CN116997565A - Lentivirus for generation of cells expressing anti-CD19 chimeric antigen receptor - Google Patents

Abstract

Compositions and methods for transducing immune cells in vivo are provided, wherein viral particles comprising polynucleotides encoding chimeric antigen receptors and multipartitioned cell surface receptors are administered to a subject.

Description

Lentivirus for the generation of cells expressing anti-CD19 chimeric antigen receptor

Related Applications

This application claims priority to and the benefit of U.S. Provisional Application No. 63/142,347, filed on January 27, 2021, and U.S. Provisional Application No. 63/185,765, filed on May 7, 2021. The contents of the foregoing patent applications are incorporated herein by reference in their entirety.

Sequence Listing

This application contains a sequence listing that has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy created on January 26, 2022 is named "UMOJ-024-02WO_SeqList_ST25.txt" and is 246KB in size.

Technical Field

The present invention generally relates to the in vivo transduction of immune cells for the treatment of cancer and/or B-cell malignancies.

Background Art

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

Instead, it would be desirable to generate therapeutic cells in vivo by delivering vectors to patients. Current methods for transducing immune cells in vivo are subject to technical, logistical, consistency, cost, and efficacy challenges. In vivo approaches have not been widely practiced to date due to the technical challenges associated with them, the main obstacles being the need to activate T cells in vivo in order to effectively engineer them, and the need to "control" the expansion of these engineered cells upon transduction.

Currently, patients with aggressive B-cell malignancies who have failed standard therapy, including chemotherapy and conventional hematopoietic stem cell transplantation (HSCT), can choose to receive autologous CAR-T cell products, which redirect their T cells against the antigen CD19 through an ex vivo preparation process. However, the preparation of these products requires a series of complex steps, which begins with collecting the patient's peripheral blood mononuclear cells through a leukapheresis procedure, and then genetically modifying the patient's T cells in a cGMP facility, which delays patient care, makes patient care risky, and complicates the logistics of reaching patient care. This is followed by the administration of lymphodepleting chemotherapy before infusing the final drug product. The medical needs of patients with relapsed/refractory B-cell malignancies are not met, both because the patient's disease is not treated and because it is impossible to prepare newer cell products or to afford the logistics time of newer cell products.

The present disclosure provides compositions and methods related to the in vivo transduction of immune cells for the treatment of cancer and/or B-cell malignancies.

Summary of the invention

In one aspect, the present disclosure provides a viral particle comprising a vector genome comprising a polynucleotide sequence encoding an anti-CD19 chimeric antigen receptor, wherein the viral particle transduces immune cells in vivo.

In some embodiments, the viral particle is a lentiviral particle.

In some embodiments, the immune cell is a T cell.

In some embodiments, the viral particle comprises a polynucleotide sequence encoding a multi-compartmental cell surface receptor.

In some embodiments, the multi-compartmental cell surface receptor is a chemically inducible cell surface receptor.

In some embodiments, the viral particle comprises a polynucleotide sequence encoding a multi-partitioned cell surface receptor, which comprises a FKBP-rapamycin complex binding domain (FRB domain) or a functional variant thereof; and the polynucleotide comprises a polynucleotide sequence encoding a FK506 binding protein domain (FKBP) or a functional variant thereof.

In some embodiments, the multi-compartmental cell surface receptor is a rapamycin-activated cell surface receptor.

In some embodiments, the viral particle comprises a sequence that confers resistance to an immunosuppressant.

In some embodiments, the viral particle comprises a sequence encoding a polypeptide that binds rapamycin and confers resistance to an immunosuppressant, wherein optionally, the polypeptide is FRB.

In some embodiments, the viral particle comprises, in 5' to 3' order on the polycistronic transcript: the polynucleotide sequence encoding the multi-partitioned cell surface receptor and the polynucleotide sequence encoding the anti-CD19 chimeric antigen receptor.

In some embodiments, the viral particle comprises, in 5' to 3' order on the polycistronic transcript: the polynucleotide sequence encoding the anti-CD19 chimeric antigen receptor and the polynucleotide sequence encoding the multi-partitioned cell surface receptor, and/or wherein the anti-CD19 chimeric antigen receptor shares at least 80%, 90%, 95% or 100% identity with SEQ ID NO:51, 79, 89, 121 or 122.

In some embodiments, the polynucleotide encoding the anti-CD19 chimeric antigen receptor and/or the polynucleotide encoding the multi-compartmented cell surface receptor are operably linked to one or more promoters.

In some embodiments, the promoter is an inducible promoter.

In some embodiments, the viral particle comprises a viral envelope comprising one or more immune cell activation proteins exposed on and/or conjugated to the surface of the viral envelope.

In some embodiments, the viral envelope comprises an anti-CD3 single-chain variable fragment exposed on and/or conjugated to the surface of the viral envelope.

In some embodiments, the viral envelope comprises Cocal glycoprotein exposed on and/or conjugated to the surface of the viral envelope.

In some embodiments, the viral envelope comprises a Cocal glycoprotein exposed on and/or conjugated to the surface of the viral envelope, optionally wherein the Cocal glycoprotein comprises an R354Q mutation compared to a reference sequence according to SEQ ID NO:5.

In some embodiments, the viral envelope comprises an anti-CD3 single-chain variable fragment and a Cocal glycoprotein exposed on and/or conjugated to the surface of the viral envelope.

In some embodiments, the viral envelope comprises an anti-CD3 single chain variable fragment sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO: 2 or 12.

In some embodiments, the viral envelope comprises a Cocal glycoprotein sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:5, 13, 19, 123, 128, 129 or 130.

In some embodiments, the promoter is the MND promoter.

In some embodiments, the viral particle comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:49.

In some embodiments, the viral particle comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:75.

In some embodiments, the viral particle comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:87.

The present disclosure provides a method of treating a disease or condition in a subject in need thereof, transducing immune cells in vivo, and/or generating immune cells expressing an anti-CD19 chimeric antigen receptor, the method comprising administering the viral particle of the present disclosure to the subject.

In some embodiments, the viral particles are administered by intraperitoneal, subcutaneous, or intranodal injection.

In some embodiments, transduced immune cells comprising the polynucleotides of the present disclosure are administered to the subject.

In another aspect, the present disclosure provides a method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of viral particles by intraperitoneal, subcutaneous or intranodal injection, wherein the viral particles transduce immune cells in vivo.

In some embodiments, the viral particles are administered by intranodal injection, optionally via the inguinal lymph nodes.

In some embodiments, the viral particles are administered by intraperitoneal injection.

The present disclosure provides a viral particle for transducing immune cells in vivo, wherein the viral particle comprises a polynucleotide comprising a polynucleotide sequence encoding a chimeric antigen receptor.

In some embodiments, the viral particle further comprises a polynucleotide sequence encoding a dominant negative TGFβ receptor.

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

In some embodiments, the ligand is rapamycin.

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

In some embodiments, the disease or disorder comprises a B-cell malignancy, a relapsed/refractory CD19-expressing malignancy, a diffuse large B-cell lymphoma (DLBCL), a Burkitt's type large B-cell lymphoma (B-LBL), a follicular lymphoma (FL), a chronic lymphocytic leukemia (CLL), an acute lymphocytic leukemia (ALL), a mantle cell lymphoma (MCL), a hematological malignancy, a colon cancer, a lung cancer, a liver cancer, a breast cancer, a kidney cancer, a prostate cancer, an ovarian cancer, a skin cancer, a melanoma, a bone cancer, a brain cancer, a squamous cell carcinoma, a leukemia, a myeloma, a B-cell lymphoma, a kidney cancer, a uterine cancer, adenocarcinoma, a pancreatic cancer, a chronic myeloid leukemia, a glioblastoma, a neuroblastoma, a medulloblastoma, a sarcoma, and any combination thereof.

In some embodiments, the disease or condition comprises diffuse large B-cell lymphoma (DLBCL).

In some embodiments, the disease or disorder comprises Burkitt's large B-cell lymphoma (B-LBL).

In some embodiments, the disease or disorder comprises follicular lymphoma (FL).

In some embodiments, the disease or condition comprises chronic lymphocytic leukemia (CLL).

In some embodiments, the disease or condition comprises acute lymphoblastic leukemia (ALL).

In some embodiments, the disease or disorder comprises mantle cell lymphoma (MCL).

The present disclosure provides a pharmaceutical composition comprising the viral particle of the present disclosure.

The present disclosure provides a kit comprising the pharmaceutical composition of the present disclosure and, optionally, a composition comprising a ligand, optionally rapamycin.

The present disclosure provides a viral particle for use in a method according to any viral particle of the present disclosure.

The present disclosure provides for use of a viral particle in a method according to any of the methods of the present disclosure.

In some embodiments, a method of treating a disease or condition associated with malignant CD19+ cells in a subject comprises administering to the subject a viral particle of the present disclosure, and after administration of the viral particle, the CD19+ B cells in the subject are depleted by at least 80%, at least 85%, at least 90%, or at least 95%, compared to a subject that did not receive the viral particle.

In some embodiments, the subject's peripheral blood is depleted of CD19+ B cells.

In some embodiments, B cell depletion is maintained in the subject for at least 7 days, at least 10 days, at least 20 days, at least 30 days, at least 40 days, at least 50 days, at least 60 days, at least 70 days, or at least 80 days after administration of the viral particle.

In some embodiments, at least 2 million, at least 4 million, at least 6 million, at least 8 million, or at least 10 million transducing units of viral particles are administered to the subject.

In some embodiments, contacting the immune cells with the ligand of the present disclosure increases the number of immune cells expressing the anti-CD19 chimeric antigen receptor in the subject by at least 10-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, or at least 1000-fold.

The present disclosure provides a polypeptide comprising a single-chain variable fragment that specifically binds to CD3 (anti-CD3 scFv) and a glycophorin A transmembrane fragment.

In some embodiments, the glycophorin A transmembrane fragment shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with HFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ (SEQ ID NO: 105).

In some embodiments, the anti-CD3 scFv shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:2 or 12.

In some embodiments, the polypeptide shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:119.

In some embodiments, the transmembrane fragment shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:13, 19, 25, 31, 37, 43 or 105.

The present disclosure provides a surface engineered lentiviral particle, which includes a polypeptide according to the present disclosure displayed on the surface of the lentiviral particle.

The present disclosure provides a method for transducing cells, the method comprising contacting the viral particle according to the present disclosure with an immune cell in vivo.

The present disclosure provides a polynucleotide comprising an anti-CD3 scFv and a glycophorin A transmembrane fragment.

In some embodiments, the glycophorin A transmembrane fragment shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:106.

In some embodiments, the anti-CD3 scFv shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:7 or 15.

In some embodiments, the polypeptide shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:120.

In some embodiments, the transmembrane segment shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:16, 22, 25, 28, 34, 40, 47 or 106.

The present disclosure provides a method for preparing virus particles, the method comprising:

a) providing cells in culture medium; and

b) transfecting the cells simultaneously or sequentially with a vector genome, a transfer plasmid and a packaging plasmid according to the present disclosure; whereby the cells express surface engineered viral particles.

The present disclosure provides a method for treating a disease or condition associated with malignant CD19+ cells, the method comprising: transducing immune cells in vivo, and/or producing viral particles expressing an anti-CD3 single-chain variable fragment exposed on the surface of a viral envelope and/or conjugated to the surface of the viral envelope, wherein the anti-CD3 single-chain variable fragment shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO: 2 or 12; and administering the viral particles to a subject.

In some embodiments, the viral particles are administered by intraperitoneal, subcutaneous, or intranodal injection.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph of 293T titers of lentiviral vectors. Amicon concentrated lentiviral vector preparations were titrated on 293T cells previously seeded in 12-well plates. Three days later, the transduced cells were evaluated for mCherry expression. Calculated viral titers are shown.

Figure 2A shows flow cytometry for measuring CD25 expression and T cells positively expressing mCherry in stimulated or unstimulated PBMCs not transduced with vector. 4 days after transduction, PBMCs were harvested and stained.

Figure 2B shows flow cytometry for measuring CD25 expression and T cells positively expressing mCherry in PBMCs that were stimulated or unstimulated and transduced with 5ul or 25ul Cocal vector. 4 days after transduction, PBMCs were harvested and stained.

Figure 2C shows flow cytometry for measuring CD25 expression and T cells positively expressing mCherry in PBMCs that were stimulated or unstimulated and transduced with 5ul or 25ul αCD3+Cocal vector. 4 days after transduction, PBMCs were harvested and stained.

Figure 2D shows flow cytometry for measuring CD25 expression and T cells positively expressing mCherry in PBMCs that were stimulated or unstimulated and transduced with 5ul or 25ul αCD3 blind-Cocal vector. 4 days after transduction, PBMCs were harvested and stained.

Fig. 3 shows the flow cytometry diagram of αCD19 CAR and 2A peptide expression.After culture in rapamycin, unstimulated PBMC cells are stained for RACR-αCD19 CAR and 2A.Unstimulated PBMCs are transduced with specified carriers (VT103, RACR-αCD19CAR MND-Cocal, RACR-αCD19 CAR CMV-Cocal, RACR-αCD19 CAR CMV-αCD3-Cocal, RACR-αCD19 CAR CMV-αCD3-(B) Cocal) every 100ul.4 days after transduction, rapamycin is added, and cells are cultured for 11 days.PBMC is then collected and stained for αCD19 CAR and 2A peptides.

Fig. 4 shows the flow cytometry diagram of αCD19 CAR and 2A peptide expression.After culture in rapamycin, PBMC cells stimulated by blinatumomab are stained for RACR-αCD19 CAR and 2A.PBMC stimulated by blinatumomab are transduced with specified carriers (RACR-αCD19CAR MND-Cocal, RACR-αCD19 CAR CMV-Cocal, RACR-αCD19 CAR CMV-αCD3-Cocal, RACR-αCD19 CAR CMV-αCD3-(B) Cocal) at each time 100ul.4 days after transduction, rapamycin is added, and cells are cultured for 11 days.PBMC is then collected and stained for αCD19 CAR and 2A peptides.

Figure 5 shows a flow cytometry graph of αCD19 CAR+CD8 T cells. Unstimulated PBMCs were transduced with RACR-αCD19CAR/αCD3-Cocal vectors as described in Example 2. Four days after transduction, rapamycin was added for culture. After 13 days, cells were stimulated with Raji cells and intracellular cytokine production was measured as described in Example 2. Live CD3+, CD8+, 2A+ cells are shown.

Figure 6 shows a flow cytometry graph of αCD19 CAR+CD4 T cells. Unstimulated PBMCs were transduced with RACR-αCD19CAR/αCD3-Cocal vectors as described in Example 2. Four days after transduction, rapamycin was added for culture. After 13 days, cells were stimulated with Raji cells and intracellular cytokine production was measured as described in Example 2. Live CD3+, CD8+, 2A+ cells are shown.

Figure 7A shows flow cytometry plots of CD8+ T cells expressing CD25. Representative flow cytometry plots of PBMCs transduced at MOI=2 with αCD19 CAR-TGFβDN and viral envelope proteins pseudotyped with Cocal or αCD3-Cocal analyzed for CD8+ T cells.

FIG. 7B shows a graph of the % CD25 T cells transduced with αCD19 CAR-TGFβDN/αCD3-Cocal viral particles at 0.5 MOI, 1.0 MOI, or 2.0 MOI and added to unstimulated PBMCs for 3 days.

Figure 8A shows flow cytometry plots of CD8+ T cells expressing 2A peptide. Representative flow cytometry plots of PBMCs transduced at MOI=2 with αCD19 CAR-TGFβDN and viral envelope proteins pseudotyped with Cocal or αCD3-Cocal analyzed for CD8+ T cells.

Figure 8B shows a graph of the % of αCD19 CAR+ T cells transduced with αCD19 CAR-TGFβDN/αCD3-Cocal viral particles at 0.5 MOI, 1.0 MOI, or 2.0 MOI and added to unstimulated PBMCs for 6 days.

Figure 9A shows a graph of the % of αCD19 CAR+CD4 T cells transduced with αCD19 CAR-TGFβDN/αCD3-Cocal viral particles at 0.5 MOI, 1.0 MOI, or 2.0 MOI and added to unstimulated PBMCs for 3 days, 6 days, or 12 days.

Figure 9B shows a graph of the % of αCD19 CAR+CD8 T cells transduced with αCD19 CAR-TGFβDN/αCD3-Cocal viral particles at 0.5 MOI, 1.0 MOI, or 2.0 MOI and added to unstimulated PBMCs for 3 days, 6 days, or 12 days.

Figure 10A shows a graph of αCD19 CAR+293T cell% transduced with αCD19 CAR-TGFβDN, αCD19 CAR-RACR or RACR-αCD19 CAR vectors in the presence of Cocal envelope protein. Also shown are the 293T titers (TU/ml) of the viral particle preparations.

Figure 10B shows a graph of αCD19 CAR+293T cell% transduced with αCD19 CAR-TGFβDN, αCD19CAR-RACR or RACR-αCD19 CAR vectors in the presence of αCD3-Cocal envelope protein. Also shown are the 293T titers (TU/ml) of the viral particle preparations.

Figure 11A shows flow cytometry for measuring CD8 T cells expressing αCD19 CAR and 2A peptide in unstimulated PBMCs transduced with MOI=1.5 αCD19 CAR-TGFβDN vector and Cocal or αCD3-Cocal pseudotyped viral envelope proteins 8 days after transduced PBMCs were harvested and stained. Live CD3+, CD8+ cells were gated.

Figure 11B shows flow cytometry for measuring CD8 T cells expressing αCD19-CAR and 2A peptide in unstimulated PBMCs transduced with MOI=1.5RACR-αCD19CAR vector and Cocal or αCD3-Cocal pseudotyped viral envelope proteins 8 days after transduced PBMCs were harvested and stained. Live CD3+, CD8+ cells were gated.

Figure 11C shows flow cytometry for measuring CD8 T cells expressing αCD19-CAR and 2A peptide in unstimulated PBMCs transduced with MOI=1.5 αCD19-RACR vector and Cocal or αCD3-Cocal pseudotyped viral envelope proteins 8 days after transduced PBMCs were harvested and stained. Live CD3+, CD8+ cells were gated.

FIG. 12 shows a schematic diagram of the study protocol described in Example 5.

Figure 13A shows flow cytometry graphs of P2A (CAR) and cell activity (CD71) in single/live/CD3+/CD8+ cells after transduction on day 8 (start of rapamycin and/or Raji treatment), day 15, and day 22 from control, i.e., MOI = 0 cells.

Figure 13B shows flow cytometry of P2A (CAR) and cell activity (CD71) in single/live/CD3+/CD8+ cells after transduction on day 8 (rapamycin and/or Raji treatment start), day 15, and day 22 from cells transduced with RACR-αCD19 CAR-directed vectors. Red arrows indicate RACR-αCD19CAR 2A+ populations.

Figure 13C shows a flow cytometry graph of P2A (CAR) and cell activity (CD71) in single/live/CD3+/CD8+ cells after transduction on day 8 (start of rapamycin and/or Raji treatment), day 15, and day 22 from cells transduced with αCD19 CAR-RACR directed vectors. Black arrows indicate αCD19 CAR-RACR 2A+ populations.

FIG. 14 shows flow cytometry plots of rapamycin enrichment for CD8+ CAR-T cells and a “sneaky” RACR-αCD19 CAR-T population (red arrows) that is only detectable upon treatment with rapamycin.

Figure 15 shows a graph of total CD8+CAR-T cells as they expand from study day 8 to day 22. As of day 22, cell expansion increased in the presence of rapamycin relative to the absence of rapamycin. Maximum expansion occurred with the addition of both rapamycin and Raji cells.

Figure 16A shows flow cytometry graphs of CD3+ (T cells) and GFP (Raji cells) in the presence and absence of rapamycin. Raji cells were reduced in the control, MOI=0 cells, and in the co-culture wells treated with rapamycin.

Figure 16B shows flow cytometry graphs of CD3+ (T cells) and GFP (Raji cells) in the presence and absence of rapamycin. Raji cells were reduced in co-culture wells transduced with RACR-αCD19 CAR directed vectors.

Figure 16C shows flow cytometry graphs of CD3+ (T cells) and GFP (Raji cells) in the presence and absence of rapamycin. Raji cells were reduced in co-culture wells transduced with αCD19 CAR-RACR directed vectors.

Figure 17A shows a graph of flow cytometry quantification of CAR+T cells/ul blood in CD3+ cells.

Figure 17B shows a graph of flow cytometry quantification of CAR+ T cells/ul blood among non-CD3+ cells.

Figure 18A shows a graph of flow cytometric quantification of the ratio of CD20+ T cells % to CD45+ T cells % at study termination.

Figure 18B shows a graph of flow cytometric quantification of the ratio of CD3+ T cells % to CD45+ T cells % at study termination.

Figure 19A shows a graph of flow cytometry quantification of B cells/ul whole blood throughout the study (Day 0-Day 30). Bars represent +/- standard error of the mean.

Figure 19B shows a graph of flow cytometric quantification of B cells in the spleen (frequency of total CD45+). The bars represent the median value for each group.

Figure 19C shows a graph of flow cytometric quantification of B cells in the bone marrow (frequency of total CD45+). The bars represent the median value for each group.

Figure 20A shows a graph of flow cytometry quantification of αCD4 CAR T cells in blood, spleen, and bone marrow at study termination on day 29. Individual values represent the frequency of CAR+ cells within the indicated T cell population after background subtraction. Bars represent the median value for each group.

Figure 20B shows a graph of flow cytometry quantification of αCD8 CAR T cells in blood, spleen, and bone marrow at study termination on day 29. Individual values represent the frequency of CAR+ cells within the indicated T cell population after background subtraction. Bars represent the median value for each group.

Figure 21A shows a graph of CAR payload integration in the blood at days 3, 10, 14, and 21. Vector copy number was determined by digital droplet PCR (ddPCR) using human as the reference genome. The bars represent the median value for each group.

Figure 21B shows a graph of CAR payload integration in the bone marrow at day 10 and day 29. Vector copy number was determined by digital droplet PCR using human as the reference genome. The bars represent the median value for each group.

FIG. 22A shows a diagram of an embodiment of a viral particle of the present disclosure.

FIG. 22B shows a diagram of an embodiment of a viral particle of the present disclosure.

Figure 23A and 23B show schematic diagrams of drug product transgenes. The transgene encodes an anti-CD19-CAR with FMC63 scFv, a short IgG4 hinge, and a 4-1BB/CD3ζ signaling domain. Anti-CD19-CAR is followed by a P2A ribosomal jump sequence and a rapamycin-activated cytokine receptor box (FRB-RACR).

Figure 24 shows a schematic diagram of the mechanism of action of the drug product. Lentiviral particles bind to T cells in vivo via anti-CD3 scFv, which simultaneously activates T cells and promotes lentiviral internalization. The capsid containing the αCD19 CAR-RACR construct is released into the cytoplasm, reverse transcribed into DNA, and integrated into the genome. Transduced T cells express anti-CD19 CAR and target tumor cells expressing CD19.

FIG25 shows a schematic diagram of a RACR system.

Figure 26A shows a representative flow chart of CD25 expression on CD8 T cells from a single donor. Live CD3+ and CD4+/CD8+ cells were gated for all samples.

Figure 26B shows the % CD25 expression for both CD8+ T cells across multiple MOIs from all three donors. Live CD3+ and CD4+/CD8+ cells were gated for all samples.

Figure 26C shows the % CD25 expression for both CD4+ T cells across multiple MOIs from all three donors. Live CD3+ and CD4+/CD8+ cells were gated for all samples.

Figure 27A shows a flow chart of αCD19 CAR+CD8+ T cells from a single donor at day 17 in the presence of +/- rapamycin. Live CD3+ and +CD8+ cells were gated among the CAR+ cells.

FIG. 27B shows the % CAR+ cells of total CD8+ T cells. Live CD3+ and +CD8+ cells were gated among the CAR+ cells.

Figure 27C shows the % of the total count of CAR+ cells relative to each well CAR+CD8+T cells. Live CD3+ and +CD8+ cells in CAR+ cells were gated. In order to calculate the total CAR+ cells per well, the total CAR counts were normalized relative to the counting beads. Similar data (not shown) were also obtained in CAR+CD4+T cells.

Figure 28 shows representative flow cytometry plots of GFP+Raji cells co-cultured for one week with either untransduced or drug product viral particle transduced T cells at an approximate E:T ratio of 1:1. The circular gate represents a Raji cell population that is absent in co-cultures with drug product viral particle transduced T cells and also decreases in response to rapamycin treatment alone. UB-VV100 represents drug product.

Figure 29A shows a representative flow cytometry diagram of drug products added to unstimulated PBMCs from a single donor at day = 0. After 3 days (day 3), the cells were washed to remove viral particles and fresh culture medium was added. The cells were then divided into two halves, and one group received rapamycin (10nM). At day 20, in the presence of Golgi inhibitors Brefeldin A and Monensin, functional responses were assessed by stimulating Raji tumor cells for 5 hours. Cells were collected and Live/Dead ^TM , surface markers and intracellular IFN-γ staining were performed. The live Raji CD3+ and CAR+ cells of all sampled samples were gated.

Figure 29B shows a representative flow cytometry diagram of the drug product added to the unstimulated PBMC from a single donor at day = 0. After 3 days (day 3), the cells were washed to remove viral particles and fresh culture medium was added. The cells were then divided into two halves, and one group received rapamycin (10nM). At the 20th day, in the presence of Golgi inhibitors Brefeldin A and Monensin, the functional response was assessed by stimulating Raji tumor cells for 5 hours. Cells were collected and Live/Dead ^TM , surface markers and intracellular TNF-α staining were performed. The live Raji CD3+ and CAR+ cells of all sampled samples were gated.

Figure 30A shows a graph of B cell populations (defined as live/single/human CD45+/CD3-CD20+) in blood, spleen, and bone marrow assessed by flow cytometry. Error bars represent +/- standard error of the mean. **** indicates p<0.0001, two-way ANOVA multiple comparisons. Bars represent median values for each group. * and *** indicate p<0.05 and <0.001, respectively, two-tailed t test. UB-VV100 represents drug product.

Figure 30B shows a graph of CAR T cells detected by peripheral leukocyte payload using ddPCR. UB-VV100 represents the drug product.

Figure 30C shows a graph of CAR T cells detected by peripheral leukocyte payload using flow cytometry. UB-VV100 represents the drug product.

FIG. 31A shows a study timeline for an illustrative lentiviral vector dose-finding study.

Figure 31B shows B cells/ul blood. Dose-dependent antigen-specific VV100 activity was observed, and there was no B cell depletion in the FITC RACR group. On day 25, B cell depletion leveled off, and on day 26, rapamycin treatment was initiated for all groups except the vehicle group. Raji cells were implanted SC on day 40.

Figure 31C shows CAR T cells/ul blood. An increase in CAR T cells/ul was observed in the 10E6 VV100 treatment group, and CAR T cells were detectable in the 2E06 VV100 treatment group after rapamycin treatment.

Figures 32A and 32B show tumor growth measured by caliper (A) using the formula (W^2×L)/2, and tumor CAR T cells analyzed by flow cytometry (B). The inhibition of tumor growth was dose-dependent; no inhibition of RAJI tumor growth was observed in mice treated with FITCRACR. The frequency of CAR T cells in tumors from mice treated with 10E ⁶ VV100 was higher.

Figure 33A shows cytometric analysis of CAR T cells in the bone marrow (left) and spleen (right) at day 81. Higher doses of UB-VV100 maintained partial B cell depletion in the bone marrow and spleen 81 days after dosing.

Figure 33B shows cytometric analysis of B cells in the bone marrow (left) and spleen (right) at day 81. Higher doses of UB-VV100 maintained partial B cell depletion in the bone marrow and spleen 81 days after dosing.

Figure 33C shows analysis of transgenic integration events normalized to DNA in blood, bone marrow, liver, ovaries, and kidneys. Error bars represent +/-1 SEM. Horizontal bars represent median values. Dashed line represents 50 vector copies/ug DNA as the FDA guidance detection threshold.

FIG. 34A shows a study timeline for an illustrative lentiviral vector in vivo efficacy study using the Nalm-6 tumor model.

FIG. 34B shows the body weight changes of different groups in the Nalm-6 efficacy study.

Figure 35A shows the total flux (p/s) of different groups in the Nalm-6 efficacy study throughout the study as an indication of tumor burden. VV100 treatment significantly reduced tumor burden measured by total flux, and by the 20th day of the study, all mice in the vehicle group died of Nalm-6 tumors, and the survival of mice treated with VV100 was extended to the 41st day of the study.

Figure 35B shows the survival curves of different groups in the Nalm-6 efficacy study throughout the study period. VV100 treatment significantly improved the survival rate, and by the 20th day of the study, all mice in the vehicle group died of Nalm-6 tumors, and the survival period of mice treated with VV100 was extended to the 41st day of the study.

Figure 36 shows the total flux data of each individual mouse in the Nalm-6 group during the entire study. The disease load of mice in the vehicle (upper left) group and the vehicle + rapamycin (upper right) group began to rise on the 10th day of the study. By the 17th day, the mice in these groups must be killed. The disease load of the mice in the VV100 group (lower left) began to decrease on the 17th day, but the impact of VV100 in this group was temporary. The disease load of all mice in the VV100 + rapamycin group (lower right) began to significantly decrease on the 17th day, and two mice maintained low disease load. The tumor load of only one mouse from this group increased after initial regression.

Figure 37 shows the bioluminescent imaging of the Nalm-6 group with the total flux heat map superimposed. From left to right: mice in the vehicle group succumbed to Nalm-6 disease on day 17, mice in the vehicle + rapamycin group succumbed to disease by day 20, disease burden in most mice in the VV100 treatment group was temporarily reduced, disease burden in mice in the VV100 + rapamycin group was significantly reduced, disease burden in most mice remained low to undetectable, and tumor burden in one mouse was partially reduced and then increased.

Figure 38A shows the average CAR T cells/ul blood. Detectable circulating CAR T cells in mice treated with UB-VV100 alone peaked at day 17, and overall levels in the blood were low. In the UB-VV100+rapamycin group, circulating CAR T cells increased over time and were significantly higher than those in the UB-VV100 alone group. CAR T cells in the peripheral blood of mouse 10 expanded significantly, increasing from 228 CAR T cells/ul at D38 to 3397.69 on day 41.

Figure 38B shows the CAR T cells/ul blood for each individual mouse. The detectable circulating CAR T cells of mice treated with UB-VV100 alone peaked at day 17, and the overall level in the blood was low. In the UB-VV100+rapamycin group, the circulating CAR T cells increased over time and were significantly higher than the cells in the UB-VV100 group alone. The CAR T cells in the peripheral blood of mouse 10 expanded significantly, from 228 CAR T cells/ul at D38 to 3397.69 on day 41.

Figure 39A shows the CAR T cell frequency of total immune cells in the bone marrow and spleen. At day 41, the CAR T cell frequency in the total immune cell population was higher in the VV100+rapamycin treatment group in both tissues, and was higher in the bone marrow than in the spleen.

Figure 39B shows the T cell frequency of human T cells in the bone marrow and spleen. On day 41, the frequency of CAR T cells in the T cell population in the bone marrow and spleen was significantly higher in the VV100+rapamycin treatment group than in the VV100 treatment group.

FIG. 40A shows CD25 activation of PBMCs by flow cytometry.

Figure 40B shows the transduction efficiency of PBMCs for CAR expression by flow cytometry.

FIG. 41A shows a flow cytometry panel of CART cells in PBMCs transduced with UB-V100 and cultured with or without 10 nM rapamycin.

Figure 41B shows a graph of T cell yield and percentage in PBMCs transduced with UB-V100 and cultured with or without 10 nM rapamycin. The summary graph combines data from 3 PBMC donors, and the error bars represent +1 SEM. **, ***, and *** represent p values of <0.01, 0.001, and 0.0001, 2-way ANOVA multiple comparisons of rapamycin treatment over time.

FIG. 42A shows flow cytometry and statistical analysis of intracellular staining of INFγ in CD8+ CAR T cells generated by UB-VV100 transduction.

FIG42B shows flow cytometry and statistical analysis of surface CD107a expression in CD8+ CAR T cells generated by UB-VV100 transduction.

FIG. 42C shows statistical analysis of Raji cell survival in the presence of PBMCs transduced with UB-VV100.

FIG. 43A shows a flow cytometry panel of PBMC samples from a B-ALL patient at the time of UB-VV100 transduction.

FIG. 43B shows a flow cytometry panel of PBMC samples from a B-ALL patient 7 days after UB-VV100 transduction.

FIG. 43C shows a flow cytometry panel of PBMC samples from a B-ALL patient 7 days after UB-VV100 transduction.

44A-44D show flow cytometry panels of PBMC samples from DLBCL patients upon UB-VV100 transduction.

Figures 45A and 45B show the circulating B cell levels of CD34-NSG mice. Human B cell levels (hCD20+ populations gated for single cells, live cells, and hCD45+ cells) were measured in the blood of CD34-NSG mice (Groups 7-10) using a flow cytometry human immune cell antibody panel. Data are shown as (A) human B cell counts (quantitative number of hCD20+ cells normalized to blood volume using counting beads) or (B) human B cell frequency (% of hCD20+ relative to hCD45+ cell populations). Data are mean ± SEM. The number of animals in each group was considered according to the study design table for an overview of sacrifice and unplanned deaths (n = 2/vehicle group/time point; n = 4/UB-VV100 group/time point). Statistics were determined using a two-way ANOVA main effect column test using Tukey's adjustment; ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. CAR=chimeric antigen receptor; h=human.

Figure 46A and 46B show the CAR-T cell levels in the blood of CD34-NSG mice. CAR-T cell levels (CAR+ populations gated for single cells, living cells, hCD45+ cells and hCD3+ cells) are measured in the blood of CD34-NSG mice (Groups 7-10) using a flow cytometry human immune cell antibody panel containing antibodies in the FMC63 region of the targeting αCD19CAR construct. Data are shown as (A) CAR-T cell frequency (CAR+% relative to hCD3+ cell colonies) or (B) the absolute number of CAR-T cells detected using counting beads normalized relative to the volume of blood. Data are mean ± SEM. The number of animals in each group considers the summary of execution and unplanned death according to the research design table (n=2/vehicle group/time point; n=4/UB-VV100 group/time point). Statistics were determined using two-way ANOVA main effects column test with Tukey's adjustment; ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. CAR = chimeric antigen receptor; h = human.

Figure 46C shows the transduction of mouse immune cells in the spleen of CD34-NSG mice.After the planned autopsy in the 4th week, the levels of CAR+ mouse immune cells (CAR+ populations gated for single cells, live cells and mCD45+ cells) were measured in the spleen (Group 7, Group 8, Group 10). CAR+ cell detection uses a flow cytometry human immune cell antibody panel containing antibodies targeting the FMC63 region of αCD19 CAR constructs. The bar is the mean ± SEM, and the midpoint represents a single mouse. For the vehicle+rapamycin group, n=2, and for the low-dose+rapamycin group and the high-dose+rapamycin group, n=4. Using one-way ANOVA, multiple comparisons were performed using Tukey adjustment to determine statistics; **=p<0.01, ***=p<0.001, ****=p<0.0001. CAR=chimeric antigen receptor, m=mouse.

Figure 46D shows the biodistribution of UB-VV100 in CD34-NSG mice. At planned autopsies 1 week or 4 weeks after treatment (Groups 7-10), ddPCR was used to measure the copies of the vector genomes integrated by UB-VV100 from the bone marrow, spleen, liver, heart, lung, kidney, brain and gonads of CD34-NSG mice. The data are shown as the copies of the amplified region (ssCD19) per ug genomic DNA. The data bars are mean ± SEM, where each point represents a single mouse. For the vehicle + rapamycin group (Group 7), n = 2, for the low dose + rapamycin group (Group 8) and the high dose + rapamycin group (Group 10), n = 4, and for the low dose - rapamycin group (Group 9), n = 3. The data of the gonads of Weeks 1 and 4 were not collected.

Figure 47 shows multiple RNA ISH staining of liver and spleen. Representative images of liver and spleen from CD34-NSG mice treated with high doses of UB-VV100 and rapamycin (TOX001_48). DAPI is shown in white, custom probes identifying the RACR sequence of UB-VV100 are shown in yellow, human CD3 RNA ISH probe pools are shown in green, mouse CD68 RNA ISH probes are shown in red, and mouse Pecam RNA ISH probes are shown in blue. Human CAR-T cells are indicated with white arrows.

Figure 48 shows that the surface CD19 detection of CAR+Nalm6 cells is reduced, but the intracellular CD19 protein level is the same as that of untransduced Nalm6 cells.Nalm6 cells are transduced with VV100 at MOI 1, 10 and 20.On the 10th day, CAR+Nalm6 cells are stained with (A) anti-CD19 antibodies (clone HIB19) to assess surface CD19 levels; and (B) are stained with anti-CD19 antibodies (clone EPR5906) bound to intracellular CD19 epitopes to determine overall CD19 protein levels.The orange curve represents the data of CAR+Nalm6 cells from gated CAR and P2A positive cells.The gray curve represents the Nalm6 cells of CD19+Nalm6 parental cells or CD19 knockouts that are not transduced.

Figure 49 shows that anti-CD19 CAR-T cells can kill CAR+Nalm6 cells in vitro. Transduced Nalm6GFP cells (VV100, MOI 10) and transduced PBMCs (VV100, MOI 5) from 3 healthy donors are co-cultured with different CAR-T and Nalm6 ratios. In order to normalize background nonspecific killing, transduced Nalm6 cells are also co-cultured with PBMCs transduced by analogs. The PBMCs transduced by analogs are treated with "empty" viral particles that make anti-CD3-scFv displayed on the surface but do not carry transgenic payloads. After 24 hours, transduced Nalm6 cells are gated as CAR+Nalm6 cells by flow cytometry based on intracellular transgene expression. The percentage of lysis is calculated based on the frequency of dead CAR+Nalm6 cells normalized by co-culture wells of PBMCs transduced with respect to analogs.

Figure 50 shows a schematic diagram of VV100 transduction in a high tumor burden model. In this high tumor burden model, 5e5 Nalm6 GFP and 5e5 PBMCs were mixed and transduced with VV100 at an MOI of 5.

Figure 51 shows that the mixed colony of Nalm6 cells and PBMC transduced with VV100 produces CAR+Nalm6 cells eliminated by anti-CD19CAR-T cells.At the 0th day, 5e5 Nalm6 GFP cells and 5e5 PBMC cells from healthy donors were mixed, and VV100 or anti-FITC CAR were transduced with MOI 5 or kept not transduced.A total of eight healthy donors were evaluated.After transduction, the frequency of (A) Nalm6 cells expressing CAR transgenes, (B) the total number of CAR+Nalm6 cells, (C) the frequency of Nalm6GFP cells in culture, and (D) the total number of living Nalm6 GFP cells were determined by flow cytometry.Each line represents the result from a single healthy donor.

Figure 52 shows that the anti-CD19 CAR-T cells that can eliminate CAR+Nalm6 cells are produced by mixing the population of Nalm6 cells and PBMCs transduced with VV100. On the 0th day, 5e5 Nalm6GFP cells and 5e5 PBMC cells from healthy donors were mixed and transduced with MOI 5 with VV100 or anti-FITC CAR or kept untransduced. A total of eight healthy donors were evaluated. After transduction, the frequency of CD3+T cells expressing CAR transgenes (A) and the total number of (B) αCD3 CAR-T cells were determined by flow cytometry. Each line represents the results from a single healthy donor.

FIG. 53 shows a study timeline for an illustrative lentiviral vector in vivo efficacy study.

Figure 54 shows a study of the survival of animals treated with UB-VV100. Vehicle (N=5), 142 (SEQ ID NO: 121) adherent 2E6 (N=5), 201 (SEQ ID NO: 122) adherent 2E6 (N=6), VPN38 20E6 (N=6), VPN38100E6 (N=6), VPN68 20E6 (N=6), VPN68 100E6 (N=6).

Figure 55 shows the T cell phenotype 3 days after UB-VV100 administration. Peripheral blood was collected from mice and analyzed by flow cytometry to determine CD71 expression. The bar graph represents the median value. *, *, **, and **** represent p values of <0.05, <0.01, and <0.0001, one-way ANOVA Tukey post-hoc comparison test.

Figures 56A and 56B show circulating CAR T cells of mice treated with UB-VV100. Blood was collected from animals during continuous weekly blood sampling. CAR T cells were enumerated by flow cytometry. Error bars are equal to +/-1SD. Vehicle (N=5), 142 (SEQ ID NO: 121) adherent 20E6 (N=5), 201 (SEQ ID NO: 122) adherent 2E6 (N=6), VPN38 20E6 (N=6), VPN38 100E6 (N=6), VPN68 20E6 (N=6), VPN68100E6 (N=6).

Figure 57 represents the average tumor burden during the entire study. All mice were monitored every two weeks by bioluminescent imaging to assess NALM-6 progression. Error bars are equal to +/-1SD. Vehicle (N=5), 142 (SEQ ID NO: 121) adherent 20E6 (N=5), 201 (SEQ ID NO: 122) adherent 2E6 (N=6), VPN38 20E6 (N=6), VPN38 100E6 (N=6), VPN68 20E6 (N=6), VPN68 100E6 (N=6). These mice received NALM=6 cells by retro-orbital injection to cause eye tumors.

Figure 58 A shows the percentage of NALM-6GFP ffLUC tumor cells present in the bone marrow at the time of sacrifice. When reaching the humane endpoint, mice were sacrificed, and their bone marrow was collected and processed for flow cytometric analysis to observe the frequency of GFP+/CD45-/living cells.

Figure 58 B shows the percentage of NALM-6GFP ffLUC tumor cells present in the spleen at the time of sacrifice. When reaching the humane endpoint, mice were sacrificed, and their spleens were collected and processed for flow cytometry analysis to observe the frequency of GFP+/CD45-/living cells.

FIG. 59 shows illustrative αCD3 scFv constructs of lentiviral surface expression plasmids of the present disclosure.

Figure 60 shows the percentage of αCD3 scFv expression in lentiviral particles with various illustrative αCD3 scFv surface constructs. 293T cells were transfected with five plasmid systems with different surface plasmid constructs. αCD3 scFv expression by production 293T cells was analyzed by flow cytometry using anti-teplizumab antibodies. Virus was harvested and used to transduce Supt1 cells, and the titer of the cells was analyzed by flow cytometry.

FIG. 61 shows the titer of Supt1 cells transduced with lentivirus including various illustrative αCD3 scFv surface constructs.

62 is a graph depicting relative light units (RLU) as a function of MOI in a T cell activation bioassay (NFAT) bioluminescent cell based assay, showing T cell activation.

Figure 63 is a graph depicting the correlation of αCD3 scFv expression with T cell activation by NFAT reporter cell assay (RLU (MOI 2)).

Figure 64 shows representative flow cytometry images of cells expressing various illustrative αCD3 scFv surface constructs and stained to visualize the αCD3 scFv.

Figure 65 shows the biodistribution of αCD3-Cocal-GFP in canine blood samples collected 24 hours after administration and before autopsy. Before autopsy: for Group 1, Group 2, Group 3, the 8th day, and for Group 4, the 29th day. Each symbol represents a single individual. The results are extrapolated to represent the number of copies of each μg of total canine DNA of the tissue sample. The dotted line represents 2 parameters of the qPCR detection as a part of the validation: the lower limit of quantification (LLOQ) of 50 copies/μg DNA (10 copies/200ngDNA) after extrapolation, and the lower limit of detection (LOD) of 25 copies/μg DNA (5 copies/200ngDNA) after extrapolation. In order to visualize the negative samples on a logarithmic scale, the samples that give undetectable results are assigned a value of 1 to be graphically represented, but the samples are still undetectable in this study.

Figure 66 shows the biodistribution of αCD3-Cocal-GFP in canine tissue.Each symbol represents a single individual.The result is extrapolated to represent the copy number of each μg total canine DNA of tissue sample.The dotted line represents 2 parameters detected by qPCR as a part of verification: the lower limit of quantification (LLOQ) of 50 copies/μg DNA (10 copies/200ng DNA) after extrapolation, and the lower limit of detection (LOD) of 25 copies/μg DNA (5 copies/200ng DNA) after extrapolation.The sample below the limit of quantification (BLQ) gives a value of 37 for graphical representation, which is the value between the LLOQ and LOD after extrapolation.In order to visualize the negative sample on a logarithmic scale, the sample giving an undetectable result is given a value of 1, to be graphically represented, but the sample is still undetectable in this study.

DETAILED DESCRIPTION

The present disclosure generally relates to a viral particle comprising a vector genome comprising a polynucleotide sequence encoding an anti-CD19 chimeric antigen receptor, wherein the viral particle transduces immune cells in vivo.

The methods and compositions described herein can facilitate administration of the viral particles directly to a subject in need of treatment.

In vitro and in vivo studies have shown that the methods and compositions described herein are able to activate and transduce unstimulated T cells that then express the αCD19 CAR, respond to rapamycin treatment with enhanced proliferation, and kill CD19-expressing B cell malignancy models.

The present disclosure provides a method for treating a disease or disorder of a subject in need, transducing immune cells in vivo and/or generating immune cells expressing anti-CD19 chimeric antigen receptors, the method comprising administering the viral particles of the present disclosure to the subject. In some embodiments, the method further comprises administering rapamycin to the subject. In some embodiments, the method of the present disclosure eliminates the need for pre-activating immune cells before administering viral particles. In some embodiments, the method includes not pre-activating immune cells in the subject before administering viral particles (e.g., within about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days before administering viral particles, or within about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks without pre-activation). In some embodiments, pre-activating immune cells includes CD3 and/or CD28 signaling in activated immune cells (e.g., T cells), optionally by administering anti-CD3 and/or anti-CD28 antibodies, respectively. Thus, in some embodiments, the methods of the present disclosure do not comprise administering a separate CD3 and/or CD28 activating agent prior to administering the viral particles.

In vivo delivery of polynucleotides

In some embodiments, a polynucleotide encoding a chimeric antigen receptor (CAR) is administered to a subject, allowing CAR to be produced in vivo. In some embodiments, the administration of such polynucleotides produces an effect similar to the direct administration of CAR in vivo. In some embodiments, the administration of such polynucleotides improves the in vivo transduction efficiency of the particles. In some embodiments, the polynucleotides are mRNA.

In some embodiments, the in vivo delivery of such polynucleotides produces 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 polypeptides causes the desired pharmacokinetics, pharmacodynamics, and/or safety profile of the encoded CAR. In some embodiments, the polynucleotides can be optimized in one or more ways to prevent immune activation, increase stability, reduce any tendency to aggregate, such as the tendency to aggregate over time, and/or avoid impurities. This optimization may 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, "Nature Medicine (Nat.Med.)". Such modifications are known in the field.

Strategies for delivering polynucleotides (e.g., mRNA) in vivo are known in the art. For a summary of strategies, see Mol. Ther. April 10, 2019; 27(4): 710-728, which is incorporated herein by reference in its entirety.

In some embodiments, the viral particles of the present disclosure can transduce T cells in vivo to express anti-CD19 CAR and target tumor cells expressing CD19.

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

(a) The virus particles bind to T cells in vivo through anti-CD3 scFv, activate T cells, and promote internalization of virus particles by interacting with Cocal glycoprotein

(b) The vector RNA genome, i.e., αCD19 CAR-FRB-RACR, is reverse transcribed into DNA, shuttled to the cell nucleus, and integrated into the genome

(c) Transduced T cells express anti-CD19 CAR and target cells expressing CD19, while also expressing the FRB and RACR systems for rapamycin-controlled cytokine signaling.

Route of administration

In some embodiments, the viral particles are administered by a route selected from the group consisting of: parenteral, intravenous, intramuscular, subcutaneous, intratumoral, intraperitoneal, and intralymphatic. In some embodiments, the viral particles are administered multiple times. In some embodiments, the viral particles are administered by intralymphatic injection of 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 intranodal injection-that is, the viral particles can be administered by injection into lymph nodes, such as inguinal lymph nodes. In some embodiments, the viral particles are administered by injecting the viral particles into the tumor site (i.e., into the tumor). In some embodiments, the viral particles are administered subcutaneously. In some embodiments, the viral particles are administered systemically. In some embodiments, the viral particles are administered intravenously. In some embodiments, the viral particles are administered intra-arterially. In some embodiments, the viral particles are lentiviral particles.

In some embodiments, the viral particles are administered by intraperitoneal, subcutaneous, or intranodal 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 intranodal injection.

In some embodiments, transduced immune cells comprising the polynucleotides of the present disclosure are administered to the subject.

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

Virus particles

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.

In some embodiments, the viral particles as described herein are retroviral particles. In some embodiments, the viral particles are lentiviral particles. In some embodiments, the viral particles are adeno-associated viral particles.

The term viral particle refers to a macromolecular complex capable of transferring nucleic acids 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 portions thereof. The term "lentiviral vector" refers to a viral vector containing structural and functional genetic elements primarily derived from a lentivirus, or portions thereof, comprising LTRs. The term "hybrid" refers to a vector, LTR or other nucleic acid containing both a retrovirus, e.g., a lentiviral sequence and a non-lentiviral sequence. In some embodiments, a hybrid vector refers to a vector or transfer plasmid comprising a retrovirus, e.g., a lentiviral sequence, for reverse transcription, replication, integration and/or packaging.

In some embodiments, the lentiviral particles of the present disclosure are replication-incompetent self-inactivating (SIN) lentiviral vector (LVV) particles, comprising:

- A surface engineered viral envelope comprising and expressing a membrane-bound anti-CD3 single chain variable fragment (scFv) and Cocal glycoprotein.

