-69, wherein the subject in need thereof has a disease or disorder, wherein the disease or disorder comprises a B-cell malignancy, a relapsed / refractory CD19-expressing malignancy, 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), a hematological malignancy, 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.

71. A kit comprising a container comprising the viral particle of any one of claims 1-62, and optionally a pharmaceutically acceptable carrier, and instructions for transducing T cells in a subject, the instructions comprising administering the viral particle to the subject.

72. A kit comprising a container comprising the viral particle of any one of claims 1-62, and optionally a pharmaceutically acceptable carrier, and instructions for treating a subject in need thereof, the instructions comprising administering the viral particle to the subject.

73. The kit of claim 71 or 72, wherein the subject has a disease or disorder.

74. The kit of any one of claims 71-73, wherein the instructions comprise administering the viral particles by intraperitoneal, subcutaneous, or intralymph node injection.

75. A method for using the viral particle of any one of claims 1-62 to transduce T cells in a subject, comprising administering the viral particle to the subject.

76. The viral particle of any one of claims 1-62 for use in a method of treating a subject having a disease or disorder, comprising administering the viral particle to the subject.

77. Use of the viral particle of any one of claims 1-62 in the preparation of a medicament for transducing T cells in a subject, comprising administering the viral particle to the subject.

78. Use of the viral particle of any one of claims 1-62 in the preparation of a medicament for treating a subject having a disease or disorder, comprising administering the viral particle to the subject.

79. A method for ex vivo transduction of a subject's T cell population, comprising contacting the T cell population with the viral particles of any one of claims 1-62 or the pharmaceutical composition of claim 63, wherein the viral particles comprise a nucleotide sequence encoding a polypeptide of interest, wherein the polypeptide of interest is expressed in the T cell population after administration, and wherein the contacting is performed ex vivo.

80. The method of claim 79, wherein the contacting is performed during a closed-loop manufacturing process.

81. The method of claim 79 or 80, wherein the T cells have not been previously contacted with an exogenous activator during the manufacturing process.

82. The viral particle of any one of claims 27, 31-45, and 47-62, wherein the Cocal glycoprotein comprises a K47Q mutation compared to SEQ ID NO:

5.

83. A method for activating a cell population in a subject, comprising administering to the subject the viral particles described in any one of claims 1-62 or the pharmaceutical composition described in claim 63, wherein the viral particles comprise a nucleotide sequence encoding a polypeptide of interest, wherein after administration, the polypeptide of interest is expressed in a first subpopulation of cells in the cell population, wherein after administration, the polypeptide of interest is not expressed in a second subpopulation of cells in the cell population, and wherein the first subpopulation of cells and the second subpopulation of cells are each activated by the viral particles.

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