- A transgene encoding:

2nd generation anti-CD19 chimeric antigen receptor (CAR), the antibody includes the binding domains FMC63 and 4-1BB and CD3ζ signaling domains;

Inducible T-cell proliferation signaling system (rapamycin-activated cytokine receptor, RACR); and

A human protein domain (FRB) derived from the mammalian target of rapamycin (mTOR) complex that binds intracellular rapamycin to confer rapamycin resistance to transduced cells.

Retroviral particles

Retroviruses include lentiviruses, gamma-retroviruses, and alpha-retroviruses, each of which can be used to deliver polynucleotides to cells using methods known in the art. Lentiviruses are complex retroviruses that contain other genes with regulatory or structural functions in addition to the common retroviral genes gag, pol, and env. The higher complexity enables the virus to regulate its life cycle, just as it does during latent infection. Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); visna-maedi virus (VMV) virus; caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). In some embodiments, the backbone is an HIV-based vector backbone (i.e., HIV cis-acting sequence elements). Retroviral particles are produced by multiple attenuation of HIV virulence genes, for example, deletion of the genes env, vif, vpr, vpu and nef, thereby rendering the vector biologically safe.

Illustrative 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 to Winter; and U.S. Pat. No. 5,994,136, each of which is incorporated herein by reference in its entirety. In general, these particles are configured to carry the necessary sequences for selecting cells containing the particles, for incorporating foreign nucleic acids into the lentiviral particles, and for transferring nucleic acids into target cells.

A commonly used lentiviral particle system is the so-called third generation system. The third generation lentiviral particle system comprises four plasmids. "Transfer plasmid" encodes a polynucleotide sequence delivered to the target cell by a lentiviral vector system. The transfer plasmid usually has one or more transgenic sequences of interest flanked by long terminal repeats (LTR), which facilitates the integration of the transfer plasmid sequence into the host genome. For safety reasons, the transfer plasmid is usually designed to make the particles produced non-replicable. For example, the transfer plasmid lacks the genetic elements necessary for producing infectious particles in the host cell. In addition, the transfer plasmid can be designed with a deletion of 3'LTR, so that the virus becomes "self-inactivating" (SIN). See Dull et al. (1998), Journal of Virology 72: 8463-71; Miyoshi et al. (1998), Journal of Virology 72: 8150-57. Viral particles can also include 3' untranslated regions (UTR) and 5' untranslated regions. The UTRs include retroviral regulatory elements that support packaging, reverse transcription, and integration of the proviral genome into cells following exposure of the cells by retroviral particles.

The third generation system usually also includes two "packaging plasmids" and one "envelope plasmid". The "envelope plasmid" usually encodes the Env gene operably connected to the promoter. In an exemplary third generation system, the Env gene is VSV-G, and the promoter is the CMV promoter. In an exemplary third generation system, the Env gene is Cocal G protein (Cocal glycoprotein), and the promoter is the promoter of MND (myeloproliferative sarcoma virus enhancer, negative control region deleted, d1587rev primer binding site replaced). In an exemplary third generation system, the Env gene is Cocal G protein (Cocal glycoprotein), and the promoter is the CMV promoter. The third generation system uses two packaging plasmids, one packaging plasmid encodes gag and pol, and the other packaging plasmid encodes rev as an additional safety feature, which is an improvement on the single packaging plasmid of the so-called second generation system. Although safer, the third generation system may be more cumbersome to use and has a lower viral titer 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.

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

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

The in vitro efficiency of retroviral or lentiviral particle systems can be evaluated in various ways 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 units of infection per milliliter (IU/mL). For example, titer can be evaluated using a functional assay performed on the cultured tumor cell line HT1080 as described below: Humbert et al. Development of Third-generation Cocal Envelope Producer Cell Lines for Robust Retroviral Gene Transfer into Hematopoietic Stem Cells and T-cells for good transfer of retroviral genes into hematopoietic stem cells and T-cells. Molecular Therapy 24: 1237–1246 (2016). When evaluating the titer of a cultured cell line that continues to divide, no stimulation is required, and therefore the titer measured is not affected by the surface engineering of retroviral particles. Other methods for evaluating the efficiency of retroviral vector systems are provided in Gaererts et al. Comparison of retroviral vector titration methods. BMC Biotechnol. 6:34 (2006).

In some embodiments, the retroviral particles and/or lentiviral particles of the present disclosure include 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. Illustrative 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.

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.

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.

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.

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

Virus envelope

In some embodiments, the retroviral particles include cell surface receptors that bind to ligands on target host cells, thereby allowing host cell transduction. The viral particles may include heterologous viral envelope glycoproteins that produce pseudotyped viral particles. For example, the viral envelope glycoprotein may be derived from RD114 or one of its variants, VSV-G, i.e., gibbon ape leukemia virus (GALV), or an amphotropic envelope, measles envelope, or baboon retrovirus envelope glycoprotein. In some embodiments, the viral envelope glycoprotein is a VSV G protein derived from a Cocal strain (Cocal glycoprotein) or a functional variant thereof.

In some embodiments, the viral envelope glycoprotein is a VSV G protein from a Cocal strain (Cocal glycoprotein), a Cocal envelope variant containing an R354Q mutation, which may be referred to as a "blinded" Cocal envelope. Illustrative Cocal envelope variants are provided, for example, in US2020/0216502 A1, which is incorporated herein by reference in its entirety.

In some embodiments, the viral particle includes a polypeptide comprising a Cocal glycoprotein that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:5.

Cocal Env:

NFLLLTFIVLPLCSHAKFSIVFPQSQKGNWKNVPSSYHYCPSSSDQNWHNDLLGITMKVKMPKTHKAIQADGWMCHAAKWITTCDFRWYGPKYITHSIHSIQPTSEQCKESIKQTKQGTWMSPGFPPQNCGYATVTDSVAVVVQATPHHVLVDEYTGEWIDSQFPNGKCETEECETVHNSTVWYSDYKVTGLCDATLVD TEITFFSEDGKKESIGKPNTGYRSNYFAYEKGDKVCKMNYCKHAGVRLPSGVWFEFVD QDVYAAAKLPECPVGATISAPTQTSVDVSLILDVERILDYSLCQETWSKIRSKQPVSPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRIDIDNPIISKMVGKISGSQTERELWTEWFPYEGVEIGPNGILKTPTGYKFPLFMIGHGMLDSDLHKTSQAEVFEHPHLAEAPKQLPEEETLFFGDTGISKNPVELIEGWFSSWKSTVVTFFFAIGVFILLYV VARIVIAVRYRYQGSNNKRIYNDIEMSRFRK (SEQ ID NO: 5).

In some embodiments, the viral particle comprises a nucleic acid sequence encoding a Cocal glycoprotein that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:10.

Cocal Env:

AATTTTCTGCTGCTGACCTTCATCGTGCTGCCTCTGTGCAGCCACGCCAAGTTTTCCATCGTGTTCCCACAGTCCCAGAAGGGCAACTGGAAGAATGTGCCCTCTAGCTACCACTATTGCCCTTCCTCTAGCGACCAGAACTGGCACAATGATCTGCTGGGCATCACAATGAAGGTGAAGATGCCCAAGACCCACAAGGCCATCCAGGCAGATGGATGGATGTGCCACGCAGCCAAGTGGATCACAACCTGTGACTTTCGGT GGTACGGCCCCAAGTATATCACACACTCCATCCACTCTATCCAGCCTACCTCCGAGCAGTGCAAGGAGTCTATCAAGCAGACAAAGCAGGGCACCTGGATGAGCCCTGGCTTCCCACCCCAG AACTGTGGCTACGCCACAGTGACCGACTCCGTGGCAGTGGTTGGTGCAGGCAACACCTCACCACGTGCTGGTGGATGAGTATACCGGCGAGTGGATCGACAGCCAGTTTCCAAACGGCAAGTGCGAGACAGAGGAGTGTGAGACCGTGCACAATTCTACAGTGTGGTACAGCGATTATAAGGTGACAGGCCTGTGCGACGCCACCCTGGTGGATACAGAGATCACCTTCTTTTCTGAGGACGGCAAGAAGGAGAG CATCGGCAAGCCCAACACCGGCTACAGATCCAATTACTTCGCCTATGAGAAGGGCGATAAGGTGTGCAAGATGAATTATTGTAAGCACGCCGGGGTGCGGCTGCCTAGCGGCGTGTGGTTTGAGTTCGTG GACCAGGACGTGTACGCAGCAGCAAAGCTGCCTGAGTGCCCAGTGGGAGCAACCATCTCCGCCCCAACACAGACCTCCGTGGACGTGTCTCTGATCCTGGATGTGGAGCGCATCCTGGACTACAGCCTGTGCCAGGAGACCTGGAGCAAGATCCGGTCCAAGCAGCCCGTGTCCCCTGTGGACCTGTCTTACCTGGCACCAAAGAACCCAGGAACCGGACCAGCCTTTACAATCATCAATGGCACCCTGAAGTACTTCGA GACCCGCTATATCCGGATCGACATCGATAACCCTATCATCAGCAAGATGGTGGGCAAGATCTCTGGCAGCCAGACAGAGAGAGCTGTGGACCGAGTGGTTCCCTTACGAGGGCGTGGAGATC GGCCCAAATGGCATCCTGAAGACACCAACCGGCTATAAGTTTCCCCTGTTCATGATCGGCCACGGCATGCTGGACAGCGATCTGCACAAGACCTCCCAGGCCGAGGTTTGAGCACCCACCTGGCAGAGGCACCAAAGCAGCTGCCTGAGGAGGAGACACTGTTCTTGGCGATACCGGCATCTCTAAGAACCCCGTGGAGCTGATCGAGGGCTGGTTTTCCTTGGAAGAGCACAGTGGTGACCTTTCTT TTTCGCCATCGGCGTGTTCATCCTGCTGTACGTGGTGGCCAGAATCGTGATCGCCGTGAGATACAGGTATCAGGGCTCCAACAATAAGAGGATCTATAATGACATCGAGATGTCTCGCTTCCGGAAG (SEQ ID NO: 10).

In some embodiments, the viral particle includes a nucleic acid sequence encoding a Cocal glycoprotein that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:104.

Cocal Env:

ATGAACTTTCTGCTGCTGACCTTCATCGTGCTGCCTCTGTGCAGCCACGCCAAGTTT

TCCATCGTGTTCCCACAGTCCCAGAAGGGCAACTGGAAGAATGTGCCCAGCTCCTA

CCACTATTGTCCTTCTAGCTCCGACCAGAACTGGCACAATGATCTGCTGGGCATCAC

CATGAAGGTGAAGATGCCTAAGACACACAAGGCCATCCAGGCAGATGGATGGATGT

GCCACGCAGCCAAGTGGATCACCACATGTGACTTTCGGTGGTACGGCCCCAAGTAT

ATCACCCACAGCATCCACTCCATCCAGCCTACAAGCGAGCAGTGCAAGGAGTCCAT

CAAGCAGACCAAGCAGGGCACATGGATGTCTCCCGGCTTCCCCCCTCAGAACTGTG

GCTACGCCACCGTGACAGATAGCGTGGCAGTGGGTGGTGCAGGCAACCCCACACCAC

GTGCTGGTGGATGAGTATACAGGCGAGTGGATCGACAGCCAGTTTCCCAACGGCAA

GTGCGAGACCGAGGAGTGTGAGACAGTGCACAATTCTACCGTGTGGTACAGCGATT

ATAAGGTGACCGGCCTGTGCGACGCCACACTGGTGGATACCGAGATCACATTCTTTT

CCGAGGACGGCAAGAAGGAGTCTATCGGCAAGCCCAACACCGGCTACAGGTCTAAT

TACTTCGCCTATGAGAAGGGCGATAAGGTGTGCAAGATGAATTATTGTAAGCACGCC

GGGGTGCGGCTGCCAAGCGGCGTGTGGTTTGAGTTCGTGGACCAGGACGTGTACGC

AGCAGCAAAGCTGCCAGAGTGCCCAGTGGGAGCAACCATCAGCGCCCCCACCCAG

ACATCTGTGGACGTGAGCCTGATCCTGGATGTGGAGAGAATCCTGGACTACTCCCTG

TGCCAGGAGACATGGTCCAAGATCCGCTCTAAGCAGCCCGTGAGCCCAGTGGACCT

GTCTTACCTGGCACCAAAGAACCCTGGAACAGGACCTGCCTTTACCATCATCAATGG

CACACTGAAGTACTTCGAGACCCGGTATATCAGAATCGACATCGATAACCCAATCAT

CTCCAAGATGGTGGGCAAGATCTCCGGCTCTCAGACCGAGAGAGAGCTGTGGACA

GAGTGGTTCCCATACGAGGGCGTGGAGATCGGCCCCAATGGCATCCTGAAGACCCC

TACAGGCTATAAGTTTCCACTGTTCATGATCGGCCACGGCATGCTGGACTCTGATCT

GCACAAGACCAGCCAGGCCGAGGTGTTTGAGCACCCACACCTGGCAGAGGCACCA

AAGCAGCTGCCCGAGGAGGAGACCCTGTTCTTTGGCGATACAGGCATCTCCAAGAA

CCCTGTGGAGCTGATCGAGGGCTGGTTTTCTAGCTGGAAGTCTACCGTGGTGACATT

CTTTTTCGCCATCGGCGTGTTCATCCTGCTGTACGTGGTGGCAAGGATCGTGATCGC

CGTGCGGTACAGATATCAGGGCAGCAACAAATAAGAGAATCTATAATGACATCGAGATGTCCAGGTTCCGCAAGTGA(SEQ ID NO:104)

In some embodiments, the viral particle comprises a polynucleotide comprising a CD8-derived signal peptide sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:1.

CD8 signal peptide: MALPVTALLLPLALLLHAARP (SEQ ID NO: 1).

In some embodiments, the viral particle comprises a nucleic acid sequence encoding a CD8-derived signal peptide that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:6.

CD8 signal peptide:

ATGGCACTGCCTGTGACAGCCCTGCTGCTGCCACTGGCCCTGCTGCTGCACGCAGCACGCCCA (SEQ ID NO: 6).

In some embodiments, the cell surface receptor is an anti-CD3 single chain variable fragment or a functional variant thereof.

Various fusion glycoproteins can be used to pseudotype lentiviral particles. Although the most commonly used example is the envelope glycoprotein from vesicular stomatitis virus (VSV-G), many other viral proteins are also used to pseudotype lentiviral particles. See Joglekar 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 make viral particles more efficient.

In some embodiments, pseudotyping of the fusion glycoprotein or a functional variant thereof facilitates targeted transduction of specific cell types, including but not limited to T cells or NK cells. In some embodiments, the fusion glycoprotein or a functional variant thereof is human immunodeficiency virus (HIV) gp160, murine leukemia virus (MLV) gp70, gibbon ape leukemia virus (GALV) gp70, feline leukemia virus (RD114) gp70, amphotropic retrovirus (Ampho) gp70, 10A1 MLV (10A1) gp70, ecotropic retrovirus (Eco) gp70, baboon 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), lymphocytic choriomeningitis virus (LCMV) GP1 and GP2, baculovirus GP64, Chikungunya virus ( 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 full-length polypeptides, functional fragments, homologs or functional variants of measles disease Paravirus CNV-G and PRV-G.

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 Vesicular Stomatitis Alagoas Virus (VSAV), Carajas Vesiculovirus (CJSV), Chandipura Vesiculovirus (CHPV), Cocal Vesiculovirus (COCV), Indiana Vesiculovirus (VSIV), Isfahan Vesiculovirus (ISFV), Maraba Vesiculovirus (MARAV), Vesicular Stomatitis New Jersey virus (VSNJV), Bas-Congo Virus (BASV). In some embodiments, the fusion glycoprotein or its functional variant is Cocal virus G protein.

In some embodiments, the viral particles are Nipah virus (NiV) envelope pseudotyped lentiviral particles ("Nipah envelope pseudotyped vectors"). In some embodiments, Nipah envelope pseudotyped vectors are pseudotyped using Nipah virus envelope glycoproteins NiV-F and NiV-G. In some embodiments, the NiV-F and/or NiV-G glycoproteins on such Nipah envelope pseudotyped vectors are modified variants. In some embodiments, the NiV-F and/or NiV-G glycoproteins on such Nipah envelope pseudotyped vectors are modified to include antigen binding domains. In some embodiments, the antigen is EpCAM, CD4, or CD8. In some embodiments, Nipah envelope pseudotyped vectors can efficiently transduce cells expressing EpCAM, CD4, or CD8. See U.S. Pat. No. 9,486,539 and Bender et al. PLoS Pathog. (2016) June; 12(6): e1005641.

Virus Envelope Antigen Binding Domain

In some embodiments, the glycoprotein on the envelope pseudotyped viral particle is modified to include an antigen binding domain. In some embodiments, the antigen is CD3. In some embodiments, the envelope pseudotyped viral particle can efficiently transduce cells expressing CD3. In some embodiments, the antigen binding domain is an anti-CD3 single chain variable fragment (scFv). In some embodiments, the antigen binding domain is an anti-CD3 humanized mouse scFv.

In some embodiments, the envelope pseudotyped viral particles are modified to include a fusion glycoprotein or a functional variant thereof and an antigen binding domain or a functional variant thereof. In some embodiments, the envelope pseudotyped viral particles are modified to include a Cocal virus G protein or a functional variant thereof and an anti-CD3 scFv or a functional variant thereof.

In some embodiments, the retroviral vector particles are surface engineered. Illustrative methods for surface engineering retroviral vector particles are provided in, for example, WO 2019/200056, PCT/US2019/062675, and US 62/916,110, each of which is incorporated herein by reference in its entirety.

In some embodiments, the retroviral particles are surface engineered to include a fusion glycoprotein or a functional variant thereof and an antigen binding domain or a functional variant thereof. In some embodiments, the retroviral particles are surface engineered to include a Cocal virus G protein or a functional variant thereof and an anti-CD3 scFv or a functional variant thereof.

The present disclosure provides various non-viral proteins capable of viral surface display. In some embodiments, the non-viral protein is a co-stimulatory molecule. Typically, in vitro lentiviral transduction requires additional exogenous activators, such as "stimbeads", such as Dynabeads ^TM human T activator αCD3/αCD28. In some embodiments, the retroviral (e.g., lentiviral) vector of the present disclosure incorporates non-viral proteins, such as one or more copies of T cell activation or co-stimulatory molecules. Incorporating T cell activation or co-stimulatory molecules into the particles can enable the particles to be activated and efficiently transduced to T cells in the absence or presence of a lower amount of exogenous activators, i.e., in the absence of stimbeads or equivalent agents.

In some embodiments, T cell activation or costimulatory molecules can be selected from the group consisting of: anti-CD3 antibodies, CD28 ligands (CD28L) and 41bb ligands (41BBL or CD137L). Various T cell activation or costimulatory molecules are known in the art, and include but are not limited to any T cell-expressing protein CD3, CD28, CD134 (also known as OX40) or 41bb (also known as 4-1BB or CD137) or TNFRSF9 specifically bound to the agent. For example, the agent specifically bound to CD3 can be an anti-CD3 antibody (e.g., OKT3, CRIS-7 or I2C) or an antigen binding fragment of an anti-CD3 antibody.

In some embodiments, the agent that specifically binds to CD3 is a single-chain Fv fragment (scFv) of an anti-CD3 antibody.

In some embodiments, the viral particle comprises a polypeptide comprising an anti-CD3 scFv (CD3 VL - connected to CD3 VH by a 3x G4S linker) that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:2.

Anti-CD3 scFv (VL-G4S x 3 linkers-VH):

DIQMTQSPSSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTSGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSK NTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSAAAKP (SEQ ID NO: 2).

The complementarity determining regions (CDRs) of this scFv are SASSSVSYMN (CDR-L1; SEQ ID NO: 133), DTSKLASG (CDR-L2; SEQ ID NO: 134), QQWSSNPFT (CDR-L3; SEQ ID NO: 135), RYTMH (CDR-H1; SEQ ID NO: 144), YINPSRGYTNYNQKVKD (CDR-H2; SEQ ID NO: 136), and YYDDHYCLDY (CDR-H3; SEQ ID NO: 137). In some embodiments, the viral particle comprises a polypeptide comprising an anti-CD3 scFv having these CDRs, wherein optionally the anti-CD3 scFv shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 2.

In some embodiments, the viral particle comprises a nucleic acid sequence encoding an anti-CD3 scFv (CD3 VL—connected to CD3 VH by a 3x G4S linker), wherein the anti-CD3 scFv shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:7.

Anti-CD3 scFv (VL-G4S x 3 linkers-VH):

GATATCCAGATGACCCAGTCCCCAAGCTCCCTGAGCGCCTCCGTGGGCGACCGGGTGACAATCACCTGCAGCGCCTCTAGCTCCGTGTCCTACATGAACTGGTATCAGCCAAAGAGATGGATCTACGATACCAGCAAGCTGGCCTCCGGCGTGCCTTCTAGGTTTTCTGGCAGCGGCTCCGGCACAGATTATACATTCACCATCTCTAGCCTGCAGCCAGAGGACATCGCCACCTACTATTGC CAGCAGTGGTCCTCTAATCCCTTTACATTCGCCAGGGCACCAAGCTGCAGATCACAAGAACCTCTGGAGGAGGAGGAAGCGGAGGAGGAGGATCCGGCGGCGGCGGCTCTCA GGTGCAGCTGGTGCAGAGCGGAGGAGGAGTGGTGCAGCCAGGCAGAAGCCTGAGGCTGTCCTGTAAGGCCTCTGGCTACACATTCACCAGATATACAATGCACTGGGTGAGGCAGGCACCAGGCAAGGGACTGGAGTGGATCGGCTACATCAACCCCTCCAGGGGCTACACCAACTATAATCAGAAGGTGAAGGATCGGTTCACCATCAGCAGGGACAACTCCAAGAATACCGCCTTCCTGCAGATGGACAGCCTGA GGCCAGAGGATACCGGCGTGTACTTTTGCGCCCGGTACTATGACGATCACTACTGTCTGGATTATTGGGGCCAGGGAACACCAGTGACCGTGAGCTCCGCCCGCAGCAAAGCCT (SEQ ID NO: 7).

In some embodiments, the viral particle comprises a polypeptide comprising an anti-CD3 scFv (CD3 VL - connected to CD3 VH by a 3x G4S linker) that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:12.

Anti-CD3 scFv (VL-G4S x 3 linkers-VH):

DIQMTQSPSSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTSGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSK NTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSAS (SEQ ID NO: 12).

The complementarity determining regions (CDRs) of this scFv are SASSSVSYMN (CDR-L1; SEQ ID NO: 133), DTSKLASG (CDR-L2; SEQ ID NO: 134), QQWSSNPFT (CDR-L3; SEQ ID NO: 135), RYTMH (CDR-H1; SEQ ID NO: 144), YINPSRGYTNYNQKVKD (CDR-H2; SEQ ID NO: 136), and YYDDHYCLDY (CDR-H3; SEQ ID NO: 137). In some embodiments, the viral particle comprises a polypeptide comprising an anti-CD3 scFv having these CDRs, wherein optionally the anti-CD3 scFv shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 12.

In some embodiments, the viral particle comprises a nucleic acid sequence encoding an anti-CD3 scFv (CD3 VL—connected to CD3 VH by a 3x G4S linker) that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:15.

Anti-CD3 scFv (VL-G4S x 3 linkers-VH):

GACATCCAGATGACCCAGTCTCCTAGCAGCCTCAGCGCTAGCGTGGGCGATAGAGTGACCATCACATGTAGCGCCAGCAGCAGCGTGTCCTACATGAACTGGTACCAGCAAACACCTGGAAAGGCCCTAAAAGGTGGATCTATGACACATCTAAGCTGGCTTCTGGAGTGCCATCTAGATTTTCTGGCAGCGGGCTCCGGCACTGATTATACATTCACCATCAGCAGCCTGCAGCCCGAGGATATCGCCACCTACTACTGT CAGCAGTGGTCCTCTAATCCCTTCACCTTCGGCCAGGGCACCAAGCTGCAGATCACCAGAACCAGCGGCGGGGGAGGAAGCGGCGGGGGAGGATCTGGCGGCGGCGGC AGCCAGGTGCAGCTGGTGCAGAGCCGGCGGCGGCGTGGTGCAACCTGGCAGAAGCCTGAGACTGAGCTGCAAGGCCTCTGGCTACACCTTCACCCGGTACACCATGCATTGGGTGCGGCAGGCCCCTGGCAAGGGCCTGGAATGGATTGGATACATCAACCCCAGCAGAGGCTACACCAACTACAACCAGAAGGTGAAGGACAGATTCACAATTTCTCGGGACAACAGCAAGAATACCGCCTTCCTGCAAATGGACTCCCTG CGCCCAGAAGATACCGGCGTGTACTTCTGCGCTAGATATTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCCCTGTGACCGTGTCCAGCGCCTCC (SEQ ID NO: 15).

In some embodiments, the viral particle comprises a polypeptide comprising an anti-CD3 scFv comprising CD3 VL connected to CD3 VH via a 3x G4S linker.

In some embodiments, the viral particle comprises a nucleic acid sequence encoding an anti-CD3 scFv comprising a CD3 VL connected to a CD3 VH via a 3x G4S linker.

In some embodiments, the viral particle includes a polypeptide comprising a Gaussia luciferase signal peptide, wherein the Gaussia luciferase signal peptide is operably linked to an anti-CD3 scFv, wherein the anti-CD3 scFv is operably linked to a hinge domain, wherein the hinge domain is operably linked to a Cocal envelope-derived transmembrane domain and a cytoplasmic tail, and the Gaussia luciferase signal peptide shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:107.

αCD3scFv_short hinge-TM-CT (pUMJ_224)

MGVKVLFALICIAVAEADIQMTQSPSSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTSGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYN QKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSASGVELIEGWFSSWKSTVVTFFFAIGVFILLYVVARIVIAVRYRYQGSNNKRIYNDIEMSRFRK(SEQ ID NO:107)

In some embodiments, the viral particle includes a nucleic acid encoding a Gaussia luciferase signal peptide, wherein the Gaussia luciferase signal peptide is operably linked to an anti-CD3 scFv, wherein the anti-CD3 scFv is operably linked to a hinge domain, wherein the hinge domain is operably linked to a Cocal envelope-derived transmembrane domain and a cytoplasmic tail, and the Gaussia luciferase signal peptide shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:108.

αCD3scFv_short hinge-TM-CT (pUMJ_224)

ATGGGCGTGAAAGTGCTGTTCGCCCTGATCTGCATCGCAGTTGCTGAAGCCGACATCCAGATGACCCAGTCTCCTAGCAGCCTCAGCGTGGGCGATAGAGTGACCATCACATGTAGCGCCAGCAGCAGCGTGTCCTACATGAACTGGTACCAGCAAACACCTGGAAAGGCCCCTAAAAGGTGGATCTATGACACATCTAAGCTGGCTTCTGGAGTGCCATCTAGATTTTCTGGCAGCG GCTCCGGCACTGATTATACATTCACCATCAGCAGCCTGCAGCCCGAGGATATCGCCACCTACTACTGTCAGCAGTGGTCCTCTAATCCCTTCACCTTCGGCCAGGGCACCAAGCTGCAGATCACCAGAACCAGCGGCGGGGGAGGAAGCGGCGGGGGAGGATCTGGCGGCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGCGGCGGCGTGGTGCAACCTGGCAGAAGCCTGAGACTGAGCTG CAAGGCCTCTGGCTACACCTTCACCCGGTACACCATGCATTGGGTGCGGCAGGCCCCTGGCAAGGGCCTGGAATGGATTGGATACATCAACCCCAGCAGAGGCTACACCAACTACAACCAGAAGGTGAAGGACAGATTCACAATTTCTCGGGACAACAGCAAGAATACCGCCTTCCTGCAAATGGACTCCCTGCGCCCAGAAGATACCGGCGTGTACTTCTGCGCTAGATATTACGACGACCAC TACTGCCTGGACTACTGGGGCCAGGGCACCCCTGTGACCGTGTCCAGCGCCTCCGGAGTGGAACTGATCGAGGGCTGGTTCAGCAGCTGGAAAAGCACCGTGGTTTACATTCTTTTTCGCCATCGGCGTGTTCATCCTGCTGTACGTGGTCGCCAGAATTGTGATCGCCGTGCGGTATAGATACCAGGGCAGCAACAAGCGGATCTACAACGACATCGAGATGAGCAGATTCAGAAAG (SEQ ID NO: 108 )

In some embodiments, the viral particle includes a polypeptide comprising a Gaussia luciferase signal peptide, wherein the Gaussia luciferase signal peptide is operably linked to an anti-CD3 scFv, wherein the anti-CD3 scFv is operably linked to a hinge domain, wherein the hinge domain is operably linked to a Cocal envelope-derived transmembrane domain and a cytoplasmic tail, and the Gaussia luciferase signal peptide shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:109.

αCD3scFv_Long Hinge_TM_CT(pUMJ_163)

MGVKVLFALICIAVAEADIQMTQSPSSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTSGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNY NQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSASSGFEHPHLAEAPKQLPEEEETLFFGDTGISKNPVELIEGWFSSWKSTVVTFFFAIGVFILLYVVARIVIAVRYRYQGSNNKRIYNDIEMSRFRK(SEQ ID NO:109)

In some embodiments, the viral particle includes a nucleic acid encoding a Gaussia luciferase signal peptide, wherein the Gaussia luciferase signal peptide is operably linked to an anti-CD3 scFv, wherein the anti-CD3 scFv is operably linked to a hinge domain, wherein the hinge domain is operably linked to a Cocal envelope-derived transmembrane domain and a cytoplasmic tail, and the Gaussia luciferase signal peptide shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:110.

αCD3scFv_Long Hinge_TM_CT(pUMJ_163)

ATGGGCGTGAAAGTGCTGTTCGCCCTGATCTGCATCGCAGTTGCTGAAGCCGACATCCAGATGACCCAGTCTCCTAGCAGCCTCAGCGCTAGCGTGGGCGATAGAGTGACCATCACATGTAGCGCCAGCAGCAGCGTGTCCTACATGAACTGGTACCAGCAAACACCTGGAAAGGCCCCTAAAAGGTGGATCTATGACACATCTAAGCTGGCTTCTGGAGTGCCATCTAGATTTTCTGGCAGCGGCCTCCGGCACT GATTATACATT CACCATCAGCAGCTGCAGCCCGAGGATATCGCCACCTACTACTGTCAGCAGTGGTCCTCTAATCCCTTCACCTTCGGCCAGGGCACCAAGCTGCAGATCACCAGAACCAGCGGCGGGGGAGGAAGCGGCGGGAGGATCTGGCGGCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGCGGCGGCGTGGTGCAACCTGGCAGAAGCCTGAGACTGAGCTGCAAGGCCTCTGGCTACACCTTCACCCGGT ACACCATGCATTGGGT GCGGCAGGCCCCTGGCAAGGGCCTGGAATGGATTGGATACATCAACCCCAGCAGAGGCTACACCAACTACAACCAGAAGGTGAAGGACAGATTCACAATTTCGGGACACAGCAAGAATACCGCCTTCCTGCAAATGGACTCCCTGCGCCCAGAAGATACCGGCGTGTACTTCTGCGCTAGATATTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCCCTGTGACCGTGTCCAGCGCCTCCGGATTCGAGCACC CCCACCTGGCCGAGGCCCCTAAGCAGCTGCCTGAAGAAGAGACACTGTTTTTCGGAGATACCGGCATCAGCAAAAACCCCGTGGAGCTGATCGAGGGCTGGTTCAGCTCTTGGAAGAGCACCGTGGTCACATTCTTTTTCGCCATCGGCGTCTTTATCCTGCTGTACGTGGTAGCCAGAATCGTGATCGCCGTGCGGTACAGATACCAGGGCAGCAACAACAAGCGGATCTACAACGACATCGAGATGAGCCGGTTC AGAAAG(SEQ ID NO:110)

In some embodiments, the viral particle includes a polypeptide comprising a Gaussia luciferase signal peptide, wherein the Gaussia luciferase signal peptide is operably linked to an anti-CD3 scFv, wherein the anti-CD3 scFv is operably linked to a linker, wherein the linker is operably linked to a transmembrane domain derived from glycophorin A and a cytoplasmic tail derived from the HIV envelope, and the Gaussia luciferase signal peptide shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:111.

αCD3scFv_hGlycophorin A_TM_HIV Env CT(pUMJ_194)

MGVKVLFALICIAVAEADIQMTQSPSSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTSGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNY NQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSASGGSTSGSGKPGSGEGSTKGPEITLIIFGVMAGVIGTILLISYGIRRLALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGACRAAIRHIPRRIRQGLERILL(SEQ ID NO:111)

In some embodiments, the viral particle includes a nucleic acid encoding a Gaussia luciferase signal peptide, wherein the Gaussia luciferase signal peptide is operably linked to an anti-CD3 scFv, wherein the anti-CD3 scFv is operably linked to a linker, wherein the linker is operably linked to a transmembrane domain derived from glycophorin A and a cytoplasmic tail derived from the HIV envelope, and the Gaussia luciferase signal peptide shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:112.

αCD3scFv_hGlycophorin A_TM_HIV Env CT(pUMJ_194)

ATGGGCGTGAAAGTGCTGTTCGCCCTGATCTGCATCGCAGTTGCTGAAGCCGACATCCAGATGACCCAGTCTCCTAGCAGCCTCAGCGTGCTAGCGTGGGCGATAGAGTGACCATCACATGTAGCGCCAGCAGCAGCGTGTCCTACATGAACTGGTACCAGCAAACACCTGGAAAGGCCCCTAAAAGGTGGATCTATGACACATCTAAGCTGGCTTCTGGAGTGCCATCTAGATTTTCTGGCAGCGGCCTCCGGCACTG ATTATACATTCACCATCAGCAGCCT GCAGCCCGAGGATATCGCCACCTACTACTGTCAGCAGTGGTCCTCTAATCCCTTCACCTTCGGCCAGGGCACCAAGCTGCAGATCACCAGAACCAGCGGCGGGGGAGGAAGCGGCGGGGGAGGATCTGGCGGCGGCGGCAGGTGCAGCTGGTGCAGAGCGGCGGCGGCGTGGTGCAACCTGGCAGAAGCCTGAGACTGAGCTGCAAGGCCTCTGGCTACACCTTTCACCCGGTACACCATGCATTGGGT GCGGCAGGCCCCTGGCAAGGGCCTGGAATG GATTGGATACATCAACCCCAGCAGAGGCTACACCAACTACAACCAGAAGGTGAAGGACAGATTCACAATTTCTCGGGACACAGCAAGAATACCGCCTTCCTGCAAATGGACTCCCTGCGCCCAGAAGATACCGGCGTGTACTTCTGCGCTAGATATTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCCCTGTGACCGTGTCCAGCGCCTCCGGAGGATCTACAAGCGGCTCTGGCAAGCCTGGCAGCGGAGAA GGCAGCACCAAGGGCC CTGAGATCACACTGATCATCTTCGGCGTGATGGCCGGCGTCATCGGCACCATCCTGCTGATCAGCTACGGCATCAGAAGACTGGCTCTGAAGTACTGGTGGAATCTGCTGCAATACTGGAGCCAGGAGCTGAAAAACAGCGCCGTGTCCCTGCTCAACGCCACCGCCATCGCCGTGGCCGAGGGCACCGACAGAGTGATCGAGGTGGTGCAGGGAGCCTGCAGAGCTATTCGGCACATCCCCAGACGGATCAGGCAGG GCCTGGAAAGAATCCCTGCTG(SEQ ID NO:112)

In some embodiments, the viral particle includes a polypeptide comprising a Gaussia luciferase signal peptide, wherein the Gaussia luciferase signal peptide is operably linked to an anti-CD3 scFv, wherein the anti-CD3 scFv is operably linked to a linker, wherein the linker is operably linked to an HIV envelope-derived transmembrane domain and a cytoplasmic tail, and the Gaussia luciferase signal peptide shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:113.

αCD3scFv_218 linker_HIV Env extracellular-TM-CT (pUMJ_195)

MGVKVLFALICIAVAEADIQMTQSPSSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTSGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNY NQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSASGGSTSGSGKPGSGEGSTKGNWLWYIRIFIIIVGSLIGLRIVFAVLSLVNRGWEALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL(SEQ ID NO:113)

In some embodiments, the viral particle includes a nucleic acid encoding a Gaussia luciferase signal peptide, wherein the Gaussia luciferase signal peptide is operably linked to an anti-CD3 scFv, wherein the anti-CD3 scFv is operably linked to a linker, wherein the linker is operably linked to an HIV envelope-derived transmembrane domain and a cytoplasmic tail, and the Gaussia luciferase signal peptide shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:114.

αCD3scFv_218 linker_HIV Env extracellular-TM-CT (pUMJ_195)

ATGGGCGTGAAAGTGCTGTTCGCCCTGATCTGCATCGCAGTTGCTGAAGCCGACATCCAGATGACCCAGTCTCCTAGCAGCCTCAGCGCTAGCGTGGGCGATAGAGTGACCATCACATGTAGCGCCAGCAGCAGCGTGTCCTACATGAACTGGTACCAGCAAACACCTGGAAAGGCCCCTAAAAGGTGGATCTATGACACATCTAAGCTGGCTTCTGGAGTGCCATCTAGATTTTCTGGCAGCGGCCTCCGGCACT GATTATACATTCACCATCAGCAGCCTGCAGC CCGAGGATATCGCCACCTACTACTGTCAGCAGTGGTCCTCTAATCCCTTCACCTTCGGCCAGGGCACCAAGCTGCAGATCACCAGAACCAGCGGCGGGGGAGGAAGCGGCGGGGGAGGATCTGGCGGCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGCGGCGGCGTGGTGCAACCTGGCAGAAGCCTGAGACTGAGCTGCAAGGCCTCTGCTACACCTTCACCCGGTACACCATGCATTGGGTGCGGCAGG CCCCTGGCAAGGGCCTGGAATGGATTGGATA CATCAACCCCAGCAGAGGCTACACCAACTACAACCAGAAGGTGAAGGACAGATTCACAATTTCTCGGGACAACAGCAAGAATACCGCCTTCCTGCAAATGGACTCCCTGCGCCCAGAAGATACCGGCGTGTACTTCTGCGCTAGATATTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCCCTGTGACCGTGTCCAGCGCCTCCGGAGGAAGCACCAGCGGCTCTGGCAAGCCTGGCAGCGGCGAGGGCTCT ACCAAGGGCAATTGGCTGTGGTAC ATCAGAATTCTTCATCATCGTGGGCAGCCTGATCGGCCTGAGAATCGTGTTCGCCGTGCTGAGCCTGGTGAACCGGGGCTGGGAAGCTCTGAAGTACTGGTGGAACCTGCTGCAATACTGGTCCCAGGAGCTGAAAAACAGCGCTGTGTCCCTGCTCAACGCCACCGCCATCGCCGTCGCCGAGGGAACAGACAGAGTGATCGAGGTGGTGCAGGGAGCCTGCAGAGCCATTCGGCACATCCCCAGACGCATCA GACAGGGCCTGGAAAGAATCCCTGCTG(SEQ ID NO:114)

In some embodiments, the viral particle includes a polypeptide comprising a Gaussia luciferase signal peptide, wherein the Gaussia luciferase signal peptide is operably linked to an anti-CD3 scFv, wherein the anti-CD3 scFv is operably linked to a triple G4S linker, wherein the triple G4S linker is operably linked to an HIV envelope-derived transmembrane domain and a cytoplasmic tail, and the Gaussia luciferase signal peptide shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:115.

αCD3scFv_G4S linker_HIV Env extracellular-TM-CT (pUMJ_196)

MGVKVLFALICIAVAEADIQMTQSPSSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTSGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNY NQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSASGGGGGSGGGGSGGGGSYIRIFIIIVGSLIGLRIVFAVLSLVNRGWEALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL(SEQ ID NO:115)

In some embodiments, the viral particle comprises a nucleic acid encoding a Gaussia luciferase signal peptide, wherein the Gaussia luciferase signal peptide is operably linked to an anti-CD3 scFv, wherein the anti-CD3 scFv is operably linked to a triple G4S linker, wherein the triple G4S linker is operably linked to an HIV envelope-derived transmembrane domain and a cytoplasmic tail, and the Gaussia luciferase signal peptide shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:116.

αCD3scFv_G4S linker_HIV Env extracellular-TM-CT (pUMJ_196)

ATGGGCGTGAAAGTGCTGTTCGCCCTGATCTGCATCGCAGTTGCTGAAGCCGACATCCAGATGACCCAGTCTCCTAGCAGCCTCAGCGTGCTAGCGTGGGCGATAGAGTGACCATCACATGTAGCGCCAGCAGCAGCGTGTCCTACATGAACTGGTACCAGCAAACACCTGGAAAGGCCCCTAAAAGGTGGATCTATGACACATCTAAGCTGGCTTCTGGAGTGCCATCTAGATTTTCTGGCAGCGGCCTCCGGCACTG ATTATACATTCACCATCAGCAGCC TGCAGCCCGAGGATATCGCCACCTACTACTGTCAGCAGTGGTCCTCTAATCCCTTCACCTTCGGCCAGGGCACCAAGCTGCAGATCACCAGAACCAGCGGCGGGGGAGGAAGCGGCGGGGGAGGATCTGGCGGCGGCGGCAGCCAGGTGCAGTGTGCAGAGCGGCGGCGGCGTGGTGCAACCTGGCAGAAGCCTGAGACTGAGCTGCAAGGCCTCTGGCTACACCTTCACCCGGTACACCATGCATTGGGT GCGGCAGGCCCCTGGCAAGGGCCTGGAA TGGATTGGATACATCAACCCCAGCAGAGGCTACACCAACTACAACCAGAAGGTGAAGGACAGATTCACAATTTCTCGGGACACAGCAAGAATACCGCCTTCCTGCAAATGGACTCCCTGCGCCCAGAAGATACCGGCGTGTACTTCTGCGCTAGATATTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCCCTGTGACCGTGTCCAGCGCCTCCGGAGGCGGTGGAGGCTCTGGTGGCGGAGGGAGCGGTGGCGG AGGCAGCTACATCAG AATCTTCATCATCATCGTGGGCAGCCTGATCGGCCTGAGAATCGTGTTCGCCGTTCTGAGCCTGGTGAACCGGGGCTGGGAAGCCCTGAAGTACTGGTGGAATCTGCTCCAGTACTGGTCTCAGGAGCTGAAGAACAGCGCCGTGTCCCTGCTGAACGCTACAGCTATCGCCGTCGCCGAGGGCACCGACAGAGTGATCGAGGTGGTGCAGGGCGCCTGCAGAGCCATCCGGCACATCCCTAGAAGGATTCGGCAA GGCCTGGAAAGAATCCTGCTG(SEQ ID NO:116)

In some embodiments, the viral particle includes a polypeptide comprising a Gaussia luciferase signal peptide, wherein the Gaussia luciferase signal peptide is operably linked to an anti-CD3 scFv, wherein the anti-CD3 scFv is operably linked to a hinge domain, wherein the hinge domain is operably linked to a Cocal envelope-derived transmembrane domain, a cytoplasmic tail, and a T2A self-cleaving peptide, wherein the T2A self-cleaving peptide is operably linked to the Cocal envelope, and the Gaussia luciferase signal peptide shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:117.

Anti-CD3scFv_Short Hinge_TM_CT_T2A_Cocal Envelope:

MGVKVLFALICIAVAEADIQMTQSPSSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTSGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYN QKVKDRFTISRDN SKNTAFLE KWITTCD FRWYGPKYITHSIHSIQPTSEQCKESIKQTKQGTWMSPGFPPQNCGYATVTDSVAVVVQATPHHVLVDEYTGEWIDSQFPNGKCETEECETVHNSTVWYSDYKVTGLCDATLVDTEITFFSEDGKKESIGKPNTGYRSNYFAYEKGDKVCKMNYCKHAGVRLPSGVWFEFVDQDVYAAAKLPECPVGATISAPTQ TSVDVSLILDVERILDYSLC QETWSKIRSKQPVSPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRIDIDNPIISKMVGKISGSQTERELWTEWFPYEGVEIGPNGILKTPTGYKFPLFMIGHGMLDSDLHKTSQAEVFEHPHLAEAPKQLPEEEETLFFGDTGISKNPVELIEGWFSSWKSTVVTFFFAIGVFILLYVVARIVIAVRYRYQGSNNKRIYNDIEMSRFRK(SEQ ID NO:11 7)

In some embodiments, the viral particle includes a nucleic acid encoding a Gaussia luciferase signal peptide, wherein the Gaussia luciferase signal peptide is operably linked to an anti-CD3 scFv, wherein the anti-CD3 scFv is operably linked to a hinge domain, wherein the hinge domain is operably linked to a Cocal envelope-derived transmembrane domain, a cytoplasmic tail, and a T2A self-cleaving peptide, wherein the T2A self-cleaving peptide is operably linked to the Cocal envelope, and the Gaussia luciferase signal peptide shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:118.

Anti-CD3scFv_Short Hinge_TM_CT_T2A_Cocal Envelope:

ATGGGCGTGAAAGTGCTGTTCGCCCTGATCTGCATCGCAGTTGCTGAAGCCGACATCCAGATGACCCAGTCTCCTAGCAGCCTCAGCGCTAGCGTGGGCGATAGAGTGACCATCACATGTAGCGCCAGCAGCAGCGTGTCCTACATGAACTGGTACCAGCAAACACCTGGAAAGGCCCCTAAAAGGTGGATCTATGACACATCTAAGCTGGCTTCTGGAGTGCCATCTAGATTTTCTGGCAGCGGCCTCCGGCACT GATTATACATTCACCATCAGCAGCCTGCAGCCCGAGGATATCGCCACCTACTACTGTCAGCAGTGG TCCTCTAATCCCTTCACCTTCGGCCAGGGCACCAAGCTGCAGATCACCAGAACCAGCGGCGGGGGAGGAAGCGGCGGGGGAGGATCTGGCGGCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGCGGCGGCGTGGTGCAACCTGGCAGAAGCCTGAGACTGAGCTGCAAGGCCTCTGGCTACACCTTCACCCGGTACACCATGCATTGGGTGCGGCAGGCCCCTGGCAAGGGCCTGGAATGGATTGGATACAT CAACCCCAGCAGAGGCTACACCAACTACAACCAGAAGGTGAAGGACAGATTCACAATTTCTCGGGACA ACAGCAAGAATACCGCCTTCCTGCAAATGGACTCCCTGCGCCCAGAAGATACCGGCGTGTACTTCTGCGCTAGATATTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCCCTGTGACCGTGTCCAGCGCCTCCGGAGTGGAACTGATCGAGGGCTGGTTCAGCAGCTGGAAAAGCACCGTGGTTTACATTCTTTTTCGCCATCGGCGTGTTCATCCTGCTGTACGTGGTCGCCAGAATTGTGATCGCCGTG CGGTATAGATACCAGGGCAGCAACAACAAGCGGATCTACAACGACATCGAGATGAGCAGAT TCAGAAAGGGATCTGGAGGGAAGGGGAAGCCTGCTGACATGCGGCGACGTGGAGGAGAACCCAGGACCAAATTTTCTGCTGCTGACCTTCATCGTGCTGCCTCTGTGCAGCCACGCCAAGTTTTCCATCGTGTTCCCACAGTCCCAGAAGGGCAACTGGAAGAATGTGCCCTCTAGCTACCACTATTGCCCTTCCTCTAGCGACCAGAACTGGCACAATGATCTGCTGGGCATCACAATGAAGGTGAAGATGCCCAAGA CCCACAAGGCCATCCAGGCAGATGGATGGATGTGCCACGCAGCCAAGTGGATCACAACCTG TGACTTTCGGTGGTACGGCCCCAAGTATATCACACACTCCATCCACTCTATCCAGGCCTACCTCCGAGCAGTGCAAGGAGTCTATCAAGCAGACAAAGCAGGGCACCTGGATGAGCCCTGGCTTCCCACCCCAGAACTGTGGCTACGCCACAGTGACCGACTCCGTGGCAGTGGTGGTGCAGGCAACACCTCACCACGTGCTGGTGGATGAGTATACCGGCGAGTGGATCGACAGCCAGTTTCCAAACGGCAAGT GCGAGACAGAGGAGTGTGAGACCGTGCACAATTCTACAGTGTGGTACAGCGATTATAAGGTGACAGG CCTGTGCGACGCCACCCTGGTGGATACAGAGATCACCTTTCTTTTCTGAGGACGGCAAGAAGGAGAGCATCGGCAAGCCCAACACCGGCTACAGATCCAATTACTTCGCCTATGAGAAGGGCGATAAGGTGTGCAAGATGAATTATTGTAAGCACGCCGGGGTGCGGCTGCCTAGCGGCGTGTGGTTTGAGTTCGTGGACCAGGACGTGTACGCAGCAGCAAAGCTGCCTGAGTGCCCAGTGGGAGCAACCATCT CCGCCCCAACACAGACCTCCGTGGACGTGTCTCTGATCCTGGATGTGGAGCGCATCCTGGACTACAGC CTGTGCCAGGAGACCTGGAGCAAGATCCGGTCCAAGCAGCCCGTGTCCCCTGTGGACCTGTCTTACCTGGCACCAAAGAACCCAGGAACCGGACCAGCCTTTACAATCATCAATGGCACCCTGAAGTACTTCGAGACCCGCTATATCCGGATCGACATCGATAACCCTATCAGCAAGATGGTGGGCAAGATCTCTGGCAGCCAGACAGAGAGAGAGCTGTGGACCGAGTGGTTCCCTTACGAGGGCGTGGAGATCGGCCC AAATGGCATCCTGAAGACACCAACCGGCTATAAGTTTCCCCTGTTCATGATCGGCCAC GGCATGCTGGACAGCGATCTGCACAAGACCTCCCAGGCCGAGGTTTGAGCACCCACACCTGGCAGAGGCACCAAAGCAGCTGCCTGAGGAGGAGACACTGTTCTTTGGCGATACCGGCATCTCTAAGAACCCCGTGGAGCTGATCGAGGGCTGGTTTTCCTCTTGGAAGAGCACAGTGGTGACCTTTCTTTTTCGCCATCGGCGTGTTCATCCTGCTGTACGTGGTGGCCAGAATCGGTGATCGCCGTGAG ATACAGGTATCAGGGCTCCAACAAATAAGAGGATCTATAATGACATCGAGATGTCTCGCTTCCGGAAG(SEQ IDNO:118)

In some embodiments, the viral particle includes a polypeptide comprising a Gaussia luciferase signal peptide, wherein the Gaussia luciferase signal peptide is operably linked to an anti-CD3 scFv, wherein the anti-CD3 scFv is operably linked to a linker, wherein the linker is operably linked to a hinge, a transmembrane domain and a cytoplasmic tail derived from glycophorin A, and the Gaussia luciferase signal peptide shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:119.

αCD3scFv_human glycophorin A hinge-TM-CT (pUMJ_232, for VV100)

MGVKVLFALICIAVAEADIQMTQSPSSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTSGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNY NQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSASHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ(SEQ ID NO:119)

In some embodiments, the viral particle includes a nucleic acid encoding a Gaussia luciferase signal peptide, wherein the Gaussia luciferase signal peptide is operably linked to an anti-CD3 scFv, wherein the anti-CD3 scFv is operably linked to a linker, wherein the linker is operably linked to a hinge, a transmembrane domain, and a cytoplasmic tail derived from glycophorin A, and the Gaussia luciferase signal peptide shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:120.

αCD3scFv_human glycophorin A hinge-TM-CT (pUMJ_232, for VV100)

ATGGGCGTGAAAGTGCTGTTCGCCCTGATCTGCATCGCAGTTGCTGAAGCCGACATCCAGATGACCCAGTCTCCTAGCAGCCTCAGCGTGGGCGATAGAGTGACCATCACATGTAGCGCCAGCAGCAGCGTGTCCTACATGAACTGGTACCAGCAAACACCTGGAAAGGCCCCTAAAAGGTGGATCTATGACACATCTAAGCTGGCTTCTGGAGTGCCATCTAGATTTTCTGGCAGCCGGC TCCGGCACTGATTATACATTCACCATCAGCAGCCTGCAGCCCGAGGATATCGCCACCTACTACTGTCAGCAGTGGTCCTCTAATCCCTTCACCTTCGGCCAGGGCACCAAGCTGCAGATCACCAGAACCAGCGGCGGGGGAGGAAGCGGCGGGGGAGGATCTGGCGGCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGCGGCGGCGTGGTGCAACCTGGCAGAAGCCTGAGACTGAGCTGCAAG GCCTCTGGCTACACCTTCACCCGGTACACCATGCATTGGGTGCGGCAGGCCCCTGGCAAGGGCCTGGAATGGATTGGATACATCAACCCCAGCAGAGGCTACACCAACTACAACCAGAAGGTGAAGGACAGATTCACAATTTCTCGGGACAACAGCAAGAATACCGCCTTCCTGCAAATGGACTCCCTGCGCCCAGAAGATACCGGCGTGTACTTCTGCGCTAGATATTACGACGACCACTACTGC CTGGACTACTGGGGCCAGGGCACCCCTGTGACCGTGTCCAGCGCCTCCCACTTCAGCGAGCCTGAGATCACCCTGATCATCTTCGGCGTGATGGCCGGAGATCGGCACAATCCTGCTGATCAGCTACGGCATCAGAAGACTGATTAAGAAATCCCCATCTGATGTGAAGCCTCTGCCTTCCTGACACCGACGTCCCCCTGAGCAGCGTGGAAATCGAGAACCCCGAAACCAGCGACCAG (SEQ ID NO: 12 0)

In some embodiments, T cell activation or costimulatory molecules are selected from the group consisting of: anti-CD3 antibodies, ligands of CD28 (e.g., CD28L) and 41bb ligands (41BBL or CD137L). CD86, also known as B7-2, is a ligand for both CD28 and CTLA-4. In some embodiments, the ligand of CD28 is CD86. CD80 is an additional ligand of CD28. In some embodiments, the ligand of CD28 is CD80. In some embodiments, the ligand of CD28 is an anti-CD28 antibody or anti-CD28 scFv coupled to a transmembrane domain to be displayed on the surface of a carrier. In some embodiments, the costimulatory molecule is CD80. Viral particles including one or more T cell activation or costimulatory molecules can be prepared by engineering a packaging cell line by the method provided in WO 2016/139463; or expressing T cell activation or costimulatory molecules by a polycis-trans electron-assisted vector described in International Patent Publication No. WO 2020/106992 A1.

In some embodiments, the viral particles include CD19 or a functional fragment thereof coupled to its native transmembrane domain or a heterologous transmembrane domain. In some embodiments, CD19 acts as a ligand for blinatumomab, thereby providing an adaptor for coupling the particles to T cells via the anti-CD3 portion of blinatumomab. In some embodiments, another type of particle surface ligand can act 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, for example, a bispecific T cell adaptor (BiTE).

The non-viral protein can be a cytokine. In certain embodiments, the cytokine can be selected from the group consisting of IL-15, IL-7 and IL-2. In the case where the non-viral protein used is a soluble protein (such as scFv or cytokine), the non-viral protein can be tethered to the surface of the lentiviral particle by fusion with a transmembrane domain, such as the transmembrane domain of CD8. Alternatively, the non-viral protein can be indirectly tethered to the lentiviral particle by using a transmembrane protein engineered to bind to a soluble protein. In addition, one or more cytoplasmic residues can be included to improve the stability of the fusion protein.

In some embodiments, the surface engineered carrier includes a transmembrane protein including a mitotic domain and/or a cytokine-based domain. In certain embodiments, the mitotic domain binds to a T cell surface antigen such as CD3, CD28, CD134, and CD137. In certain embodiments, the mitotic domain binds to a CD3ε chain.

CD28 is one of the proteins expressed on T cells, which provides the co-stimulatory signals required for T cell activation and survival. T cell stimulation by CD28 plus the T cell receptor (TCR) can provide a potent signal for the production of various interleukins (specifically, IL-6).

CD134, also known as OX40, is a member of the TNFR receptor superfamily and, unlike CD28, is not constitutively expressed on resting naive T cells. OX40 expression is dependent on full activation of T cells; in the absence of CD28, OX40 expression is delayed and expression levels are reduced fourfold.

CD137, also known as 4-1BB, is a member of the tumor necrosis factor (TNF) receptor family. CD137 can be expressed by activated T cells, but the degree of expression on CD8 is greater than the degree of expression on CD4 T cells. In addition, CD137 expression is present on dendritic cells, follicular dendritic cells, natural killer cells, granulocytes and vascular wall cells at inflammatory sites. A characteristic activity of CD137 is its co-thorn activity to activated T cells. The cross-linking of CD137 can enhance T cell proliferation, IL-2 secretion, survival and cytolytic activity.

The mitotic domain may include all or part of an antibody or other molecule that specifically binds to a T cell surface antigen. The antibody may activate TCR or CD28. The antibody may bind to TCR, CD3 or CD28. Examples of such antibodies include: OKT3, 15E8 and TGN1412. Other suitable antibodies include: anti-CD28: CD28.2, 10F3; anti-CD3/TCR: UCHT1, YTH12.5, TR66. The mitotic domain may include a binding domain from OKT3, 15E8, TGN1412, CD28.2, 10F3, UCHT1, YTH12.5 or TR66. The mitotic domain may include all or part of a co-stimulatory molecule, such as OX40L and 41BBL. For example, the mitotic domain may include a binding domain from OX40L or 41BBL.

In some embodiments, the carrier comprises an anti-CD3ε antibody or antigen-binding fragment thereof coupled to a transmembrane domain. An illustrative anti-CD3ε antibody is OKT3. OKT3, also known as Muromonab-CD3, is a monoclonal antibody targeting the CD3ε chain.

In certain embodiments, the carrier includes a ligand or its functional fragment of 4-1BB coupled with 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, which serves as a ligand of the 4-1BB of the co-stimulatory receptor molecule in T lymphocytes.In addition to promoting T lymphocyte proliferation, 4-1BBL has been shown to reactivate an incompetent T lymphocytes.

transduction enhancer spacer domain

The mitotic transduction enhancer and/or cytokine-based transduction enhancer may include a "spacer sequence" for connecting 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 "coupled to" refers to chemical connection, direct C-terminal fusion of two proteins to the N-terminal; chemical connection to non-peptide space; chemical connection to polypeptide space; and C-terminal fusion of two proteins to the N-terminal through a peptide bond to a polypeptide spacer, such as a spacer sequence.

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

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

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

In some embodiments, the viral particle comprises a polypeptide comprising a CD8 hinge that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:3.

CD8 hinge:TTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASD(SEQ ID NO:3)

In some embodiments, the viral particle comprises a nucleic acid sequence encoding a CD8 hinge that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:8.

CD8 hinge:

ACCACAACCCCTGCCCCAAGGCCACCTACACCCGCCCCTACCATCGCCTCTCAGCC ACTGAGCCTGAGGCCAGAGGCATCCAGGCCTGCCGCAGGGGGGGCCGTGCACACC CGGGGCCTGGACTTTGCTCTGAT (SEQ ID NO: 8).

In some embodiments, the viral particle includes a polypeptide comprising a short hinge derived from a Cocal glycoprotein, wherein the short hinge is operably linked to a transmembrane domain, wherein the transmembrane domain is operably linked to a cytoplasmic tail, and wherein the short hinge shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO: 13.

Short Hinge - TM - CT from Cocal Env:

GVELIEGWFSSWKSTVVTFFFAIGVFILLYVVARIVIAVRYRYQGSNNKRIYNDIEMSRFRK (SEQ ID NO: 13).

In some embodiments, the viral particle comprises a nucleic acid sequence encoding a short hinge, wherein the short hinge is operably linked to the transmembrane domain, wherein the transmembrane domain is operably linked to the cytoplasmic tail derived from Cocal glycoprotein, and the short hinge shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:16.

Short Hinge - TM - CT from Cocal Env:

GGAGTGGAACTGATCGAGGGCTGGTTCAGCAGCTGGAAAAGCACCGTGGTTACATTCTTTTTCGCCATCGGCGTGTTCATCCTGCTGTACGTGGTCGCCAGAATTGTGATCGCCGTGCGGTATAGATACCAGGGCAGCAACAAGCGGATCTACAACGACATCGAGATGAGCAGATTCAGAAAG (SEQ ID NO: 16).

In some embodiments, the viral particle comprises a polypeptide comprising a long hinge, wherein the long hinge is operably linked to a transmembrane domain, wherein the transmembrane domain is operably linked to a cytoplasmic tail derived from a Cocal glycoprotein, and wherein the long hinge shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO: 19.

Long Hinge -TM - CT from Cocal Env:

SGFEHPHLAEAPKQLPEEETLFFGDTGISKNPVELIEGWFSSWKSTVVTFFFAIGVFILLYVVARIVIAVRYRYQGSNNKRIYNDIEMSRFRK (SEQ ID NO: 19).

In some embodiments, the viral particle comprises a nucleic acid sequence encoding a long hinge, wherein the long hinge is operably linked to a transmembrane domain, wherein the transmembrane domain is operably linked to a cytoplasmic tail derived from a Cocal glycoprotein, and the long hinge shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:22.

Long Hinge -TM - CT from Cocal Env:

TCCGGATTCGAGCACCCCCACCTGGCCGAGGCCCCTAAGCAGCTGCCTGAAGAAGAGACACTGTTTTTCGGAGATACCGGCATCAGCAAAAACCCCGTGGAGCTGATCGAGGGCTGGTTCAGCTCTTGGAAGAGCACCGTGGTCACATTCTTTTTCGCCATCGGCGTCTTTATCCTGCTGTACGTGGTAGCCAGAATCGTGATCGCCGTGCGGTACAGATACCAGGGCAGCAACAACAAGCGGATCTACAACGACATCG AGATGAGCCGGTTCAGAAAG (SEQ ID NO: 22).

In some embodiments, the viral particle includes a polypeptide comprising a 218 linker, wherein the 218 linker is operably linked to the transmembrane domain of the extracellular domain of human glycoprotein A, wherein 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 218 linker shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:25.

218 linker_human glycophorin A extracellular-TM_HIV Env CT:

SGGSTSGSGKPGSGEGSTKGPEITLIIFGVMAGVIGTILLISYGIRRLALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL (SEQ ID NO: 25).

In some embodiments, the viral particle includes a nucleic acid sequence encoding a 218 linker, wherein the 218 linker 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 the cytoplasmic tail derived from the HIV viral envelope, and the 218 linker shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:28.

218 linker_human glycophorin A extracellular-TM_HIV Env CT:

TCCGGAGGATCTACAAGCGGCTCTGGCAAGCCTGGCAGCGGAGAAGGCAGCACCAAGGGCCCTGAGATCACACTGATCATCTTCGGCGTGATGGCCGGCGTCATCGGCACCATCCTGCTGATCAGCTACGGCATCAGAAGACTGGCTCTGAAGTACTGGTGGAATCTGCTGCAATACTGGAGCCAGGAGCTGAAAAACAGCGCCGTGTCCCTGCTCAACGCCACCGCCATCGCCGTGGCCGAGGGCACCGACAGAGT GATCGAGGTGGTGCAGGGAGCCTGCAGAGCTATTCGGCACATCCCCAGACGGATCAGGCAGGGCCTGGAAAGAATCCTGCTG (SEQ ID NO: 28).

In some embodiments, the viral particle comprises a polypeptide comprising a 218 linker operably linked to the HIV viral envelope transmembrane domain and the cytoplasmic tail, wherein the 218 linker shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:31.

218 Connector_HIV Env-TM-CT:

SGGSTSGSGKPGSGEGSTKGNWLWYIRIFIIIVGSLIGLRIVFAVLSLVNRGWEALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL (SEQ ID NO: 31).

In some embodiments, the viral particle comprises a nucleic acid sequence encoding a 218 linker operably linked to the HIV viral envelope transmembrane domain and the cytoplasmic tail, wherein the 218 linker shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:34.

218 Connector_HIV Env-TM-CT:

TCCGGAGGAAGCACCAGCGGCTCTGGCAAGCCTGGCAGCGGCGAGGGCTCTACCAAGGCAATTGGCTGTGGTACATCAGAATCTTCATCATCGTGGGCAGCCTGATCGGCCTGAGAATCGTGTTCGCCGTGCTGAGCCTGGTGAACCGGGGCTGGGAAGCTCTGAAGTACTGGTGGAACCTGCTGCAATACTGGTCCCAGGAGCTGAAAAACAGCGCTGTGTCCCTGCTCAACGCCACCGCCATCGCC GTCGCCGAGGGAACAGACAGAGTGATCGAGGTGGTGCAGGGAGCCTGCAGAGCCATTCGGCACATCCCCAGACGCATCAGACAGGGCCTGGAAAGAATCCTGCTG (SEQ ID NO: 34).

In some embodiments, the viral particle comprises a polypeptide comprising a triple G4S linker operably linked to the HIV viral envelope transmembrane domain and the cytoplasmic tail, wherein the triple G4S linker shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:37.

G4Sx3 Connector_HIV Env-TM-CT:

SGGGGGSGGGGSGGGGSYIRIFIIIVGSLIGLRIVFAVLSLVNRGWEALKYWWNLLQYW SQELKNSAVSLLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL(SEQ ID NO:37)

In some embodiments, the viral particle comprises a nucleic acid sequence encoding a triple G4S linker operably linked to the HIV viral envelope transmembrane domain and the cytoplasmic tail, wherein the triple G4S linker shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:40.

G4Sx3 Connector_HIV Env-TM-CT:

TCCGGAGGCGGTGGAGGCTCTGGTGGCGGAGGGCGGTGGCGGAGGCAGCTACATCAGAATCTTCATCATCATCGTGGGCAGCCTGATCGGCCTGAGAATCGTGTTCGCCGTTCTGAGCCTGGTGAACCGGGGCTGGGAAGCCCTGAAGTACTGGTGGAATCTGCTCCAGTACTGGTCTCAGGAGCTGAAGAACAGCGCCGTGTCCCTGCTGAACGCTACAGCTATCGCCGTCGCCGAGGGCACCGACAGA GTGATCGAGGTGGTGCAGGGCGCCTGCAGAGCCATCCGGCACATCCCTAGAAGGATTCGGCAAGGCCTGGAAAGAATCCTGCTG (SEQ ID NO: 40).

In some embodiments, the viral particle includes a polypeptide comprising a Ser-Gly peptide operably linked to a small extracellular domain, a transmembrane and a cytoplasmic tail sequence derived from human glycophorin A, the Ser-Gly peptide sharing 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: 97. The underlined sequence represents a transmembrane domain fragment.

Human Glycophorin A TM_CT:

SGHFSEPE ITLIIFGVMAGVIGTILLISYGI RRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ(SEQ ID NO:97)

In some embodiments, the viral particle comprises a nucleic acid sequence encoding a Ser-Gly peptide operably linked to a small extracellular domain, a transmembrane and a cytoplasmic tail sequence derived from human glycophorin A, and the Ser-Gly peptide shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:98.

218 linker_human glycophorin A extracellular-TM_HIV Env CT:

TCCGGACACTTCAGCGAGCCTGAGATCACCCTGATCATCTTCGGCGTGATGGCCGGAGTGATCGGCACAATCCTGCTGATCAGCTACGGCATCAGAAGACTGATTAAGAAATCCCCATCTGATGTGAAGCCTCTGCCTTCTCCTGACACCGACGTCCCCCTGAGCAGCGTGGAAATCGAGAACCCCGAAACCAGCGACCAG (SEQ ID NO: 98).

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

Human Glycophorin A Hinge-TM-CT:

HFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ(SEQID NO:105)

In some embodiments, the viral particle comprises a nucleic acid sequence encoding a hinge operably linked to the glycophorin A transmembrane domain and the cytoplasmic tail, wherein the hinge shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:106.

Human Glycophorin A Hinge-TM-CT:

CACTTCAGCGAGCCTGAGATCACCCTGATCATCTTCGGCGTGATGGCCGGAGTGATCGGCACAATCCTGCTGATCAGCTACGGCATCAGAAGACTGATTAAGAAATCCCCATCTGATGTGAAGCCTCTGCCTTCTCCTGACACCGACGTCCCCCTGAGCAGCGTGGAAATCGAGAACCCCGAAACCAGCGACCAG (SEQ ID NO: 106)

In some embodiments, the viral particle comprises a polypeptide comprising a short hinge operably linked to the Cocal glycoprotein transmembrane domain and the cytoplasmic tail, wherein the short hinge shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:43.

Short Hinge - TM-CT from Cocal Env_T2A:

GVELIEGWFSSWKSTVVTFFFAIGVFILLYVVARIVIAVRYRYQGSNNKRIYNDIEMSRFRKGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 43).

In some embodiments, the viral particle comprises a nucleic acid sequence encoding a short hinge, wherein the short hinge is operably linked to the Cocal glycoprotein transmembrane domain and the cytoplasmic tail, wherein the cytoplasmic tail is operably linked to the T2A linker, and the short hinge shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:47.

Short Hinge - TM-CT from Cocal Env_T2A:

GGAGTGGAACTGATCGAGGGCTGGTTCAGCAGCTGGAAAAGCACCGTGGTTTACATTCTTTTTCGCCATCGGCGTGTTCATCCTGCTGTACGTGGTCGCCAGAATTGTGATCGCCGTGCGGTATAGATACCAGGGCAGCAACAAGCGGATCTACAACGACATCGAGATGAGCAGATTCAGAAAGGGATCTGGAGAGGGAAGGGGAAGCCTGCTGACATGCGGCGACGTGGAGGAGAACCCAGGACCA(SEQ ID NO:47).

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

CD4 TM and cytoplasmic tail_T2A:

MALIVLGGVAGLLLFIGLGIFFCVRCRHRRRQGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 4).

In some embodiments, the viral particle 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 CD4-derived transmembrane domain and the cytoplasmic tail share 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:9.

CD4 TM and cytoplasmic tail_T2A:

ATGGCACTGATCGTGCTGGGAGGAGTGGCAGGACTGCTGCTGTTCATCGGACTGGGCATCTTCTTTTGCGTGCCTGTAGGCACCGGAGAAGGCAGGGATCTGGAGAGGGAAGGGGAAGCCTGCTGACATGCGGCGACGTGGAGGAGAACCCAGGACCA (SEQ ID NO: 9).

In some embodiments, the viral particle comprises a polypeptide comprising a Gaussia luciferase-derived signal peptide sequence, which shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:11.

Gaussia luciferase SP: MGVKVLFALICIAVAEA (SEQ ID NO: 11).

In some embodiments, the viral particle comprises a nucleic acid sequence encoding a Gaussia luciferase-derived signal peptide, wherein the Gaussia luciferase-derived signal peptide sequence shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:14.

Gaussia luciferase SP:

ATGGGCGTGAAAGTGCTGTTCGCCCTGATCTGCATCGCAGTTGCTGAAGCC (SEQ ID NO: 14).

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

The viral particles of the present invention may include a cytokine-based transduction enhancer located in the viral envelope. In some embodiments, the cytokine-based transduction enhancer is derived from a host cell during viral particle production. In some embodiments, the cytokine-based transduction enhancer is prepared by a host cell and expressed at the cell surface. When the nascent viral particle buds out from the host cell membrane, the cytokine-based transduction enhancer can be incorporated into the viral envelope as part of the lipid bilayer derived from the packaging cell.

Cytokine-based transduction enhancers may include a cytokine domain and a transmembrane domain. The transduction enhancer may have a structure C-S-TM, 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.

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

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 the proliferation of reactive T cells. It is secreted as a single glycosylated polypeptide and requires a cleavage signal sequence for its activity. Solution NMR shows that the structure of IL2 includes a bundle of 4 helices (referred to as A-D) flanked by 2 shorter helices and several poorly defined loops. The residues in helix A and the loop region between helices A and B are important for receptor binding.

Virus particle envelope expression cassette

In some embodiments, the viral particles of the present disclosure include a viral envelope expression cassette encoding the following in 5' to 3' order:

(a) CD8-derived signal peptide

(b) Anti-CD3 scFV

(c) CD8-derived hinge domain

(d) CD4 transmembrane domain and cytoplasmic tail

(e) T2A connector

(f) Cocal glycoprotein

In some embodiments, the viral particles of the present disclosure include a viral envelope expression cassette encoding the following in 5' to 3' order: (a) a Gaussia luciferase-derived signal peptide

(b) Anti-CD3 scFV

(c) Short hinge domain

(d) Cocal glycoprotein-derived transmembrane domain and cytoplasmic tail

In some embodiments, the viral particles of the present disclosure include a viral envelope expression cassette encoding the following in 5' to 3' order: (a) a Gaussia luciferase-derived signal peptide

(b) Anti-CD3 scFV

(c) Long hinge domain

(d) Cocal glycoprotein-derived transmembrane domain and cytoplasmic tail

In some embodiments, the viral particles of the present disclosure include a viral envelope expression cassette encoding the following in 5' to 3' order: (a) a Gaussia luciferase-derived signal peptide

(b) Anti-CD3 scFV

(c)218 connector

(d) Transmembrane domain derived from the extracellular domain of human glycophorin A

(e) HIV viral envelope-derived cytoplasmic tail

In some embodiments, the viral particles of the present disclosure include a viral envelope expression cassette encoding the following in 5' to 3' order: (a) a Gaussia luciferase-derived signal peptide

(b) Anti-CD3 scFV

(c)218 connector

(d) HIV viral envelope-derived transmembrane domain and cytoplasmic tail

In some embodiments, the viral particles of the present disclosure include a viral envelope expression cassette encoding the following in 5' to 3' order: (a) a Gaussia luciferase-derived signal peptide

(b) Anti-CD3 scFV

(c) Triple G4S connector

(d) HIV viral envelope-derived transmembrane domain and cytoplasmic tail

In some embodiments, the viral particles of the present disclosure include a viral envelope expression cassette encoding the following in 5' to 3' order: (a) a Gaussia luciferase-derived signal peptide

(b) Anti-CD3 scFV

(c) Short hinge domain

(d) Cocal glycoprotein-derived transmembrane domain and cytoplasmic tail

(e) T2A connector

(f) Cocal glycoprotein

Adeno-associated virus

In certain embodiments, the viral particle is an adeno-associated virus (AAV) particle. AAV is a single-stranded DNA virus of 4.7 kb. Recombinant particles based on AAV have excellent clinical safety because wild-type AAV is non-pathogenic and has no etiological relationship with any known disease. In addition, AAV provides the ability to carry out very efficient gene delivery and sustained transgenic expression in many tissues. "AAV particles" means particles derived from adeno-associated virus serotypes, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAVrh.74. AAV vectors can have AAV wild-type genes that are entirely or partially deleted, such as one or more of rep and/or cap genes, but retain functional flanking reverse terminal repeats (ITRs). Functional ITR sequences are essential for the rescue, replication and packaging of AAV virions. Thus, AAV vectors are defined herein as comprising at least those sequences required for replication and packaging (e.g., functional ITRs) of the virus in cis. ITRs do not need to be wild-type nucleotide sequences and can be altered, for example, by inserting, deleting or substituting nucleotides, as long as the sequence provides functional rescue, replication and packaging. AAV particles may include other modifications including, but not limited to, one or more modified capsid proteins (e.g., VP1, VP2 and/or VP3). For example, the capsid protein may be modified to change tropism and/or reduce immunogenicity.

The serotype of a recombinant AAV particle is determined by its capsid. International Patent Publication No. WO2003042397A2 discloses various capsid sequences, including those of AAV1, AAV2, AAV3, AAV8, AAV9, and rh10. International Patent Publication No. WO2013078316A1 discloses the polypeptide sequence of VP1 from AAVrh74. Many different naturally occurring or genetically modified AAV capsid sequences are known in the art.

Gene delivery virus particles that can be used to practice the present disclosure can be constructed using methods known in the field of molecular biology. Generally, the viral vector carrying the transgene is assembled by a polynucleotide encoding the transgene, a suitable regulatory element, and the elements required 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 helper plasmids or viruses. Examples of viral packaging cells include, but are not limited to, HeLa cells, SF9 cells (optionally with baculovirus helper vectors), 293 cells, etc. Detailed protocols for producing such replication-deficient recombinant viruses can be found, for example, in the following: 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 which are incorporated herein by reference.

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 hereby incorporated in its entirety.

Chimeric Antigen Receptor

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 causes one or more CARs to be expressed in transduced cells.

CAR is an artificial membrane-bound protein that guides T lymphocytes to antigens and stimulates T lymphocytes to kill cells displaying antigens. See, for example, Eshhar, U.S. Patent No. 7,741,465. In general, CAR is a genetically engineered receptor, and the genetically engineered receptor includes an extracellular domain, an optional linker, a transmembrane domain, and an intracellular (cytoplasmic) domain that binds to an antigen, such as an antigen on a cell, and the intracellular domain includes a costimulatory domain and/or a signal transduction domain that transmits an activation signal to an immune cell. In the case of CAR, a single receptor can be programmed to recognize a specific antigen, and when bound to the antigen, activates immune cells to attack and destroy cells carrying the antigen. When these antigens are present on tumor cells, immune cells expressing CAR can target and kill tumor cells. In the case of meeting all other conditions, when CAR is expressed on the surface of, for example, a T lymphocyte, and the extracellular domain of CAR binds to an antigen, the intracellular signal transduction domain transmits a signal to T lymphocytes to activate and/or proliferate, and if the antigen is present on the cell surface, the cells expressing the antigen are killed. Since T lymphocytes require two signals, namely, a primary activation signal and a co-stimulatory signal, in order to maximize activation, CAR can include a stimulatory domain and a co-stimulatory domain, so that the binding of the antigen to the extracellular domain causes the transmission of the primary activation signal and the co-stimulatory signal.

In some embodiments, the expression of the polycistronic transgenic payload is driven by the MND promoter.MND promoter (myeloproliferative sarcoma virus enhancer, negative control region deletion, dl587rev primer binding site replacement) is a virus-derived synthetic promoter containing a modified Moloney mouse leukemia virus (MoMuLV) LTR U3 region with myeloproliferative sarcoma virus enhancer 13, and has high expression in human CD34+ stem cells, lymphocytes and other tissues.In some embodiments, four separate proteins are expressed and separated by ribosome jumps and cleaved 2A peptide sequences during induction translation.In some embodiments, CAR is a second generation CAR comprising FMC63 mouse anti-human CD19 scFv connected to a 4-1BB costimulatory domain and a CD3ζ intracellular signaling domain.

CAR intracellular domain

In some embodiments, the intracellular domain of CAR is or includes an intracellular domain or motif of a protein expressed on the surface of a T lymphocyte and triggering the activation and/or proliferation of the T lymphocyte. Such a domain or motif can transmit a primary antigen binding signal, which is necessary for activating T lymphocytes in response to the binding of the antigen to the extracellular part of CAR. Typically, this domain or motif includes or is ITAM (immunoreceptor tyrosine-based activation motif). Suitable ITAM-containing polypeptides for CAR include, for example, ζCD3 chain (CD3ζ) or its ITAM-containing portion. In some embodiments, the intracellular domain is a CD3ζ intracellular signaling domain. In some embodiments, the intracellular domain is from a lymphocyte receptor chain, a TCR/CD3 complex protein, an Fc receptor subunit, or an IL-2 receptor subunit. In some embodiments, the intracellular signaling domain of CAR can be derived from, for example, a signaling domain of OO3ζ, CD3ε, CD22, CD79a, CD66d, or CD39. "Intracellular signaling domain" refers to a portion of a CAR polypeptide that is involved in transducing messages for effective CAR binding to a target antigen into the interior of an immune effector cell to elicit effector cell functions, e.g., activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors to CAR-bound target cells, or other cellular responses elicited following antigen binding to an extracellular CAR domain.

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

In some embodiments, the viral particle comprises a polypeptide comprising a CAR whose intracellular domain comprises a CD3 zeta domain, wherein the CD3 zeta domain shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:54.

CD3ζ:

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 54).

In some embodiments, the viral particle comprises a nucleic acid encoding an intracellular domain of a CAR, wherein the intracellular domain comprises a CD3 zeta domain, and the CD3 zeta domain shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:66.

CD3ζ:

CGCGTGAAGTTCAAGCCGGTCCGCCGATGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGGGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGA CACGATGGCCTGTACCAGGGCCTGAGCACCGCCACAAAGGACACATATGATGCCCTGCACATGCAGGCCCTGCCACCTAGG (SEQ ID NO: 66).

In some embodiments, CAR further includes one or more costimulatory domains or motifs, for example, as a part of the intracellular domain of a polypeptide.Costimulatory molecules are well-known cell surface molecules other than antigen receptors or Fc receptors, and the cell surface molecules provide a second signal required for effectively activating T lymphocytes and acting after binding to an antigen. One or more costimulatory domains or motifs can be, for example, 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: 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 the intracellular domain of ZAP70.

In some embodiments, the costimulatory domain is the intracellular domain of 4-1BB.

In some embodiments, the viral particle comprises a polypeptide comprising a CAR whose intracellular domain comprises a co-stimulatory 4-1BB polypeptide sequence, and the co-stimulatory 4-1BB polypeptide sequence shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:53.

4-1BB signal domain: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 53).

In some embodiments, the viral particle comprises a nucleic acid encoding an intracellular domain of a CAR, wherein the intracellular domain comprises a co-stimulatory 4-1BB sequence, and the co-stimulatory 4-1BB sequence shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:65.

4-1BB signaling domain:

AAGAGAGGCAGGAAGAAGCTGCTGTATATCTTTAAGCAGCCCTTCATGCGCCCTGTGCAGACCACACAGGAGGAGGACGGCTGCAGCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGCGAGCTG (SEQ ID NO: 65).

In some embodiments, the viral particle comprises a polypeptide comprising a CAR whose intracellular domain comprises an IgG4 linker, wherein the IgG4 linker is operably linked to a CD28-derived transmembrane domain, wherein the CD28-derived transmembrane domain is operably linked to a co-stimulatory 4-1BB polypeptide, wherein the co-stimulatory 4-1BB polypeptide is operably linked to a CD3ζ domain, and the IgG4 linker shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 80.

IgG4 linker-CD28 TM_4-1BB-CD3ζ:

ESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:8 0).

In some embodiments, the viral particle comprises a nucleic acid encoding an intracellular domain of a CAR, the intracellular domain comprising an IgG4 linker, the IgG4 linker being operably linked to a CD28-derived transmembrane domain, the CD28-derived transmembrane domain being operably linked to a co-stimulatory 4-1BB polypeptide, the co-stimulatory 4-1BB polypeptide being operably linked to a CD3 ζ domain, the IgG4 linker sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 86.

IgG4 linker-CD28 TM_4-1BB-CD3ζ:

GAGTCTAAGTATGGCCCTCCATGCCCCCCTTGTCCTATGTTCTGGGTGCTGGTGGTGGTGGGAGGCGTGCTGGCCTGTTACTCCCTGCTGGTGACCGTGGCCTTTATCATCTTCTGGGTGAAGCGCGGCCGGAAGAAGCTGCTGTATATCTTTAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAGGACGGCTGCAGCTGTAGGTTTCCAGAGGAGGAGGAGGATGCGAGCTGCGCGTG AAGTTCTCTCGGAGCGCCGATGCCCCTGCCTACCAGCAGGG ACAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGAGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATTCCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTGAGCACCGCCACAAAAGGACACCTA TGATGCCCTGCACATGCAGGCCCTGCCACCCAGG (SEQ ID NO:86).

In some embodiments, the viral particle comprises a polypeptide comprising a CAR whose intracellular domain comprises an IgG4 linker, wherein the IgG4 linker is operably linked to a CD28-derived transmembrane domain, wherein the CD28-derived transmembrane domain is operably linked to a co-stimulatory 4-1BB polypeptide, wherein the co-stimulatory 4-1BB polypeptide is operably linked to a CD3ζ domain, and wherein the IgG4 linker shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:90.

IgG4 linker-CD28 TM_4-1BB-CD3ζ_P2A:

ESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLK QAGDVEENPGP (SEQ ID NO:90).

In some embodiments, the viral particle comprises a nucleic acid encoding an intracellular domain of a CAR, the intracellular domain comprising an IgG4 linker, the IgG4 linker being operably linked to a CD28-derived transmembrane domain, the CD28-derived transmembrane domain being operably linked to a co-stimulatory 4-1BB polypeptide, the co-stimulatory 4-1BB polypeptide being operably linked to a CD3 ζ domain, the IgG4 linker sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 95.

IgG4 linker-CD28 TM_4-1BB-CD3ζ_P2A:

GAGTCCAAGTACGGCCCACCCTGCCCTCCATGTCCCATGTTTTGGGTGCTGGTGGTG

GTGGGAGGCGTGCTGGCCTGTTATTCCCTGCTGGTGACCGTGGCCTTCATCATCTTT

TGGGTGAAGCGCGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAG

ACCCGTGCAGACCACACAGGAGGAGGACGGCTGCAGCTTGTAGGTTCCCAGAGGAG

GAGGAGGGAGGATGCGAGCTGAGGGTGAAGTTTTCCCGGTCTGCCGATGCCCCTGC

CTATCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAATCTGGGCAGGCGCGAGG

AGTACGACGTGCTGGATAAGAGGAGAGGAAGGGACCCTGAGATGGGAGGCAAGCC

AAGGCGCAAGAACCTCAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATG

GCCGAGGCCTACTCCGAGATCGGCATGAAGGGAGAGCGGAGAAGGGGGCAAGGGAC

ACGATGGCCTGTATCAGGGCCTGAGCACCGCCACAAAGGACACCTACGATGCACTG

CACATGCAGGCCCTGCCACCTAGAGGATCTGGAGCCACAAACTTCAGCCTGCTGAAGCAGGCCGGCGATGTGGAGGAGAATCCTGGACCA (SEQ ID NO:95).

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

CAR transmembrane region

The transmembrane region can be any transmembrane region that can be incorporated into a functional CAR, for example, a transmembrane region from a CD4 or CD8 molecule.

In some embodiments, the transmembrane domain of the CAR can be derived from CD8, the 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), CD 160. CD19, IL2Rβ, IL2Rγ, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2 , CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, 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 transmembrane domain. In some embodiments, the transmembrane domain of CAR can be derived from the transmembrane domain of CD28.

CAR linker region

The optional linker of the CAR positioned between the extracellular domain and the transmembrane domain can be a polypeptide of about 2 to 100 amino acids in length. The linker may include or may be composed of flexible residues such as glycine and serine so that adjacent protein domains can move freely relative to each other. For example, when it is desired to ensure that two adjacent domains do not interfere with each other spatially, a longer linker can be used. The linker may be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (eg, T2A), 2A-like linkers or their functional equivalents and combinations thereof.

In some embodiments, the linker is a P2A self-cleaving peptide. In some embodiments, the viral particle comprises a polypeptide comprising a P2A linker that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:55.

P2A self-cleaving peptide: GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 55).

In some embodiments, the viral particle comprises a nucleic acid encoding a P2A linker that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:67.

P2A self-cleaving peptide:

GGATCTGGAGCCACCAACTTTAGCCTGCTGAAGCAGGCAGGCGATGTGGAGGAGAATCCAGGACCT (SEQ ID NO: 67).

In some embodiments, the viral particle comprises a nucleic acid encoding a P2A linker that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:69.

P2A self-cleaving peptide:

GGCTCCGGCGCCACCAACTTCTCCCTGCTGAAGCAGGCCGGCGATGTGGAAGAAAATCCAGGACCA (SEQ ID NO: 69).

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 IgG4.

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

IgG4 linker-CD28 TM: ESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 52).

In some embodiments, the linker is an IgG4 linker operably linked to a CD28-derived transmembrane domain, and the linker shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:64.

IgG4 Linker-CD28 TM:

GAGTCTAAGTATGGCCCACCCTGCCCTCCATGTCCAATGTTCTGGGTGCTGGTGGTGGTGGGAGGCGTGCTGGCCTGTTACTCCCTGCTGGTGACCGTGGCCTTTATCATCTTCTGGGTG (SEQ ID NO: 64).

CAR extracellular domain

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. A single-chain Fv domain may comprise, for example, a VL connected to a VH by a flexible linker, wherein the VL and the VH are from an antibody that binds to the antigen.

In some embodiments, the extracellular domain of CAR may contain any polypeptide bound to a desired antigen (e.g., prostate neoantigen). The extracellular domain may include a portion of scFv, an antibody, or an alternative scaffold. CAR may also be engineered to bind to two or more desired antigens, which may be arranged in series and separated by a linker sequence. For example, one or more domain antibodies, scFv, alpaca VHH antibodies, or other antibody fragments of VH alone may be organized in series by a linker to provide bispecific or multispecific CAR.

The antigen bound by the extracellular domain of the polypeptide can be any antigen of interest, for example, it can be an antigen on a tumor cell. The tumor cell can be a cell in a solid tumor or a blood cancer cell. The antigen can be an antigen expressed on cells of any tumor or cancer type, for example, lymphoma, lung cancer, breast cancer, prostate cancer, adrenocortical carcinoma, thyroid cancer, nasopharyngeal carcinoma, melanoma, such as malignant melanoma, skin cancer, colorectal cancer, desmoid tumor, desmoplastic small round cell tumor, endocrine tumor, Ewing 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 may be chronic lymphocytic leukemia (small lymphocytic lymphoma), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, Waldenstrom's macroglobulinemia ( macroglobulinemia), splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal zone B-cell lymphoma, MALT lymphoma, nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma, T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, aggressive NK-cell leukemia, adult T-lymphocytic leukemia/lymphoma, extranodal NK/T-lymphocytic lymphoma, nasal type, enteropathy type T-lymphocytic lymphoma, hepatosplenic T-lymphocytic lymphoma, blastic NK-cell lymphoma, mycosis fungoides, Sezary syndrome syndrome, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T-lymphocytic lymphoma, peripheral T-lymphocytic lymphoma (unspecified), anaplastic large cell lymphoma, Hodgkin lymphoma, or non-Hodgkin lymphoma. In some embodiments where the cancer is chronic lymphocytic leukemia (CLL), the B cells of CLL have a normal karyotype. In some embodiments where the cancer is chronic lymphocytic leukemia (CLL), the B cells of CLL carry a 17p deletion, an 11q deletion, a 12q trisomy, a 13q deletion, or a p53 deletion.

In some embodiments, the antigen is expressed on a B-cell malignancy cell, a relapsed/refractory CD19-expressing malignancy cell, a diffuse large B-cell lymphoma (DLBCL) cell, a Burkitt-type large B-cell lymphoma (B-LBL) cell, a follicular lymphoma (FL) cell, a chronic lymphocytic leukemia (CLL) cell, an acute lymphocytic leukemia (ALL) cell, a mantle cell lymphoma (MCL) cell, a hematological malignancy cell, a colon cancer cell, a lung cancer cell, a liver cancer cell, a breast cancer cell, a kidney cancer cell, a prostate cancer cell, an ovarian cancer cell, a skin cancer cell, a melanoma cell, a bone cancer cell, a brain cancer cell, a squamous cell cancer cell, a leukemia cell, a myeloma cell, a B-cell lymphoma cell, a renal cancer cell, a uterine cancer cell, an adenocarcinoma cell, a pancreatic cancer cell, a chronic myeloid leukemia cell, a glioblastoma cell, a neuroblastoma cell, a medulloblastoma cell, or a sarcoma cell.

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 activation factor (BAFF), 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, calreticulin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD19, CD20, CD34, CD45, CD99, CD1 17. Chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), giant cystic disease fluid protein (GCDFP-15), HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, muscle-specific actin (MSA), neurofilaments, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptonemal breakdown, thyroglobulin, thyroid transcription factor-1, vascular endothelial growth factor receptor (VEGFR), dimer form of pyruvate kinase isozyme M2 type (tumor M2-PK), abnormal ras protein or abnormal p53 protein.

In some embodiments, the antigen is CD19.

In some embodiments, the CAR comprises an extracellular domain comprising an FMC63 scFv binding domain for CD19 binding.

In some embodiments, the viral particle comprises a polypeptide comprising a CAR whose extracellular domain includes a signal peptide that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:50.

Signal peptide (human CSF2R): MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 50).

In some embodiments, the viral particle comprises a polynucleotide encoding a CAR whose extracellular domain comprises an αCD19 scFv (CD19 VL connected to CD19VH), wherein the αCD19 scFv shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:51.

CD19 VL_Connector_VH:

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLK MNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO: 51).

The complementarity determining regions (CDRs) of this scFv are RASQDISKYLN (CDR-L1; SEQ ID NO: 138), HTSRLHS (CDR-L2; SEQ ID NO: 139), QQGNTLPYT (CDR-L3; SEQ ID NO: 140), DYGV (CDR-H1; SEQ ID NO: 141), VIWGSETTYYNSALKS (CDR-H2; SEQ ID NO: 142), HYYYGGSYAMDY (CDR-H3; SEQ ID NO: 143). 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 shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO: 51.

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 shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO: 51 or 89.

In some embodiments, the viral particle comprises a nucleic acid encoding a signal peptide of the extracellular domain of CAR, wherein the signal peptide shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:62.

Signal peptide (human CSF2R):

ATGCTGCTGCTGGTGACCTCCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCC (SEQ ID NO: 62).

In some embodiments, the viral particle comprises a nucleic acid encoding an extracellular domain of a CAR, wherein the extracellular domain comprises an αCD19 scFv that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:63.

αCD19 scFv (VL_Linker_VH):

GACATCCAGATGACACAGACCACAAGCTCCCTGTCTGCCAGCCTGGGCGACAGAGTGACCATCTCCTGTAGGGCCTCTCAGGATATCAGCAAGTACCTGAACTGGTATCAGCAGAAGCCAGATGGCACAGTGAAGCTGCTGATCTACCACACCTCCAGGCTGCACTCTGGAGTGCCAAGCCGGTTCTCCGGATCTGGAAGCGGCACCGACTATTCCCTGACAATCTCTAACCTGGAGCAGGAGGATATCGCCACATACTTT TGCCAGCAGGGCAATACCCTGCCATATACATTCGGCGGAGGAACCAAGCTGGAGATCACCGGATCCACATCTGGAAGCGGCAAGCCAGGAAGCGGAGAGGGATCCACA AAGGGAAGGTGAAGCTGCAGGAGAGCGGACCAGGACTGGTGGCACCATCCCAGTCTCTGAGCGTGACCTGTACAGTGTCCGGCGTGTCTCTGCCTGACTACGGCGTGTCCTGGATCAGGCAGCCACCTAGGAAGGGACTGGAGTGGCTGGGCGTGATCTGGGGCTCTGAGACCACATACTATAATTCTGCCCTGAAGAGCCGCCTGACCATCATCAAGGACAACTCCAAGTCTCAGGTGTTTCTGAAGATGAATA GCCTGCAGACCGACGATACAGCCATCTACTATTGCGCCAAGCACTACTATTACGGCGGCTCCTACGCCATGGATTATTGGGGCCAGGGCACCTCCGTGACAGTGTCTAGC (SEQ ID NO: 63).

In some embodiments, the viral particle comprises a polypeptide comprising a CAR whose extracellular domain comprises an αCD19 scFv that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:79.

αCD19 scFv: MLLLVTSLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLG VIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO:79).

In some embodiments, the viral particle comprises a nucleic acid encoding an extracellular domain of a CAR, wherein the extracellular domain comprises an αCD19 scFv that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:85.

αCD19 scFv:

ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAGCTGCCACACCCTGCCTTCCTGCTGATCCCAGATATCCAGATGACACAGACCACATCCTCTCTGTCCGCCTCTCTGGGCGACAGAGTGACCATCTCTTGTAGGGCCAGCCAGGATATCTCCAAGTACCTGAACTGGTATCAGCAGAAGCCTGACGGCACAGTGAAGCTGCTGATCTACCACACCTCTAGGCTGCACAGCGGGTGCCATCCCGGTTCACG GATCCGGATCTGGAACAGACTATTCTCTGACCATCAGCAACCTGGAGCAGGAGGATATCGCCACATACTTTTGCCAGCAGGGCAATACCCTGCCATATACATTCGCGGGAGGAACCAAGCTGGAGATCACCGGAAGCACATCCGGA TCTGGCAAGCCAGGATCCGGAGAGGGATCTACAAAGGGAGAGGTGAAGCTGCAGGAGAGCGGACCAGGACTGGTGGCACCCAGCCAGTCCCTGTCTGTGACCTGTACAGTGTCTGGCGTGAGCCTGCCCGATTACGGCGTGTCCTGGATCAGACAGCCACCAAGGAAGGGACTGGAGTGGCTGGGCGTGATCTGGGGCTCTGAGACCACATACTATAATAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACA GCAAGTCCCAGGTGTTTCTGAAGATGAATAGCCTGCAGACCGACGATACAGCCATCTACTATTGCGCCAAGCACTACTATTACGGCGGCTCCTACGCCATGGATTATTGGGGCCAGGGCACCTCCGTGACAGTGAGCTCC (SEQ ID NO: 85).

In some embodiments, the viral particle comprises a polynucleotide encoding a CAR whose extracellular domain comprises an αCD19 scFv that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:89.

αCD19 scFv:

MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSAL KSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO:89).

The complementarity determining regions (CDRs) of this scFv are RASQDISKYLN (CDR-L1; SEQ ID NO: 138), HTSRLHS (CDR-L2; SEQ ID NO: 139), QQGNTLPYT (CDR-L3; SEQ ID NO: 140), DYGV (CDR-H1; SEQ ID NO: 141), VIWGSETTYYNSALKS (CDR-H2; SEQ ID NO: 142), HYYYGGSYAMDY (CDR-H3; SEQ ID NO: 143). 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 shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO: 89.

In some embodiments, the viral particle comprises a nucleic acid encoding an extracellular domain of a CAR, wherein the extracellular domain comprises an αCD19 scFv that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:94.

αCD19 scFv:

ATGCTGCTGCTGGTGACATCCCTGCTGCTGTGCGAGCTGCCACACCCAGCCTTCCTGCTGATCCCCGATATCCAGATGACCCAGACCACAAGCTCCCTGAGCGCCTCCCTGGGCGACAGGGTGACAATCTCTTGTCGGGCCAGCCAGGATATCTCCAAGTATCTGAATTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTATCACACATCTAGACTGCACAGCGGCGTGCCTTCCAGGTTTTCTGGCAGCG GCTCCGGCACCGACTACTCTCTGACAATCAGCAACCTGGAGCAGGAGGATATCGCCACCTATTTCTGCCAGCAGGGCAATACCCTGCCTTACACATTTGGCGGCGGCACAAAGCTGGAGATCACCGGCTCTAC

AAGCGGATCCGGCAAGCCAGGATCCGGAGAGGGATCTACCAAGGGAGAGGTGAAG

CTGCAGGAGAGCGGACCTGGACTGGTGGCACCATCTCAGAGCCTGTCCGTGACCTG

TACAGTGTCTGGCGTGAGCCTGCCAGATTATGGCGTGAGCTGGATCAGGCAGCCAC

CTAGGAAGGGACTGGAGTGGCTGGGCGTGATCTGGGGCTCCGAGACCACATACTAT

AACAGCGCCCTGAAGTCCCGCCTGACCATCATCAAGGACAACTCTAAGAGCCAGGT

GTTCCTGAAGATGAATTCCCTGCAGACCGACGATACAGCCATCTACTATTGCGCCAA

GCACTACTATTACGGCGGCTCTTATGCCATGGATTACTGGGGCCAGGGCACCAGCGTGACAGTGTCTAGC (SEQ ID NO: 94).

In some embodiments, the TAA or TSA is a cancer/testis (CT) antigen, e.g., 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.

In some embodiments, the TAA or TSA is a carbohydrate or ganglioside, for example, fuc-GM1, GM2 (carcinoembryonic antigen immunogenicity-1; OFA-I-1); GD2 (OFA-I-2), GM3, GD3, etc.

In some embodiments, the TAA or TSA is α-actinin-4, Bage-1, BCR-ABL, Bcr-Abl fusion protein, β-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-All, hsp70-2, KIAAO205, Mart2, Mum-1, 2 and 3, neo-PAP, myosin class I, OS-9, pml-RARα fusion protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, Gage 3, 4, 5, 6, 7, GnTV, Herv-K-mel, Lage-1, NA-88, NY-Eso-1/Lage-2, SP17, SSX-2, TRP2-Int2, gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, RAGE, GAGE-1, GAGE-2, p15(58), RAGE, SCP-1, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, 13-Catenin, Mum-1, p16, TAGE, PSMA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, 13HCG, BCA225, BTAA, CD68\KP1, CO-029, FGF-5, G250, Ga733(EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG 72, 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 γ alternative 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), galactosidase, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene) or Ral-B. Other tumor-associated antigens and tumor-specific antigens are known to those skilled in the art.

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

In some embodiments, the antigen is not considered to be a TSA or TAA, but 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, for example, a growth factor, a cytokine or an interleukin associated with angiogenesis or angiogenesis. These 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 local hypoxic environment for tumors. Thus, in some embodiments, the antigen is a hypoxia-related factor, for example, HIF-1α, HIF-1β, HIF-2a, HIF-2β, HIF-3α or HIF-3β. Tumors can also cause local damage to normal tissues, resulting in the release of molecules called damage-associated molecular pattern molecules (DAMPs, also known as alarmins). Thus, in some embodiments, the antigen is a DAMP, for example, heat shock protein, chromatin-associated protein high mobility group box 1 (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.

In some embodiments of the polypeptides described herein, the extracellular domain is linked to the transmembrane domain directly or through a linker, spacer, or hinge polypeptide sequence, such as a sequence from CD28 or a sequence from CTLA4.

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

Rapamycin-activated cell surface receptor (RACR)

In some embodiments, the viral particle comprises a polynucleotide sequence encoding a multi-compartment cell surface receptor. In some embodiments, the multi-compartment cell surface receptor is a proliferation receptor.

In some embodiments, the multi-compartmental cell surface receptor is a rapamycin-activated cell surface receptor (RACR).

In some embodiments, the multi-compartmental cell surface receptor is a chemically inducible cell surface receptor.

In some embodiments, the multi-compartment 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 multi-compartment 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.

In some embodiments, the viral particle comprises a RACR polypeptide comprising a signal peptide operably linked to FKBP12, wherein the signal peptide shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:57.

Signal peptide-FKBP12:

MPLGLLWLGLALLGALHAQAGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKL(SEQ ID NO:57)

In some embodiments, the viral particle comprises a RACR polypeptide comprising an IL-2Rγ transmembrane domain, wherein the IL-2Rγ transmembrane domain is operably linked to a cytoplasmic domain, and the IL-2Rγ transmembrane domain shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:58.

IL-2RγTM-cytoplasmic domain:

GEGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET (SEQ ID NO: 58).

In some embodiments, the viral particle comprises a RACR polypeptide comprising a P2A self-cleaving peptide that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:55.

P2A: GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:55).

In some embodiments, the viral particle comprises a RACR polypeptide comprising a signal peptide operably linked to a FRB, wherein the signal peptide shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:59.

Signal peptide-FRB:

MALPVTALLLPLALLLHAARPILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK (SEQ ID NO: 59).

In some embodiments, the viral particle comprises a RACR polypeptide comprising an IL-2Rβ transmembrane domain, wherein the IL-2Rβ transmembrane domain is operably linked to a cytoplasmic domain, and the IL-2Rβ transmembrane domain shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:60.

IL-2RβTM-cytoplasmic domain:

GKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGE ERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV (SEQ ID NO: 60).

In some embodiments, the viral particle comprises a nucleic acid encoding a signal peptide operably linked to FKBP12, wherein the signal peptide shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:70.

Signal peptide-FKBP12:

ATGCCTCTGGGACTGCTGTGGCTGGGACTGGCCCTGCTGGGCGCCCTGCACGCCCAGGCCGGCGTGCAGGTGGAGACAATCAGCCCTGGCGACGGCAGAACCTTTCCAAAGAGGGGCCAGACATGCGTGGTGCACTACACCGGCATGCTGGAGGATGGCAAGAAGTTCGACTCCTCTCGCGATCGGAACAAGCCCTTTAAGTTCATGCTGGGCAAGCAGGAAGTGATCAGAGGCTGGGAGGGAGGGCGTGGCCCAGATGT CTGTGGGCCAGAGGGCCAAGCTGACAATCAGCCCAGACTATGCATACGGAGCAACCGGACACCCTGGAATCATCCCACCACACGCCACACTGGTGTTCGATGTGGAGCTGCTGAAGCTG (SEQ ID NO: 70).

In some embodiments, the viral particle comprises a nucleic acid encoding an IL-2Rγ transmembrane domain, wherein the IL-2Rγ transmembrane domain is operably linked to a cytoplasmic domain, and the IL-2Rγ transmembrane domain shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:71.

IL-2RγTM-cytoplasmic domain:

GGCGAGGGCTCTAACACCAGCAAGGAGAATCCATTTCTGTTCGCACTGGAGGCAGTGGTCATCTCCGTGGGCTCTATGGGCCTGATCATCTCCCTGCTGTGCGTGTACTTTTGGCTGGAGAACAATGCCAAGGATCCCCACCCTGAAGAACCTGGAGGACCTGGTGACCGAGTACCACGGCAATTTCAGCGCCTGGTCCGGCGTGTCTAAGGGACTGGCAGAGTCCCTGCAGCCAGATTATTCTGAGCGGCTGTGCCTGGTG AGCGAGATCCCTCCAAAGGGAGGCGCCCTGGGAGAGGGACCAGGAGCCAGCCCCTGCAACCAGCACTCCCCTTACTGGGCCCCCCCTTGTTATACCCTGAAGCCAGAGACA (SEQ ID NO: 71).

In some embodiments, the viral particle comprises a nucleic acid encoding a P2A self-cleaving peptide that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:72.

P2A:

GGCTCTGGCGCCACCAACTTCAGCCTGCTGAAGCAAGCCGGCGACGTGGAAGAAA ACCCAGGACCA (SEQ ID NO: 72).

In some embodiments, the viral particle comprises a nucleic acid encoding a signal peptide operably linked to a FRB, wherein the signal peptide shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:73.

Signal peptide-FRB:

ATGGCACTGCCAGTGACCGCCCTGCTGCTGCCTCTGGCCCTGCTGCTGCACGCAGCCAGACCCATCCTGTGGCACGAAATGTGGCATGAAGGCCTGGAGGAGGCAAGCAGACTGTACTTTGGCGAGAGAAATGTGAAAGGAATGTTTGAGGTGCTGGAGCCTCTGCACGCCATGATGGAGAGGGGCCCTCAGACCCTGAAGGAGACATCCTTTAACCAGGCCTACGGCAGAGACCTGATGGAGGCCCAGGAGTGG TGCAGGAAGTATATGAAGAGCGGAAATGTGAAAGACCTGCTGCAGGCCTGGGATCTGTACTACCACGTGTTCCGCCGGATCTCTAAG (SEQ ID NO: 73).

In some embodiments, the viral particle includes a nucleic acid encoding an IL-2Rβ transmembrane domain, which is operably linked to a cytoplasmic domain, and the IL-2Rβ transmembrane domain shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:74.

IL-2RβTM-cytoplasmic domain:

GGCAAGGATACAATCCCTTGGCTGGGACACCTGCTGGTGGGACTGAGCGGAGCCTTTGGCTTCATCATCCTGGTGTATCTGCTGATCAACTGCAGAAATACAGGCCCATGGCTGAAGAAGGTGCTGAAGTGTAACACCCTGACCCATCCAAGTTCTTTTCTCAGCTGAGCTCCGAGCACGGCGGCGATGTGCAGAAGTGGCTGTCTAGCCCCTTTCCTTCCTCTAGCTTCAGCCCTGGAGGACTGGCACCTGAGAGA TCTCCCCACTGGAGGTGCTGGAGAGGGACAAGGTGACCCAGCTGCTGCTGCAGCAGGATAAGGTGCCAGAGCCCGCCTCCCTGTCCTCTAACCACAGCCTGACCTCCTGCTTTACAAATCAGGGCTACTTCTTTTCCACCTGCCAGACGCACTGGAGATCGAGGCATGTCAGGTGTATTTCACATACGATCCCTATAGCGAGGAGGACCCTGATGAG GGAGTGGCCGGCGCCCCAACCGGAAGCTCCCCTCAGCCACTGCAGCCACTGAGCGGAGAGGACGATGCATATTGTACATTTCCTTCCCGCGACGATCTGCTGCTGTTCTCCAAGCCTGCTGGGAGGACCATCTCCACCCAGCACCGCACCTGGAGGATCCGGGGCAGGGGAGGAGCGGATGCCTCCATCTCTGCAGGAGAGAGTGCCAAGGGACTGGGATCCACAGCCTCTGGGA CCACCTACCCCTGGAGTGCCAGACCTGGTGGATTTCCAGCCACCCCCTGAGCTGGTGCTGCGGGAGGCAGGAGAGGAGGTGCCAGACGCAGGACCTAGAGAGGGCGTGAGCTTTCCCTGGTCCAGGCCACCAGGACAGGGAGAGTTCCGCGCCCTGAACGCCCGGCTGCCCCTGAATACAGACGCCTACCTGTCTCTGCAGGAGCTGCAGGGCCAGGATCCTACCCACCTGGTG(SEQ ID NO:74)。

In some embodiments, the viral particle includes a RACR polypeptide comprising FKBP12, wherein the FKBP12 is operably linked to an IL-2Rγ domain, wherein the IL-2Rγ domain is operably linked to a P2A peptide, and wherein the FKBP12 shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:77.

RACRg(FKBP12_IL-2Rγ)_P2A：

MPLGLLWLGLALLGALHAQAGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGEGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLC LVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPETGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 77).

In some embodiments, the viral particle comprises a RACR polypeptide comprising a FRB, wherein the FRB is operably linked to an IL-2Rβ domain, wherein the IL-2Rβ domain is operably linked to a P2A peptide, and wherein the FRB shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:78.

RACRb(FRB_IL-2Rβ)_P2A:

MALPVTALLLPLALLLHAARPILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQ QDKVPEPASL SSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGPSPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALLNARLPLNTDAYLSLQELQGQDPTHLVGSGATNFSLLKQAGDVE ENPGP (SEQ ID NO:78).

In some embodiments, the viral particle includes a nucleic acid encoding FKBP12, wherein the FKBP12 is operably linked to the IL-2Rγ domain, the IL-2Rγ domain is operably linked to the P2A peptide, and the FKBP12 shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:83.

RACRg(FKBP12_IL-2Rγ)_P2A：

ATGCCACTGGGACTGCTGTGGCTGGGACTGGCCCTGCTGGGCGCCCTGCACGCCCAGGCCGGCGTGCAGGTGGAGACAATCAGCCCTGGCGACGGACGCACCTTTCCAAAGAGGGGACAGACATGCGTGGTGCACTACACCGGCATGCTGGAGGATGGCAAGAAGTTCGACAGCTCCAGAGATAGGAATAAGCCCTTTAAGTTCATGCTGGGCAAGCAGGAAGTGATCAGGGGATGGGAGGAGGGAGTGGCACAGATG TCTGTGGGACAGCGGGCCAAGCTGACAATCAGCCCAGACTATGCATACGGAGCAACCGGACACCCTGGAATCATCCCACCTCACGCCACACTGGTGTTTGATGTGGAGCTGCTGAAGCTGGGCGAGGGCAGCAACACCTCCAAGGAGAATCCA TTTCTGTTCGCCCTGGAGGCCGTGGTCATCTCTGTGGGCAGCATGGGCCTGATCATCTCCCTGCTGTGCGTGTACTTTTGGCTGGAGCGCACAATGCCACGGATCCCCACCCTGAAGAACCTGGAGGACCTGGTGACCGAGTACCACGGCAATTTCTCCGCCTGGTCTGGCGTGAGCAAGGGACTGGCAGAGTCTCTGCAGCCAGATTATAGCGAGCGGCTGTGCCTGGTGAGCGAGATCCCACCCAAGGGAGGCG CCCTGGGAGAGGGACCAGGAGCCTCCCCTTGCAACCAGCACTCTCCTTACTGGGCCCCTCCATGTTATACCCTGAAGCCAGAGACAGGCAGCGGAGCTACTAACTTCTCCCTGCTGAAGCAAGCAGGCGACGTGGAAGAAAATCCTGGACCA (SEQ ID NO: 83).

In some embodiments, the viral particle comprises a nucleic acid encoding a FRB, wherein the FRB is operably linked to an IL-2Rβ domain, wherein the IL-2Rβ domain is operably linked to P2A, and wherein the FRB shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:84.

RACRb(FRB_IL-2Rβ)_P2A:

ATGGCACTGCCAGTGACCGCCCTGCTGCTGCCTCTGGCCCTGCTGCTGCACGCAGCCAGACCCATCCTGTGGCACGAAATGTGGCATGAAGGCCTGGAGGAGGCAAGCAGGCTGTACTTTGGCGAGCGGAATGTGAAAGGAATGTTTGAAGTGCTGGAGCCTCTGCACGCCATGATGGAGAGGGGCCCTCAGACCCTGAAGGAGAGACATCCTTTAACCAGGCCTACGGCAGAGACCTGATGGAGGCCCAGGA GTGGTGCAGGAAGTATATGAAGTCTGGAAATGTGAAAGACCTGCTGCAGGCCTGGGATCTGTATTATCACGTGTTCAGGCGCATCTCT AAGGGCAAGGATACAATCCCTTGGCTGGGACACCTGCTGGTGGGACTGAGCGGAGCCTTTGGCTTCATCATCCTGGTGTATCTGCTGATCAACTGCCGCAATACAGGCCCATGGCTGAAGAAGGTGCTGAAGTGTAACCCCGACCCTTCCAAGTTCTTTTCTCAGCTGTCTAGCGAGCACGGCGGCGATGTGCAGAAGTGGCTGTCCTCTCCATTTCCCAGCTCCTCTTTCAGCCCAGGAGGACTGGCACCAGA GATCTCCCCACTGGAGGTGCTGGAGAGGGACAAGGTGACCCAGCTGCTGCTGCAGCAGGATAAGGTGCCTGAGCCAGCCTCC CTGAGCTCCAACCACTCCCTGACCTCTTGCTTTACAAATCAGGGCTACTTCTTTTCCACCTGCCAGACGCACTGGAGATCGAGGCATGTCAGGTGTATTTCACACATCGATCCCTATAGCGAGGAGGACCCTGATGAGGGAGTGGCCGGCGCCCCAACCGGATCTAGCCCACAGCCTCTGCAGCCACTGAGCGGAGAGGACGATGCATATTGTACATTTCCTTCCCGCGACGATCTGCTGCTGTTCTCTCCAAGCCTGCT GGGAGGACCAAGCCCACCTTCCACCGCACCAGGCGGCTCCGGGGCAGGGGAGGAGCGGATGCCACCCTCTCTGCAGGAG AGAGTGCCAAGGGACTGGGATCCACAGCCACTGGGACCTCCAACCCCTGGAGTGCCAGACCTGGTGGATTTCCAGCCCCCTCCAGAGCTGGTGCTGAGAGAGGCAGGAGAGGAGGTGCCTGACGCAGGACCAAGAGAGGGCGTGAGCTTTCCTTTGGTCCAGGCCACCTGGACAGGGAGAGTTCAGAGCCCTGAACGCCAGGCTGCCCCTGAATACAGACGCCTACCTGTCTCTGCAGGAGCTGCAGGGCCAG GATCCTACACACCTGGTCGGATCTGGCGCCACCAACTTTAGCCTGCTGAAGCAGGCAGGCGACGTGGAAGAGAACCCTGGACCA (SEQ ID NO: 84).

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

In some embodiments, the ligand is rapamycin.

In some embodiments, the FRB domain and FKBP together with rapamycin form a ternary complex that sequesters rapamycin in the transduced cell.

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 (rapamycin analog, rapalog). In some embodiments, the rapamycin includes a variant of rapamycin with one or more of the following modifications relative to rapamycin: demethylation, elimination or replacement of a methoxy group at C7, C42 and/or C29; elimination, derivatization or replacement of a hydroxyl group at C13, C43 and/or C28; reduction, elimination or derivatization of a ketone at C14, C24 and/or C30; replacement of a 6-membered tubular ring by a 5-membered propyl ring; and replacement of a cyclohexyl ring by an alternative substitution or a substituted cyclopentyl ring by a cyclohexyl ring. Therefore, in some embodiments, the rapamycin analog is everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus, CCI-779, C20-methallyl rapamycin, C16-(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).

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.

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 cell growth and/or survival. In some embodiments, the IL2Rγ domain and the IL2Rβ domain are heterodimerized in the presence of rapamycin to promote cell growth and/or survival. In some embodiments, the IL2Rγ domain and the IL2Rβ domain are heterodimerized in the presence of rapamycin to promote T cell activation.

Cytoplasmic FRB

In some embodiments, the vector genome includes a polynucleotide sequence that confers resistance to an immunosuppressant.

In some embodiments, polynucleotides conferring resistance to immunosuppressants are combined with rapamycin. In some embodiments, polynucleotides conferring resistance to immunosuppressants encode cytoplasmic ("naked") FRB domains. Naked FRB domains are approximately 100 amino acid domains extracted from mTOR protein kinases. 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 continue to be activated and give the transduced T cells a proliferation advantage over natural T cells.

In some embodiments, the viral particle comprises a polypeptide comprising a cytoplasmic FRB domain that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:56.

Naked FRB:

EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK (SEQ ID NO: 56).

In some embodiments, the viral particle comprises a nucleic acid encoding a cytoplasmic FRB domain that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:68.

Naked FRB:

GAGATGTGGCACGAGGGACTGGAGGAGGCAAGCAGGCTGTACTTTGGCGAGCGGAATGTGAAGGGCATGTTCGAGGTGCTGGAGCCACTGCACGCAATGATGGAGAGGGGACCACAGACCCTGAAGGAGACATCCTTCAACCAGGCATACGGAAGGGACCTGATGGAGGCACAGGAGTGGTGCCGGAAGTATATGAAGTCTGGCAATGTGAAGGACCTGCTGCAGGCCTGGGATCTGTATTACCACGTGTTTAGA AGGATCAGCAAG (SEQ ID NO:68).

In some embodiments, the viral particle comprises a polypeptide comprising a cytoplasmic FRB domain, wherein the cytoplasmic FRB domain is operably linked to the P2A peptide, and the cytoplasmic FRB domain shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:76.

Naked FRB_P2A:

MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 76).

In some embodiments, the viral particle comprises a nucleic acid encoding a cytoplasmic FRB domain, wherein the cytoplasmic FRB domain is operably linked to the P2A peptide, and the cytoplasmic FRB domain shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:82.

Naked FRB_P2A:

ATGGAGATGTGGCACGAGGGACTGGAGGAGGCAAGCAGACTGTACTTTGGCGAGAGGAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCACTGCACGCAATGATGGAGAGGGGACCACAGACCCTGAAGGAGACATCTTTCAACCAGGCATACGGAAGGGACCTGATGGAGGCACAGGAGTGGTGCCGGAAGTATATGAAGAGCGGCAATGTGAAGGACCTGCTGCAGGCCTGGGATCTGTACTATCACGTGTTTCGG AGAATCTCCAAGGGCTCTGGCGCCACCAACTTCTCCCTGCTGAAGCAGGCCGGCGATGTGGAGGAGAATCCTGGACCA (SEQ ID NO: 82).

In some embodiments, the viral particle comprises a polypeptide comprising a cytoplasmic FRB domain, wherein the cytoplasmic FRB domain is operably linked to the P2A peptide, and the cytoplasmic FRB domain shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:88.

Naked FRB_P2A:

MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLM EAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 88).

In some embodiments, the viral particle comprises a nucleic acid encoding a cytoplasmic FRB domain, wherein the cytoplasmic FRB domain is operably linked to a P2A peptide, and wherein the cytoplasmic FRB domain shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:93.

Naked FRB_P2A:

ATGGAGATGTGGCACGAGGGACTGGAGGAGGCATCCAGACTGTACTTCGGCGAGAGGAACGTGAAGGGCATGTTTGAGGTGCTGGAGCCACTGCACGCCATGATGGAGAGAGGCCCCCAGACCCTGAAGGAGACATCTTTCAACCAGGCCTATGGAAGGGACCTGATGGAGGCACAGGAGTGGTGCCGGAAGTACATGAAGAGCGGCAATGTGAAGGACCTGCTGCAGGCCTGGGATCTGTACTATCACGTGTTCCGG AGAATCAGCAAGGGCTCCGGCGCCACCAACTTTAGCCTGCTGAAGCAGGCAGGCGACGTGGAGGAGAATCCAGGACCT (SEQ ID NO:93).

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

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

In some embodiments, the ligand is rapamycin or a rapamycin analog described herein.

TGF-β double negative (TGF-βDN)

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

In some embodiments, the viral particles of the present disclosure include a polynucleotide sequence of a dominant negative TGF-β. In some embodiments, the viral particles include a polypeptide comprising a dominant negative TGF-β that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO: 91.

TGFβDN:

MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKRRR (SEQ ID NO: 91).

In some embodiments, the viral particle comprises a nucleic acid encoding a dominant negative TGF-β that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:96.

TGFβDN:

ATGGGAAGAGGACTGCTGAGGGGACTGTGGCCACTGCACATCGTGCTGTGGACCAGGATCGCCTCTACAATCCCACCCCACGTGCAGAAGAGCGTGAACAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAGTTTCCCCAGCTGTGCAAGTTCTGTGACGTGCGCTTTTCCACCTGTGATAACCAGAAGTCCTGCATGTCTAATTGTAGCATCACATCCATCTGCGAGAAGCCTCAGGAGGTGTGCGTGGCCGTG TGGCGGAAGAACGACGAGAATATCACCCTGGAGACAGTGTG CCACGATCCCAAGCTGCCTTATCACGACTTCATCCTGGAGGATGCCGCCTCTCCTAAGTGTATCATGAAGGAGAAGAAGAAGCCAGGCGAGACCTTCTTTATGTGCAGCTGTTCCTCTGACGAGTGCAACGATAATCATCTTCTCCGAGGAGTACAACACCTCTAATCCTGACCTGCTGCTGGTCATCTTTCAGGTGACAGGCATCTCCCTGCTGCCTCCACTGGGCGTGGCCATCTCTGTGATCATCATCTTTTT ATTGTTACAGAGTGAACAGGCAGCAGAAGCGCCGGCGCTAG (SEQ ID NO:96).

Vector genome

Payload plasmid

In some embodiments, the viral particles of the present disclosure include a polynucleotide sequence encoding the following in 5' to 3' order on a polycistronic transcript:

(a) MND promoter;

(b) a CAR comprising a polypeptide that binds to CD19;

(c) a cytoplasmic FRB domain or a portion thereof;

(d) RACR cell surface receptor; and

(e) WPRE sequence.

In some embodiments, the viral particles of the present disclosure include a polynucleotide sequence encoding the following in 5' to 3' order:

(a) a CAR comprising a polypeptide that binds to CD19;

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

(c) RACR cell surface receptor.

In some embodiments, the viral particle comprises a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:35.

MND Promoter:

GAACAGAGAAACAGGAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGTTGGAACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTTGAACTA ACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCTAGC (SEQ ID NO: 35).

In some embodiments, the viral particle comprises a polypeptide sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:49.

αCD19 CAR_FRB_RACR lentiviral vector:

MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSAL KSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSESKYGPPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRP VQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQ AYGRRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGSGATNFSLLKQAGDVEENPGPMPLGLLWLGLALLGALHAQAGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKF DSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGEGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPETGSGATNFSLLKQAGDVEENPGPMAL PVTALLLPLALLLHAARPILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHV FRRISKGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPS TAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV (SEQ ID NO: 49).

In some embodiments, the viral particle comprises a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:61.

αCD19 CAR_FRB_RACR lentiviral vector:

ATGCTGCTGCTGGTGACCTCCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCCGACATCCAGATGACACAGACCACAAGCTCCCTGTCTGCCAGCCTGGGCGACAGAGTGACCATCTCCTGTAGGGCCTCTCAGGATATCAGCAAGTACCTGAACTGGTATCAGCAGAAGCCAGATGGCACAGTGAAGCTGCTGATCTACCACACCTCCAGGCTGCACTCTGGAGTGCCAAGCCGGTTCTCCG GATCTGGAAGCGGCACCGACTATTCCCTGACAATCTCTAACCTGGAGCAGGAGGATATCGCCACATACTTTTGCCAGCAGGGCAATACCCTGCCATATACATTCGGCG GAGGAACCAAGCTGGAGATCACCGGATCCACATCTGGAAGCGGCAAGCCAGGAAGCGGAGAGGGATCCACAAAGGGAGGTGAAGCTGCAGGAGAGCGGACCAGGACTGGTGGCACCATCCCAGTCTCTGAGCGTGACCTGTACAGTGTCCGGCGTGTCTCTGCCTGACTACGGCGTGTCCTGGATCAGGCAGCCACCTAGGAAGGGACTGGAGTGGCTGGGCGTGATCTGGGGCACTTCTGAGACCACAT ATAATTCTGCCCTGAAGAGCCGCCTGACCATCATCATCAAGGACAACTCCAAGTCTCAGGTGTTTCTGAAGATGAATAGCCTGCAGACCGACGATACAGCCATCTACTATTGCGCC

AAGCACTACTATTACGGCGGCTCCTACGCCATGGATTATTGGGGCCAGGGCACCTCC

GTGACAGTGTCTAGCGAGTCTAAGTATGGCCCACCCTGCCCTCCATGTCCAATGTTC

TGGGTGCTGGTGGTGGTGGGAGGCGTGCTGGCCTGTTACTCCCTGCTGGTGACCGT

GGCCTTTATCATCTTCTGGGTGAAGAGAGGCAGGAAGAAGCTGCTGTATATCTTTAA

GCAGCCCTTCATGCGCCCTGTGCAGACCACACAGGAGGAGGACGGCTGCAGCTGT

CGGTTTCCAGAGGAGGAGGAGGGAGGATGCGAGCTGCGCGTGAAGTTCAGCCGGT

CCGCCGATGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTATAACGAGCTGAAT

CTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGGGACCCAG

AGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTACAATGAGCT

GCAGAAGGACAAGATGGCCGAGGCCTATTCTGAGATCGGCATGAAGGGAGAGAGG

CGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTGAGCACCGCCACAAAGG

ACACATATGATGCCCTGCACATGCAGGCCCTGCCACCTAGGGGATCTGGAGCCACCA

ACTTAGCCTGCTGAAGCAGGCAGGCGATGTGGAGGAGAATCCAGGACCTGAGATG

TGGCACGAGGGACTGGAGGAGGCAAGCAGGCTGTACTTTGGCGAGCGGAATGTGA

AGGGCATGTTCGAGGTGCTGGAGCCACTGCACGCAATGATGGAGAGGGGACCACA

GACCCTGAAGGAGACATCCTTCAACCAGGCATACGGAAGGGACCTGATGGAGGCA

CAGGAGTGGTGCCGGAAGTATATGAAGTCTGGCAATGTGAAGGACCTGCTGCAGGC

CTGGGATCTGTATTACCACGTGTTTAGAAGGATCAGCAAGGGCTCCGGCGCCACCA

ACTTCTCCCTGCTGAAGCAGGCCGGCGATGTGGAAGAAAATCCAGGACCAATGCCT

CTGGGACTGCTGTGGCTGGGACTGGCCCTGCTGGGCGCCCTGCACGCCCAGGCCGG

CGTGCAGGTGGAGACAATCAGCCCTGGCGACGGCAGAACCTTTCCAAAGAGGGGC

CAGACATGCGTGGTGCACTACACCGGCATGCTGGAGGATGGCAAGAAGTTCGACTC

CTCTCGCGATCGGAACAAGCCCTTTAAGTTCATGCTGGGCAAGCAGGAAGTGATCA

GAGGCTGGGAGGAGGGCGTGGCCCAGATGTCTGTGGGCCAGAGGGCCAAGCTGAC

AATCAGCCCAGACTATGCATACGGAGCAACCGGACACCCTGGAATCATCCCACCAC

ACGCCACACTGGTGTTCGATGTGGAGCTGCTGAAGCTGGGCGAGGGCTCTAACACC

AGCAAGGAGAATCCATTTCTGTTCGCACTGGAGGCAGTGGTCATCTCCGTGGGCTC

TATGGGCCTGATCATCTCCCTGCTGTGCGTGTACTTTTGGCTGGAGAGAACAATGCC

AAGGATCCCCACCCTGAAGAACCTGGAGGACCTGGTGACCGAGTACCACGGCAATT

TCAGCGCCTGGTCCGGCGTGTCTAAGGGACTGGCAGAGTCCCTGCAGCCAGATTAT

TCTGAGCGGCTGTGCCTGGTGAGCGAGATCCCTCCAAAGGGAGGCGCCCTGGGAG

AGGGACCAGGAGCCAGCCCCTGCAACCAGCACTCCCCTTACTGGGCCCCCCCTTGT

TATACCCTGAAGCCAGAGACAGGCTCTGGCGCCACCAACTTCAGCCTGCTGAAGCA

AGCCGGCGACGTGGAAGAAAACCCAGGACCAATGGCACTGCCAGTGACCGCCCTG

CTGCTGCCTCTGGCCCTGCTGCTGCACGCAGCCAGACCCATCCTGTGGCACGAAAT

GTGGCATGAAGGCCTGGAGGAGGCAAGCAGACTGTACTTTGGCGAGAGAAATGTG

AAAGGAATGTTTGAGGTGCTGGAGCCTCTGCACGCCATGATGGAGAGGGGCCCTCA

GACCCTGAAGGAGACATCCTTTAACCAGGCCTACGGCAGAGACCTGATGGAGGCCC

AGGAGTGGTGCAGGAAGTATATGAAGAGCGGAAATGTGAAAGACCTGCTGCAGGC

CTGGGATCTGTACTACCACGTGTTCCGCCGGATCTCTAAGGGCAAGGATACAATCCC

TTGGCTGGGACACCTGCTGGTGGGACTGAGCGGAGCCTTTGGCTTCATCATCCTGG

TGTATCTGCTGATCAACTGCAGAAATACAGGCCCATGGCTGAAGAAGGTGCTGAAG

TGTAACACCCCTGACCCATCCAAGTTCTTTTCTCAGCTGAGCTCCGAGCACGGCGG

CGATGTGCAGAAGTGGCTGTCTAGCCCCTTTCCTTCCTCTAGCTTCAGCCCTGGAGG

ACTGGCACCTGAGATCTCCCCACTGGAGGTGCTGGAGAGGGACAAGGTGACCCAG

CTGCTGCTGCAGCAGGATAAGGTGCCAGAGCCCGCCTCCCTGTCCTCTAACCACAG

CCTGACCTCCTGCTTTACAAATCAGGGCTACTTCTTTTTCCACCTGCCAGACGCACT

GGAGATCGAGGCATGTCAGGTGTATTTCACATACGATCCCTATAGCGAGGAGGACCC

TGATGAGGGAGTGGCCGGCGCCCCAACCGGAAGCTCCCCTCAGCCACTGCAGCCA

CTGAGCGGAGAGGACGATGCATATTGTACATTTCCTTCCCGCGACGATCTGCTGCTG

TTCTCTCCAAGCCTGCTGGGAGGACCATCTCCACCCAGCACCGCACCTGGAGGATC

CGGGGCAGGGGAGGAGCGGATGCCTCCATCTCTGCAGGAGAGAGTGCCAAGGGAC

TGGGATCCACAGCCTCTGGGACCACCTACCCCTGGAGTGCCAGACCTGGTGGATTT

CCAGCCACCCCCTGAGCTGGTGCTGCGGGAGGCAGGAGAGGAGGTGCCAGACGCA

GGACCTAGAGAGGGCGTGAGCTTTCCCTGGTCCAGGCCACCAGGACAGGGAGAGT

TCCGCGCCCTGAACGCCCGGCTGCCCCTGAATACAGACGCCTACCTGTCTCTGCAGGAGCTGCAGGGCCAGGATCCTACCCACCTGGTGTGA (SEQ ID NO: 61).

In some embodiments, the viral particles of the present disclosure include a polynucleotide sequence encoding the following in 5' to 3' order on a polycistronic transcript:

(a) MND promoter;

(b) a cytoplasmic FRB domain or a portion thereof;

(c) RACR cell surface receptor;

(d) a CAR comprising a polypeptide that binds to CD19; and

(e) WPRE sequence.

In some embodiments, the viral particles of the present disclosure include a polynucleotide sequence encoding the following in 5' to 3' order:

(a) a cytoplasmic FRB domain or a portion thereof;

(b) RACR cell surface receptor; and

(c) CAR, comprising a polypeptide that binds to CD19.

In some embodiments, the viral particle comprises a polypeptide sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:75.

FRB_RACR_αCD19 CAR lentiviral vector

MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGSGATNFSLLKQAGDVEENPGPMPLGLLWLGLALLGALHAQAGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAK LTISPDYAYGATGHPGIIPPHATLVFDVELLKLGEGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCL VSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPETGSGATNFSLLKQAGDVEENPGPMALPVTALLLPLALLLHAARPILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTP DPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPY SEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGPSPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLVGSGATNFSLLKQAGDVEENPGPMLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDR VTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPG SGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:75)

In some embodiments, the viral particle comprises a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:81.

FRB_RACR_αCD19 CAR lentiviral vector

ATGGAGATGTGGCACGAGGGACTGGAGGAGGCAAGCAGACTGTACTTTGGCGAGAGGAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCACTGCACGCAATGATGGAGAGGGGACCACAGACCCTGAAGGAGACATCTTTCAACCAGGCATACGGAAGGGACCTGATGGAGGCACAGGAGTGGTGCCGGAAGTATATGAAGAGCGGCAATGTGAAGGACCTGCTGCAGGCCTGGGATCTGTACTATCACGTGTTTCGG AGAATCTCCAAGGGCTCTG

GCGCCACCAACTTCTCCCTGCTGAAGCAGGCCGGCGATGTGGAGGAGAATCCTGGA

CCAATGCCACTGGGACTGCTGTGGCTGGGACTGGCCCTGCTGGGCGCCCTGCACGC

CCAGGCCGGCGTGCAGGTGGAGACAATCAGCCCTGGCGACGGACGCACCTTTCCA

AAGAGGGGACAGACATGCGTGGTGCACTACACCGGCATGCTGGAGGATGGCAAGA

AGTTCGACAGCTCCAGAGATAGGAATAAGCCCTTTAAGTTCATGCTGGGCAAGCAG

GAAGTGATCAGGGGATGGGAGGAGGGAGTGGCACAGATGTCTGTGGGACAGCGGG

CCAAGCTGACAATCAGCCCAGACTATGCATACGGAGCAACCGGACACCCTGGAATC

ATCCCACCTCACGCCACACTGGTGTTTGATGTGGAGCTGCTGAAGCTGGGCGAGGG

CAGCAACACCTCCAAGGAGAATCCATTTCTGTTCGCCCTGGAGGCCGTGGTCATCTC

TGTGGGCAGCATGGGCCTGATCATCTCCCTGCTGTGCGTGTACTTTTGGCTGGAGCG

CACAATGCCACGGATCCCCACCCTGAAGAACCTGGAGGACCTGGTGACCGAGTACC

ACGGCAATTTCTCCGCCTGGTCTGGCGTGAGCAAGGGACTGGCAGAGTCTCTGCAG

CCAGATTATAGCGAGCGGCTGTGCCTGGTGAGCGAGATCCCACCCAAGGGAGGCGC

CCTGGGAGAGGGACCAGGAGCCTCCCCTTGCAACCAGCACTCTCCTTACTGGGCCC

CTCCATGTTATACCCTGAAGCCAGAGACAGGCAGCGGAGCTACTAACTTCTCCCTGC

TGAAGCAAGCAGGCGACGTGGAAGAAAATCCTGGACCAATGGCACTGCCAGTGAC

CGCCCTGCTGCTGCCTCTGGCCCTGCTGCTGCACGCAGCCAGACCCATCCTGTGGC

ACGAAATGTGGCATGAAGGCCTGGAGGAGGCAAGCAGGCTGTACTTTGGCGAGCG

GAATGTGAAAGGAATGTTTGAAGTGCTGGAGCCTCTGCACGCCATGATGGAGAGGG

GCCCTCAGACCCTGAAGGAGACATCCTTTAACCAGGCCTACGGCAGAGACCTGATG

GAGGCCCAGGAGTGGTGCAGGAAGTATATGAAGTCTGGAAATGTGAAAGACCTGCT

GCAGGCCTGGGATCTGTATTATCACGTGTTCAGGCGCATCTCTAAGGGCAAGGATAC

AATCCCTTGGCTGGGACACCTGCTGGTGGGACTGAGCGGAGCCTTTGGCTTCATCAT

CCTGGTGTATCTGCTGATCAACTGCCGCAATACAGGCCCATGGCTGAAGAAGGTGCT

GAAGTGTAACACCCCCGACCCTTCCAAGTTCTTTTTCCAGCTGTCTAGCGAGCACG

GCGGCGATGTGCAGAAGTGGCCTTCCCTCTCCATTTCCCAGCTCCTCTTTCAGCCCAG

GAGGACTGGCACCAGAGATCTCCCCACTGGAGGTGCTGGAGAGGGACAAGGTGAC

CCAGCTGCTGCTGCAGCAGGATAAGGTGCCTGAGCCAGCCTCCCTGAGCTCCAACC

ACTCCCTGACCTCTTGCTTTACAAATCAGGGCTACTTCTTTTTCCACCTGCCAGACG

CACTGGAGATCGAGGCATGTCAGGTGTATTTCACATACGATCCCTATAGCGAGGAGG

ACCCTGATGAGGGAGTGGCCGGCGCCCCAACCGGATCTAGCCCACAGCCTCTGCAG

CCACTGAGCGGAGAGGACGATGCATATTGTACATTTCCTTCCCGCGACGATCTGCTG

CTGTTCTCTCCAAGCCTGCTGGGAGGACCAAGCCCACCTTCCACCGCACCAGGCGG

CTCCGGGGCAGGGGAGGAGCGGATGCCACCCTCTCTGCAGGAGAGAGTGCCAAGG

GACTGGGATCCACAGCCACTGGGACCTCCAACCCCTGGAGTGCCAGACCTGGTGG

ATTTCCAGCCCCCTCCAGAGCTGGTGCTGAGAGAGGCAGGAGAGGAGGTGCCTGA

CGCAGGACCAAGAGAGGGCGTGAGCTTTCCTTTGGTCCAGGCCACCTGGCAGGGA

GAGTTCAGAGCCCTGAACGCCAGGCTGCCCCTGAATACAGACGCCTACCTGTCTCT

GCAGGAGCTGCAGGGCCAGGATCCTACACACCTGGTCGGATCTGGCGCCACCAACT

TTAGCCTGCTGAAGCAGGCAGGCGACGTGGAAGAGAACCCTGGACCAATGCTGCT

GCTGGTGACCAGCCTGCTGCTGTGCGAGCTGCCACAACCCTGCCTTCCTGCTGATCCC

AGATATCCAGATGACACAGACCACATCCTCTCTGTCCGCCTCTCTGGGCGACAGAGT

GACCATCTCTTGTAGGGCCAGCCAGGATATCTCCAAGTACCTGAACTGGTATCAGCA

GAAGCCTGACGGCACAGTGAAGCTGCTGATCTACCACACCTCTAGGCTGCACAGCG

GAGTGCCATCCCGGTTCACGGATCCGGATCTGGAACAGACTATTCTCTGACCATCA

GCAACCTGGAGCAGGAGGATATCGCCACATACTTTTGCCAGCAGGGCAATACCCTG

CCATATACATTCGGCGGAGGAACCAAGCTGGAGATCACCGGAAGCACATCCGGATC

TGGCAAGCCAGGATCCGGAGAGGGATCTACAAAGGGAGAGGTGAAGCTGCAGGAG

AGCGGACCAGGACTGGTGGCACCCAGCCAGTCCCTGTCTGTGACCTGTACAGTGTC

TGGCGTGAGCCTGCCCGATTACGGCGTGTCCTGGATCAGACAGCCACCAAGGAAGG

GACTGGAGTGGCTGGGCGTGATCTGGGGCTCTGAGACCACATACTATAATAGCGCCC

TGAAGTCCCGGCTGACCATCATCAAGGACAACAGCAAGTCCCAGGTGTTTCTGAAG

ATGAATAGCCTGCAGACCGACGATACAGCCATCTACTATTGCGCCAAGCACTACTAT

TACGGCGGCTCCTACGCCATGGATTATTGGGGCCAGGGCACCTCCGTGACAGTGAG

CTCCGAGTCTAAGTATGGCCCTCCATGCCCCCCTTGTCCTATGTTCTGGGTGCTGGTG

GTGGGTGGGAGGCGTGCTGGCCTGTTACTCCCTGCTGGTGACCGTGGCCTTTATCATC

TTCTGGGTGAAGCGCGGCCGGAAGAAGCTGCTGTATATCTTTAAGCAGCCCTTCATG

AGACCTGTGCAGACCACACAGGAGGAGGACGGCTGCAGCTGTAGGGTTTCCAGAGG

AGGAGGAGGGAGGATGCGAGCTGCGCGTGAAGTTCTCTCGGAGCGCCGATGCCCC

TGCCTACCAGCAGGGACAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAG

AGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGAGACCCAGAGATGGGAGGCAA

GCCTCGGAGAAAGAACCCACAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAAG

ATGGCCGAGGCCTATTCCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGG

GACACGATGGCCTGTACCAGGGCCTGAGCACCGCCACAAAGGACACCTATGATGCCCTGCACATGCAGGCCCTGCCACCCAGGTGA (SEQ ID NO: 81).

In some embodiments, the viral particles of the present disclosure include a polynucleotide sequence encoding the following in 5' to 3' order on a polycistronic transcript:

(a) MND promoter;

(b) a cytoplasmic FRB domain or a portion thereof;

(c) a CAR comprising a polypeptide that binds to CD19;

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

(e) WPRE sequence.

In some embodiments, the viral particles of the present disclosure include a polynucleotide sequence encoding the following in 5' to 3' order:

(a) a cytoplasmic FRB domain or a portion thereof;

(b) a CAR comprising a polypeptide that binds to CD19; and

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

In some embodiments, the viral particle comprises a polypeptide sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:87.

FRB_αCD19 CAR_TGFbDN lentiviral vector

MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGSGATNFSLLKQAGDVEENPGPMLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISN LEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSESKYGPPCPPCPMFWVLVVV GGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMGRGLLRGLW PLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKRRR (SEQ ID NO: 87).

In some embodiments, the viral particle comprises a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:92.

FRB_αCD19 CAR_TGFbDN lentiviral vector

ATGGAGATGTGGCACGAGGGACTGGAGGAGGCATCCAGACTGTACTTCGGCGAGAGGAACGTGAAGGGCATGTTTGAGGTGCTGGAGCCACTGCACGCCATGATGGAGAGAGGCCCCCAGACCCTGAAGGAGACATCTTTCAACCAGGCCTATGGAAGGGACCTGATGGAGGCACAGGAGTGGTGCCGGAAGTACATGAAGAGCGGCAATGTGAAGGACCTGCTGCAGGCCTGGGATCTGTACTATCACGTGTTCCGG AGAATCAGCAAGGGCTCCGGCGCCACCAACTTTAGCCTGCTGAAGCAGGCAGGCGACGTGGAGGAGAATCCAGG

ACCTATGCTGCTGCTGGTGACATCCCTGCTGCTGTGCGAGCTGCCACACCCAGCCTT

CCTGCTGATCCCCGATATCCAGATGACCCAGACCACAAGCTCCCTGAGCGCCTCCCT

GGGCGACAGGGTGACAATCTCTTGTCGGGCCAGCCAGGATATCTCCAAGTATCTGA

ATTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTATCACACATCTA

GACTGCACAGCGGCGTGCCTTCCAGGTTTTCTGGCAGCGGCTCCGGCACCGACTAC

TCTCTGACAATCAGCAACCTGGAGCAGGAGGATATCGCCACCTATTTCTGCCAGCAG

GGCAATACCCTGCCTTACACATTTGGCGGCGGCACAAAAGCTGGAGATCACCGGCTC

TACAAGCGGATCCGGCAAGCCAGGATCCGGAGAGGGATCTACCAAGGGAGAGGTG

AAGCTGCAGGAGAGCGGACCTGGACTGGTGGCACCATCTCAGAGCCTGTCCGTGA

CCTGTACAGTGTCTGGCGTGAGCCTGCCAGATTATGGCGTGAGCTGGATCAGGCAG

CCACCTAGGAAGGGACTGGAGTGGCTGGGCGTGATCTGGGGCTCCGAGACCACATA

CTATAACAGCGCCCTGAAGTCCCGCCTGACCATCATCAAGGACAACTCTAAGAGCC

AGGTGTTCCTGAAGATGAATTCCCTGCAGACCGACGATACAGCCATCTACTATTGCG

CCAAGCACTACTATTACGGCGGCTCTTATGCCATGGATTACTGGGGCCAGGGCACCA

GCGTGACAGTGTCTAGCGAGTCCAAGTACGGCCCACCCTGCCCTCCATGTCCCATGT

TTTGGGTGCTGGTGGTGGTGGGAGGCGTGCTGGCCTGTTATTCCCTGCTGGTGACC

GTGGCCTTCATCATCTTTTGGGTGAAGCGCGGCCGGAAGAAGCTGCTGTACATCTTC

AAGCAGCCCTTCATGAGACCCGTGCAGACCACACAGGAGGAGGACGGCTGCAGCT

GTAGGTTCCCAGAGGAGGAGGAGGGAGGATGCGAGCTGAGGGTGAAGTTTTCCCG

GTCTGCCGATGCCCCTGCCTATCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGA

ATCTGGGCAGGCGCGAGGAGTACGACGTGCTGGATAAGAGGAGAGGAAGGGACCC

TGAGATGGGAGGCAAGCCAAGGCGCAAGAACCCTCAGGAGGGCCTGTATAATGAG

CTGCAGAAGGACAAGATGGCCGAGGCCTACTCCGAGATCGGCATGAAGGGAGAGC

GGAGAAGGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACCGCCACAAA

GGACACCTACGATGCACTGCACATGCAGGCCCTGCCACCTAGAGGATCTGGAGCCA

CAAACTTCAGCCTGCTGAAGCAGGCCGGCGATGTGGAGGAGAATCCTGGACCAATG

GGAAGAGGACTGCTGAGGGGACTGTGGCCACTGCACATCGTGCTGTGGACCAGGA

TCGCCTCTACAATCCCACCCCACGTGCAGAAGAGCGTGAACAATGACATGATCGTG

ACCGATAACAATGGCGCCGTGAAGTTTCCCCAGCTGTGCAAGTTCTGTGACGTGCG

CTTTTCCACCTGTGATAACCAGAAGTCCTGCATGTCTAATTGTAGCATCACATCCATC

TGCGAGAAGCCTCAGGAGGTGTGCGTGGCCGTGTGGCGGAAGAACGACGAGAATA

TCACCCTGGAGACAGTGTGCCACGATCCCAAGCTGCCTTATCACGACTTCATCCTGG

AGGATGCCGCCTCTCCTAAGTGTATCATGAAGGAGAAGAAGAAGCCAGGCGAGACC

TTCTTTATGTGCAGCTGTTCCTCTGACGAGTGCAACGATAATATCATCTTCTCCGAGG

AGTACAACACCTCTAATCCTGACCTGCTGCTGGTCATCTTTCAGGTGACAGGCATCT

CCCTGCTGCCTCCACTGGGCGTGGCCATCTCTGTGATCATCATCTTTTATTGTTACAGAGTGAACAGGCAGCAGAAGCGCCGGCGCTAG (SEQ ID NO:92).

In some embodiments, the viral particles of the present disclosure include a polynucleotide sequence encoding the following in 5' to 3' order:

(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 a polynucleotide sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:121.

In some embodiments, the viral particle comprises a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:121.

In some embodiments, the viral particles of the present disclosure include a polynucleotide sequence encoding the following in 5' to 3' order:

(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) CD3ζ; (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 same as SEQ ID NO: 122 shares a polynucleotide sequence of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity.

In some embodiments, the viral particle comprises a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:122.

Helper plasmid

In some embodiments, the viral particles of the present disclosure include a polynucleotide sequence encoding the following in 5' to 3' order on a polycistronic transcript:

(a) gap protein; and

(b) Pol protein.

In some embodiments, the viral particle comprises a gap protein amino acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:99.

Gag:

MGARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRELERFAVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALDKIEEEQNKSKKKAQQAAADTGHSNQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAA EWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTH NPPIPVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGPGHKARVLAEAMSQVTNPATIMIQKGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAPP EESFRFGEETTTPSQKQEPIDKELYPLASLRSLFGSDPSSQ(SEQ ID NO:99)

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

Pol:

FFREDLAFPQGKAREFSSEQTRANSPTRRELQVWGRDNNSLSEAGADRQGTVSFSFPQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMNLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLLTQIGCTLNFPISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKEGKISKIG PENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPH PAGLKQKKSVTVLDVGDAYFSVPLDKDFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQCSMTKILEPFRKQNPDIVIYQYMDDLYVGSDLEIGQHRTKIEELRQHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIVLPEKDSWTVNDIQKLVGKLNWASQIYAGIKVRQLCKLLRGTKALT EVVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGQGQWTYQIYQEPFKN LKTGKYARMKGAHTNDVKQLTEAVQKIATESIVIWGKTPKFKLPIQKETWEAWWTEYWQATWIPEWEFVNTPPLVKLWYQLEKEPIIGAETFYVDGAANRETKLGKAGYVTDRGRQKVVPLTDTTNQKTELQAIHLALQDSGLEVNIVTDSQYALGIIQAQPDKSESELVSQIIEQLIKKEKVYLAWVPAHKGIGGN EQVDKLVSAGIRKVLFLDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVASC DKCQLKGEAMHGQVDCSPGIWQLDCTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAGRWPVKTVHTDNGSNFTSTTVKAACWWAGIKQEFGIPYNPQSQGVIESMNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIVDIIATDIQTKELQKQITKIQNFRVYYRDSRDPV WKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMAGDDCVASRQDED(SEQ ID NO:100)

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

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

In some embodiments, the viral particles of the present disclosure include a polynucleotide sequence encoding the following in 5' to 3' order:

(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 a polynucleotide sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:131.

In some embodiments, the viral particle comprises a gag-pol nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:131.

In some embodiments, the viral particle comprises a Rev protein amino acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:102.

Rev Protein:

MAGRSGDSDDLKAVRLIKFLYQSNPPPNPEGTRQARRNRRRRWRERQRQIHSISERILSTYLGRSAEPVPLQLPPLERLTLDCNEDCGTSGTQGVGSPQILVESPTILESGAKE*(SEQ ID NO:102)

In some embodiments, the viral particle comprises a Rev nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:103.

Rev nucleic acid sequence:

ATGGCCGGCAGAAGCGGCGACAGCGACGAGGATCTGCTGAAAGCCGTGCGGCTGATCAAGTTCCTGTACCAGAGCAACCCTCCTCCTAACCCCGAGGGCACCAGACAGGCTAGACGGAACCGCAGAAGAAGGTGGCGGGAACGGCAAAGACAGATCCACTCTATCAGCGAGAGAATCCTGAGCACCTACCTGGGAAGATCCGCCGAGCCTGTCCCCCTGCAGCTGCCTCCACTGGAAAGACTGACCCTGGATTGTA ATGAGGACTGCGGCACAAGCGGAACCCAGGGCGTGGGCAGCCCCCAGATTCTGGTGGAATCCCCTACAATCCTCGAGTTCTGGCGCCAAGGAATGA (SEQ ID NO: 103)

In some embodiments, the viral particle comprises a Rev nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:125.

In some embodiments, the viral particles of the present disclosure include a polynucleotide sequence encoding the following in 5' to 3' order:

(a) RSV promoter; (b) HXB3 Rev; (c) HIV-1 polyA LTR, and a polynucleotide sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:132.

In some embodiments, the viral particle comprises a gag-pol nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:132.

Cocal Envelope Plasmid

In some embodiments, the viral particle comprises nucleic acid encoding a cocal envelope, an anti-CD3 scFv.

In some embodiments, the viral particle comprises a cocal envelope and an anti-CD3 scFv nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:128.

In some embodiments, the viral particles of the present disclosure include a polynucleotide sequence encoding the following in 5' to 3' order:

(a) MND promoter;

(b) CD8-derived signal peptide;

(c) anti-CD3 scFv;

(d) CD8-derived hinge;

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

(f) T2A;

(g) Cocal coating;

(h) WPRE; and

(i) a polyA signal, and a polynucleotide sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:128.

In some embodiments, the viral particles of the present disclosure include a polynucleotide sequence encoding the following in 5' to 3' order:

(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 a polynucleotide sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO: 129.

In some embodiments, the viral particle comprises a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:129.

In some embodiments, the viral particles of the present disclosure include a polynucleotide sequence encoding the following in 5' to 3' order:

(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 a polynucleotide sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO: 123.

In some embodiments, the viral particle comprises a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:123.

In some embodiments, the viral particles of the present disclosure include a polynucleotide sequence encoding the following in 5' to 3' order:

(a) MND promoter; (b) Cocal envelope; (c) transmembrane domain; (d) cytoplasmic tail domain; (e) WPRE; (f) BGH polyA signal, and a polynucleotide sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:130.

In some embodiments, the viral particle comprises a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:130.

Anti-CD3 plasmid

In some embodiments, the viral particle comprises an anti-CD3 nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:126.

In some embodiments, the viral particles of the present disclosure include a polynucleotide sequence encoding the following in 5' to 3' order:

(a) human CMV enhancer and CMV promoter; (b) human β-globin intron; (c) Gaussia luciferase 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 a polynucleotide sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO: 126.

In some embodiments, the viral particle comprises an anti-CD3 nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:127.

In some embodiments, the viral particles of the present disclosure include a polynucleotide sequence encoding the following in 5' to 3' order:

(a) human CMV enhancer and CMV promoter; (b) human β-globin intron; (c) Gaussia luciferase signal peptide; (d) anti-CD3 VL chain; (e) G4S linker; (f) anti-CD3 VH chain; (g) glycophorin A transmembrane domain; (h) glycophorin A cytoplasmic tail domain; (i) BGH polyA signal, and a polynucleotide sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:127.

Gene Editing

Many gene editing methods are known in the art, and other methods are constantly produced. The methods and compositions disclosed herein can deliver a variety of genetic payloads, including polynucleotides for insertion into the genome and/or gene editing system (CRISPR-Cas, meganucleases, homing endonucleases, zinc finger enzymes, etc.) of target cells. In an embodiment, polynucleotides (e.g., transgenic), enzymes and/or guide RNAs are delivered with one, two, three or more vectors of the same type (e.g., slow virus, AAV, etc.) or one, two, three or more vectors of different types (including a combination of, for example, non-viral vectors 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 are also within the scope of the present disclosure.

Targeting immune cells

Non-limiting examples of cells that can be targets of the viral particles described herein include T lymphocytes, dendritic cells (DCs), Treg cells, B cells, natural killer cells, and macrophages.

T cells

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 subsets of T cells, each with different functions. T cells can be distinguished from other lymphocytes, such as B cells and NK cells, by the presence of the T cell receptor (TCR) on 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 and beta (beta) chain. When the TCR engages with 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.

In some embodiments, the cells used in the methods provided herein are primary T lymphocytes (e.g., primary human T lymphocytes). The primary T lymphocytes used in the methods provided herein can be naive T lymphocytes or MHC-restricted T lymphocytes. In some embodiments, T lymphocytes are CD4+. In other embodiments, T lymphocytes are CD8+. In some embodiments, primary T lymphocytes are tumor infiltrating lymphocytes (TIL). In some embodiments, primary T lymphocytes have been separated from tumor biopsies or have been amplified from T lymphocytes isolated from tumor biopsies. In some embodiments, primary T lymphocytes have been separated from T lymphocytes isolated from peripheral blood, cord blood or lymph or have been amplified from the T lymphocytes. In some embodiments, relative to a specific individual, for example, the recipient of the T lymphocytes, the T lymphocytes are allogeneic. In certain other embodiments, relative to certain individuals, for example, the recipient of the T lymphocytes, the T lymphocytes are not allogeneic. In some embodiments, relative to a specific individual, for example, the recipient of the T lymphocytes, the T lymphocytes are autologous.

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

NK cells

Natural killer (NK) cells are cytotoxic lymphocytes that constitute a major component of the innate immune system. NK cells typically comprise approximately 10% to 15% of the mononuclear cell fraction in normal peripheral blood. NK cells do not express T cell antigen receptors (TCR), CD3, or surface immunoglobulin (Ig) B cell receptors, but typically express surface markers CD16 (FcγRIII) and CD56 in humans. NK cells are cytotoxic; and small granules in their cytoplasm contain special proteins, such as perforin and proteases known as granzymes. When released in close proximity to cells planned for killing, perforin forms holes in the cell membrane of target cells through which granzymes and related molecules can enter, thereby inducing apoptosis. A granzyme, granzyme B (also known as granzyme 2 and cytotoxic T lymphocyte-associated serine esterase 1), is a serine protease that is critical for rapidly inducing apoptosis of target cells in cell-mediated immune responses.

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

Among other activities, NK cells play a role in host rejection of tumors. Since many cancer cells have reduced or no MHC class I expression, the cancer cells can become targets of NK cells. Natural killer cells can be activated by cells lacking major histocompatibility complex (MHC) proteins or showing reduced levels of MHC proteins. In addition to participating in direct cytotoxic killing, NK cells also play a role in cytokine production, which may be important for controlling cancer and infection. Activated and amplified NK cells and LAK cells have been used in both ex vivo and in vivo treatments of patients with advanced cancer, where some success has been achieved in resisting bone marrow-related diseases such as leukemia; breast cancer and certain types of lymphomas.

Immune cell activation

In some embodiments, the particles are administered to a subject to cause activation of immune cells. In some embodiments, the activation of immune cells is mediated by the binding of CAR to both immune cells and cells expressing specific antigens.

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 the group consisting of CD71, CD25 and any combination thereof.

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.

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.

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

In some embodiments, administration of the viral particles to a subject reduces the number of immune cells (eg, T cells) in the G0 phase and/or increases the number of immune cells (eg, T cells) in a non-G0 phase.

In some embodiments, administration of the viral particles to a subject increases the number and/or percentage of immune cells that are metabolically competent for transduction of the vector.

In some embodiments, administration of the viral particles in a subject causes immune cells to accumulate in lymph nodes. In some embodiments, administration of the viral particles in a subject causes immune cells to accumulate at a tumor site.

In some embodiments, the viral particles are lentiviral particles. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells here are subsets of immune cells in vivo, and the subsets can be recognized by at least one antigen-specific binding domain of CAR. In some embodiments, the immune cells reside in lymph nodes.

Dosage form and dosing regimen

Virus particles

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

Viral particles can also be delivered according to viral titer (TU/mL). The amount of lentivirus injected directly is determined by total TU and can vary based on the volume that can be feasibly injected into the site and the type of tissue to be injected. In some embodiments, the viral titer delivered is about 1×10 ⁵ to 1×10 ⁶ , about 1×10 ⁵ to 1×10 ⁷ , 1×10 ⁵ to 1×10 ⁷ , about 1×10 ⁶ to 1×10 ⁹ , about 1×10 ⁷ to 1×10 ¹⁰ , about 1×10 ⁷ to 1×10 ¹¹ , or about 1×10 ⁹ to 1×10 ¹¹ TU, or more can be used per injection. In some embodiments, the virus titer delivered is about 1×10 ⁶ to 1×10 ⁷ , about 1×10 ⁶ to 1×10 ⁸ , 1×10 ⁶ to 1×10 ⁹ , about 1×10 ⁷ to 1×10 ¹⁰ , about 1×10 ⁸ to 1×10 ¹¹ , about 1×10 ⁸ to 1×10 ¹² , or about 1×10 ¹⁰ to 1×10 ¹² , or more can be used per injection. For example, a brain injection site may only allow injection of a very small volume of virus, so a high titer preparation would be preferred, using about 1×10 ⁶ to 1×10 ⁷ , about 1×10 ⁶ to 1×10 ⁸ , 1×10 ⁶ to 1×10 ⁹ , about 1×10 ⁷ to 1×10 ¹⁰ , about 1×10 ⁸ to 1×10 ¹¹ , about 1×10 ⁸ to 1×10 ¹² , or about 1×10 ¹⁰ to 1×10 ¹² or more TU per injection. However, systemic delivery can accommodate much larger TUs, and a load of about 1×10 ⁸ , about 1×10 ⁹ , about 1×10 ¹⁰ , about 1×10 ¹¹ , about 1×10 ¹² , about 1×10 ¹³ , about 1×10 ¹⁴ , or about 1×10 ¹⁵ can be delivered.

In some embodiments, the vector is administered at a dose of between about 1×10 ¹² and 5×10 ¹⁴ vector genomes (vg) per kilogram of vector (vg) (vg/kg) of the subject's total body mass. In some embodiments, the vector is administered at a dose of between about 1×10 ¹³ vg/kg and 5×10 ¹⁴ vg/kg. In some embodiments, the vector is administered at a dose of between about 5×10 ¹³ vg/kg and 3×10 ¹⁴ vg/kg. In some embodiments, the vector is administered at a dose of between about 5×10 ¹³ vg/kg and 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 ¹⁴ vg/kg, less than about 1×10 ¹⁵ vg/kg, less than about 3×10 ¹⁵ vg/kg, less than about 5×10 ¹⁵ vg/kg, or less than about 7×10 ¹⁵ vg/kg.

In some embodiments, the vector is administered at a dose of between about 1×10 ¹² and 5×10 ¹⁴ vector particles (vp) per kilogram of vector (vp) of the subject's total body mass (vp/kg). In some embodiments, the vector is administered at a dose of between about 1×10 ¹³ vp/kg and 5×10 ¹⁴ vp/kg. In some embodiments, the vector is administered at a dose of between about 5×10 ¹³ vp/kg and 3×10 ¹⁴ vp/kg. In some embodiments, the vector is administered at a dose of between about 5×10 ¹³ vp/kg and 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 3×10 ¹⁵ vp/kg, less than about 5×10 ¹⁵ vp/kg, or less than about 7×10 ¹⁵ vp/kg.

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

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

In some embodiments, B cells are depleted in the subject 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.

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

Rapamycin

(sirolimus, rapamycin) is available as an oral solution or tablet and is FDA-approved for the following indications:

-Prevent organ rejection in kidney transplants

-Limited use in kidney transplantation

-Treatment of patients with lymphangioleiomyomatosis

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

In some embodiments, rapamycin is administered at a dose between about 0.1 mg/m ² and 100 mg/m ² of the surface area of the subject. In some embodiments, the subject is a human. In some embodiments, rapamycin is administered at a dose between about 0.5 mg/m ² and 50 mg/m ^2. In some embodiments, rapamycin is administered at a dose between about 0.5 mg/m ² and 10 mg/m ^2. In some embodiments, rapamycin is administered at a dose between about 0.5 mg/m ² and 3 mg/m ^2. In some embodiments, rapamycin is administered at a dose between about 0.5 mg/m ² and 5 mg/m ^2. In some embodiments, rapamycin is administered at a dose between about 1 mg/m ² and 5 mg/m ^2. In some embodiments, rapamycin is administered at a dose between about 2 mg/m ² and 6 mg/m ^2. In some embodiments, rapamycin is administered at a dose of about 1 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 between about 2 mg/m ² and 6 mg/m ^2. In some embodiments, rapamycin is administered at a dose between about 3 mg/m ² and 9 mg/m ^2. In some embodiments, rapamycin is administered at a dose between about 4 mg/m ² and 12 mg/m ^2. In some embodiments, rapamycin is administered at a dose between about 5 mg/m ² and 15 mg/m ^2. In some embodiments, rapamycin is administered at a dose between about 6 mg/m ² and 20 mg/m ^2. In some embodiments, rapamycin is administered at a dose between about 10 mg/m ² and 50 mg/m ^2. In some embodiments, the dose of rapamycin is the total dose over a 24 hour period.

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

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

In some embodiments, after the first administration of the viral particles, the first dose of rapamycin is administered at about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 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 at about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 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 at about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 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.

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 or in a specific organ/region of the 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 the 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 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks after the first dose of rapamycin (after administration of the viral particles), 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 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months after the first dose of rapamycin (after administration of the viral particles), 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 administration of rapamycin increases the percentage of immune cells (e.g., CAR T cells) transduced by viral particles in a subject or in a specific organ/region of the 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 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 increase is assessed by the percentage of immune cells transduced by viral particles 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks after the first dose of rapamycin (after administration of viral particles), 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 the viral particles at 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months after the first dose of rapamycin (after administration of the viral particles), wherein the reference percentage is the percentage of immune cells transduced by the viral particles on the day of the first dose of rapamycin. In some embodiments, the percentage is the percentage of immune cells transduced by the viral particles to the total immune cells in the subject or in a specific organ/region of the subject. In some embodiments, the percentage is the percentage of immune cells transduced by the viral particles to the same type of immune cells (e.g., T cells) in the subject or in a specific organ/region of the subject.

Pharmaceutical compositions and formulations

The formulations and compositions of the present disclosure may include any number of viral particles in combination with optionally one or more additional agents (polypeptides, polynucleotides, compounds, etc.) formulated into a pharmaceutically acceptable or physiologically acceptable composition for administration to cells, tissues, organs or animals alone or in combination with one or more other therapeutic modalities. In some embodiments, the one or more additional agents further increase the transduction efficiency of the vector.

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

In some embodiments, compositions and formulations of viral particles used in accordance with the present disclosure can be prepared for storage by mixing viral particles having a desired degree of purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions (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 surface-active agents (surfactants), buffers, isotonic agents, salts, amino acids, sugars, stabilizers, and/or antioxidants are used in the formulations.

Suitable pharmaceutically acceptable surfactants include, but are not limited to, polyethylene-sorbitan-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.

Isotonic agents are used to provide isotonic formulations.Isotonic formulations are liquids, or by solid forms, such as the liquid of freeze-dried form reorganization, and represent some other solutions compared therewith, such as physiological saline and serum with the same tension solution.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 and any component of the group from amino acids, sugars, salts and combinations thereof.In certain embodiments, isotonic agents are usually used in a total amount of about 5mM to about 350mM.

The limiting examples of salt include cation sodium, potassium, calcium or magnesium and anion chloride, phosphate, citrate, succinate, sulfate or any combination thereof. The limiting examples of amino acid include arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline. The limiting examples of sugar according to the present invention include trehalose, sucrose, mannitol, sorbitol, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine (also referred to as "methylglucamine"), galactosamine and neuraminic acid and combinations thereof. The limiting examples of stabilizers include amino acids and sugars as described above, and commercially available cyclodextrins and dextran of any kind and molecular weight known in the art. The limiting examples of antioxidants include excipients such as methionine, benzyl alcohol or any other excipients for minimizing oxidation.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to humans. The preparation of aqueous compositions containing proteins as active ingredients is well known in the art. Typically, such compositions are prepared as injectables in the form of liquid solutions or suspensions; however, solid forms suitable for solution or suspension in liquid prior to injection can also be prepared. The formulations can also be emulsified.

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. Except in the case where any conventional media or agents are incompatible with the active ingredient, their use in pharmaceutical compositions is contemplated. Supplementary active ingredients may also be incorporated into the composition.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, etc. that are physiologically compatible, including pharmaceutically acceptable cell culture media. In some embodiments, the composition including the carrier is suitable for parenteral administration, for example, intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersants and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersants. The use of such media and medicaments for pharmaceutically active substances is well known in the art. Except for the case where any conventional media or medicaments are incompatible with the transduced cells, its use in the pharmaceutical composition of the present disclosure is contemplated.

The composition may further include one or more polypeptides, polynucleotides, vectors including the polypeptides and the polynucleotides, compounds that are formulated into pharmaceutically acceptable or physiologically acceptable solutions for administration to cells or animals alone or in combination with one or more other therapeutic modalities to improve the transduction efficiency of the vector. It should also be understood that, if desired, the compositions of the present disclosure may also be administered in combination with other agents, such as, for example, cytokines, growth factors, hormones, small molecules, or various pharmaceutically active agents. There is no practical limitation on other components that may also be included in the composition, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended therapy.

The present disclosure also provides a pharmaceutical composition, which includes 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 includes a lentiviral vector, the lentiviral vector including an expression cassette disclosed herein, for example, wherein the expression cassette includes one or more polynucleotide sequences encoding one or more chimeric antigen receptors (CAR) and variants thereof.

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

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

In various embodiments, the pharmaceutical composition contains a pharmaceutically acceptable vehicle (e.g., carrier, diluent, and excipient) for a formulation that can be injected. Exemplary excipients include poloxamer. The formulation buffer for viral vectors generally contains salts for preventing aggregation and other excipients for reducing the viscosity of viral particles (e.g., poloxamer). These can specifically be isotonic, sterile saline solutions (monosodium phosphate or disodium phosphate, sodium, potassium, calcium or magnesium chloride, etc. or mixtures of such salts) that allow the formation of injectable solutions after adding sterile water or saline according to the circumstances, or dried, especially freeze-dried compositions. In some embodiments, the formulation is stable for storage and use when frozen (e.g., below 0°C, about -60°C, or about -72°C). In some embodiments, the formulation is a frozen solution.

The formulation of the pharmaceutical compositions of the present disclosure, pharmaceutically acceptable excipients and carrier solutions are well known to those skilled in the art, as is the development of appropriate dosing and treatment regimens for using the specific compositions described herein in a variety of treatment regimens, including, for example, oral, parenteral, intravenous, intranasal, intraperitoneal, and intramuscular administration and formulation.

In some cases, it will be desirable to deliver parenterally, intravenously, intramuscularly or intraperitoneally the compositions disclosed herein, such as U.S. Patent Nos. 5,543,158, 5,641,515 and 5,399,363 (each of which is incorporated herein by reference in its entirety). Solutions of active compounds in the form of free alkali or pharmaceutically acceptable salts can be prepared in water appropriately mixed with surfactants such as hydroxypropylcellulose. Dispersants can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oils. Under normal storage and use conditions, these preparations contain preservatives that prevent the growth of microorganisms.

The pharmaceutical form suitable for injectable use comprises sterile aqueous solution or dispersion and sterile powder (U.S. Patent No. 5,466,468, which is specifically incorporated herein by reference in its entirety) for the temporary preparation of sterile injectable solution or dispersion. In all cases, the form should be sterile and should be fluid to the extent that there is easy injection. The carrier should be stable under manufacturing and storage conditions and should be preserved for the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.), a suitable mixture thereof and/or vegetable oil. Suitable fluidity can be maintained, for example, by using coatings such as lecithin, by maintaining the required particle size in the case of a dispersant and by using a surfactant. The prevention of the effect of microorganisms can be promoted by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In certain embodiments, isotonic agents, such as sugar or sodium chloride, are added. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

For example, for parenteral administration in aqueous solution, if necessary, the solution should be properly buffered, and first make the liquid diluent isotonic with enough saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, in view of the disclosure, those skilled in the art will know the sterile aqueous medium that can be adopted. For example, a dose can be dissolved in 1ml isotonic NaCl solution, and added to 1000ml subcutaneous infusion fluid or injected at the proposed infusion site (see, for example, "Remington: Pharmaceutical Science and Practice (Remington:TheScience and Practice of Pharmacy)", the 20th edition of Baltimore, Maryland: Lippincott Williams and Wilkins Publishing Company (Baltimore, Md.: Lippincott Williams & Wilkins), 2005). Some changes in dosage will inevitably occur according to the condition of the subject being treated. The person responsible for administration will determine the appropriate dosage for individual subjects in any case. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by the FDA's Office of Biologics Standards.

In some embodiments, the present disclosure provides formulations or compositions suitable for delivery of viral vector systems (ie, viral-mediated transduction), including but not limited to retroviral (eg, lentiviral) vectors.

Combination therapy

The present disclosure further contemplates that one or more additional agents that enhance the transduction efficiency of the viral particles may be used.

In some embodiments, the method further comprises administering one or more anti-cancer therapies to the subject.

In some embodiments, the one or more anti-cancer therapies are selected from the group consisting of autologous stem cell transplantation (ASCT), radiation, surgery, chemotherapeutic agents, immunomodulators, and targeted cancer therapies.

In some embodiments, the one or more anticancer therapies are selected from the group consisting of lenalidomide, thalidomide, pomalidomide, bortezomib, carfilzomib, elotuzumab, ixazomib, melphalan, dexamethasone, vincristine, cyclophosphamide, hydroxydaunorubicin, prednisone, rituximab, imatinib, dasatinib, nilotinib, bosutinib, ponati nib), bafetinib, saracatinib, tozasertib or danusertib, cytarabine, daunorubicin, idarubicin, mitoxantrone, hydroxyurea, decitabine, cladribine, fludarabine, topotecan, etoposide 6-thioguanine, corticosteroids, methotrexate, 6-mercaptopurine, azacitidine, arsenic trioxide, and all-trans retinoic acid, or any combination thereof.

disease

The present disclosure also provides a viral particle that can be used to treat a disease, disorder or condition. In some embodiments, the disease or condition is cancer. In some embodiments, the cancer is a hematological malignancy or a solid tumor. In some embodiments, the subject is relapsed or refractory to treatment with a previous anticancer therapeutic agent.

In some embodiments, the therapeutic application of the viral particles disclosed herein is the treatment of CD19-expressing B-cell malignancies for which other non-CAR T cell treatment options have failed.

Hematologic malignancies

In some embodiments, the cancer is a hematological malignancy.

In some embodiments, the hematological malignancy is lymphoma, B-cell malignancy, Hodgkin's lymphoma, non-Hodgkin's lymphoma, DLBLC, FL, MCL, marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), CLL, ALL, AML, Waldenstrom's Macroglobulinemia or T-cell lymphoma.

In some embodiments, the solid tumor is lung cancer, liver cancer, cervical cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer, melanoma, glioblastoma, prostate cancer, esophageal cancer or gastric cancer. WO2019057124A1 discloses cancers suitable for treatment with T cell redirection therapeutic agents that bind to CD19.

In some embodiments, the hematological malignancies include multiple myeloma, smoldering multiple myeloma, monoclonal gammopathy of undetermined significance (MGUS), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma (BL), follicular lymphoma (FL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, plasma cell leukemia, light chain amyloidosis (AL), precursor B-cell lymphoblastic leukemia, precursor B-cell lymphoblastic leukemia, acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic lymphocytic leukemia (CLL), B-cell malignancies, chronic myeloid leukemia (CML), hairy cell leukemia (HCL), blastic plasmacytoid dendritic cell neoplasm, Hodgkin lymphoma, non-Hodgkin lymphoma, marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), plasma cell leukemia, anaplastic large cell lymphoma (ALCL), leukemia or lymphoma.

In some embodiments, the at least one genetic abnormality is a translocation between chromosomes 8 and 21, a translocation or inversion of chromosome 16, a translocation between chromosomes 15 and 17, a change in chromosome 11, or a mutation in fin-related tyrosine kinase 3 (FLT3), nuclear phosphoprotein (NPM1), isocitrate dehydrogenase 1 (IDH1), isocitrate dehydrogenase 2 (IDH2), DNA (cytosine-5)-methyltransferase 3 (DNMT3A), CCAAT/enhancer binding protein alpha (CEBPA), U2 small nuclear RNA auxiliary factor 1 (U2AF1), enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), maintenance of chromosome structure 1A (SMC1A), or maintenance of chromosome structure 3 (SMC3).

In some embodiments, the hematological malignancy is ALL.

In some embodiments, the ALL is B-cell lineage ALL, T-cell lineage ALL, adult ALL, or pediatric ALL.

In some embodiments, the subject with ALL has the Philadelphia chromosome or is resistant or has acquired resistance to treatment with a BCR-ABL kinase inhibitor.

Ph chromosome is present in about 20% of adults with ALL and a small percentage of children with ALL, and is associated with poor prognosis. When recurring, patients with Ph+ positive ALL can be in a tyrosine kinase inhibitor (TKI) regimen, and therefore may be resistant to TKI. Therefore, the method described herein can be applied to subjects with resistance to selective or partially selective BCR-ABL inhibitors. Exemplary BCR-ABL inhibitors are, for example, imatinib, dasatinib, nilotinib, bosutinib, ponatinib, bafatinib, sacatinib, tauzacer or darusher.

In some embodiments, the subject has ALL associated with t(v;11q23)(MLL rearranged), t(1;19)(q23;p13.3); TCF3-PBX1(E2A-PBX1), t(12;21)(p13;q22); ETV6-RUNX1(TEL-AML1) or t(5;14)(q31;q32); IL3-IGH chromosomal rearrangements.

Chromosomal rearrangements can be identified using well-known methods, such as fluorescence in situ hybridization, karyotyping, pulsed-field gel electrophoresis or sequencing.

In some embodiments, the hematological malignancy is smoldering multiple myeloma, MGUS, ALL, DLBLC, BL, FL, MCL, Waldenstrom's macroglobulinemia, plasma cell leukemia, AL, precursor B-cell lymphoblastic leukemia, precursor B-cell lymphoblastic leukemia, myelodysplastic syndrome (MDS), CLL, B-cell malignancies, CML, HCL, blastic plasmacytoid dendritic cell neoplasm, Hodgkin lymphoma, non-Hodgkin lymphoma, MZL, MALT, plasma cell leukemia, ALCL, leukemia, or lymphoma.

In some embodiments, the cancer is diffuse large B-cell lymphoma (DLBCL). In some embodiments, the cancer is Burkitt's large B-cell lymphoma (B-LBL). In some embodiments, the cancer is follicular lymphoma (FL). In some embodiments, the cancer is chronic lymphocytic leukemia (CLL). In some embodiments, the cancer is acute lymphocytic leukemia (ALL). In some embodiments, the cancer is mantle cell lymphoma (MCL).

Solid Tumors

In some embodiments, the cancer is a solid tumor.

In some embodiments, the solid tumor is prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), liver cancer, cervical cancer, colon cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, melanoma, esophageal cancer, gastric cancer, stomach cancer, kidney cancer, bladder cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma, head and neck cancer, glioma, glioblastoma, colorectal cancer, thyroid cancer, epithelial cancer, or adenocarcinoma.

In some embodiments, the prostate cancer is recurrent prostate cancer. In some embodiments, the prostate cancer is refractory prostate cancer. In some embodiments, the prostate cancer is malignant prostate cancer. In some embodiments, the prostate cancer is castration-resistant prostate cancer.

definition

Unless otherwise defined, all terms used herein (including technical terms and scientific terms) have the same meaning as the meaning commonly understood by those skilled in the art to which the present disclosure belongs. It will be further understood that terms, such as those defined in common dictionaries, should be interpreted as having the same meaning as that in the context of the present application and the related art, and unless clearly defined as such in this article, should not be interpreted as idealized or over-formal meaning. The terms used in the specification are only for the purpose of describing specific embodiments, and are not intended to be restrictive. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In the case of a conflict of terms, this specification shall prevail.

In the context of two or more nucleic acid or polypeptide sequences, the term "identical" or "identity" percentage refers to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides (i.e., when compared and aligned for maximum correspondence over a comparison window or designated region, sharing at least about 80% identity with a reference sequence over a designated region, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, as measured using the following sequence comparison algorithm or by manual alignment and visual inspection). Such sequences are then referred to as "substantially identical". This definition also relates to the complement of a particular sequence. In some embodiments, identity is present over a region of at least about 25 amino acids or nucleotides in length, e.g., over a region of 50, 100, 200, 300, 400 amino acids or nucleotides in length, or over the full-length reference sequence.

In order to carry out sequence comparison, usually a sequence serves as a reference sequence compared with a test sequence. When using a sequence comparison algorithm, test sequence and reference sequence are input into a computer, subsequence coordinates are specified (if necessary), and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. Then, the sequence comparison algorithm calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the program parameters. In certain embodiments, BLAST and BLAST 2.0 algorithms and default parameters are used.

As used herein, a "comparison window" comprises reference to any number of segments of consecutive positions, the number being selected from the group consisting of 20 to 600, typically about 50 to about 200, more typically about 100 to about 150, wherein a sequence can be compared to a reference sequence of the same number of consecutive positions after the two sequences are optimally aligned. Methods of sequence alignment for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), the search for similarity method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Avenue, Madison, Wisconsin), and the like. Dr., Madison, WI) or manual alignment and visual inspection (see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology (1995 Supplement)). Examples of algorithms suitable for determining sequence identity and sequence similarity percentages are BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Journal of Molecular Biology 215:403-410 (1990) and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1977), respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (on the World Wide Web ncbi.nlm.nih.gov/).

An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid has immunological cross-reactivity with antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is generally substantially identical to a second polypeptide, for example where the two peptides differ only in conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent hybridization conditions. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences.

As used in the description of the invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

Also as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination when interpreted in the alternative (or).

As used herein, "administration" refers to local and systemic administration, for example, including enteral, parenteral, pulmonary and topical/transdermal administration. The route of administration of the drug component (e.g., carrier) used in the methods described herein includes, for example, oral (oral (P.O.)) administration, nasal administration or inhalation administration, administration in the form of a suppository, topical contact, transdermal delivery (e.g., via a transdermal patch), intrathecal (IT) administration, intravenous ("iv") administration, intraperitoneal ("ip") administration, intramuscular ("im") administration, intralesional administration or subcutaneous ("sc") administration, or implanting a sustained release device, e.g., a micro-osmotic pump, a reservoir formulation, etc., into a subject. Administration can be carried out by any route, including parenteral and transmucosal (e.g., oral, nasal, vaginal, rectal or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intrarenal, intraurethral, intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, iontophoresis, and intracranial, etc. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.

The terms "systemic administration" and "systemically administered" refer to a method of administering a pharmaceutical ingredient or composition to a mammal so that the pharmaceutical ingredient or composition is delivered to a site in the body, including a target site of drug action, via the circulatory system. Systemic administration includes, but is not limited to, oral, intranasal, rectal, and parenteral (e.g., other than through the digestive tract, such as intramuscular, intravenous, intraarterial, transdermal, and subcutaneous) administration.

The term "co-administration" or "simultaneous administration", when used, for example, with respect to a pharmaceutical ingredient (e.g., a carrier) and/or an analog thereof and another active agent (e.g., a multispecific antibody), refers to the administration of a pharmaceutical ingredient and/or an analog and an active agent so that both can achieve a physiological effect simultaneously. However, the two agents do not need to be administered simultaneously. In some embodiments, the administration of one agent may precede the administration of the other. Simultaneous physiological effects do not necessarily require that the two agents be present in the circulation at the same time. However, in some embodiments, co-administration typically results in the presence of two agents in the body (e.g., in plasma) at a significant fraction (e.g., 20% or more, e.g., 30% or 40% or more, e.g., 50% or 60% or more, e.g., 70%, or 80% or 90% or more) of their maximum serum concentrations at any given dose.

The term "effective amount" or "pharmaceutically effective amount" refers to the amount and/or dosage and/or dosage regimen of one or more pharmaceutical ingredients (eg, carrier) required to produce the desired result.

The phrase "reason for administration" refers to the actions taken by a medical professional (e.g., a physician) or a person controlling the medical care of a subject to control the administration of the agent/compound at issue to a subject and/or to permit the administration of the agent/compound. The reason for administration may involve diagnosis and/or determination of an appropriate therapeutic agent or prophylactic regimen, and/or prescribing a particular agent/compound for a subject. Such prescribing may include, for example, drawing up a prescription form, annotating a medical record, and the like.

As used herein, the terms "treating" and "treatment" refer to delaying the onset of the disease or condition to which the term applies, or one or more symptoms of such disease or condition, slowing or reversing the progression of the disease or condition or its symptoms, reducing the severity of the disease or condition or its symptoms, or alleviating or preventing the disease or condition or its symptoms. The terms "treating" and "treatment" also include preventing, alleviating, ameliorating, reducing, inhibiting, eliminating and/or reversing one or more symptoms of the disease or condition.

The term "alleviate" refers to reducing or eliminating one or more symptoms of the pathology or disease, and/or reducing the incidence or delaying the onset of one or more symptoms of the pathology or disease or reducing its severity and/or preventing the pathology or disease. In some embodiments, the reduction or elimination of one or more symptoms of the pathology or disease can include, for example, a measurable and sustained reduction in tumor volume.

As used herein, the phrase "consisting essentially of refers to the genus or species of active agent recited in the method or composition, and may further include other agents that themselves are not substantially active for the recited indication or purpose.

The terms "subject", "individual" and "patient" refer interchangeably to mammals, preferably humans or non-human primates, but also to domestic mammals (e.g., dogs or cats), laboratory mammals, and agricultural mammals. In various embodiments, the subject can be a human (e.g., an adult male, an adult female, an adolescent male, an adolescent female, a male child, a female child).

As used herein, the term "viral particle" refers to a macromolecular complex capable of delivering a foreign nucleic acid molecule into a cell independent of another agent. The particle can be a viral particle or a non-viral particle. Viral particles include retroviral particles and lentiviral particles. Non-viral particles are limited to liposomes, nanoparticles, and other encapsulation systems for delivering polynucleotides into cells.

The abbreviation "α" or "anti" before the gene name refers to an antibody or an antigen-binding fragment of an antibody (such as scFv) that specifically binds to a target. For example, αCD19 refers to an anti-CD19 antibody or an antigen-binding fragment thereof, and αCD3 refers to an anti-CD3 antibody or an antigen-binding fragment thereof.

As used herein, the term "expression cassette" or "vector genome" refers to a DNA fragment that is capable of driving, in an appropriate setting, the expression of a polynucleotide ("transgene" or "payload") encoding a polypeptide (e.g., a chimeric antigen receptor) incorporated into the expression cassette. When introduced into a host cell, the expression cassette is particularly capable of directing the cell's machinery to transcribe the transgene into RNA, which is then typically further processed and ultimately translated into a polypeptide. The expression cassette may be included in a particle (e.g., a viral particle). Typically, the term expression cassette does not include the polynucleotide sequences of 5' to 5' ITR and 3' to 3' ITR.

The term "transgene" or "payload" refers to the nucleic acid itself that is transferred. The transgene can be a naked nucleic acid molecule (such as a plasmid) or RNA. The transgene can include a polynucleotide encoding one or more polypeptides (e.g., a chimeric antigen receptor). The transgene can include a polynucleotide encoding one or more heterologous proteins (e.g., a chimeric antigen receptor), one or more capsid proteins, and other proteins necessary for transducing the polynucleotide into the target cell.

The term "derived" is used to indicate that a cell has been obtained from its biological source and grown or otherwise manipulated in vitro (eg, cultured in a growth medium to expand a population and/or generate a cell line).

The term "transduction" refers to the introduction of nucleic acid into a cell or host organism by particles (e.g., lentiviral particles). Therefore, the introduction of a transgene into a cell by a viral particle can be referred to as the "transduction" of a cell. The transgene may or may not be integrated into the genomic nucleic acid of the transduced cell. If the introduced transgene becomes integrated into the nucleic acid (genomic DNA) of the recipient cell or organism, the transgene can be stably maintained in the cell. Alternatively, the introduced transgene can be present in the recipient cell or host organism extrachromosomally or only transiently. Therefore, "transduced cells" refer to cells into which a transgene is introduced by means of transduction. Therefore, "transduced" cells are cells introduced into a polynucleotide.

The term "transduction efficiency" is the expression of the proportion of cells that express or transduce a transgene when a cell culture is contacted with particles. In some embodiments, efficiency can be expressed as the number of cells that express a transgene when a given number of cells is contacted with a given number of particles. In some embodiments, "relative transduction efficiency" is the proportion of cells transduced by a given number of viral particles under one condition relative to the proportion of cells transduced by the same number of particles under another condition involving a similar number of cells of the same cell type. Relative transduction efficiency is most often used to compare the effects of a transduction efficiency modulator on cells and/or animals treated with or without the modulator.

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All publications and patents mentioned herein are hereby incorporated by reference in their entirety, as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In the event of a conflict, the present application, including any definitions herein, will control. However, reference to any reference, article, publication, patent, patent publication, and patent application cited herein is not and should not be taken as an admission or any form of suggestion that it constitutes valid prior art or forms part of the common general knowledge in any country in the world.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Examples

The following examples are presented in order to provide one of ordinary skill in the art with a description of how the compositions and methods described herein may be used, prepared, and evaluated, and are intended to be fully illustrative of the invention and are not intended to be limiting as to the scope of the invention.

Example 1: Lentiviral particle production and in vitro transduction

To generate lentiviral particles, a VT103 transgenic plasmid expressing mCherry (a red fluorescent protein derived from Discosoma) instead of a chimeric antigen receptor (CAR) and containing a rapamycin-activated cytokine receptor (RACR) was co-transfected into 293 cells with an envelope plasmid encoding Cocal G protein and a membrane-tethered anti-CD3 antibody.

Virus production

1.2e6 293T cells were seeded into TC-treated 6-well plates in a total volume of 2.5 ml complete DMEM medium. After 24 hours, the cells were transfected. (The protocol is written for 1 well of a 6-well plate; all reagents should be at room temperature)

The following DNA was added to 500ul serum-free OptiMEM ^TM medium: 2ug transfer plasmid, 1ug Gag/pol plasmid, 1ug REV plasmid, 1ug Cocal envelope plasmid. Then 15ul (15ug) PEI was added to the medium/DNA mixture. The mixture was mixed evenly and incubated at room temperature for 20 minutes. The medium/DNA/PEI mixture was added to 2.5ml fresh complete DMEM medium. The inoculation medium in the well containing 293T was removed and replaced with fresh medium containing transfection reagent and placed in a humidified incubator at 37°C. After 48 hours, the supernatant was collected and filtered through a 0.45um PVDF filter. The supernatant was concentrated using an Amicon-Ultra 15 100K column and centrifuged at 3000xg for 30 minutes at 4°C. The virus was then stored at 4°C before use.

293T transduction titer

1e5 293T cells were inoculated into 12-well plates treated with TC in 1ml complete DMEM medium. After 24 hours, the empty wells were counted 3X to calculate the titer. The virus was then added to the wells in the following amounts: 2ul, 1ul, 0.5ul, 0.2ul, 0.1ul, 0.05ul virus per well. The virus was diluted 1:100 and then added to the 293T cells. After 3 days, the 293T cells were collected for analysis by flow cytometry. The medium was removed, the cells were washed in PBS, and then the cells were washed in trypsin and incubated in a 37°C incubator for about 3-5 minutes. The cells were resuspended in 1ml FACS buffer and about 100-200ul were added to a 96-well V-bottom plate. Flow cytometry analysis was performed for mCherry expression.

293T titer calculation:

TU/ml = (number of cells at transduction x mCherry + % x 100) / (vector volume in ul x 1000)

PBMC transduction and staining for flow cytometry

PBMCs were thawed, resuspended in 10 ml RPMI complete medium, and PBMCs were diluted to 1e6 cells/ml in complete RPMI medium. IL-2 was added to a final concentration of 50 U/ml. PBMCs were divided into 3x groups of 15 ml each for different stimulations:

Group #1 - blinatumomab was added to a final concentration of 1 ng/ml

Group #2 - Add 15e6 αCD3/αCD28 Dynabeads

Group #3 - IL-2 only

For each group, 5ml of stimulation solution was added to 3x wells in a 6-well plate that was not TC treated, and incubated at 37°C for 48 hours. The beads were removed from the applicable wells, and the cells were counted and pelleted. The cells were then resuspended in 1e6 cells/ml of fresh complete RPMI culture medium with a final concentration of 50U/ml IL-2. 1ml (1e6 cells) was added to the wells of a 12-well plate that was not TC treated. 5ul and 25ul Amicon concentrated preparations were added to the plate, and then placed in a 37°C incubator.

5ul = multiplicity of infection (MOI) ~ 0.25

25ul＝MOI～1

After 4 days, cells are resuspended in the hole in 96-well V bottom plates with 200ul.Then cells are washed with 200ul FACS buffer.Cell pellet is resuspended in the 100ul PBS containing Live/Dead ^TM stain (1:1000), and incubated 20 minutes at 4°C, then washed again in 200ul FACS buffer.Cell is resuspended in 50ul surface antibody mixture (anti-CD3-PerCP, anti-CD19-FITC, anti-CD56-APC and anti-CD25-BV421), incubated 20 minutes at 4°C, washed in 200ul FACS buffer, and resuspended in 100ul FACS buffer, and analyzed by flow cytometry.

Results and Conclusions

As shown in Figure 1, lentiviral particles packaged with the αCD3-Cocal envelope plasmid (SEQ ID NO: 129) were successfully packaged and exhibited similar 293T titers compared to vectors produced with conventional Cocal envelopes. Minimal transduction was observed with vectors pseudotyped with a "blinded" Cocal envelope containing the R354Q mutation. This was expected because the R354Q mutation has been shown to restrict binding of the VSV-G (approximately 70% homologous to Cocal) envelope protein to the low-density lipoprotein receptor.

To assess vector-induced activation and transduction, concentrated vector preparations were added to human PBMCs stimulated with IL-2 alone, blinatumomab + IL-2, or anti-CD3/anti-CD28 beads + IL-2, as described above. 4 days later, PBMCs were harvested and stained for flow cytometry analysis as described above. αCD3-Cocal pseudotyped vectors activated and transduced unstimulated human PBMCs.

CD25 expression of T cells was analyzed (Fig. 2). Blinatumomab and αCD3/αCD28 beads effectively activated T cells, as demonstrated by greatly increased CD25 expression. αCD3-Cocal, but not conventional Cocal pseudotyped vectors (Fig. 2B), was also able to activate unstimulated PBMCs to a similar degree to that of blinatumomab alone (Fig. 2C). The increased levels of activation induced by the αCD3-Cocal envelope corresponded to the increased levels of mCherry transgene expression in unstimulated PBMCs. Although similar levels of activation were detected on T cells stimulated with blinded Cocal envelopes, high levels of mCherry transgene expression were not detected (Fig. 2D). αCD3-Cocal pseudotyped lentiviral vectors activated unstimulated PBMCs and caused successful transduction and transgene expression.

Off-target transduction in culture was examined by mCherry expression on NK cells (live, CD3-, CD19-, CD56+) and B cells (live, CD3-, CD56-, CD19+) (data not shown). Very low levels of mCherry expression were seen in both NK cells and B cells, suggesting that off-target transduction is a very rare event in these cultures.

This data shows that the αCD3-Cocal vector can be successfully packaged at 293T titers similar to those of vectors expressing conventional Cocal envelopes. When added to unstimulated human PBMCs, the vector induces T cell activation, as shown by increased CD25 expression and transduction. Unstimulated PBMCs transduced with the αCD3-Cocal vector had the highest transduction levels. The results show that viral vector particles expressing αCD3-Cocal effectively activate and transduce unstimulated PBMCs.

Vectors pseudotyped with αCD3-Cocal containing a "blinding" mutation located in the Cocal coding sequence (R345Q) were analyzed for their ability to transduce unstimulated PBMCs. Although activation was induced by these particles, the amount of mCherry expression was very low, indicating that this vector transduces T cells at low levels.

Other cells in the PBMC cultures were analyzed for expression of mCherry as an indication of off-target transduction by the vector. No transduction of NK cells or B cells was observed.

The data show that particles containing the αCD3-Cocal envelope activate and transduce unstimulated PBMCs in vitro and, therefore, these particles are suitable candidates for in vivo CAR T cell manufacturing.

Example 2: Comparison of Cocal vs. αCD3-Cocal vs. αCD3-Cocal (blinded) viral particle envelope and in vitro transduction of T cells

This study evaluated the in vitro transduction of T cells on the surface of lentiviral particles engineered with Cocal glycoprotein and a packaged transgene containing αCD19 CAR plus RACR components. The experimental readout was the transduction of unstimulated PBMCs as measured by flow cytometry by CAR+T cells. This study also incorporated staining with an anti-2A flow reagent specific for the 2A cleavage peptide in the vector transgene.

Another objective of this study was to examine vector particle production using two envelope plasmid backbones. One envelope plasmid backbone used the CMV promoter and the other envelope plasmid backbone used the MND promoter. The plasmid containing the MND promoter also had a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) sequence. The WPRE sequence was included to enhance transcription of the viral particle payload.

In addition to examining the % CAR+ T cells, the ability of viral particles to secrete cytokines after stimulation with CD19+ Raji cells was also analyzed.

PBMC transduction and staining for CAR+ cells by flow cytometry

PBMCs were thawed, resuspended in 10 ml RPMI complete medium, and PBMCs were diluted to 1e6 cells/ml in complete RPMI medium. IL-2 was added to a final concentration of 50 U/ml. PBMCs were divided into 2x groups for different stimulations:

Group #1 - blinatumomab was added to a final concentration of 1 ng/ml

Group #2 - IL-2 only

For each group, 5 ml of stimulation solution was added to a 6-well plate that was not TC treated and incubated at 37°C for 3 days. The cells were counted and pelleted. The cells were then resuspended in 1e6 cells/ml of fresh complete RPMI medium with a final concentration of 50 U/ml of IL-2. 1 ml (1e6 cells) was added to the wells of a 24-well plate that was not TC treated. 100 ul of Amicon concentrated preparation was added to the plate and then placed in a 37°C incubator.

After 4 days, rapamycin was added to a final concentration of 10 nM. After 11 days (15 days after the virus), the cells were mixed and 200ul was added to the wells in the 96-well V-bottom plate. The cells were then washed with 200ul FACS buffer. The cell pellet was resuspended in 100ul PBS containing Live/Dead ^TM stain (1:1000) and incubated at 4°C for 20 minutes, followed by another wash in 200ul FACS buffer. The cells were resuspended in 50ul FACS buffer + CD19-FITC conjugate (2ug/ml), incubated at 4°C for 20 minutes, washed in 200ul FACS buffer, resuspended in 100ul BD Cytofix/Cytoperm ^TM , and incubated at 4°C for 20 minutes.

The cells were then washed in 1x BD Biosciences Perm/Wash ^™ buffer ("Perm Wash"), resuspended in 50 ul 1x Perm Wash with anti-2A-af647 (1:100), incubated at 4°C for 20 minutes, washed in 1x Perm Wash buffer, resuspended in 100 ul FACS buffer, and analyzed by flow cytometry.

Raji stimulation for cytokine production

13 days after transduction, 250ul PBMCs were added to 100,000 Raji cells in 96-well V-bottom plates. Cells were pelleted and resuspended in 100ul cell stimulation medium containing Golgi inhibitors Brefeldin A (1:1000) and Monensin (1:1000). Cells were briefly centrifuged into pellets, incubated for 5 hours, washed with 200ul FACS buffer, resuspended in 100ul PBS containing Live/Dead ^TM stain (1:1000), incubated at 4°C for 20 minutes, washed in 200ul FACS buffer, resuspended in 50ul surface antibody mixture diluted in FACS buffer, and incubated at 4°C for 20 minutes.

Diluted surface antibody cocktail used:

a. Anti-CD3-Percp 1:100

b. Anti-CD4-BV650

c. Anti-CD8-BV605

After incubation, cells were washed in 200ul FACS buffer, resuspended in 100ul BD Biosciences Cytofix/Cytoperm ^™ , incubated at 4°C for 20 minutes, washed in 1x Perm Wash buffer, resuspended in 50ul intracellular antibody cocktail diluted in 1x PermWash, and incubated at 4°C for 20 minutes.

Diluted intracellular antibody cocktail used:

a. Anti-2A-af647

b. Anti-IL-2-PEcy7

c. Anti-IFNg-BV421

d. Anti-GzmB-FITC

e. Anti-TNFa-PE

After incubation, cells were washed in 1x Perm Wash buffer, resuspended in 100 ul FACS buffer, and analyzed by flow cytometry.

Results and Conclusions

Cells were transduced with the following plasmids:

1. VT103 transgenic plasmid expressing mCherry instead of anti-CD19 scFv CAR as well as MND promoter and Cocal surface envelope (SEQ ID NO: 10) protein (VT103 MND-Cocal)

2. A transgenic plasmid containing the αCD19 CAR plus the RACR components and the MND promoter and the Cocal surface envelope (SEQ ID NO: 10) protein (RACR-αCD19 MND-local)

3. A transgenic plasmid containing the αCD19 CAR plus the RACR components as well as the CMV promoter and the Cocal surface envelope (SEQ ID NO: 10) protein (RACR-αCD19 CMV-local)

4. A transgenic plasmid containing αCD19 CAR plus RACR components and CMV promoter and αCD3-Cocal surface envelope protein (SEQ ID NO: 129) (RACR-αCD19 CMV-αCD3-Cocal)

5. Transgenic plasmid containing αCD19 CAR plus RACR components and CMV promoter and αCD3-blinded Cocal variant surface envelope protein (RACR-αCD19 CMV-αCD3-(B)Cocal)

11 days after rapamycin addition (10nM final concentration), PBMC was collected and analyzed by flow cytometry for αCD19 CAR and intracellular 2A expression. After culturing with rapamycin, both unstimulated (Fig. 3) and blinatumomab-stimulated (Fig. 4) PBMC cells showed that the expression of αCD19 CAR was enhanced. Staining with anti-2A reagents was successful, and compared to CD19-FITC reagents, better separation of positive cells and negative cells was provided (Fig. 3 and Fig. 4, bottom figure). Data show that staining 2A peptides is a feasible alternative for surface expression staining of CAR.

Stimulation assays were performed to determine CAR T cell function. 100,000 PBMCs from unstimulated RACR-αCD19 CAR/αCD3-Cocal transduced wells were co-cultured with 100,000 Raji cells in the presence of Golgi inhibitors Brefeldin A and Monensin for 5 hours. The cells were then collected, and stained for surface markers and intracellular cytokines, and analyzed by flow cytometry. Under Raji stimulation, two CD8 (Fig. 5) and CD4 (Fig. 6) CAR+T cells easily produced IFNg, IL-2 and TNFa at levels similar to those of the control stimulated by PMA+ ionomycin. As another control, there was no cytokine production in the negative control wells of PBMC alone. These data show that the vector particles of αCD3-Cocal pseudotypes encoding RACR-αCD19 CAR payloads can successfully transduce unstimulated PBMCs. In addition, culturing with rapamycin for about 2 weeks greatly enhanced the surface expression of αCD19 CAR. CAR+ cells were still highly functional after rapamycin culture, as demonstrated by the production of IFNg, IL-2, and TNFa after Raji stimulation. Thus, unstimulated PBMCs transduced with the RACR-αCD19/αCD3-Cocal vector and cultured in rapamycin were functional and produced IFNg, IL-2, and TNFa after Raji cell stimulation.

αCD3-Cocal pseudotyped viral particles can be packaged with RACR-αCD19 CAR payload. After prolonged culture in rapamycin, the particles were able to transduce unstimulated PBMCs. After stimulation with CD19+Raji cells, αCD19CAR-transduced T cells efficiently produced IFNg, IL-2, and TNFa cytokines, indicating their potent Th1-like phenotype and high functionality.

This study also showed that vector packaging and 293T transduction were better using envelope expressed in the MND promoter backbone compared to CMV driven envelope plasmids.

Example 3: Comparison of Cocal and αCD3-Cocal Virus Particle Films Using αCD19 CAR-TGFβ Payload and In Vitro Transduction of T Cells

The purpose of this study was to determine the effect of αCD3-Cocal pseudotyped viral particle payloads on the detectability and function of lentiviral particles containing the following payload vectors: αCD19 CAR-TGFβ payload and RACR-αCD19CAR payload. The ability of the two payload designs to activate and transduce unstimulated human PBMCs was evaluated compared to conventional Cocal pseudotyped vector viral particles containing the same payload.

Virus production

28e6 293T cells were seeded into 16x T175 flasks each with 28e6 293T cells (8x per vector) in a total volume of 25 ml complete DMEM medium. After 24 hours, the cells were transfected. (The protocol was written for 1x T175 flask scale; all reagents should be at 37°C).

The following DNA is added to 1ml serum-free OptiMEM ^TM medium (without additives): 12ug transfer plasmid, 6ug Gag/pol plasmid, 6ug REV plasmid, 6ug envelope plasmid. Then 90ul (90ug) PEI is added to the medium/DNA mixture. The mixture is mixed evenly and incubated at room temperature for 20 minutes. Then the medium/DNA/PEI mixture is added to 25ml fresh complete DMEM medium. The inoculation medium in the hole containing 293T is removed and replaced with fresh medium containing transfection reagent and placed in a humidified incubator at 37°C. After 48 hours, the supernatant is collected and stored in a refrigerator and replaced with fresh DMEM medium. The next day, (after 72 hours) the supernatant is collected and filtered through a 0.45um PVDF filter. The supernatant is concentrated using an Amicon-Ultra 15 100K column and centrifuged at 3000xg for 30 minutes at 4°C. The virus is then stored at 4°C before use.

List of virus preparations used for research:

1. αCD19 CAR-TGFbDN/MND-Cocal, titer = 4e7 TU/ml

2. αCD19 CAR-TGFbDN/CMV-αCD3-Cocal, titer = 1.8e7 TU/ml

PBMC transduction and staining for flow cytometry

Thaw 50e6 PBMCs and dilute to 1e6 cells/ml in complete RPMI medium. Add IL-2 to a final concentration of 50U/ml. Divide PBMCs into 2x groups for different stimulations:

Group #1 - IL-2 only (50 U/ml)

Group #2 – added αCD3/αCD28 Dynabeads

Each group was incubated overnight at 37°C. The beads were removed from the applicable wells, and the cells were counted and pelleted. The cells were then resuspended in 1e6 cells/ml of fresh complete RPMI medium with a final concentration of 50U/ml IL-2. 500ul (5e5 cells) were added to the wells of the non-TC treated 48-well plate. Amicon concentrated preparations of MOI=2, 1 and 0.5 (based on 293T titers) were added to the plate and then placed in a 37°C incubator.

a.αCD19 CAR-TGFb/αCD3-Cocal (50ul, 25ul and 12.5ul)

b.αCD19 CAR-TGFb/Cocal (25ul, 12.5ul, 6.25ul)

After 3 days, the carrier is washed off and replaced with 500ul fresh RPMI culture medium+IL-2 (50U/ml). The cells are mixed, and 100ul is added to the hole in the 96-well V bottom plate to activate flow cytometry analysis. The cells are then washed with 200ul FACS buffer. The cell pellet is resuspended in the 100ul PBS containing Live/Dead ^TM stain (1:1000), and incubated at 4°C for 20 minutes, then washed again in 200ul FACS buffer. The cells are resuspended in 50ul FACS buffer+surface stain mixture, incubated at 4°C for 30 minutes, washed in 200ul FACS buffer.

Diluted surface stain mixture to use:

a.CD19-FITC 1:50

b. Anti-CD3-Percp 1:100

c. Anti-CD4-BV650 1:200

d. Anti-CD8-BV605 1:200

e. Anti-CD25-PECy7 1:100

The cell pellet was then resuspended in 100ul BD Biosciences Cytofix/Cytoperm ^TM , incubated at 4°C for 20 minutes, washed in 1x Perm Wash buffer, resuspended in 50ul 1x Perm Wash with anti-2A-af647 (1:100), incubated at 4°C for 30 minutes, washed in 1x Perm Wash buffer, resuspended in 100ul FACS buffer and analyzed by flow cytometry.

After 3 days, after CD25 analysis, cells were mixed and 100ul was added to wells in 96-well V-bottom plates for transduction flow cytometry analysis. Cells were washed with 200ul FACS buffer, resuspended in 100ul PBS containing Live/Dead ^™ stain (1:1000), incubated at 4°C for 20 minutes, washed in 200ul FACS buffer, resuspended in 50ul FACS buffer + surface stain mixture, and incubated at 4°C for 30 minutes.

Diluted surface stain mixture to use:

a.CD19-FITC 1:50

b. Anti-CD3-Percp 1:100

c. Anti-CD4-BV650 1:200

d. Anti-CD8-BV605 1:200

After incubation, cells were washed in 200ul FACS buffer, resuspended in 100ul BD Biosciences Cytofix/Cytoperm ^TM , incubated at 4°C for 20 minutes, washed in 1x Perm Wash buffer, resuspended in 50ul 1x Perm Wash with anti-2A-af647 (1:100), incubated at 4°C for 30 minutes, washed in 1x Perm Wash buffer, resuspended in 100ul FACS buffer, and analyzed by flow cytometry.

Results and Conclusions

In order to evaluate whether αCD19 CAR-TGFβDN/αCD3-Cocal viral vector particles can activate human T cells, several MOI vector particles were added to human PBMC. After 3 days, the virus was removed, and fresh culture medium was provided to the cells, and the activation marker CD25 was analyzed. αCD19 CAR-TGFβDN/αCD3-Cocal particles effectively activate both CD4 and CD8 T cells (Figures 7A and 7B). In addition, CD25 upregulation is dose-dependent (Figure 7B). Compared with αCD3-Cocal particles, the lowest level of CD25 induced by conventional Cocal envelope pseudotyped viral particles (Figure 7A) was found.

To examine transduction, samples were analyzed for αCD19 CAR and 2A expression for a total of 6 days after vector addition. Similar to CD25 expression on day 3, αCD3-Cocal pseudotyped particles were able to transduce unstimulated PBMCs, while Cocal pseudotyped particles were not (Figure 8A). In addition, transduction of both CD4 T cells and CD8 T cells occurred in a dose-dependent manner (Figure 8B). The data show that αCD19 CAR-TGFβDN/αCD3-Cocal particles effectively activate and transduce unstimulated PBMCs in vitro.

In addition to analyzing the αCD19 CAR expression on unstimulated PBMCs on the 6th day after transduction, CAR expression was also analyzed on the 3rd day (when analyzing activation) and again on the 12th day to determine the best time for CAR expression analysis. For both CD4 (Figure> 9A) T cells and CD8 (Figure 9B) T cells across all MOIs tested, it was found that the percentage of T cells expressing αCD19 CAR peaked on the 6th day and remained relatively stable until the 12th day (Figure 9). These data show that it is optimal to check the transduction efficiency measured by flow cytometry as measured by CAR surface expression about 1 week after transduction.

This study demonstrates the ability of the αCD3-Cocal envelope construct to deliver a payload consisting of an αCD19 CAR to unstimulated PBMCs in vitro. The αCD3-Cocal envelope induces activation of T cells, as measured by CD25 expression, and this activation is associated with transduction, as measured by the percentage of T cells expressing the αCD19 CAR and 2A peptide. In addition, activation and transduction occur in a dose-dependent manner. It was also found in this study that αCD19 CAR surface expression peaked at approximately day 6, and was similar on day 12, as analyzed by flow cytometry. This data further supports the use of αCD3-Cocal pseudotyped vectors to deliver CAR payloads to unstimulated PBMCs in vitro and in vivo.

Example 4: Comparison of αCD19 CAR-RACR targeting and in vitro transduction of T cells

The aim of this study was to evaluate the construct placement with the highest αCD19 CAR expression after transduction.

Construct orientation A) FRB-P2A-RACR-P2A-αCD19 CAR (SEQ ID NO: 81)

Construct orientation B): αCD19 CAR-P2A-FRB-P2A-RACR (SEQ ID NO: 61)

Virus production

28e6 293T cells were seeded into 6x T175 flasks with 28e6 293T cells each in a total volume of 25 ml complete DMEM medium (1x per vector). After 24 hours, the cells were transfected. (The protocol was written for 1x T175 flask scale; all reagents should be at 37°C).

The following DNA is added to 2.5ml serum-free DMEM medium (without additives): 30ug transfer plasmid, 15ug Gag/pol plasmid, 15ug REV plasmid and 15ug envelope plasmid (SEQ ID NO: 130 or 128). Then 225ul (225ug) PEI is added to the medium/DNA mixture. The mixture is mixed evenly and incubated at room temperature for 20 minutes. Then the medium/DNA/PEI mixture is added to 25ml fresh complete DMEM medium. The inoculation medium in the hole containing 293T is removed and replaced with fresh medium containing transfection reagent and placed in a humidified incubator at 37°C. After 48 hours, the supernatant is collected and stored in a refrigerator and replaced with fresh DMEM medium. The next day, (after 72 hours) the supernatant is collected and filtered through a 0.45um PVDF filter. The supernatant is concentrated using an Amicon-Ultra 15100K column and centrifuged at 3000xg for 30 minutes at 4°C. The virus was then stored at 4 °C before use.

Viral vector particles used in the study:

1.αCD19 CAR-TGFbDN/Cocal (SEQ ID NO:92), titer=5.7e7 TU/ml

2.αCD19 CAR-TGFbDN/αCD3-Cocal (SEQ ID NO:92), titer=4.1e7 TU/ml

3. RACR-αCD19 CAR/Cocal, titer = 1.3e7 TU/ml

4. RACR-αCD19 CAR/αCD3-Cocal, titer = 4.5e6 TU/ml

5. αCD19 CAR-RACR/Cocal, titer = 2.8e7 TU/ml

6. αCD19 CAR-RACR/αCD3-Cocal, titer = 1e7 TU/ml

PBMC transduction and staining for flow cytometry

Thaw PBMCs and dilute to 1e6 cells/ml in complete RPMI medium. Add IL-2 to a final concentration of 50 U/ml. Divide PBMCs into 2x groups for different stimulations:

Group #1 - IL-2 only (50 U/ml)

Group #2 - added +αCD3/αCD28 Dynabeads

Each group was incubated overnight at 37°C. The beads were removed from the applicable wells, and the cells were counted and pelleted. The cells were then resuspended in 1e6 cells/ml of fresh complete RPMI medium with a final concentration of 50U/ml IL-2. 500ul (5e5 cells) were added to the wells of the non-TC treated 48-well plates. The Amicon concentrated preparation of MOI=1.5 (based on 293T titer) was added to the plate and then placed in a 37°C incubator. Transgenic, envelope protein, 293 titer, and ul required to reach 1.5MOI are shown in Table 1.

Table 1

Group genetically modified Encapsulation 293T titer MOI = 1.5 ul required 1 αCD19 CAR TGFb Cocal 5.70E+07 13.2 2 αCD19 CAR TGFb αCD3-Cocal 4.10E+07 18.3 3 RACR-αCD19 CAR Cocal 1.30E+07 57.7 4 RACR-αCD19 CAR αCD3-Cocal 4.50E+06 166.7 5 αCD19 CAR-RACR Cocal 2.80E+07 26.8 6 αCD19 CAR-RACR αCD3-Cocal 1.00E+07 75.0

After 3 days, carrier is washed off, and replaced with 500ul fresh RPMI culture medium+IL-2 (50U/ml).Cell is mixed, and 100ul is added in the hole in 96-well V bottom plate, to activate flow cytometry analysis.Then the cell is washed with 200ul FACS buffer.Cell pellet is resuspended in the 100ul PBS containing Live/Dead ^TM stain (1:1000), and incubated 20 minutes at 4 ℃, and washed again in 200ul FACS buffer.Cell is resuspended in 50ul FACS buffer+surface stain mixture (see below), incubated 30 minutes at 4 ℃, and washed in 200ul FACS buffer.

Diluted surface stain mixture to use:

a.CD19-FITC 1:50

b. Anti-CD3-Percp 1:100

c. Anti-CD4-BV650 1:200

d. Anti-CD8-BV605 1:200

e. Anti-CD25-PECy7 1:100

The cell pellet was then resuspended in 100ul BD Biosciences Cytofix/Cytoperm ^TM , incubated at 4°C for 20 minutes, washed in 1x Perm Wash buffer, resuspended in 50ul 1x Perm Wash with anti-2A-af647 (1:100), incubated at 4°C for 30 minutes, washed in 1x Perm Wash buffer, resuspended in 100ul FACS buffer and analyzed by flow cytometry.

After 5 days after CD25 analysis (day 8 after vector addition), cells were mixed and 100ul was added to wells in 96-well V-bottom plates for transduction flow cytometry analysis. Cells were washed with 200ul FACS buffer, resuspended in 100ul PBS containing Live/Dead ^™ stain (1:1000), incubated at 4°C for 20 minutes, washed in 200ul FACS buffer, resuspended in 50ul FACS buffer + surface stain mixture (see below), and incubated at 4°C for 30 minutes.

Diluted surface stain mixture to use:

a.CD19-FITC 1:50

b. Anti-CD3-Percp 1:100

c. Anti-CD4-BV650 1:200

d. Anti-CD8-BV605 1:200

After incubation, cells were washed in 200ul FACS buffer, resuspended in 100ul BD Biosciences Cytofix/Cytoperm ^TM , incubated at 4°C for 20 minutes, washed in 1x Perm Wash buffer, resuspended in 50ul 1x Perm Wash with anti-2A-af647 (1:100), incubated at 4°C for 30 minutes, washed in 1x Perm Wash buffer, resuspended in 100ul FACS buffer, and analyzed by flow cytometry.

Results and Conclusions

The viral vector particles containing the three transgenic plasmids described above are packaged with Cocal or αCD3-Cocal envelope proteins, and the preparation is titrated on 293T cells (Figure 10). The viral vector particles containing αCD19 CAR-TGFbDN transgenes show a higher titer than the titer of the preparation containing αCD19 CAR-RACR in either direction. When two constructs containing RACR are compared, higher titers (constructs toward B) are achieved when αCD19 CAR is directed before RACR components. This is true for both Cocal pseudotyped (Figure 10A) and αCD3-Cocal pseudotyped (Figure 10B) viral particles.

Two analyzed directions: αCD19 CAR-RACR and RACR-αCD19CAR particles activated unstimulated T cells (data not shown) with αCD3-Cocal envelope pseudotypes. In the presence of anti-CD3/anti-CD28 dynabead+IL-2 or IL-2 alone, concentrated viral vector particles were added to PBMC. After 3 days, PBMC was washed, beads were removed, and PBMC was resuspended in fresh culture medium containing IL-2. The activation performed by CD25 expression was then analyzed by flow cytometry. Cocal pseudotyped carriers did not induce significant increases in CD25. In contrast, robust CD25 increases were seen on both CD8 T cells and CD4 T cells (data not shown). Similar CD25 levels were seen in the RACR-αCD19 CAR and αCD19 CAR-RACR directed constructs. The results showed that both RACR-αCD19 CAR and αCD19CAR-RACR directed vector particles pseudotyped with αCD3-Cocal effectively activated unstimulated T cells.

αCD19 CAR and 2A expression in T cells 8 days after vector transduction (5 days after detection activation) were analyzed to evaluate T cell transduction.αCD19 CAR surface expression (Figure 11A) on cells transduced with αCD19 CAR-TGFbDN was detected.αCD19 CAR surface expression and intracellular expression of 2A peptides were not detected in T cells transduced with RACR-αCD19 CAR constructs (Figure 11B).In contrast, αCD19 CAR surface expression and intracellular expression of 2A peptides were detected in T cells transduced with αCD19 CAR-RACR constructs (Figure 11C).The lack of transduction seen when using RACR-αCD19 CAR construct particles cannot be rescued by adding anti-CD3/anti-CD28 stimulating beads before viral transduction (data not shown).Similar results were seen in CD4 T cells (data not shown). The data show that αCD19 CAR and RACR orientation are important considerations and that αCD19-RACR orientation results in higher detectability and manufacturability.

Surprisingly, this data showed that constructs with both CAR 5' and RACR 3' increased the 293T titer of viral particles and the ability to detect CAR on transduced T cells by flow cytometry. The αCD19-RACR transgene was efficiently encapsidated by the αCD3-Cocal envelope, and this particle transduced unstimulated PBMCs in vitro.

Example 5: Comparison of viral particle payload gene sequencing and in vitro transduction of T cells

The purpose of this study is to determine the effect of order on the detectability and function of lentiviral payloads containing the following functional elements: Frb-RACR and αCD19-CAR. When rapamycin is added to the culture, the Frb-RACR element provides a selective advantage to the cells. The αCD19-CAR element targets T cells to CD19+ target cells. Together, the two elements produce CAR-T cells that are enriched with rapamycin and are cytotoxic to CD19+ target cells. The difference between the two payload designs evaluated is the order in which the elements are expressed in the polycistronic transcript. This study tested the functional aspects of payloads in two orientations to understand whether differences in expression affect 1) payload detection by flow cytometry (2A antibody), 2) CAR-T cell rapamycin response, and 3) CAR-T cell CD19+ cytotoxicity.

Two payload designs were evaluated:

1. MND promoter–FRB--P2A–RACRβ–P2A–RACRγ–P2A–CAR (SEQ ID NO: 81)

2. MND promoter-CAR-P2A--FRB--P2A-RACRβ-P2A-RACRγ (SEQ ID NO:61)

Study plan

Table 2

*Add fresh media to wells as needed, add 10 nM rapamycin to wells treated with rapamycin. Media is added on days 10, 14, 15, and 20.

Results and Conclusions

PBMCs were treated with each of the RACR-αCD19-CAR and αCD19-CAR-RACR vectors in equal amounts. Although transduced with the same amount of infection units, on the 8th day after transduction, recognizable 2A+ colonies were only visible in αCD19-CAR-RACR constructs (Figure 13C, the 8th day: black arrows). Rapamycin selection (10nM) revealed that on the 15th and 22nd days, 2A+ cell colonies (Figure 13B, red arrows, Figure 14, red arrows) appeared in cells transfected with RACR-αCD19-CAR vectors. In contrast, regardless of rapamycin treatment, αCD19-CAR-RACR T cells form well-defined 2A+ colonies (Figure 13C, black arrows).

Rapamycin enrichment for αCD19-RACR-CAR-T cells was easily detected at day 15 and continued to be enriched until day 22. When Raji cells were added with rapamycin, this enrichment was greatly enhanced (data not shown). Enrichment is defined as the increase in the percentage of CAR-T cells over time. Enrichment can occur by a decrease in the abundance of non-CAR-T cells and/or an increase in the abundance of CAR-T cells.

The expansion of CAR-T cells was measured using cell counting, culture volume, and flow cytometry population frequency to estimate total CAR-T cells. The gating requirements for the 15th and 22nd days were different; however, in each case, cells not transduced were used as biological negative controls. At the 15th day, there was almost no rapamycin-driven amplification of cells; however, by the 22nd day, the rapamycin-driven amplification of cells was significant (Figure 15). The activation by adding Raji cells by CAR produced a larger amplification than the amplification driven by rapamycin, and both rapamycin addition and Raji cell addition occurred maximum amplification (Figure 15). This shows that rapamycin is necessary for cell expansion.

To evaluate the cytotoxicity of RACR-αCD19 CAR and αCD19 CAR-RACR vectors against CD19-positive cells, 5,000 Raji GFP::ffluc cells were added to the culture on day 8 after transduction in the presence and absence of rapamycin. The 5,000 Raji cell spike corresponds to a 1.3:1 (effector: T cell) ratio of αCD19 CAR-RACR. Since RACR-αCD19 CAR was not detected at day 8, the exact E:T of these samples is unknown, but it is probably also about 1.3:1. After 1 week of co-culture of Raji cells, Raji cells were eradicated with two αCD19 CAR vectors that were independent of rapamycin addition (Figures 16B and 16C). In the presence of rapamycin, Raji cells were reduced even in the absence of αCD19-CAR, indicating that rapamycin alone has a profound effect on Raji cell biology (Figure 16A).

These data show that the detection of payload by flow cytometry varies with orientation.αCD19 CAR-RACR can be detected across multiple conditions and time points.RACR-αCD19 CAR is not detectable 8 days after transduction, and 15 days after transduction/activation, the most detectable vector is treated with rapamycin.The vector genomes sorted with 5' anti-CD19 CAR and 3'RACR produce superior cell amplification, and their detectability is more robust and predictable by flow cytometry.Surprisingly, the viral particles whose vector genomes have polynucleotide sequences encoding anti-CD19 chimeric antigen receptors in 5' to 3' order and polynucleotide sequences encoding receptors (RACR) thereafter make the transduction efficiency of T cells higher than the transduction efficiency of viral particles whose vector genomes have two polynucleotide sequences in another order (receptor 5' to anti-CD19 CAR).

The data also show that rapamycin is enriched for CAR-T cells containing two payloads. Rapamycin amplification is most obvious between the 15th and 22nd days of the study. By the 22nd day, compared to non-rapamycin treated cells, non-rapamycin treated αCD19-CAR-T cells expanded 4.3 times (data not shown). Maximum T cell amplification was observed in CAR-T cells treated with Raji cells and rapamycin. The data further show that the payload of both αCD19 CAR-RACR and RACR-αCD19 CAR is cytotoxic to CD19 positive Raji cells, and the growth of the cells is negatively affected by 10nM rapamycin.

Example 6: In vivo transduction of T cells with lentiviral vectors encoding anti-CD19

TGFβ-DN CAR

This study evaluated the in vivo transduction of cells with the surface of lentiviral particles engineered with Cocal glycoprotein and anti-CD3 scFv (SEQ ID NO: 129) described in Example 1. The lentiviral particles contain polynucleotides encoding anti-CD19 CAR and dominant negative TGFβ receptors, which are designed to provide resistance to TGFβ signaling. The lentiviral particles were delivered to CD34+ humanized mice by intraperitoneal or subcutaneous injection. The mice used in the study were immunocompromised and contained implanted human hematopoietic stem cells that produced circulating human T cells and B cells.

Study Design

Virus preparations, animal strains, cell lines

Umoja Biopharma (Umoja Biopharma) prepared conventional Cocal and engineered αCD3-Cocal enveloped lentiviral particles (SEQ ID NO: 92) carrying anti-CD19 TGFβ-DN CAR payload using PEI-mediated transient transfection of adherent 293T cells. These preparations were concentrated using Amicon filters. According to institutional guidelines (Fred Hutchinson Cancer Research Center), 19 female CD34+HSC humanized mice (Jackson laboratory) were raised 19 weeks after implantation.

Study plan

19 female CD34+ humanized mice were acclimated for one week after receiving. On day -7, blood from all mice was collected for flow cytometry analysis to quantify the degree of humanization. Mice were randomized into the treatment groups described in Table 3 based on their overall human CD3 levels.

Table 3: Study treatment groups

Research timeline

On study day 0 (SD0), mice were dosed with viral particles according to the table above, and peripheral blood was collected once a week for the duration of the study for analysis by flow cytometry.

At SD28, mice were sacrificed, and peripheral blood, spleen, and bone marrow were collected from each mouse for flow cytometric analysis and histology.

Results and Conclusions

Mice were randomized to treatment groups using blood analysis by flow cytometry to the abundance of human CD3+ cells. In order to quantify the frequency of human CD3+ cells in peripheral blood, the following gating strategy was used: the score of hCD3+ was multiplied by the humanized score (hCD45+) to obtain the relative abundance of hCD3+. Counting beads were used by flow cytometry to quantify human B cells, T cells and CAR+ cells.

Some weight loss occurred throughout the study, but all groups recovered to a large extent and no clear trends were observed. At study termination, spleen weights were similar in all groups.

Throughout the study, T cells, B cells, CD71+T cells (markers of activation) and CAR+ cells in peripheral blood were quantified. Throughout the study, the number of T cells fluctuated, and the activation level also fluctuated, but no obvious trend was observed (data not shown). Although the abundance of human B cells gradually decreased in all groups during the study due to drift in the CD34 humanized mouse model, overall B cell depletion was observed in the groups administered with αCD3-Cocal and cocal IP by the 7th day (data not shown). On the 14th day, the level of CAR+T cells was detected only in the blood of mice treated with αCD3-Cocal by IP approach, which was higher than the background level (data not shown).

CAR+ cells were detected by flow cytometry only in CD3+T cells from mice administered with αCD3-Cocal IP (Figure 17A), and not detected in any non-CD3+ cells (Figure 17B), indicating that CAR transduction is selective for CD3+T cells. Flow cytometry data from peripheral blood on the 14th day showed that the CAR+ population of T cells in the group administered with αCD3-Cocal IP was strongly enriched for CD8+ cells, while non-CAR+ populations had approximately equal proportions of CD4 and CD8 (data not shown).

On day 14 of the study, ddPCR of WPRE in blood pellets confirmed that CAR was detected only in mice administered with αCD3-Cocal IP (data not shown). At the end of the study, B cells (Figure 18A) and T cells (Figure 18B) in the peripheral blood, bone marrow, and spleen of mice were evaluated by flow cytometry. Complete B cell depletion was observed in mice treated with αCD3-cocal particles by the IP route, indicating that T cells transduced by αCD3-Cocal engineered lentiviral particles have strong killing potential. In contrast, incomplete B cell depletion was observed in mice treated with Cocal particles (non-αCD3) by the IP route, which was most obvious in the bone marrow.

In summary, when delivered intraperitoneally, 9 million TU of αCD3-Coca engineered lentiviral particles successfully transduced T cells in vivo and caused rapid and complete B cell depletion in all tissues analyzed.

Overview: No off-target transduction was observed (Figure 17). In mice that received αCD3-Cocal engineered particles by IP, B cells were completely undetectable in the blood starting on day 7 after injection. In the blood, bone marrow, and spleen, B cells were undetectable 4 weeks after the study was terminated. Compared to CAR negatives, on day 14 after injection, CAR-positive cells from the blood of mice treated with αCD3-Cocal engineered particles by IP were enriched for CD8+ cells, indicating the expansion and enrichment of cytotoxic effector cells. Detection of the WPRE sequence with ddPCR confirmed the in vivo transduction of CD3+T cells in the blood of mice treated with αCD3-Cocal particles by IP. Peripheral blood B cell depletion was also observed in mice that received Cocal particles by IP (Figure 3A), but CAR+ cells were not detected by flow cytometry or ddPCR.

Example 7: In vivo transduction of T cells with lentiviral vectors encoding anti-CD19

CAR and anti-B cell activity

The purpose of this study was to evaluate the in vivo transduction of anti-CD19 CAR T cells in CD34 humanized NSG mice using an αCD19 CAR-RACR payload (SEQ ID NO: 121) packaged in an αCD3-Cocal envelope (SEQ ID NO: 128) and a helper plasmid including gag/pol (SEQ ID NO: 131) and Rev protein (SEQ ID NO: 132). In this study, the ability of αCD19 CAR-RACRαCD3-Cocal lentiviral particles to deplete B cells in a CD34 humanized model was evaluated. Another study objective was to determine whether rapamycin administration altered the process of B cell depletion or the expansion of CAR T cells.

Virus preparation and QC data

The lentivirus was concentrated by ultracentrifugation, titrated on 293T cells, cryopreserved, and stored at -80°C before use. Mycoplasma was tested for all preparations used in animal studies and the preparations were certified as negative for contamination. Endotoxin activity was less than 1 EU/mL for all batches.

αCD19 CAR-TGFBαCD3-Cocal virus particles with a titer of 1.6x 10^8/mL were thawed at room temperature or in hand. 290uL of virus was diluted with 1mL Hanks'Balanced Salt Solution (HBSS) and kept on ice. Each mouse was given 250uL/injection intraperitoneally (4 mice in total). 4.7mLαCD19 CAR-RACRαCD3-Cocal virus particles with a titer of 4x 10^7TU/mL were thawed, diluted with 0.5mL HBSS, and kept on ice. Each mouse was given 250uL/injection intraperitoneally (16 mice in total).

Study plan

Female HuNSG mice engrafted with CD34+ HSC at 21 and 16 weeks of age were used in this study. Mice were rested for 1 week after arrival at the facility before the study began. Mice were bled and randomized into treatment groups as described in Table 4 based on engraftment parameters:

Table 4

Processing Timeline:

Mice in all study groups were treated with virus or vehicle on the same schedule (on study day 0) and then injected with rapamycin or vehicle starting on study day 2.

Tissue Collection Schedule:

Mice in Study Groups 1-4 were divided into two equal endpoint groups, A and B, to allow for more frequent blood draws and two terminal collections. Mice in Study Group 5 were assigned the same endpoint schedule as the "B" endpoint group.

Group A finish timetable:

Blood was collected from the A endpoint group on study day 3, and mice were sacrificed on study day 10. Approximately 75% of the spleen and 1 femur were collected from all mice and fixed in 10% neutral buffered formalin (NBF) at approximately 20x tissue volume for 72 hours, transferred to 70% ethanol, and kept at 4°C for processing and paraffin embedding. Terminal blood, a small piece of spleen, and 1 femur were placed in PBS on ice for flow cytometry.

Group B end timetable:

Blood was collected from the B endpoint group once a week. Body weight was measured twice a week over the length of the study. Mice in group B were collected on day 29. Approximately 75% of the spleen and 1 femur were collected from all mice and fixed in 10% neutral buffered formalin (NBF) for 72 hours with approximately 20x tissue volume, transferred to 70% ethanol, kept at 4°C, for processing and paraffin embedding. Terminal blood, a small section of spleen and 1 femur were placed in PBS on ice for flow cytometry. The study timeline is shown in Table 5.

Table 5

Results and Conclusions

For analysis by flow cytometry, all cells were gated by debris exclusion, singlet discrimination, live discrimination, and expression of human CD45. B cells were defined as human CD20+CD3-. T cells were defined as human CD3+CD20-. CAR+ events were defined as CD19-FITC positive or anti-2A peptide APC positive. Negative gates were set by stained samples from mice that did not receive the virus. Positive staining was validated using cultured CAR T cells. T cells were further analyzed for expression of CD71 as a primary marker of activation.

Surprisingly, mice treated with αCD19 CAR-RACR and αCD19 CAR-TGFβ began to show severe B cell depletion 7 days after virus administration, and reached the lowest point with almost no detectable B cells when the study was terminated. In contrast, mice treated with vehicle showed a gradual decrease in circulating B cells over time, as reported in the CD34 humanized model (Figure 19A). Spleen (Figure 19B) and bone marrow (Figure 19C) were collected from mice on the 10th and 29th days of the study, and B cell colonies were evaluated. On the 10th day of the study, compared with mice treated with vehicle, the B cell colonies in the spleen of mice treated with lentivirus were reduced by about 70% (Figure 19B, left figure). On the 29th day of the study, compared with the spleen B cell colonies in mice treated with vehicle, the spleen B cell colonies in mice treated with lentivirus were reduced by>95% (Figure 19B, right figure). The B cell population in the bone marrow was equal between all study groups at day 10 and decreased by 70% in the lentivirus-treated group at day 29 of the study (Figure 19C, right). B cells were first significantly depleted from the blood starting at day 7, then from the spleen at day 10, and from the bone marrow at day 29. B cell depletion was consistent with anti-CD19 CAR activity. Rapamycin did not affect the course of B cell depletion in lentivirus-treated mice or vehicle-treated mice.

The rate of humanization remained relatively constant during the length of the study and did not differ between the study groups. Throughout the study, little change was observed in CD4 T cells and CD8 T cells in all groups (data not shown). T cell activation was assessed by CD71 expression during the study (data not shown). On the 10th and 14th days of the study, CD4 T cells in mice treated with rapamycin showed lower CD71 expression than those in mice not treated with rapamycin. Lentivirus administration did not affect CD71 expression in CD4 T cells. On the 3rd day after lentivirus administration, CD71 expression in CD8 T cells of mice treated with lentivirus was increased compared to mice treated with vehicle, which is consistent with αCD3-Cocal-dependent T cell activation.

Plasma was collected from mice by centrifugation at the specified time point, and its human cytokine production was analyzed to determine whether T cell activation and B cell depletion were associated with cytokine release. Low levels of human cytokines close to the detection threshold were observed, and there was no difference between the research groups. Cytokines IL-13, IL-1β, IL-4 were all below the detection threshold. Cytokines IFNγ, IL-10, IL-12p70, IL-2, IL-6, IL-8 and TNFα were detected at low levels and were equal between groups (data not shown).

CAR expression in T cells in the blood was evaluated on days 3, 7, 10, 14, 23 and 29 of the study, and CAR expression in T cells in the spleen and bone marrow was evaluated on days 10 and 29 of the study. The CAR+ population was observed only in the CD8+ fractions of blood, spleen and bone marrow on day 29 of the study. The background noise of P2A+CAR+ was subtracted by using the average of vehicle-treated mice as a baseline. With background subtraction, 5-10% CD8 T cells in the blood were CAR+, 5-20% in the spleen were CAR+, and 10-40% in the bone marrow were CAR+ (Figure 20). Although the assumed CAR population was observed at other time points in all lentiviral-treated groups, the difference between the population and the background events was not enough to represent a true CAR T cell.

Digital droplet PCR (ddPCR) was used to assess vector integration of blood, spleen, and bone marrow populations relative to the human genome by targeting the WPRE sequence. Detectable levels of vector were found in the blood at days 3, 10, 14, and 21 ( FIG. 21A ). Vector integration was detectable in the bone marrow at days 10 and 29 ( FIG. 21B ). The vector copy number in the blood is approximately 10 times that in the bone marrow, because T cells account for a large fraction of human white blood cells in the blood, but a very small fraction of human white blood cells in the bone marrow.

Data show that in a CD34 humanized model with endogenous B cells as the sole source of antigen, intraperitoneal administration of 9 million transduction units (TU) of αCD19 CARαCD3-Cocal envelope lentivirus resulted in rapid and complete B cell depletion. B cell depletion was similar in mice treated with αCD19 CARαCD3-Cocal envelope lentivirus compared to mice treated with αCD19-TGFβαCD3-Cocal lentivirus.

Example 8: Clinical studies on safety, efficacy and pharmacokinetics

Research objectives

The primary objectives of the study are to evaluate and characterize the tolerability and safety profile of the drug product and rapamycin in adult and pediatric patients with B-ALL and B-lineage lymphomas and to evaluate the antitumor activity of the drug product and rapamycin in adult and pediatric patients with B-lineage hematologic malignancies.

Secondary objectives of the study are to evaluate the complete response rate and durability of response of the drug product in B-ALL and aggressive B-lineage lymphomas, to evaluate progression-free survival and overall survival in adult and pediatric patients with B-lineage hematologic malignancies treated with the drug product, and to evaluate the pharmacokinetics of the drug product.

Exploratory objectives of the study are to explore biomarkers of response and toxicity to the drug product, explore the immunogenicity of the drug product, explore the pharmacodynamics of the drug product, evaluate the contribution of drug insertion site and frequency to the safety/efficacy/PK of the drug product, and evaluate the impact of the tumor microenvironment on antitumor activity and PK/PD.

A qualified strength test for measuring the functional viral particles in the drug product (DP) will be performed to determine the dosage. The strength of the drug product will be reported in units of transduction units per milliliter (TU/mL) derived from the transduction of HOS cells (human osteosarcoma cell line), and the integration of payload components (e.g., viral packaging sequences) will be quantified by PCR measurement of genomic DNA. The measurement of TU/mL (referred to as "functional titer") of viral integration into susceptible recipient cell lines using molecular readout is a conventionally used measurement of the strength of the viral product because it determines the concentration of functional units of the virus present in the formulation. The most accurate and quantitative measurement of the strength of this stage of drug product development is the ability of viral particles to transduce human cells (as measured by the proposed assay), because transduction means functional viral particles.

In order to understand the biological effect of the drug product, each batch of drug product will be qualitatively evaluated to measure the expression and/or function of all elements that contribute to the biological activity of this product. The proposed drug product characterization plan will include measuring the expression and/or function of αCD3-cocal surface engineering, anti-CD19 CAR and RACR-FRB systems. Specifically, this product characterization work will involve in vitro transduction of primary human unstimulated PBMCs and measurement of: T cell activation, CAR expression, RACR expression and/or RACR function, and CAR activity in response to antigens. The relationship between these functional readouts and the quantitative dose in TU will be evaluated in nonclinical pharmacology studies and during clinical development.

Table 6: Summary of Phase 1 Clinical Studies

Drug products

Description of the drug product and proposed mechanism of action

The drug product is an investigational replication-incompetent, self-inactivating (SIN) lentiviral vector (LVV) designed to transduce T cells in vivo to express an anti-CD19 CAR and target CD19-expressing tumor cells.

Drug products have a multi-step mechanism of action:

-LVV binds to T cells in vivo via anti-CD3 scFv, which activates T cells and promotes lentiviral internalization by interacting with Cocal glycoprotein

-The payload RNA genome, i.e., αCD19 CAR-FRB-RACR, is reverse transcribed into DNA, shuttled to the nucleus, and integrated into the genome

- Transduced T cells express an anti-CD19 CAR and target cells expressing CD19, and also express the FRB and RACR systems for rapamycin-controlled cytokine signaling

The five plasmids used to prepare the drug product LVV include:

1) Gag-Pol helper plasmid: expresses viral gag and pol genes (SEQ ID NO: 124). These helper plasmids encode viral structural proteins as well as proteins necessary for reverse transcription and integration into the target genome.

2) Rev helper plasmid: encodes the viral protein Rev, which is required for nuclear export of the unspliced packageable RNA genome in the producing cell line (SEQ ID NO: 125).

3) αCD3 scFv plasmid: encoding anti-CD3 scFv (SEQ ID NO: 127).

4) Cocal helper plasmid: encoding Cocal envelope glycoprotein (SEQ ID NO: 123).

5) αCD19 CAR-FRB-RACR: Encodes a transgene to be delivered to activated T cells, which comprises two chimeric receptor systems: 1) a second-generation anti-CD19 CAR comprising FMC63 scFv fused to 4-1BB and CD3ζ intracellular signaling domains; and 2) the RACR-FRB system (SEQ ID NO: 122) (Figure 25).

Lentiviral particle delivery and in vivo T cell interactions

Lentiviral particles deliver genetic payloads to T cells by intranodal injection or delivery to the interstitial space (e.g., SC or IP injection), which drain to local lymph nodes. Like other LVVs, the size of the drug product viral particles is expected to be 80-120nm, and thus, after administration, are carried from the interstitial fluid into the lymph, allowing them to be transported directly to secondary lymphoid tissues (i.e., lymphatic vessels and lymph nodes). It is expected that either route of administration will cause drug product engagement and transduction of CD3+T cells in the lymph nodes.

The ability of the drug product to efficiently deliver genetic payloads to T cells in vivo is enabled by surface engineering of the lentiviral particles, which contain expression of anti-CD3 scFv and Cocal glycoprotein. Specifically:

-The anti-CD3 scFv on the surface of the LVV particles is designed to bind to and activate T cells via CD3. CD3 stimulation activates T cells by initiating a signaling cascade within the cell, leading to cell cycle progression and transcriptional/metabolic changes associated with activation. This state renders T cells permissive to LVV transduction.

- Cocal is a fusion glycoprotein that, following receptor binding and viral particle internalization, functions to fuse the viral membrane with the endosomal membrane following endosome acidification, thereby releasing the viral capsid into the cytoplasm. Thus, Cocal complements the action of CD3, as CD3-mediated T cell activation leads to upregulation of one of the Cocal receptors, the low-density lipoprotein receptor (LDLR), and supports the internalization of LVV.

In vivo transduction

After capsid delivery to the T cell cytoplasm:

1) The lentiviral particle capsid is transported to the T cell nuclear envelope/membrane. During this transport, reverse transcription of the viral genome and formation of the pre-integration complex (PIC) occur within the capsid. Efficient reverse transcription of the viral particle payload is achieved by increasing the pool of cellular nucleotides produced by drug product-mediated T cell activation through binding to CD3. During the nuclear membrane transport process, capsid disassembly occurs, thereby releasing the PIC to bind to the endogenous cellular chromatin regulator LEDGF that is ubiquitous in host cells.

2) PIC binds to LEDGF to tether the PIC to the active chromatin region of the host cell, and the reverse transcribed payload cassette is integrated into the genome of the transduced cell through the activity of the lentiviral INT protein on the viral long terminal repeat (LTR) sequence. The use of lentiviral mechanisms to affect integration tends to favor active genes and is therefore generally safer than other non-targeted integration methods.

Surface expression of payload

The drug product payload consists of an approximately 7 kb RNA genome that is reverse transcribed into a gene expression cassette to drive the expression of the αCD19 CAR, FRB, and RACR components, which provide drug-regulated immune cell activation, expansion, and targeting signals (Figure 23).

The expression of the polycistronic transgenic payload is driven by the MND promoter (SEQ ID NO: 35). The MND promoter (myeloproliferative sarcoma virus enhancer, negative control region deletion, dl587rev primer binding site replacement) is a virus-derived synthetic promoter containing a modified Moloney mouse leukemia virus (MoMuLV) LTR U3 region with myeloproliferative sarcoma virus enhancer 13, and has high expression in human CD34+ stem cells, lymphocytes and other tissues. Four separate proteins are expressed and separated by ribosome jumps and cleaved 2A peptide sequences during induction of translation. CAR is a second-generation CAR consisting of FMC63 mouse anti-human CD19 scFv connected to a 4-1BB costimulatory domain and a CD3ζ intracellular signaling domain.

After integration of the drug product transgene (Figure 23) into the genome of the cell, the transgene promoter (MND) drives expression of the payload open reading frames encoding four different proteins separated by 2A ribosomal skipping peptide sequences.

CAR and RACR receptors drive the transmission of complementary signals, which regulate T cell survival, proliferation and "activation" of anti-tumor effector properties (Figure 24). The expression and binding of anti-CD19 CAR and CD19 on normal and malignant B cells in lymphoid tissue and peripheral blood will produce signals that replicate TCR and costimulatory receptor engagement. This is because the CAR construct contains CD3ζ ("signal 1") and 4-1BB ("signal 2") costimulatory signaling domains, which are necessary for T cell immune responses.

Unique to drug products, administration of rapamycin acts as a ligand for dimerization of the RACR subunits and provides IL-2/15 cytokine-like pro-survival/proliferation signals ("signal 3") to viral particle-transduced T cells. Together, CAR and RACR receptor-mediated signals enhance the in vivo expansion and maintenance of CD19-targeted CAR T cell populations.

RACR Function

The RACR components RACRγ and RACRβ are different fusion proteins expressed as transmembrane receptor proteins. RACRγ includes a fusion between an extracellularly localized FK binding protein (FKBP) and the transmembrane and cytoplasmic tail of the common cytokine receptor γ subunit, and RACRβ includes a fusion between an extracellularly localized FKBP rapamycin binding (FRB) domain and the transmembrane and cytoplasmic tail of IL2RB.

Rapamycin is an FDA-approved mTOR inhibitor immunosuppressant for many clinical settings. Rapamycin induces dimerization of two RACR components, thereby triggering IL-2/IL-15-like signaling in transduced cells. The transgene comprises a naked FRB domain, i.e., 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 allows the transduced T cells to be continuously activated, and gives the transduced T cells a proliferation advantage over natural T cells (Figure 25). Therefore, the RACR system provides a survival/proliferation advantage for transduced T cells by providing IL2/15 signaling, while untransduced T cells are subject to the inhibitory effect of rapamycin mTOR inhibition.

In vivo pharmacology

To evaluate the ability of the drug product LVV to transduce T cells in vivo, a humanized mouse model was used. NSG mice transplanted with human umbilical cord blood CD34+ stem cells were obtained. These mice have approximately 25-50% human CD45+ immune cells in circulation and active production of human B cells and T cells from the bone marrow. At approximately 20 weeks after transplantation, the human CD45+ fraction in these humanized mice typically contains approximately 60-80% B cells and 20-40% T cells; therefore, these mice are considered to be an appropriate model for transducing human T cells in vivo with depletion of B cells as a readout of anti-CD19 CAR activity.

As shown in Figure 30, in order to evaluate the function of the drug product in vivo, female CD34 humanized mice (n=3 or 4/group) were treated by intraperitoneal injection with approximately 10 million TU of drug product, and the B cell population obtained by continuous blood sampling was quantified by flow cytometry. Rapid depletion of B cells in peripheral blood was observed, reaching a minimum point where almost no B cells could be detected 14 days after injection, thereby indicating anti-CD19 CAR activity. By flow cytometry analysis, mice killed 29 days after drug product administration showed spleen and bone marrow B cell population depletion. Drug product payload integration was detected by ddPCR analysis in leukocytes collected from peripheral blood, and the expression of anti-CD19 CAR was detected by flow cytometry.

These in vivo pharmacology studies demonstrated that the drug product LVV was able to transduce a detectable population of CAR T cells in vivo and resulted in near-complete depletion of B cells.

Example 9: Overview of dose-finding studies of lentiviral vectors encoding anti-CD19 CAR in CD34+ humanized mice

Lentivirus was prepared using adherent production and titrated on 293T cells by flow cytometry. Raji GFP FFLUC cells expressing luciferase and GFP were obtained from the Jensenlab (SCRI) at Scottish Enterprise Research, cultured by Umoja Biopharma, and delivered to the Fred Hutchinson Cancer Research Center in PBS on ice for injection.

Twenty human NSG CD34+ female animals were randomized into 5 groups based on their human B cell levels. On study day 0, different doses of UB-VV100 viral particles, FITC RACR particles as control or vehicle were injected IP according to Table 7 below.

Table 7: Study groups

Group N Dosage (IP) Virus IP Rapamycin IP Raji SC cells 1 4 10 million UB-VV100 1mg/kg 2 million 2 4 2 million UB-VV100 1mg/kg 2 million 3 4 400,000 UB-VV100 1mg/kg 2 million 4 4 10 million FITC RACR 1mg/kg 2 million 5 4 0 Medium not applicable 2 million

The UB-VV100 virus particle includes SEQ ID NOs: 121, 128, 131 and 132.

Starting at D4, mice were blooded once a week after orbital reflex until D53, and their blood was analyzed by flow cytometry. On the 26th day, all groups except the vehicle group began to receive rapamycin at a dosage of 1mg/kg, 3 times a week. On the 40th day, 100ul mixtures of 2 million RAJI GFP ffLUC and Matrigel at a ratio of 1:1 were implanted subcutaneously in all mice. Tumors were measured 3 times a week with a digital caliper to monitor tumor growth, using the formula (W^2×L)/2 to calculate tumor volume. From the 59th day to the 70th day, tumors were measured twice a week. From the 59th day to the 70th day, mice were switched to the rapamycin schedule twice a week, and then received their last dose from the 73rd day to the 77th day, switching back to three times a week. All mice were killed on the 81st day of the study. Bone marrow, spleen, peripheral blood and tumor were collected, processed into single cell suspensions, and analyzed by flow cytometry. The schematic diagram of the research timeline is shown in Figure 31A.

result

Blood B cell populations were monitored by flow cytometry as a surrogate for anti-CD19 CAR T cell activity. Animals treated with 10 million TU of UB-VV100 demonstrated 95% B cell depletion relative to vehicle-treated controls as of study day 11, maintaining that level of depletion until study day 25, after which B cells further declined to the point where circulating B cells were barely detectable by flow cytometry. This second phase of B cell decline was associated with rapamycin administration, which began on study day 26. Animals treated with 2 million TU of UB-VV100 demonstrated approximately 75% B cell depletion relative to vehicle-treated controls as of study day 18, which was maintained until study day 25, and then declined to barely detectable levels as of study day 32. This second phase of B cell decline was also associated with rapamycin administration.

Animals treated with vehicle only showed a gradual decline in circulating B cells, which is usually seen in CD34 humanized mice over time and observed in previous studies. In vehicle-treated mice, a sharp decline in B cells was observed on the 32nd day of the study, and the sharp decline was associated with rapamycin administration. However, vehicle-treated mice did not receive rapamycin, so this decline cannot be explained by the rapamycin effect, and since the B cell levels of vehicle-treated mice increased again by the 32nd day of the study, this decline was not significant. Compared with vehicle-treated animals, animals treated with 400,000 TU of UB-VV100 did not show B cell depletion at early time points, but there was a trend of B cell reduction at later time points. Relative to the vehicle, animals treated with cocal pseudotyped lentiviral particles encoding anti-FITC CAR surface engineering did not show B cell depletion, indicating that B cell depletion depends on the expression of anti-CD19 CAR (Figure 31B).

Throughout the study, circulating CAR T cells were measured by flow cytometry. In animals treated with 400,000 TU of UB-VV100 particles or 10 million TU of lentiviral particles encoding anti-FITC CAR, no circulating CAR T cells were observed. Circulating CAR T cells were observed in animals treated with 10 million TU of UB-VV100 starting on day 18, and increased until day 46, after which the total number of CAR T cells gradually decreased. In mice administered 2 million TU, CAR T cells were only detectable after rapamycin administration; the number peaked on day 39 and fell back to about baseline by day 53 (Figure 31C).

Since complete depletion of endogenous B cells was observed in animals treated with 2 million and 10 million TU of UB-VV100, animals were implanted with subcutaneous Raji tumors to evaluate the ability of potential circulating CAR T cells to eliminate malignant CD19-expressing cells. Tumor growth was monitored using the formula (W^2×L)/2, and it was found that groups receiving UB-VV100 at a dose of 10E ⁶ or 2E ⁶ showed reduced tumor implantation and proliferation compared to animals treated with vehicle or lentiviral particles encoding anti-FITC CAR (Figure 32A).

After study termination, tumor CAR T cell infiltration of tumors was assessed by flow cytometry and CAR T cells were found to be more abundant in tumors from mice treated with ^10E6 UB-VV100 particles (Figure 32B).

At day 81, all mice in the study were sacrificed and their bone marrow and spleen were analyzed by flow cytometry to measure the percentage of CAR T-positive cells present in the human T cell population in the bone marrow and spleen. Partial B cell depletion was observed in the bone marrow of mice dosed with 10 million TU of UB-VV100, and significant B cell depletion was observed in the spleen compared to mice treated with the vehicle (Figure 33).

All mice were weighed once a week throughout the study and the percentage of body weight change compared to their arrival body weight was calculated. No body weight loss was observed after UB-VV100 treatment, throughout rapamycin administration and after Raji tumor implantation in all the different treatment groups (data not shown).

Transduction events in blood, bone marrow, spleen, liver, ovaries, and kidneys were analyzed by ddPCR. Transgene integration was detected in the bone marrow and spleen of mice treated with 2 million or 10 million TUs 81 days after VV100 administration, and no transgene was detected in the bone marrow and spleen of animals treated with 400,000 TUs, indicating that 400,000 TUs is below the minimum effective dose in the current study design model (Figure 33C). The VV100 transgene was detected in the liver, kidney, and possibly ovaries of mice treated with 10 million TUs and sacrificed on day 81 of the study.

In this study, non-T cell transduction events were also evaluated. Flow cytometry was used to detect FMC63+ populations in CD3-human fractions, mouse CD45+ fractions, and the FMC63 expression of human CD45-, mouse CD45- fractions in spleen, blood and bone marrow was evaluated. Possibly except for the mouse CD45+ fraction of spleen, no clear non-T cell transduction population was observed in the analyzed organs (data not shown).

in conclusion

Dose-dependent B cell depletion was observed in the peripheral blood of UB-VV100-treated CD34 humanized mice. This B cell depletion was maintained for more than 81 days and was partially maintained in the bone marrow and spleen.

Compared to only gradual B cell depletion in mice treated with vehicle or control particles loaded with anti-FITC CAR (usually observed over time in the CD34+ model), a dramatic drop in B cells occurred in mice treated with 10 million TU of UB-VV100 or 2 million TU of UB-VV100, confirming that the B cell depletion was CAR-mediated and specific for the CD19 antigen (Figure 31).

The addition of rapamycin enhanced UB-VV100-mediated cell depletion and expanded the CAR-T cell population. Before the addition of rapamycin, CAR T cells were only evident in the 10 million TU UB-VV100 group, while after the addition of rapamycin, a clear CAR T cell population appeared in the blood of mice treated with 2 million TU UB-VV100 (Figure 31C).

The results further showed that the circulation of a large number of CAR T cells was not required for anti-B cell effector activity and that treatment with VV100 inhibited SC Raji tumor growth in a dose-dependent manner ( FIG. 32 ).

No significant off-target events were detected in the blood, bone marrow, or spleen by flow cytometry.

Example 10: Efficacy of lentiviral vector encoding anti-CD19 CAR in the Nalm-6 systemic tumor model in PBMC humanized mice

The purpose of this study was to evaluate the efficacy of UB-VV100 and the effect of rapamycin administration after UB-VV100 administration in the Nalm-6 systemic tumor model in PBMC humanized mice.UB-VV100 viral particles include SEQ ID NOs: 121, 128, 131 and 132.

Research Overview

Lentivirus was prepared using adherent production and titrated on 293T cells by flow cytometry.Nalm-6 GFP FFLUC cells expressing luciferase and GFP were obtained from the Jackson Laboratory at Scottish Enterprise Research, cultured by Umoja Biopharma, and delivered in PBS for injection.

Twenty-four MHC I/II KO NSG female mice were used in the study. On study day -5 (D-5), mice were implanted with 500,000 Nalm-6 cells via retro-orbital injection.

On the -1 day of the study, mice were humanized with 10,000,000 PBMCs by IP injection. On the same day, all mice were imaged with an IVIS imager 15 minutes after d-luciferin injection (15 mg/kg) to detect Nalm-6 disease load. According to tumor bioluminescence (total flux) levels, all mice from each tumor group were randomized to 4 queues according to Table 8 below.

Table 8: Study groups

On study day 0, mice from groups 3 and 4 were treated with 500ul PBS IP containing 20 million viral particles of UB-VV100. Groups 1 and 2 received vehicle (PBS) IP injections. Starting on study day 3, all mice were imaged twice a week (Tuesday, Friday) for the remainder of the study. On study day 4, mice in groups 2 and 4 began to receive 1 mg/kg rapamycin treatment by IP injection 3 times a week (Monday, Wednesday, Friday). The study timeline is shown in Figure 34A. On study days 10, 13, 17, and 20, mice with high disease burden had to be sacrificed due to weight loss and signs of distress.

The percentage of body weight loss was monitored during the study (Figure 34B). Due to the rapid expansion of tumor cells, only mice in the vehicle group and vehicle + rapamycin group had significant weight loss. On study day 41, all mice that survived to the end of the study were sacrificed. Bone marrow, spleen, peripheral blood and tumors were collected, processed into single cell suspensions, and analyzed by flow cytometry.

result

UB-VV100 treatment significantly reduced tumor burden measured by tumor bioluminescence (photons/second) (Figure 35A) and improved survival in the Nalm-6 tumor model (Figure 35B). Mice treated with UB-VV100 had their survival extended to study day 41. In contrast, all mice in the vehicle group and vehicle + rapamycin group died of disease between study days 17 and 20.

As shown in Figure 36, the disease burden of mice in the vehicle group (upper left) and the vehicle + rapamycin (upper right) group began to increase on study day 10. By day 17, the mice in these groups had to be sacrificed. The disease burden of mice in the UB-VV100 group (lower left) began to decrease on day 17; however, the effect of UB-VV100 in this group was temporary. The disease burden of all mice in the UB-VV100 + rapamycin group (lower right) began to decrease significantly on day 17, and two mice maintained a low disease burden. Only one mouse from this group had an increase in tumor burden after the initial regression.

As shown in Figure 37, mice in the vehicle group succumbed to Nalm-6 disease on day 17; mice in the vehicle + rapamycin group succumbed to disease by day 20. Disease burden was temporarily reduced in most mice in the UB-VV100-treated group; and disease burden was significantly reduced in mice in the UB-VV100 + rapamycin group, with disease burden remaining low to undetectable in most mice (only one mouse had a partial reduction in tumor burden, which subsequently increased).

As shown in Figure 38, CAR T cells expanded significantly over time in the peripheral blood of mice treated with UB-VV100 + rapamycin. In contrast, in mice treated with UB-VV100 alone, CAR T cells showed a small increase, peaked at day 17, then declined and remained stable for the rest of the study.

On day 17 of the study, tumor regression was observed in the VV100 group, however, after day 20, tumor burden began to increase. Mice in the VV100+rapamycin group began to regress tumors on day 17, with significant clearance of tumor burden in two mice and a temporary decrease in tumor burden detected by bioluminescent imaging in one mouse, followed by an increase (Figure 37). CAR T cells significantly expanded over time in the peripheral blood of mice treated with VV100+rapamycin. In mice treated with only VV100, CAR T cells showed a small increase, peaking on day 17, then declining, and remaining stable for the rest of the study (Figure 38).

As shown in Figure 39, the frequency of CAR T cells in the total immune cell population was higher in the UB-VV100+rapamycin treatment group, and was higher in the bone marrow than in the spleen at day 41 (Figure 39A). In addition, the frequency of CAR T cells in the T cell population in the bone marrow and spleen was significantly higher in the UB-VV100+rapamycin treatment group than in the UB-VV100 treatment group (Figure 39B). Therefore, in this study, rapamycin treatment resulted in enrichment of CAR+T cells.

The total CAR T population was higher in the bone marrow than in the spleen, and the percentage of CAR T cells present in the total T cell population was higher in the VV100+rapamycin group than in the VV100 alone group ( FIG. 39 ).

Bone marrow staining with a panel including P2A and CD19 confirmed that Nalm-6 tumor cells were not transduced with the UB-VV100 lentiviral vector (data not shown).

in conclusion

The data showed that VV100 significantly reduced NALM-6 tumor burden and prolonged survival. In addition, rapamycin expanded the CAR T cell population in the NALM-6 group of mice, and this expansion was inversely proportional to the tumor burden.

Example 11: In vitro pharmacology studies

The results of other in vitro pharmacology studies are summarized here.UB-VV100 virus particles include SEQ ID NOs:122, 123, 127, 101 and 103.

As shown in Figure 40A and Figure 40B, PBMCs from 3 donors were transduced with lentiviruses pseudotyped with UB-VV100 or Cocal without anti-CD3 scFv. 3 days later, CD25 activation was assessed by flow cytometry (Figure 40A). 7 days later, the transduction of CAR expression was assessed by flow cytometry (Figure 40B). The data of CD25 are shown in Figures representing all activation markers measured (i.e., CD71 and CD69; data not shown). Figures are gated for CD3+ live singlets from infection multiplicity (MOI) 5. The summary diagram is a combined data from 3 donors, and the error bars represent ± 1SEM. The results show that anti-CD3 scFv promotes the activation and transduction of T cells.

As shown in Figures 41A and 41B, PBMCs were transduced with UB-VV100 at a multiplicity of infection of 10. Starting on day 3, cells were split into separate cultures and treated with or without 10 nM rapamycin. CAR T cell transduction and expansion were assessed by flow cytometry and cell enumeration. Representative flow charts were gated for live CD3+ singlets. The results show that the RACR engine and rapamycin drive the in vitro enrichment and proliferation of CAR T cells.

The cytotoxicity of transduced CAR T cells against Raji cells was assessed. In the case of 2mM monensin, 5mg/mL brefeldin A and 2mg/mL CD107a Ab, PBMCs transduced with UB-VV100 from 2 donors were co-cultured with CD19 knockout (KO) or CD19-expressing Raji-GFP tumor cells for 5 hours. The cytotoxicity of CD8 T cells was assessed by intracellular staining of INFγ (Figure 42A) and surface CD107a (Figure 42B). In another group of experiments (Figure 42C), UB-VV100-transduced PBMCs were co-cultured with CD19-KO or CD19-expressing Raji-GFP tumor cells for 48 hours, and the killing potential was assessed by (live Raji-GFP/total Raji-GFP). In order to analyze the results, representative flow charts are gated for CD8+CD3+ live singlets. **, ***, and **** indicate p values < 0.01, 0.001, and 0.0001, respectively, by 2-way ANOVA multiple comparison test. The results showed that UB-VV100-transduced CAR T cells exhibited CD19-dependent cytotoxicity against Raji cells in vitro.

Transduction of patient-derived T cells

PBMC samples were collected from a B-ALL patient (male, 23 years old) at diagnosis and before starting treatment. The hematology report showed that the patient was in the blast stage, with 62% blasts in the blood, 95% blasts in the bone marrow, and a white blood cell count of 205.7 x 10 ⁹ cells/L. T cells accounted for <4% of the total live PBMCs (Figure 43A). The cells were transduced with twice-daily treatments of 2.5UB-VV100 transduction units per live PBMC. T cell activation and transduction were assessed by flow cytometry on the 7th day. (Figure 43B) The results showed that UB-VV100 effectively transduced T cells even when the T cell fraction accounted for 4% of the total PBMCs.

PBMC samples were collected from DLBCL patients (male, 70 years old) at diagnosis and before starting treatment. At the time of collection, the white blood cell count was 11.3 x 10 ⁹ cells/L (Figure 44A). The cells were transduced with 2.5UB-VV100 transduction units (TU) per live PBMC twice a day. T cell activation and transduction were assessed by flow cytometry on the 7th day (Figure 44B). The results show that UB-VV100 effectively transduces T cells in PBMC populations derived from DLBCL patients.

in conclusion

The results of this example show that: 1) UB-VV100 viral particles activate and transduce T cells from healthy human PBMCs in a surface engineering-dependent manner; 2) UB-VV100 viral particles transduce T cells from PBMCs of patients with B-cell malignancies; and 3) UB-VV100-transduced CAR T cells exhibit CD19-specific anti-tumor activity in vitro.

Example 12: Safety and biodistribution of UB-VV100 in mice

Research Overview

The aim of this study was to evaluate UB-VV100 tolerability and compare the effects of time and dose on vector biodistribution in three mouse models (wild-type C57BL/6J mice, NSG CD34 humanized mice [strain NOD/SCID/IL2Rγnull (NSG) - humanized with CD34+ umbilical cord blood] and NSG MHCI/IIDKO mice [strain NOD.Cg-Prkdc ^scid H2-K1 ^tm1Bpe H2-Ab1 ^em1Mvw H2-D1 ^tm1Bpe Il2rg ^tm1Wjl /SzJ] with PBMC humanization). NSG MHCI/IIDKO mice are NOD SCID-IL-2 receptor γ-deficient (an immunodeficient model lacking mature B cells, T cells and NK cells (natural killer cells), monocytes and complement). These mice also lack endogenous MHC I and II expression. CD34-NSG mice are NOD SCID-IL-2 receptor gamma deficient (an immunodeficient model lacking mature B cells, T cells and NK cells (natural killer cells), monocytes and complement) and are implanted with CD34+ cells to reconstitute the human immune system.

UB-VV100 viral particles include SEQ ID NOs: 122, 123, 126, 101, and 103. The presence and number of CD19-directed CAR+T cells in peripheral blood, spleen tissue, and bone marrow were measured by flow cytometry. B cell aplasia in the blood was assessed by flow cytometry, which is an indicator of UB-VV100 activity and an early alternative measurement of the presence of CAR+T cells. Vector integration was determined by ddPCR analysis of genomic DNA extracted from mouse tissues. Multiple RNA in situ hybridization (ISH) was performed to assess the overall expression level of transgenic RNA and determine the identity of transduced cells within each tissue.

UB-VV100 vector particles were prepared using suspended HEK293T clone A3 cells. After virus collection, the material was purified using a Mustang Q XT5 system, concentrated, and sterile filtered through a 0.22uM filter. The virus preparation was formulated in CTS ^TM OpTmizer ^TM (ThermoFisher Scientific), and aliquoted and stored at -80 ° C in disposable volumes. Before being administered to subjects, all virus preparations were tested for mycoplasma and endotoxin. Rapamycin was administered to all animals three times a week (M, W, F) starting on the 5th day of the study and continued until the end of the study.

Subjects were administered ultrapure grade phosphate buffered saline (PBS), USP, pH 7.4 as a vehicle control for UB-VV100 and rapamycin administration.All mice were randomized into cohorts according to the study groups shown in Table 9.

Table 9: Study Groups

result

UB-V100 was administered to CD34 humanized NSG mice carrying both human T cells and human B cells to evaluate its ability to generate CAR-T cells targeting CD19 in vivo across the following two dose ranges: a low dose of 20 million TU/animal and a high dose of 100 million TU/animal. Mice were treated with vehicle and rapamycin (Group 7), low dose UB-VV100 (20 million TU/mouse) and rapamycin (Group 8), low dose UB-VV100 (20 million TU/mouse) without rapamycin (Group 9) or high dose UB-VV100 (100 million TU/mouse) and rapamycin (Group 10). The ability of the anti-CD19 CAR-T cells produced to cause endogenous B cell depletion was then assessed to confirm on-target activity. The activity of the subsets in the first 4 weeks after UB-V100 administration was analyzed because the level of humanization was greatly reduced after this period of time.

Compared to vehicle-treated controls, UB-VV100 caused a dose-dependent depletion of human CD20+ B cells in the blood of CD34-NSG mice treated with low or high doses of UB-VV100 (Figure 45). B cell levels in extracted blood were measured over time (hCD20+ cell population gated for single cells, live cells, and hCD45+) weekly starting on study day 7 before dosing and after UB-VV100 administration. While human B cell levels were depleted over time in vehicle-treated mice due to loss of humanization (Group 7), a significant dose-dependent depletion of human B cell frequency occurred in UB-VV100-treated mice (Figure 45).

Compared to vehicle and rapamycin treated controls (Group 1), no depletion of endogenous circulating mouse B cells was observed in wild-type mice treated with low doses of UB-VV100 and rapamycin (20 million TU/mouse, Group 2) (data not shown). These findings are consistent with the fact that the FMC63 binder on the anti-human CD19 CAR construct expressed by UB-VV100 is not expected to cross-react with mouse CD19.

Anti-FMC63 antibodies were used in human immune cell flow cytometry panels to detect anti-CD19 CAR-T cells produced by UB-VV100. CAR-T cell levels were measured in the blood from week 1 to week 12 and in the spleen and bone marrow at planned autopsy time points (CAR+ populations gated for single cells, live cells, hCD45+ cells, and hCD3+ cells). Anti-FMC63 antibodies were also used with mouse immune cell flow cytometry panels to assess CAR+ immune cell populations in the spleen and bone marrow (groups 3-10) of humanized mice during wild-type mice (groups 1-2) and planned autopsy time points.

Significantly increased CAR-T cells were detected in the blood of CD34-NSG mice treated with high dose (100 million TU/animal) UB-VV100 and rapamycin (Group 10) compared to vehicle controls (Group 7), with the highest levels occurring at week 4 (Figure 54A). CAR-T cell levels were also significantly higher in the spleens of high dose UB-VV100 CD34-NSG mice and were detected in the spleens of some mice treated with low dose (20 million TU/animal) UB-VV100 and rapamycin (Group 8).

No staining of CAR+ mouse T cells (mCD3+ cells) was detected in the blood, bone marrow, and spleen of wild-type C57BL/6J mice treated with low doses (20 million TU/animal) of UB-VV100 above the levels in vehicle-treated animals at any time point. These findings are consistent with the prediction that the αCD3 scFv molecules present on the surface of UB-VV100 viral particles do not bind to mouse CD3 and therefore will not activate or transduce mouse T cells.

In order to determine which cell types in each ddPCR positive tissue integrate UB-VV100 payload, RNAscope ^TM LS multiplex fluorescence ISH assay was performed on a multi-tissue array containing spleen, ovary, liver, kidney and lung. The assay was performed on tissues from CD34-NSG mice; one vehicle-treated (Group 7) was used as a negative control, and four were treated with high-dose UB-VV100 (Group 10). 4-Multiple staining contains custom RNA ISH probes targeting the RACR region of UB-VV100, RNA ISH probes targeting human T cells (hCD3), RNA ISH probes targeting mouse macrophages/monocytes (mCD68), and RNA ISH probes targeting mouse endothelial cells (mPecam). The cell markers used for this assessment were selected based on the comments of pathologists on the RACR+ cell morphology in the test tissue during the initial assay development of the RACR RNA ISH probe. Visual scoring was performed by assigning a single score to each sample based on the main staining pattern in the entire sample by qualified scientists. The percentage of cells positive for each marker, as well as the percentage of RACR+ cells doubly positive for other cell type markers, were scored visually based on the number of cells with >1 spot/cell and binned into categories (0%, 1-10%, etc.).

All samples passed the negative control stain through quality control, and vehicle-treated mouse tissues (TOX001_39) were negative for RACR mRNA expression in all tissues. Across tissues from the UB-VV100 high-dose group, RACR positive staining was highest in the spleen (11-20% of all mice) and CD80 (1-10% of all mice). No RACR positive cells were observed in the heart. No or very few RACR positive rates (0%, <5 cells, or <1%) occurred in the ovaries, kidneys, and lungs. These results are greatly correlated with the levels of vector genomes detected by ddPCR in the same tissues (data not shown).

The analysis of the double positive rate of RACR+ cells in each tissue was carried out to classify the cell types. In all mice, the vast majority of RACR+ cells were co-positive for mCD68 or hCD3, indicating that the detected vector genome was due to the transduction of immune cells (macrophages or T cells) (Figure 47). In the spleen, it was noted that the staining of three cell type markers overlapped (probably due to the high cell density of the tissue); CAR-T cells were detected in the spleen of all UB-VV100-treated mice, but CAR+ macrophages were more common. In the liver, the vast majority of RACR+ cells were macrophages and some rarer RACR+ endothelial cells, but it was noted that there was an overlap between Pecam and CD68 signals (Figure 47). Rare RACR+ endothelial cell transduction was also observed in some tissues. 1 to 3 RACR+ cells negative for all markers were identified on the external ovarian lining or uterine lining of some mice, which may be due to the result of direct exposure due to the route of administration (intraperitoneal). With the exception of cells lining the uterus/ovary of one mouse and a few cells in the kidney, no unclassified RACR-positive cell types were observed.

In order to further enhance the function of UB-VV100 lentiviral particles, T cell co-stimulatory ligands were incorporated into the surface of the particles to initiate co-stimulation in conjunction with particle binding, T cell activation and transduction. In vitro, the incorporation of one or more co-stimulatory ligands on the surface of the particles enhanced the binding of lentiviral particles to T cells and the activation of T cells, thereby enhancing the proliferation and activation of transduced CAR+T cells. In addition, compared with CAR-T cells without co-stimulatory ligands, CAR-T cells produced with UB-VV100 lentiviral particles displaying co-stimulatory ligands exhibited a less differentiated central memory-like phenotype and enhanced CAR-mediated proliferation and tumor killing in vitro. It was observed that in a humanized NSG mouse model of B cell malignancies, UB-VV100 lentiviral particles engineered on the surface of co-stimulatory ligands produced CAR T cells with enhanced anti-tumor activity in vivo. For example, UB-VV100 lentiviral particles are optimized by incorporating the T cell co-stimulatory ligand CD80, which triggers CD28 co-stimulation during T cell activation and transduction. The presence of CD80 on the particle surface enhances lentiviral particle binding to T cells and activation of T cells, thereby enhancing the proliferation and activation of CAR+T cells in vivo. In a humanized NSG mouse model of B-cell malignancies, CAR T cells generated with CD80-containing lentiviral particles enhanced antitumor activity.

The results suggest that the collective mechanism of action of UB-VV100 lentiviral particles initiating anti-tumor immune responses can be enhanced by the expression of a combination of surface-displayed ligands that engage T cell activation and co-stimulatory pathways, both of which are required to enable T cell transduction while optimizing their immunophenotype and function.

The UB-VV100 toxicology study further demonstrated good safety and biodistribution properties using intranodal administration in dogs. The study design is depicted in Table 10.

Table 10: Study Groups

Conc. = concentration; No. = number; TU = transduction unit; LN = lymph node; IP = intraperitoneal;

Adm = administration.

All tissues and blood samples of the control animals in Group 1 were negative for αCD3-Cocal-GFP. Group 2, Group 3 and Group 4 were negative or below the quantitative lower limit (Figure 66) for brain, ovary, testis, heart, adrenal gland, spinal cord, chest, kidney, lung, thymus, injection site and liver. αCD3-Cocal-EGFP was well tolerated in canines by using 3.98e8 TU/mL and 3.98e9 TU/mL in the nodal and intraperitoneal respectively. There was no effect on toxicological endpoints (clinical signs, body weight, food consumption and clinical pathology) in life, and there was no post-mortem macroscopic and microscopic discovery. αCD3-Cocal-GFP was quantitatively shown by qPCR in blood (by intraperitoneal), superficial inguinal lymph nodes, medial iliac lymph nodes and lumbar lymph nodes (by in the nodal) and spleen (by intraperitoneal and in the nodal).

Intranodal administration of UB-VV100 lentiviral particles to dogs was well tolerated and resulted in transduction that was largely restricted to the injected lymph nodes, with transduction of downstream draining lymph nodes reduced by approximately 90% and no transduction in non-immune organs.

in conclusion

In this study, three mouse models (wild-type C57BL/6J mice, CD34 humanized NSG mice, and PBMC humanized NSG MHCI/IIDKO mice) were evaluated. UB-VV100 was well tolerated at a dose of 20 million TU per animal in all three mouse models and at a dose of 100 million TU per animal in CD34-NSG mice. Importantly, histopathological tissue evaluation performed by a board-certified pathologist did not reveal any microscopic findings clearly related to UB-VV100 treatment.

UB-VV100 treatment results in the production of human CAR-T cells in vivo in the blood and spleen of CD34 and PBMC humanized NSG mice. In wild-type C57BL/6J mice lacking human T cells, the target of UB-VV100, no evidence of any UB-VV100 bioactivity (CAR-T cell production, B cell depletion) was detected. Since PBMC humanized mice lack human B cells, the target of the viral αCD19 CAR payload, the complete UB-VV100 bioactivity was only evaluated in CD34 humanized NSG mice. During the first 28 days after UB-VV100 was injected intraperitoneally into CD34-NSG mice, a significant dose-dependent depletion of circulating B cells was observed over time (Figure 45).

Due to the high sensitivity of the assay, the biodistribution of UB-VV100 was first evaluated on genomic DNA extracted from tissues using ddPCR, and then multiple RNA ISH was performed to identify the cell types of transduced cells in ddPCR positive tissues. In all three mouse models of UB-VV100 injected intraperitoneally with 20 million TU, the presence of detectable vector copies was largely confined to the liver and spleen (Figures 46C and 46D), and the main transduced cell types observed were human T cells and mouse macrophages. Rare transduction of tissue epithelial cells was observed in some tissues. Vector integration occurs in a dose-dependent manner in CD34-NSG mice (the only model treated at two dose levels). At a dose of 20 million TU of UB-VV100, no transduction (Figure 46C) higher than the vehicle control was observed in the heart, brain, kidney, and lung.

In summary, in this study, the main tissues in which transduction events were detected were liver and spleen, and the main transduced cell types observed were human T cells and mouse macrophages (Figure 47). Rare transduced non-immune cells could be identified in these tissues and the outer ovarian lining, which was attributed to the IP administration route. At low doses, transduction above vehicle control levels was not measured in the heart, brain, kidney, and lung. Importantly, no histopathological findings clearly associated with UB-VV100 administration were observed.

The data showed that the CD34 humanized NSG mouse model transduced with UB-V100 had complete biological activity (CAR-T cell generation and B cell aplasia), and the mice displayed a higher and more stable level of humanization.

Example 13: Transduction of CD19+ B cells with VV100

VV100 is engineered to selectively activate and transduce T cells in vivo.VV100 lentiviral drug products include SEQ ID NO: 122, 123, 127, 124 and 125. Selective T cell activation is achieved by expressing anti-CD3-scFv on the viral envelope.Slow virus encodes anti-CD19-CAR for tumor targeting by FMC63 scFv, short IgG4 hinge (SEQ ID NO: 95) and 4-1BB/CD3ζ signaling domain. It also encodes rapamycin-activated cytokine receptor box (FRB-RACR) to promote the survival and proliferation of CAR-T cells in the presence of rapamycin (Figure 23B). The purpose of this study is to evaluate the therapeutic application of VV100 for the treatment of B-cell malignancies expressing CD19 by intranodal injection.

Epitope masking occurs when tumor B cells are inadvertently transduced to express an anti-CD19 CAR, and the expression of the CAR renders the CD19 epitope invisible, or "masked" by the CAR T cell so that it cannot be recognized. The potential mechanism driving epitope masking is the binding of the anti-CD19 CAR to CD19 on the surface of tumor cells in cis. This phenomenon was first reported in pediatric B-ALL patients treated with the anti-CD19 CAR T cell product CTL019 (Ruella M et al. Nature Medicine 2018; 24(10): 1499-1503). CTL019 encodes a 45-amino acid long CD8a hinge. It is speculated that the longer length of the CD8a hinge achieves epitope masking by providing the flexibility required to approach the CAR binding epitope. In order to minimize the risk of epitope masking by VV100, the anti-CD19 CAR was intentionally designed to encode a short IgG4 hinge (14 amino acids). Structural analysis of CD19 suggests membrane distal localization of the CAR epitope and is therefore inaccessible for cis binding by CARs with a short hinge. To assess whether epitope masking occurs in the context of VV100, Nalm6 cells, a B-ALL tumor cell line, were transduced with VV100 to model the transduced CD19+ tumor cell scenario. The study sought to answer two questions: (1) Do CAR+Nalm6 cells show a significant loss of surface CD19 detection by flow cytometry? (2) Can anti-CD19 CAR-T cells kill CAR+Nalm6 cells?

result

In order to determine the impact of transducing CD19+B cells with VV100, Nalm6, i.e., B-ALL tumor cell line, was used as a model. By the 10th day, 37.6% of CAR+Nalm6 cells were produced by transduction with MOI 1 using VV100, and even higher transduction efficiency was caused by transduction with MOI 10 and 20, with 70% of CAR+Nalm6 cells. Next, CAR+Nalm6 cells were stained with anti-CD19 antibodies (clone HIB19) to assess surface CD19 levels by flow cytometry (Figure 48A). HIB19 antibodies are combined with the same CD19 epitopes as anti-CD19 CAR FMC63 scFv. Using this antibody, it was observed that, compared with untransduced Nalm6 parental cells, the surface CD19 detection of CAR+Nalm6 cells was reduced, and surface CD19 MFI was lower (Figure 56A) with higher MOI transduction. When CAR+Nalm6 cells were stained with different anti-CD19 antibodies (clone EPR5906) recognizing intracellular CD19 epitopes, it was observed that the overall CD19 protein level was similar to that of untransduced Nalm6 cells, which confirmed that CD19 protein was still expressed in CAR+Nalm6 cells (Figure 48 B). Data show that the surface CD19 detection of CAR+Nalm6 cells is reduced because the CD19 epitope recognized by HIB19 antibody is blocked by the cis anti-CD19 CAR binding to CD19. However, the surface CD19 level on CAR+Nalm6 cells is still detectable and is higher than the level of Nalm6 cells knocked out by CD19.

To determine whether anti-CD19 CAR-T cells can kill CAR+Nalm6 cells in the presence of reduced surface CD19 detection, an in vitro killing assay was established. Nalm6 GFP cells were transduced with VV100 at MOI 10, and 88% of CAR+Nalm6 cells were produced in the presence of reduced surface CD19 detection, as observed in the above studies. PBMCs from 3 healthy donors were transduced with VV100 to produce 7.8% to 19% of CAR-T cells. The transduced Nalm6 cells were then co-cultured with the transduced PBMCs at different CAR-T/Nalm6 ratios. In order to normalize background nonspecific killing, the transduced Nalm6 cells were also co-cultured with the PBMCs transduced with the mocks. After 24 hours, the transduced Nalm6 cells were gated as CAR+Nalm6 cells by flow cytometry based on intracellular transgene expression (detected with anti-P2A antibody). The percentage of lysis is calculated as the frequency of CAR+Nalm6 cells that died when co-cultured with CAR-T cells minus the frequency of CAR+Nalm6 cells that died when co-cultured with PBMCs transduced with mocks. As shown in Figure 49, anti-CD19 CAR-T cells are able to kill CAR+Nalm6 cells. Importantly, the percentage of lysis is similar between CAR+Nalm6 cells and untransduced Nalm6 parental cells, and the percentage of lysis is higher than the percentage of lysis of Nalm6 cells knocked out by CD19 in all 3 healthy donors. The data show that epitope masking and subsequent antigen escape do not occur when Nalm6 cells are transduced with VV100. However, despite the reduction in surface CD19 detection, anti-CD19 CAR-T cells can still mediate CD19 antigen-specific cytolytic activity against CAR+Nalm6 cells in vitro.

When VV100 is injected into the patient, tumor cells can be exposed to viral particles. In order to model the scenario in which there are a large number of tumor B cells during transduction, 5e5 Nalm6 GFP cells and 5e5 healthy donor PBMCs are mixed and transduced with VV100 at MOI 5 (Figure 50). In this model, anti-CD19 CAR-T cells and CAR+Nalm6 cells are produced in the same well by VV100 transduction. It can then be assessed whether anti-CD19 CAR-T cells eliminate CAR+Nalm6 cells, or whether CAR+Nalm6 escapes detection and grows unconstrained. In order to understand whether anti-CD19 CAR-T cells from multiple healthy donors can kill CAR+Nalm6 cells, PBMCs from 8 healthy donors were studied in a single experiment. Healthy donors represent a wide age range with different percentages of natural and memory T cell subsets in PBMC samples.

In all 8 healthy donors, mixed populations of Nalm6 cells and PBMCs transduced with VV100 or unrelated anti-FITC CAR produced both CAR+Nalm6 cells and CAR-T cells (Figures 51 and 52). On the 3rd day, in the case of reduced surface CD19 detection, 51.7%-67.9% of anti-CD19CAR+Nalm6 cells were produced with VV100 transduction. As of the 7th day, the frequency and total number of anti-CD19 CAR+Nalm6 cells decreased until all anti-CD19CAR+Nalm6 cells of 7 healthy donors in 8 healthy donors were eliminated (Figure 51B). On the contrary, no Nalm6 cells were eliminated with unrelated CAR transduction, indicating that the killing of CAR+Nalm6 cells was mediated by anti-CD19 CAR-T cells combined with CD19 epitopes.

As of the 7th day, healthy donor 66BW did not eliminate Nalm6 cells, however, this donor did show control of CAR- and CAR+Nalm6 cells (Figure 51). It is speculated that this is due to 66BW not producing enough CAR-T cell numbers to eliminate rapidly dividing Nalm6 cells. On the 7th day, compared with other healthy donors, the frequency (2.03%) and total number (1.39e4) of CAR-T cells of 66BW were the lowest (Figure 52). Compared with transduction (4.78e6 cells) with anti-FITC CAR, on the 10th day, transduction with VV100 in this donor still produced fewer Nalm6 cells (7.31e5 cells), and the ratio of CAR+Nalm6 and CAR-Nalm6 cells remained constant over time. This indicates that donor 66BW has similar levels of control for CAR+Nalm6 and CAR-Nalm6, and any apparent reduction in killing is not due to the expression of CAR by Nalm6 cells, but rather to an overall defect in T cell effector function specific to this donor (Figures 51A and 51B). Overall, this data confirms that anti-CD19 CAR-T cells can target and kill CAR+Nalm6 cells after VV100 treatment, indicating that epitope masking does not occur when CD19+B cells are transduced with VV100.

in conclusion

Surface CD19 detection was reduced in Nalm6 cells transduced with VV100, but the reduced level was higher than that of CD19 knockout Nalm6 cells. In an in vitro killing assay, anti-CD19 CAR-T cells could kill CAR+Nalm6, with similar percentages of lysis between CAR+Nalm6 and CAR-Nalm6 cells. When a mixed population of Nalm6 and PBMCs was transduced with VV100, anti-CD19 CAR-T cells could eliminate CAR+Nalm6 cells in the same well.

Example 14: Comparison of different versions of UB-VV100 against the Nalm-6 systemic tumor model

To evaluate the efficacy of different versions of UB-VV100 in a humanized NSG mouse model, UB-VV100 produced by suspension versus adherent methods, UB-VV100 produced with different payload versions, and UB-VV100 produced with different anti-CD3 scFv versions were compared and analyzed.

This study directly compared the efficacy of adherent UB-VV100 particles to suspension UB-VV100 particles. To perform this experiment, seven different treatment groups were analyzed: Two groups of mice were treated with 20E+06 TU of UB-VV100 produced using 293T adherent cells using a 4-plasmid system comparing a payload containing WPRE (142) (SEQ ID NO: 121) and a payload not containing WPRE (201) (SEQ ID NO: 122). Two groups of mice were treated with 20E+06 or 100E+06 TU of VPN38, a suspension vector produced using a 5-plasmid system using transgenes SEQ ID NO: 122 and αCD3 scFv (SEQ ID NO: 126). Two groups of mice were treated with 20E+06 or 100E+06 TU of VPN68, a 5-plasmid suspension vector containing an αCD3 scFv binder (SEQ ID NO: 127).

The aims of this study are to:

1) Determine whether the adherent vector prepared with transgene SEQ ID NO:121 is equivalent to the adherent vector prepared with transgene SEQ ID NO:122.

2) To determine if efficacy is reduced during the change in preparation from adherent to suspended vectors.

3) To evaluate whether dose escalation to 100E+06TU could overcome the potential loss of activity/PK shift observed with the suspension batches relative to the adherent batches.

4) The anti-CD3 scFv binder (SEQ ID NO: 127) was compared to the version (SEQ ID NO: 126).

Research Overview

48 female 6-8 week old mice NSG MHC class I and class II KO mice were used in the study. Lentivirus was prepared using 293T adherent production (142 (SEQ ID NO: 121), 201 (SEQ ID NO: 122)) titrated on 293T adherent cells. Suspension batches were produced on 293T suspension cells (VPN38 and VPN68) and titrated on SUPT1 cells by ddPCR. UB-VV100 virus preparations were negative for mycoplasma with endotoxin activity less than 2EU/ml, respectively.

On the morning of infection, the virus was thawed at room temperature and diluted in PBS. The virus preparation was kept on ice and allowed to equilibrate to room temperature before 500 ul was injected per mouse (IP).

Study plan

On the 5th day of the study, 5E05 Nalm-6 GFP FFLUC tumor cells were transplanted into 40 mice by retro-orbital injection. On the morning of the 1st day of the study, IVIS imaging was used to assess tumor load, and 10 million human PBMCs were injected into mice by IP injection to ensure successful implantation. Mice were assigned a research group at this time and were distributed to 7 treatment groups based on tumor load. On the 0th day of the study, mice were treated with UB-VV100 or vehicle by IP (Table 11). At the beginning of the study on the 4th day, all mice were treated by intraperitoneal injection with 1mg/kg rapamycin on Monday/Wednesday/Friday schedule. The research timeline is described in Figure 53.

Table 11: Study Groups

result

During the study, the survival rate of the animals was monitored. It was found that all animals treated with the vehicle died as of day 22 of the study, while animals treated with UB-VV100 of the attached or suspended batches showed survival after this time point (Figure 54). During the study, 40% of the animals were sacrificed due to retro-orbital tumor development. In addition, 5 animals receiving VPN68 of 100 million TU were treated due to acute weight loss shortly after the flooding event.

T cell populations on day 3 were analyzed to assess T cell activation shortly after vector administration. It was found that CD25 expression was low in all study groups (data not shown). In animals treated with various tested batches of UB-VV100 T cells, CD71 expression in the east test batch was higher than that in T cells of vehicles treated animals (Figure 55). It was found that animals treated with 100E06 TU of VPN68 showed higher CD71 expression than animals treated with 100E6 TU VPN38. The trend that emerged was that CD71 expression in T cells of animals treated with 100E06 TU was higher than that of animals treated with 20E06 TU of suspension batches. In summary, these findings indicate that both adherent particles and suspension particles promote activation, as measured by the upregulation of CD71, and that the upregulation is dose-dependent. Animals treated with VPN68 showed the highest activation (highest CD71 expression %) (Figure 55).

During the study, continuous weekly blood draws were performed to assess CAR T cell expansion. It was found that animals treated with all formulations of UB-VV100 produced CAR T cells, and in the remaining surviving animals, the cells expanded between the 14th and 42nd days of the study, and then shrank within the 42nd and 49th days (Figure 56A). Animals treated with 20E06 TU adherent materials with 142 payloads (SEQ ID NO: 121) showed CART cell expansion between the 14th and 28th days, and the cells shrank thereafter. Animals treated with adherent formulations 142 (SEQ ID NO: 121), VPN38 20E06 TU, VPN38100E06 TU, VPN68 20E06 TU, and VPN68 100E06 TU had a peak circulating CAR T cell concentration of >150 CAR T cells/μL of blood, while animals treated with adherent formulations 201 reached a peak of <100 CAR T cells/μL of blood. Animals treated with both adherent formulations, as well as animals treated with 100E06 TU of VPN68 and VPN38, had readily detectable CAR T cells by study day 14; in contrast, animals treated with 20E06 TU of VPN38 and VPN68 had no detectable CAR T cells at this early time point. In summary, all formulations of UB-VV100 promoted upregulation of CD71 by study day 3 and produced detectable CAR T cells by study day 21, but animals treated with 20E6 TU of suspension particles exhibited a 1-week delay in detecting CAR T cells compared to animals treated with adherent formulations or at higher doses (Figure 56B).

Disease progression was monitored by bioluminescent imaging after injection of d-luciferin by the IP route. Bioluminescent imaging was performed twice a week throughout the study. Mice in the control group reached a humane endpoint around day 25 and had to be sacrificed due to disease burden and weight loss. About 40% of the mice developed ocular tumors and had to be sacrificed early. The tumor burden of mice treated with 20E6UB-VV100 VPN38 and VPN68 was controlled, and the life of the mice was extended compared to the vehicle, however, no tumor reduction was observed. Compared with mice treated with 20E6 UB-VV100, the tumor reduction of mice treated with 100E6 UB-VV100VPN38 and VPN68 was increased. The greatest tumor reduction was observed in mice treated with 100E6 VPN68 (Figure 57). Mice treated with 20E6 adherent batches of UB-VV100 (142 (SEQ ID NO: 121) or 201 (SEQ ID NO: 122) payload) had greater tumor reduction than mice treated with the suspension batch of 20E6 TU (Figure 57).

When mice reached humane endpoints, spleen and bone marrow were collected and processed for analysis, including analysis of tumor burden by flow cytometry. To assess tumor burden, the percentage of NALM-6 cells (live, CD45-, GFP+) was measured. It was observed that mice in the vehicle control group had NALM-6 cells, which accounted for more than 20% of cells in their spleen and approached 80% in the bone marrow. After UB-VV100 treatment, tumors in spleen and bone marrow were greatly reduced (Figure 58). The maximum tumor reduction was observed in the bone marrow and spleen in mice treated with 100E6 VPN68 (Figure 58).

in conclusion

Improved survival was observed in mice treated with UB-VV100 relative to controls. During the study, 40% of the animals developed retro-orbital tumors and many of these animals had to be sacrificed based on criteria recommended by the attending veterinarian, despite the low tumor burden in peripheral organs and the absence of other sacrifice criteria. Mice treated with 100E6 TU UB-VV100 VPN68 experienced cage hemorrhage events and had to be sacrificed shortly thereafter due to weight loss.

All versions of UB-VV100 tested showed anti-tumor activity, with prolonged survival compared to mice treated with vehicle alone, however, mice treated with VPN68 showed the most significant tumor control. At sacrifice, mice treated with UB-VV100 presented tumor reduction in the spleen and bone marrow compared to vehicle-treated mice. Taken together, these results suggest that all formulations of UB-VV100 have some activity. The most significant tumor reduction/control was observed in non-retro-orbital sites with treatment of the adherent batch at 20E+06TU and the suspension batch at 100E+06TU.

The activation of T cells after UB-VV100 administration (using activation markers CD25 and CD71) was examined to examine the difference in efficacy. On day 3 of the study, no difference in CD25 expression was found in any group. However, all mice treated with UB-VV100 showed the highest expression level of CD71 on T cells compared to mice treated with the vehicle. Specifically, at a dose of 20E6 UB-VV100, higher expression of CD71 was observed in mice treated with adherent particles compared to suspended particles. This finding suggests that the adherent carrier of 20E+06TU is more active than the suspended carrier of 20E+06TU. When examining VPN38 and VPN68, it was observed that the construct for αCD3 scFv expression present in VPN68 (SEQ ID NO: 127) was significantly associated with higher levels of activation measured by CD71 at higher dose levels. Additionally, higher CD71 levels were present in mice treated with 100E6 UB-VV100 compared to 20E6 suspended particles, suggesting that upregulation of CD71 was dose-dependent.

All formulations of UB-VV100 generated detectable CAR T cells that expanded between study days 14 and 21 and contracted during the period from day 42 to day 49. Consistent with the lower expression of CD71 detected on study day 3, animals treated with 20E+06 TU of suspended particles showed a delay of 1 week in detecting CAR T cells compared to animals treated with either adherent formulation that generated detectable CAR T cells by study day 14.

In summary, it was found that switching from plasmid SEQ ID NO: 121 to plasmid SEQ ID NO: 122 had little effect on in vivo efficacy, the suspended particles appeared to be less potent and less active than the adherent particles, and this difference could be overcome by increasing the dose. In addition, switching from plasmid SEQ ID NO: 126 to plasmid SEQ ID NO: 127 was associated with greater activation of T cells and better tumor clearance at day 3 in animals treated with 100E+06TU. In the systemic Nalm-6 tumor model, dose escalation to 100E+06TU will likely be necessary to produce consistent and predictable efficacy.

abbreviation

Sequence Listing

<110> Umoja Biopharma, Inc

Scharenberg, Andrew

Crisman, Ryan

Nicolai, Christopher

Sullivan, Alessandra

Michels, Kathryn

K. Michels (Ryu, Byoung)

Shon Green

L. Beitz, Laurie

Hernandez Lopez, Susana

<120> Lentivirus for generating cells expressing anti-CD19 chimeric antigen receptor

<130> TPG03262A

<150> 63/142,347

<151> 2021-01-27

<150> 63/185,765

<151> 2021-05-07

<160> 144

<170> PatentIn Version 3.5

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85 90 95

Ser Ile His Ser Ile Gln Pro Thr Ser Glu Gln Cys Lys Glu Ser Ile

100 105 110

Lys Gln Thr Lys Gln Gly Thr Trp Met Ser Pro Gly Phe Pro Pro Gln

115 120 125

Asn Cys Gly Tyr Ala Thr Val Thr Asp Ser Val Ala Val Val Val Gln

130 135 140

Ala Thr Pro His His Val Leu Val Asp Glu Tyr Thr Gly Glu Trp Ile

145 150 155 160

Asp Ser Gln Phe Pro Asn Gly Lys Cys Glu Thr Glu Glu Cys Glu Thr

165 170 175

Val His Asn Ser Thr Val Trp Tyr Ser Asp Tyr Lys Val Thr Gly Leu

180 185 190

Cys Asp Ala Thr Leu Val Asp Thr Glu Ile Thr Phe Phe Ser Glu Asp

195 200 205

Gly Lys Lys Glu Ser Ile Gly Lys Pro Asn Thr Gly Tyr Arg Ser Asn

210 215 220

Tyr Phe Ala Tyr Glu Lys Gly Asp Lys Val Cys Lys Met Asn Tyr Cys

225 230 235 240

Lys His Ala Gly Val Arg Leu Pro Ser Gly Val Trp Phe Glu Phe Val

245 250 255

Asp Gln Asp Val Tyr Ala Ala Ala Lys Leu Pro Glu Cys Pro Val Gly

260 265 270

Ala Thr Ile Ser Ala Pro Thr Gln Thr Ser Val Asp Val Ser Leu Ile

275 280 285

Leu Asp Val Glu Arg Ile Leu Asp Tyr Ser Leu Cys Gln Glu Thr Trp

290 295 300

Ser Lys Ile Arg Ser Lys Gln Pro Val Ser Pro Val Asp Leu Ser Tyr

305 310 315 320

Leu Ala Pro Lys Asn Pro Gly Thr Gly Pro Ala Phe Thr Ile Ile Asn

325 330 335

Gly Thr Leu Lys Tyr Phe Glu Thr Arg Tyr Ile Arg Ile Asp Ile Asp

340 345 350

Asn Pro Ile Ile Ser Lys Met Val Gly Lys Ile Ser Gly Ser Gln Thr

355 360 365

Glu Arg Glu Leu Trp Thr Glu Trp Phe Pro Tyr Glu Gly Val Glu Ile

370 375 380

Gly Pro Asn Gly Ile Leu Lys Thr Pro Thr Gly Tyr Lys Phe Pro Leu

385 390 395 400

Phe Met Ile Gly His Gly Met Leu Asp Ser Asp Leu His Lys Thr Ser

405 410 415

Gln Ala Glu Val Phe Glu His Pro His Leu Ala Glu Ala Pro Lys Gln

420 425 430

Leu Pro Glu Glu Glu Thr Leu Phe Phe Gly Asp Thr Gly Ile Ser Lys

435 440 445

Asn Pro Val Glu Leu Ile Glu Gly Trp Phe Ser Ser Trp Lys Ser Thr

450 455 460

Val Val Thr Phe Phe Phe Ala Ile Gly Val Phe Ile Leu Leu Tyr Val

465 470 475 480

Val Ala Arg Ile Val Ile Ala Val Arg Tyr Arg Tyr Gln Gly Ser Asn

485 490 495

Asn Lys Arg Ile Tyr Asn Asp Ile Glu Met Ser Arg Phe Arg Lys

500 505 510

<210> 6

<211> 63

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding CD8 signal peptide

<400> 6

atggcactgc ctgtgacagc cctgctgctg ccactggccc tgctgctgca cgcagcacgc 60

cca 63

<210> 7

<211> 744

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding anti-CD3 scFv construct

<400> 7

gatatccaga tgacccagtc cccaagctcc ctgagcgcct ccgtgggcga ccgggtgaca 60

atcacctgca gcgcctctag ctccgtgtcc tacatgaact ggtatcagca gacacctggc 120

aaggccccaa agagatggat ctacgatacc agcaagctgg cctccggcgt gccttctagg 180

ttttctggca gcggctccgg cacagattat acattcacca tctctagcct gcagccagag 240

gacatcgcca cctactattg ccagcagtgg tcctctaatc cctttacatt cggccagggc 300

accaagctgc agatcacaag aacctctgga ggaggaggaa gcggagggaggatccggc 360

ggcggcggct ctcaggtgca gctggtgcag agcggaggag gagtggtgca gccaggcaga 420

agcctgaggc tgtcctgtaa ggcctctggc tacacattca ccagatatac aatgcactgg 480

gtgaggcagg caccaggcaa gggactggag tggatcggct acatcaaccc ctccaggggc 540

tacaccaact ataatcagaa ggtgaaggat cggttcacca tcagcaggga caactccaag 600

aataccgcct tcctgcagat ggacagcctg aggccagagg ataccggcgt gtacttttgc 660

gcccggtact atgacgatca ctactgtctg gattattggg gccagggaac accagtgacc 720

gtgagctccg ccgcagcaaa gcct 744

<210> 8

<211> 135

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding CD8 hinge

<400> 8

accacaaccc ctgccccaag gccacctaca cccgccccta ccatcgcctc tcagccactg 60

agcctgaggc cagaggcatc caggcctgcc gcagggggggg ccgtgcacac ccggggcctg 120

gactttgcctctgat 135

<210> 9

<211> 159

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding cd tm, cytoplasmic tail and T2a linker construct

<400> 9

atggcactga tcgtgctggg aggagtggca ggactgctgc tgttcatcgg actgggcatc 60

ttcttttgcg tgcgctgtag gcaccggaga aggcagggat ctggagaggg aaggggaagc 120

ctgctgacat gcggcgacgt ggaggagaac ccaggacca 159

<210> 10

<211> 1533

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation of polynucleotide encoding cocal env in the laboratory

<400> 10

aattttctgc tgctgacctt catcgtgctg cctctgtgca gccacgccaa gttttccatc 60

gtgttcccac agtcccagaa gggcaactgg aagaatgtgc cctctagcta ccactattgc 120

ccttcctcta gcgaccagaa ctggcacaat gatctgctgg gcatcacaat gaaggtgaag 180

atgcccaaga cccacaaggc catccaggca gatggatgga tgtgccacgc agccaagtgg 240

atcacaacct gtgactttcg gtggtacggc cccaagtata tcacacactc catccactct 300

atccagccta cctccgagca gtgcaaggag tctatcaagc agacaaagca gggcacctgg 360

atgagccctg gcttcccacc ccagaactgt ggctacgcca cagtgaccga ctccgtggca 420

gtggtggtgc aggcaacacc tcaccacgtg ctggtggatg agtataccgg cgagtggatc 480

gacagccagt ttccaaacgg caagtgcgag acagaggagt gtgagaccgt gcacaattct 540

acagtgtggt acagcgatta taaggtgaca ggcctgtgcg acgccaccct ggtggataca 600

gagatcaccttcttttctga ggacggcaag aaggagagca tcggcaagcc caacaccggc 660

tacagatcca attacttcgc ctatgagaag ggcgataagg tgtgcaagat gaattattgt 720

aagcacgccg gggtgcggct gcctagcggc gtgtggtttg agttcgtgga ccaggacgtg 780

tacgcagcag caaagctgcc tgagtgccca gtggggagcaa ccatctccgc cccaacacag 840

acctccgtgg acgtgtctct gatcctggat gtggagcgca tcctggacta cagcctgtgc 900

caggagacct ggagcaagat ccggtccaag cagcccgtgt cccctgtgga cctgtcttac 960

ctggcaccaa agaacccagg aaccggacca gcctttacaa tcatcaatgg caccctgaag 1020

tacttcgaga cccgctatat ccggatcgac atcgataacc ctatcatcag caagatggtg 1080

ggcaagatct ctggcagcca gacagagaga gagctgtgga ccgagtggtt cccttacgag 1140

ggcgtggaga tcggcccaaa tggcatcctg aagacaccaa ccggctataa gtttcccctg 1200

ttcatgatcg gccacggcat gctggacagc gatctgcaca agacctccca ggccgaggtg 1260

tttgagcacc cacacctggc agaggcacca aagcagctgc ctgaggagga gacactgttc 1320

tttggcgata ccggcatctc taagaacccc gtggagctga tcgagggctg gttttcctct 1380

tggaagagca cagtggtgac cttctttttc gccatcggcg tgttcatcct gctgtacgtg 1440

gtggccagaa tcgtgatcgc cgtgagatac aggtatcagg gctccaacaa taagaggatc 1500

tataatgaca tcgagatgtc tcgcttccgg aag 1533

<210> 11

<211> 17

<212> PRT

<213> Unknown

<220>

<223> Gaussia species luciferase

<400> 11

Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu

1 5 10 15

Ala

<210> 12

<211> 245

<212> PRT

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Anti-CD3 scFv construct

<400> 12

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Gln Ile Thr Arg Thr Ser Gly Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu

115 120 125

Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu

130 135 140

Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp

145 150 155 160

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile Asn

165 170 175

Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Val Lys Asp Arg Phe

180 185 190

Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Ala Phe Leu Gln Met Asp

195 200 205

Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys Ala Arg Tyr Tyr

210 215 220

Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Pro Val Thr

225 230 235 240

Val Ser Ser Ala Ser

245

<210> 13

<211> 62

<212> PRT

<213> Cocal virus

<400> 13

Gly Val Glu Leu Ile Glu Gly Trp Phe Ser Ser Trp Lys Ser Thr Val

1 5 10 15

Val Thr Phe Phe Phe Ala Ile Gly Val Phe Ile Leu Leu Tyr Val Val

20 25 30

Ala Arg Ile Val Ile Ala Val Arg Tyr Arg Tyr Gln Gly Ser Asn Asn

35 40 45

Lys Arg Ile Tyr Asn Asp Ile Glu Met Ser Arg Phe Arg Lys

50 55 60

<210> 14

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> Prepared in the laboratory - Polynucleotide encoding the Gaussia species luciferase signal peptide

<400> 14

atgggcgtga aagtgctgtt cgccctgatc tgcatcgcag ttgctgaagc c 51

<210> 15

<211> 735

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding anti-CD3 scFv construct

<400> 15

gacatccaga tgacccagtc tcctagcagc ctcagcgcta gcgtgggcga tagagtgacc 60

atcacatgta gcgccagcag cagcgtgtcc tacatgaact ggtaccagca aacacctgga 120

aaggccccta aaaggtggat ctatgacaca tctaagctgg cttctggagt gccatctaga 180

ttttctggca gcggctccgg cactgattat acattcacca tcagcagcct gcagcccgag 240

gatatcgcca cctactactg tcagcagtgg tcctctaatc ccttcacctt cggccagggc 300

accaagctgc agatcaccag aaccagcggc gggggaggaa gcggcggggg aggatctggc 360

ggcggcggca gccaggtgca gctggtgcag agcggcggcg gcgtggtgca acctggcaga 420

agcctgagac tgagctgcaa ggcctctggc tacaccttca cccggtacac catgcattgg 480

gtgcggcagg cccctggcaa gggcctggaa tggattggat acatcaaccc cagcagaggc 540

tacaccaact acaaccagaa ggtgaaggac agattcacaa tttctcggga caacagcaag 600

aataccgcct tcctgcaaat ggactccctg cgcccagaag ataccggcgt gtacttctgc 660

gctagatatt acgacgacca ctactgcctg gactactggg gccagggcac ccctgtgacc 720

gtgtccagcgcctcc 735

<210> 16

<211> 186

<212> DNA

<213> Artificial sequence

<220>

<223> Prepared in the laboratory - Polynucleotide encoding the short hinge-TM-CT from Cocal Env

<400> 16

ggagtggaac tgatcgaggg ctggttcagc agctggaaaa gcaccgtggt tacattcttt 60

ttcgccatcg gcgtgttcat cctgctgtac gtggtcgcca gaattgtgat cgccgtgcgg 120

tatagatacc agggcagcaa caacaagcgg atctacaacg acatcgagat gagcagattc 180

agaaag 186

<210> 17

<400> 17

000

<210> 18

<211> 0

<212> PRT

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Anti-CD3 scFv construct

<400> 18

000

<210> 19

<211> 93

<212> PRT

<213> Cocal virus

<400> 19

Ser Gly Phe Glu His Pro His Leu Ala Glu Ala Pro Lys Gln Leu Pro

1 5 10 15

Glu Glu Glu Thr Leu Phe Phe Gly Asp Thr Gly Ile Ser Lys Asn Pro

20 25 30

Val Glu Leu Ile Glu Gly Trp Phe Ser Ser Trp Lys Ser Thr Val Val

35 40 45

Thr Phe Phe Phe Ala Ile Gly Val Phe Ile Leu Leu Tyr Val Val Ala

50 55 60

Arg Ile Val Ile Ala Val Arg Tyr Arg Tyr Gln Gly Ser Asn Asn Lys

65 70 75 80

Arg Ile Tyr Asn Asp Ile Glu Met Ser Arg Phe Arg Lys

85 90

<210> 20

<400> 20

000

<210> 21

<211> 0

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding anti-CD3 scFv construct

<400> 21

000

<210> 22

<211> 279

<212> DNA

<213> Artificial sequence

<220>

<223> Prepared in the laboratory - Polynucleotide encoding the long hinge-TM-CT from Cocal Env

<400> 22

tccggattcg agcaccccca cctggccgag gcccctaagc agctgcctga agaagagaca 60

ctgtttttcg gagataccgg catcagcaaa aaccccgtgg agctgatcga gggctggttc 120

agctcttgga agagcaccgt ggtcacattc tttttcgcca tcggcgtctt tatcctgctg 180

tacgtggtag ccagaatcgt gatcgccgtg cggtacagat accagggcag caacaacaag 240

cggatctaca acgacatcga gatgagccgg ttcagaaag 279

<210> 23

<400> 23

000

<210> 24

<211> 0

<212> PRT

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Anti-CD3 scFv construct

<400> 24

000

<210> 25

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Human glycophorin A extracellular-TM_HIV Env CT construct

<400> 25

Ser Gly Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly

1 5 10 15

Ser Thr Lys Gly Pro Glu Ile Thr Leu Ile Ile Phe Gly Val Met Ala

20 25 30

Gly Val Ile Gly Thr Ile Leu Leu Ile Ser Tyr Gly Ile Arg Arg Leu

35 40 45

Ala Leu Lys Tyr Trp Trp Asn Leu Leu Gln Tyr Trp Ser Gln Glu Leu

50 55 60

Lys Asn Ser Ala Val Ser Leu Leu Asn Ala Thr Ala Ile Ala Val Ala

65 70 75 80

Glu Gly Thr Asp Arg Val Ile Glu Val Val Gln Gly Ala Cys Arg Ala

85 90 95

Ile Arg His Ile Pro Arg Arg Ile Arg Gln Gly Leu Glu Arg Ile Leu

100 105 110

Leu

<210> 26

<400> 26

000

<210> 27

<211> 0

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding anti-CD3 scFv

<400> 27

000

<210> 28

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation of the human glycophorin A extracellular-TM_HIV Env CT construct in the laboratory

Polynucleotide

<400> 28

tccggaggat ctacaagcgg ctctggcaag cctggcagcg gagaaggcag caccaagggc 60

cctgagatca cactgatcat cttcggcgtg atggccggcg tcatcggcac catcctgctg 120

atcagctacg gcatcagaag actggctctg aagtactggt ggaatctgct gcaatactgg 180

agccaggagc tgaaaaacag cgccgtgtcc ctgctcaacg ccaccgccat cgccgtggcc 240

gagggcaccg acagagtgat cgaggtggtg cagggagcct gcagagctat tcggcacatc 300

cccagacgga tcaggcaggg cctggaaaga atcctgctg 339

<210> 29

<400> 29

000

<210> 30

<211> 0

<212> PRT

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Anti-CD3 scFv construct

<400> 30

000

<210> 31

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - 218 linker_HIV Env-TM-CT construct

<400> 31

Ser Gly Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly

1 5 10 15

Ser Thr Lys Gly Asn Trp Leu Trp Tyr Ile Arg Ile Phe Ile Ile Ile

20 25 30

Val Gly Ser Leu Ile Gly Leu Arg Ile Val Phe Ala Val Leu Ser Leu

35 40 45

Val Asn Arg Gly Trp Glu Ala Leu Lys Tyr Trp Trp Asn Leu Leu Gln

50 55 60

Tyr Trp Ser Gln Glu Leu Lys Asn Ser Ala Val Ser Leu Leu Asn Ala

65 70 75 80

Thr Ala Ile Ala Val Ala Glu Gly Thr Asp Arg Val Ile Glu Val Val

85 90 95

Gln Gly Ala Cys Arg Ala Ile Arg His Ile Pro Arg Arg Ile Arg Gln

100 105 110

Gly Leu Glu Arg Ile Leu Leu

115

<210> 32

<400> 32

000

<210> 33

<211> 0

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding anti-CD3 scFv construct

<400> 33

000

<210> 34

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding 218 linker_HIV Env-TM-CT construct

<400> 34

tccggaggaa gcaccagcgg ctctggcaag cctggcagcg gcgagggctc taccaagggc 60

aattggctgt ggtacatcag aatcttcatc atcatcgtgg gcagcctgat cggcctgaga 120

atcgtgttcg ccgtgctgag cctggtgaac cggggctggg aagctctgaa gtactggtgg 180

aacctgctgc aatactggtc ccaggagctg aaaaacagcg ctgtgtccct gctcaacgcc 240

accgccatcg ccgtcgccga gggacagac agagtgatcg aggtggtgca gggagcctgc 300

agagccattc ggcacatccc cagacgcatc agacagggcc tggaaagaat cctgctg 357

<210> 35

<211> 353

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic MND promoter

<400> 35

gaacagagaa acaggagaat atgggccaaa caggatatct gtggtaagca gttcctgccc 60

cggctcaggg ccaagaacag ttggaacagc agaatatggg ccaaacagga tatctgtggt 120

aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc ggtcccgccc 180

tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc tgaaatgacc 240

ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc gcgcttctgc 300

tccccgagct ctatataagc agagctcgtt tagtgaaccg tcagatcgct agc 353

<210> 36

<211> 0

<212> PRT

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Anti-CD3 scFv construct

<400> 36

000

<210> 37

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Prepared in the laboratory - G4Sx3 linker_HIV Env-TM-CT construct

<400> 37

Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

1 5 10 15

Ser Tyr Ile Arg Ile Phe Ile Ile Ile Val Gly Ser Leu Ile Gly Leu

20 25 30

Arg Ile Val Phe Ala Val Leu Ser Leu Val Asn Arg Gly Trp Glu Ala

35 40 45

Leu Lys Tyr Trp Trp Asn Leu Leu Gln Tyr Trp Ser Gln Glu Leu Lys

50 55 60

Asn Ser Ala Val Ser Leu Leu Asn Ala Thr Ala Ile Ala Val Ala Glu

65 70 75 80

Gly Thr Asp Arg Val Ile Glu Val Val Gln Gly Ala Cys Arg Ala Ile

85 90 95

Arg His Ile Pro Arg Arg Ile Arg Gln Gly Leu Glu Arg Ile Leu Leu

100 105 110

<210> 38

<400> 38

000

<210> 39

<211> 0

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding anti-CD3 scFv construct

<400> 39

000

<210> 40

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding the G4Sx3 linker_HIV Env-TM-CT construct

<400> 40

tccggaggcg gtggaggctc tggtggcgga gggagcggtg gcggaggcag ctacatcaga 60

atcttcatca tcatcgtggg cagcctgatc ggcctgagaa tcgtgttcgc cgttctgagc 120

ctggtgaacc ggggctggga agccctgaag tactggtgga atctgctcca gtactggtct 180

caggagctga agaacagcgc cgtgtccctg ctgaacgcta cagctatcgc cgtcgccgag 240

ggcaccgaca gagtgatcga ggtggtgcag ggcgcctgca gagccatccg gcacatccct 300

agaaggattc ggcaaggcct ggaaagaatc ctgctg 336

<210> 41

<400> 41

000

<210> 42

<211> 0

<212> PRT

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Anti-CD3 scFv construct

<400> 42

000

<210> 43

<211> 83

<212> PRT

<213> Artificial sequence

<220>

<223> Prepared in the lab - Short hinge-TM-CT from Cocal Env_T2A construct

<400> 43

Gly Val Glu Leu Ile Glu Gly Trp Phe Ser Ser Trp Lys Ser Thr Val

1 5 10 15

Val Thr Phe Phe Phe Ala Ile Gly Val Phe Ile Leu Leu Tyr Val Val

20 25 30

Ala Arg Ile Val Ile Ala Val Arg Tyr Arg Tyr Gln Gly Ser Asn Asn

35 40 45

Lys Arg Ile Tyr Asn Asp Ile Glu Met Ser Arg Phe Arg Lys Gly Ser

50 55 60

Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn

65 70 75 80

Pro Gly Pro

<210> 44

<211> 0

<212> PRT

<213> Cocal virus

<400> 44

000

<210> 45

<400> 45

000

<210> 46

<211> 0

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding anti-CD3 scFv construct

<400> 46

000

<210> 47

<211> 249

<212> DNA

<213> Artificial sequence

<220>

<223> Prepared in the laboratory - Encoding short hinge-TM-CT from Cocal Env_T2A construct

Polynucleotide

<400> 47

ggagtggaac tgatcgaggg ctggttcagc agctggaaaa gcaccgtggt tacattcttt 60

ttcgccatcg gcgtgttcat cctgctgtac gtggtcgcca gaattgtgat cgccgtgcgg 120

tatagatacc agggcagcaa caacaagcgg atctacaacg acatcgagat gagcagattc 180

agaaagggat ctggagagggg aaggggaagc ctgctgacat gcggcgacgt ggaggagaac 240

ccaggacca 249

<210> 48

<211> 0

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation of polynucleotide encoding cocal env in the laboratory

<400> 48

000

<210> 49

<211> 1296

<212> PRT

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - CD19 CAR_FRB_RACR lentiviral vector construct

<400> 49

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser

20 25 30

Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser

35 40 45

Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly

50 55 60

Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr

85 90 95

Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln

100 105 110

Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

115 120 125

Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser

130 135 140

Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala

145 150 155 160

Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu

165 170 175

Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu

180 185 190

Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser

195 200 205

Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln

210 215 220

Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr

225 230 235 240

Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

245 250 255

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Glu Ser Lys Tyr Gly

260 265 270

Pro Pro Cys Pro Pro Cys Pro Met Phe Trp Val Leu Val Val Val Gly

275 280 285

Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile

290 295 300

Phe Trp Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

305 310 315 320

Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser

325 330 335

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys

340 345 350

Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln

355 360 365

Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu

370 375 380

Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg

385 390 395 400

Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met

405 410 415

Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly

420 425 430

Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp

435 440 445

Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly

450 455 460

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

465 470 475 480

Pro Gly Pro Glu Met Trp His Glu Gly Leu Glu Glu Ala Ser Arg Leu

485 490 495

Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu Pro

500 505 510

Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr Ser

515 520 525

Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp Cys

530 535 540

Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Leu Gln Ala Trp

545 550 555 560

Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Gly Ser Gly Ala

565 570 575

Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro

580 585 590

Gly Pro Met Pro Leu Gly Leu Leu Trp Leu Gly Leu Ala Leu Leu Gly

595 600 605

Ala Leu His Ala Gln Ala Gly Val Gln Val Glu Thr Ile Ser Pro Gly

610 615 620

Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr

625 630 635 640

Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg

645 650 655

Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly

660 665 670

Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu

675 680 685

Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile

690 695 700

Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu

705 710 715 720

Gly Glu Gly Ser Asn Thr Ser Lys Glu Asn Pro Phe Leu Phe Ala Leu

725 730 735

Glu Ala Val Val Ile Ser Val Gly Ser Met Gly Leu Ile Ile Ser Leu

740 745 750

Leu Cys Val Tyr Phe Trp Leu Glu Arg Thr Met Pro Arg Ile Pro Thr

755 760 765

Leu Lys Asn Leu Glu Asp Leu Val Thr Glu Tyr His Gly Asn Phe Ser

770 775 780

Ala Trp Ser Gly Val Ser Lys Gly Leu Ala Glu Ser Leu Gln Pro Asp

785 790 795 800

Tyr Ser Glu Arg Leu Cys Leu Val Ser Glu Ile Pro Pro Lys Gly Gly

805 810 815

Ala Leu Gly Glu Gly Pro Gly Ala Ser Pro Cys Asn Gln His Ser Pro

820 825 830

Tyr Trp Ala Pro Pro Cys Tyr Thr Leu Lys Pro Glu Thr Gly Ser Gly

835 840 845

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

850 855 860

Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala

865 870 875 880

Leu Leu Leu His Ala Ala Arg Pro Ile Leu Trp His Glu Met Trp His

885 890 895

Glu Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val

900 905 910

Lys Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg

915 920 925

Gly Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg

930 935 940

Asp Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly

945 950 955 960

Asn Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe

965 970 975

Arg Arg Ile Ser Lys Gly Lys Asp Thr Ile Pro Trp Leu Gly His Leu

980 985 990

Leu Val Gly Leu Ser Gly Ala Phe Gly Phe Ile Ile Leu Val Tyr Leu

995 1000 1005

Leu Ile Asn Cys Arg Asn Thr Gly Pro Trp Leu Lys Lys Val Leu

1010 1015 1020

Lys Cys Asn Thr Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Ser

1025 1030 1035

Ser Glu His Gly Gly Asp Val Gln Lys Trp Leu Ser Ser Pro Phe

1040 1045 1050

Pro Ser Ser Ser Phe Ser Pro Gly Gly Leu Ala Pro Glu Ile Ser

1055 1060 1065

Pro Leu Glu Val Leu Glu Arg Asp Lys Val Thr Gln Leu Leu Leu

1070 1075 1080

Gln Gln Asp Lys Val Pro Glu Pro Ala Ser Leu Ser Ser Asn His

1085 1090 1095

Ser Leu Thr Ser Cys Phe Thr Asn Gln Gly Tyr Phe Phe Phe His

1100 1105 1110

Leu Pro Asp Ala Leu Glu Ile Glu Ala Cys Gln Val Tyr Phe Thr

1115 1120 1125

Tyr Asp Pro Tyr Ser Glu Glu Asp Pro Asp Glu Gly Val Ala Gly

1130 1135 1140

Ala Pro Thr Gly Ser Ser Pro Gln Pro Leu Gln Pro Leu Ser Gly

1145 1150 1155

Glu Asp Asp Ala Tyr Cys Thr Phe Pro Ser Arg Asp Asp Leu Leu

1160 1165 1170

Leu Phe Ser Pro Ser Leu Leu Gly Gly Pro Ser Pro Pro Ser Thr

1175 1180 1185

Ala Pro Gly Gly Ser Gly Ala Gly Glu Glu Arg Met Pro Pro Ser

1190 1195 1200

Leu Gln Glu Arg Val Pro Arg Asp Trp Asp Pro Gln Pro Leu Gly

1205 1210 1215

Pro Pro Thr Pro Gly Val Pro Asp Leu Val Asp Phe Gln Pro Pro

1220 1225 1230

Pro Glu Leu Val Leu Arg Glu Ala Gly Glu Glu Val Pro Asp Ala

1235 1240 1245

Gly Pro Arg Glu Gly Val Ser Phe Pro Trp Ser Arg Pro Pro Gly

1250 1255 1260

Gln Gly Glu Phe Arg Ala Leu Asn Ala Arg Leu Pro Leu Asn Thr

1265 1270 1275

Asp Ala Tyr Leu Ser Leu Gln Glu Leu Gln Gly Gln Asp Pro Thr

1280 1285 1290

His Leu Val

1295

<210> 50

<211> 22

<212> PRT

<213> Homo sapiens

<400> 50

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro

20

<210> 51

<211> 245

<212> PRT

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - VL-linker-VH construct

<400> 51

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly

100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys

115 120 125

Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser

130 135 140

Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser

145 150 155 160

Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile

165 170 175

Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu

180 185 190

Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn

195 200 205

Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr

210 215 220

Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

225 230 235 240

Val Thr Val Ser Ser

245

<210> 52

<211> 40

<212> PRT

<213> Artificial sequence

<220>

<223> Prepared in the laboratory - IgG4 linker-CD28 TM construct

<400> 52

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp Val

1 5 10 15

Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr

20 25 30

Val Ala Phe Ile Ile Phe Trp Val

35 40

<210> 53

<211> 42

<212> PRT

<213> Homo sapiens

<400> 53

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 54

<211> 112

<212> PRT

<213> Homo sapiens

<400> 54

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 55

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> P2A synthetic construct

<400> 55

Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro

20

<210> 56

<211> 89

<212> PRT

<213> Homo sapiens

<400> 56

Glu Met Trp His Glu Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly

1 5 10 15

Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu Pro Leu His Ala

20 25 30

Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln

35 40 45

Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr

50 55 60

Met Lys Ser Gly Asn Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr

65 70 75 80

Tyr His Val Phe Arg Arg Ile Ser Lys

85

<210> 57

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Signal peptide-FKBP12 synthetic construct

<400> 57

Met Pro Leu Gly Leu Leu Trp Leu Gly Leu Ala Leu Leu Gly Ala Leu

1 5 10 15

His Ala Gln Ala Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly

20 25 30

Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly

35 40 45

Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys

50 55 60

Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu

65 70 75 80

Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile

85 90 95

Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro

100 105 110

Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu

115 120 125

<210> 58

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> IL-2Rγ TM-cytoplasmic domain synthetic construct

<400> 58

Gly Glu Gly Ser Asn Thr Ser Lys Glu Asn Pro Phe Leu Phe Ala Leu

1 5 10 15

Glu Ala Val Val Ile Ser Val Gly Ser Met Gly Leu Ile Ile Ser Leu

20 25 30

Leu Cys Val Tyr Phe Trp Leu Glu Arg Thr Met Pro Arg Ile Pro Thr

35 40 45

Leu Lys Asn Leu Glu Asp Leu Val Thr Glu Tyr His Gly Asn Phe Ser

50 55 60

Ala Trp Ser Gly Val Ser Lys Gly Leu Ala Glu Ser Leu Gln Pro Asp

65 70 75 80

Tyr Ser Glu Arg Leu Cys Leu Val Ser Glu Ile Pro Pro Lys Gly Gly

85 90 95

Ala Leu Gly Glu Gly Pro Gly Ala Ser Pro Cys Asn Gln His Ser Pro

100 105 110

Tyr Trp Ala Pro Pro Cys Tyr Thr Leu Lys Pro Glu Thr

115 120 125

<210> 59

<211> 114

<212> PRT

<213> Artificial sequence

<220>

<223> Signal peptide-FRB synthetic construct

<400> 59

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Ile Leu Trp His Glu Met Trp His Glu Gly Leu

20 25 30

Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met

35 40 45

Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly Pro Gln

50 55 60

Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met

65 70 75 80

Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys

85 90 95

Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile

100 105 110

Ser Lys

<210> 60

<211> 315

<212> PRT

<213> Homo sapiens

<400> 60

Gly Lys Asp Thr Ile Pro Trp Leu Gly His Leu Leu Val Gly Leu Ser

1 5 10 15

Gly Ala Phe Gly Phe Ile Ile Leu Val Tyr Leu Leu Ile Asn Cys Arg

20 25 30

Asn Thr Gly Pro Trp Leu Lys Lys Val Leu Lys Cys Asn Thr Pro Asp

35 40 45

Pro Ser Lys Phe Phe Ser Gln Leu Ser Ser Glu His Gly Gly Asp Val

50 55 60

Gln Lys Trp Leu Ser Ser Pro Phe Pro Ser Ser Ser Phe Ser Pro Gly

65 70 75 80

Gly Leu Ala Pro Glu Ile Ser Pro Leu Glu Val Leu Glu Arg Asp Lys

85 90 95

Val Thr Gln Leu Leu Leu Gln Gln Asp Lys Val Pro Glu Pro Ala Ser

100 105 110

Leu Ser Ser Asn His Ser Leu Thr Ser Cys Phe Thr Asn Gln Gly Tyr

115 120 125

Phe Phe Phe His Leu Pro Asp Ala Leu Glu Ile Glu Ala Cys Gln Val

130 135 140

Tyr Phe Thr Tyr Asp Pro Tyr Ser Glu Glu Asp Pro Asp Glu Gly Val

145 150 155 160

Ala Gly Ala Pro Thr Gly Ser Ser Pro Gln Pro Leu Gln Pro Leu Ser

165 170 175

Gly Glu Asp Asp Ala Tyr Cys Thr Phe Pro Ser Arg Asp Asp Leu Leu

180 185 190

Leu Phe Ser Pro Ser Leu Leu Gly Gly Pro Ser Pro Pro Ser Thr Ala

195 200 205

Pro Gly Gly Ser Gly Ala Gly Glu Glu Arg Met Pro Pro Ser Leu Gln

210 215 220

Glu Arg Val Pro Arg Asp Trp Asp Pro Gln Pro Leu Gly Pro Pro Thr

225 230 235 240

Pro Gly Val Pro Asp Leu Val Asp Phe Gln Pro Pro Pro Glu Leu Val

245 250 255

Leu Arg Glu Ala Gly Glu Glu Val Pro Asp Ala Gly Pro Arg Glu Gly

260 265 270

Val Ser Phe Pro Trp Ser Arg Pro Pro Gly Gln Gly Glu Phe Arg Ala

275 280 285

Leu Asn Ala Arg Leu Pro Leu Asn Thr Asp Ala Tyr Leu Ser Leu Gln

290 295 300

Glu Leu Gln Gly Gln Asp Pro Thr His Leu Val

305 310 315

<210> 61

<211> 3891

<212> DNA

<213> Artificial sequence

<220>

<223> Polynucleotide encoding CD19 CAR_FRB_RACR lentiviral vector construct

<400> 61

atgctgctgc tggtgacctc cctgctgctg tgcgagctgc ctcacccagc ctttctgctg 60

atccccgaca tccagatgac acagaccaca agctccctgt ctgccagcct gggcgacaga 120

gtgaccatct cctgtagggc ctctcaggat atcagcaagt acctgaactg gtatcagcag 180

aagccagatg gcacagtgaa gctgctgatc taccacacct ccaggctgca ctctggagtg 240

ccaagccggt tctccggatc tggaagcggc accgactatt ccctgacaat ctctaacctg 300

gagcaggagg atatcgccac atacttttgc cagcagggca ataccctgcc atatacattc 360

ggcggaggaa ccaagctgga gatcaccgga tccacatctg gaagcggcaa gccaggaagc 420

ggagaggggat ccacaaaggg agaggtgaag ctgcaggaga gcggaccagg actggtggca 480

ccatcccagt ctctgagcgt gacctgtaca gtgtccggcg tgtctctgcc tgactacggc 540

gtgtcctgga tcaggcagcc acctaggaag ggactggagt ggctgggcgt gatctggggc 600

tctgagacca catactataa ttctgccctg aagagccgcc tgaccatcat caaggacaac 660

tccaagtctc aggtgtttct gaagatgaat agcctgcaga ccgacgatac agccatctac 720

tattgcgcca agcactacta ttacggcggc tcctacgcca tggattattg gggccagggc 780

acctccgtga cagtgtctag cgagtctaag tatggcccac cctgccctcc atgtccaatg 840

ttctgggtgc tggtggtggt gggaggcgtg ctggcctgtt actccctgct ggtgaccgtg 900

gcctttatca tcttctgggt gaagagaggc aggaagaagc tgctgtatat ctttaagcag 960

cccttcatgc gccctgtgca gaccacacag gaggaggacg gctgcagctg tcggtttcca 1020

gaggaggagg agggaggatg cgagctgcgc gtgaagttca gccggtccgc cgatgcccct 1080

gcctaccagc agggccagaa ccagctgtat aacgagctga atctgggccg gagagaggag 1140

tacgacgtgc tggataagag gaggggaagg gacccagaga tgggaggcaa gcctcggaga 1200

aagaacccac aggagggcct gtacaatgag ctgcagaagg acaagatggc cgaggcctat 1260

tctgagatcg gcatgaaggg agagaggcgc cggggcaagg gacacgatgg cctgtaccag 1320

ggcctgagca ccgccacaaa ggacacatat gatgccctgc acatgcaggc cctgccacct 1380

aggggatctg gagccaccaa ctttagcctg ctgaagcagg caggcgatgt ggaggagaat 1440

ccaggacctg agatgtggca cgagggactg gaggaggcaa gcaggctgta ctttggcgag 1500

cggaatgtga agggcatgtt cgaggtgctg gagccactgc acgcaatgat ggagagggga 1560

ccacagaccc tgaaggagac atccttcaac caggcatacg gaagggacct gatggaggca 1620

caggagtggt gccggaagta tatgaagtct ggcaatgtga aggacctgct gcaggcctgg 1680

gatctgtatt accacgtgtt tagaaggatc agcaagggct ccggcgccac caacttctcc 1740

ctgctgaagc aggccggcga tgtggaagaa aatccaggac caatgcctct gggactgctg 1800

tggctgggac tggccctgct gggcgccctg cacgcccagg ccggcgtgca ggtggagaca 1860

atcagccctg gcgacggcag aacctttcca aagaggggcc agacatgcgt ggtgcactac 1920

accggcatgc tggaggatgg caagaagttc gactcctctc gcgatcggaa caagcccttt 1980

aagttcatgc tgggcaagca ggaagtgatc agaggctggg aggagggcgt ggcccagatg 2040

tctgtgggcc agagggccaa gctgacaatc agcccagact atgcatacgg agcaaccgga 2100

caccctggaa tcatcccacc acacgccaca ctggtgttcg atgtggagct gctgaagctg 2160

ggcgagggct ctaacaccag caaggagaat ccatttctgt tcgcactgga ggcagtggtc 2220

atctccgtgg gctctatggg cctgatcatc tccctgctgt gcgtgtactt ttggctggag 2280

agaacaatgc caaggatccc caccctgaag aacctggagg acctggtgac cgagtaccac 2340

ggcaatttca gcgcctggtc cggcgtgtct aagggactgg cagagtcct gcagccagat 2400

tattctgagc ggctgtgcct ggtgagcgag atccctccaa agggaggcgc cctgggagag 2460

ggaccaggag ccagcccctg caaccagcac tccccttat gggccccccc ttgttatacc 2520

ctgaagccag agacaggctc tggcgccacc aacttcagcc tgctgaagca agccggcgac 2580

gtggaagaaa acccaggacc aatggcactg ccagtgaccg ccctgctgct gcctctggcc 2640

ctgctgctgc acgcagccag acccatcctg tggcacgaaa tgtggcatga aggcctggag 2700

gaggcaagca gactgtactt tggcgagaga aatgtgaaag gaatgtttga ggtgctggag 2760

cctctgcacg ccatgatgga gaggggcct cagaccctga aggagacatc ctttaaccag 2820

gcctacggca gagacctgat ggaggcccag gagtggtgca ggaagtatat gaagagcgga 2880

aatgtgaaag acctgctgca ggcctgggat ctgtactacc acgtgttccg ccggatctct 2940

aagggcaagg atacaatccc ttggctggga cacctgctgg tgggactgag cggagccttt 3000

ggcttcatca tcctggtgta tctgctgatc aactgcagaa atacaggccc atggctgaag 3060

aaggtgctga agtgtaacac ccctgaccca tccaagttct tttctcagct gagctccgag 3120

cacggcggcg atgtgcagaa gtggctgtct agcccctttc cttcctctag cttcagccct 3180

ggaggactgg cacctgagat ctccccactg gaggtgctgg agagggacaa ggtgacccag 3240

ctgctgctgc agcaggataa ggtgccagag cccgcctccc tgtcctctaa ccacagcctg 3300

acctcctgct ttacaaatca gggctacttc tttttccacc tgccagacgc actggagatc 3360

gaggcatgtc aggtgtattt cacatacgat ccctatagcg aggaggaccc tgatgaggga 3420

gtggccggcg ccccaaccgg aagctcccct cagccactgc agccactgag cggagaggac 3480

gatgcatatt gtacatttcc ttcccgcgac gatctgctgc tgttctctcc aagcctgctg 3540

ggaggaccat ctccacccag caccgcacct ggaggatccg gggcagggga ggagcggatg 3600

cctccatctc tgcaggagag agtgccaagg gactggggatc cacagcctct gggaccacct 3660

acccctggag tgccagacct ggtggatttc cagccacccc ctgagctggt gctgcgggag 3720

gcaggagagg aggtgccaga cgcaggacct agagagggcg tgagctttcc ctggtccagg 3780

ccaccaggac agggagtt ccgcgccctg aacgcccggc tgcccctgaa tacagacgcc 3840

tacctgtctc tgcaggagct gcagggccag gatcctaccc acctggtgtg a 3891

<210> 62

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding signal peptide (human CSF2r)

<400> 62

atgctgctgc tggtgacctc cctgctgctg tgcgagctgc ctcacccagc ctttctgctg 60

atcccc 66

<210> 63

<211> 735

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding CD19 scFv (VL-linker-VH)

<400> 63

gacatccaga tgacacagac cacaagctcc ctgtctgcca gcctgggcga cagagtgacc 60

atctcctgta gggcctctca ggatatcagc aagtacctga actggtatca gcagaagcca 120

gatggcacag tgaagctgct gatctaccac acctccaggc tgcactctgg agtgccaagc 180

cggttctccg gatctggaag cggcaccgac tattccctga caatctctaa cctggagcag 240

gaggatatcg ccacatacttttgccagcag ggcaataccc tgccatatac attcggcgga 300

ggaaccaagc tggagatcac cggatccaca tctggaagcg gcaagccagg aagcggagag 360

ggatccacaa agggagaggt gaagctgcag gagagcggac caggactggt ggcaccatcc 420

cagtctctga gcgtgacctg tacagtgtcc ggcgtgtctc tgcctgacta cggcgtgtcc 480

tggatcaggc agccacctag gaagggactg gagtggctgg gcgtgatctg gggctctgag 540

accacatact ataattctgc cctgaagagc cgcctgacca tcatcaagga caactccaag 600

tctcaggtgt ttctgaagat gaatagcctg cagaccgacg atacagccat ctactattgc 660

gccaagcact actattacgg cggctcctac gccatggatt attggggcca gggcacctcc 720

gtgacagtgt ctagc 735

<210> 64

<211> 120

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding the IgG4 linker-CD28 TM portion of the vector construct

<400> 64

gagtctaagt atggcccacc ctgccctcca tgtccaatgt tctgggtgct ggtggtggtg 60

ggaggcgtgc tggcctgtta ctccctgctg gtgaccgtgg cctttatcat cttctgggtg 120

<210> 65

<211> 126

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding the "4-1BB signaling domain portion of the vector construct

<400> 65

aagagaggca ggaagaagct gctgtatatc tttaagcagc ccttcatgcg ccctgtgcag 60

accacacagg aggaggacgg ctgcagctgt cggtttccag aggaggagga gggaggatgc 120

gagctg 126

<210> 66

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding the CD3ζ portion of the vector construct

<400> 66

cgcgtgaagt tcagccggtc cgccgatgcc cctgcctacc agcagggcca gaaccagctg 60

tataacgagc tgaatctggg ccggagagag gagtacgacg tgctggataa gaggagggga 120

agggacccag agatgggagg caagcctcgg agaaagaacc cacaggaggg cctgtacaat 180

gagctgcaga aggacaagat ggccgaggcc tattctgaga tcggcatgaa gggagagagg 240

cgccggggca agggacacga tggcctgtac cagggcctga gcaccgccac aaaggacaca 300

tatgatgccc tgcacatgca ggccctgcca cctagg 336

<210> 67

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding the P@A self-cleaving peptide portion of the vector construct

<400> 67

ggatctggag ccaccaactt tagcctgctg aagcaggcag gcgatgtgga ggagaatcca 60

ggacct 66

<210> 68

<211> 267

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding the naked FRB portion of the vector construct

<400> 68

gagatgtggc acgagggact ggaggaggca agcaggctgt actttggcga gcggaatgtg 60

aagggcatgt tcgaggtgct ggagccactg cacgcaatga tggagagggg accacagacc 120

ctgaaggaga catccttcaa ccaggcatac ggaagggacc tgatggaggc acaggagtgg 180

tgccggaagt atatgaagtc tggcaatgtg aaggacctgc tgcaggcctg ggatctgtat 240

taccacgtgt ttagaaggat cagcaag 267

<210> 69

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding the P2A self-cleaving peptide portion of the vector construct

<400> 69

ggctccggcg ccaccaactt ctccctgctg aagcaggccg gcgatgtgga agaaaatcca 60

ggacca 66

<210> 70

<211> 378

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding the signal peptide FKBP12 portion of the vector construct

<400> 70

atgcctctgg gactgctgtg gctgggactg gccctgctgg gcgccctgca cgcccaggcc 60

ggcgtgcagg tggagacaat cagccctggc gacggcagaa cctttccaaa gaggggccag 120

acatgcgtgg tgcactacac cggcatgctg gaggatggca agaagttcga ctcctctcgc 180

gatcggaaca agccctttaa gttcatgctg ggcaagcagg aagtgatcag aggctggggag 240

gagggcgtgg cccagatgtc tgtgggccag agggccaagc tgacaatcag cccagactat 300

gcatacggag caaccggaca ccctggaatc atcccaccac acgccacact ggtgttcgat 360

gtggagctgc tgaagctg 378

<210> 71

<211> 375

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Encoding the "IL-2RγTM-cytoplasmic domain portion" of the vector construct

Polynucleotide

<400> 71

ggcgagggct ctaacaccag caaggagaat ccatttctgt tcgcactgga ggcagtggtc 60

atctccgtgg gctctatggg cctgatcatc tccctgctgt gcgtgtactt ttggctggag 120

agaacaatgc caaggatccc caccctgaag aacctggagg acctggtgac cgagtaccac 180

ggcaatttca gcgcctggtc cggcgtgtct aagggactgg cagagtcct gcagccagat 240

tattctgagc ggctgtgcct ggtgagcgag atccctccaa agggaggcgc cctgggagag 300

ggaccaggag ccagcccctg caaccagcac tccccttat gggccccccc ttgttatacc 360

ctgaagccag agaca 375

<210> 72

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding the P2A self-cleaving peptide portion of the vector construct

<400> 72

ggctctggcg ccaccaactt cagcctgctg aagcaagccg gcgacgtgga agaaaaccca 60

ggacca 66

<210> 73

<211> 342

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding the signal peptide FRB portion of the vector construct

<400> 73

atggcactgc cagtgaccgc cctgctgctg cctctggccc tgctgctgca cgcagccaga 60

cccatcctgt ggcacgaaat gtggcatgaa ggcctggagg aggcaagcag actgtacttt 120

ggcgagagaa atgtgaaagg aatgtttgag gtgctggagc ctctgcacgc catgatggag 180

aggggccctc agaccctgaa ggagacatcc tttaaccagg cctacggcag agacctgatg 240

gaggcccagg agtggtgcag gaagtatatg aagagcggaa atgtgaaaga cctgctgcag 300

gcctgggatc tgtactacca cgtgttccgc cggatctcta ag 342

<210> 74

<211> 945

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Encoding the "IL-2RβTM-cytoplasmic domain portion" of the vector construct

Polynucleotide

<400> 74

ggcaaggata caatcccttg gctgggacac ctgctggtgg gactgagcgg agcctttggc 60

ttcatcatcc tggtgtatct gctgatcaac tgcagaaata caggcccatg gctgaagaag 120

gtgctgaagt gtaacacccc tgacccatcc aagttctttt ctcagctgag ctccgagcac 180

ggcggcgatg tgcagaagtg gctgtctagc ccctttcctt cctctagctt cagccctgga 240

ggactggcac ctgagatctc cccactggag gtgctggaga gggacaaggt gacccagctg 300

ctgctgcagc aggataaggt gccagagccc gcctccctgt cctctaacca cagcctgacc 360

tcctgcttta caaatcaggg ctacttcttt ttccacctgc cagacgcact ggagatcgag 420

gcatgtcagg tgtatttcac atacgatccc tatagcgagg aggaccctga tgagggagtg 480

gccggcgccc caaccggaag ctcccctcag ccactgcagc cactgagcgg agaggacgat 540

gcatattgta catttccttc ccgcgacgat ctgctgctgt tctctccaag cctgctggga 600

ggaccatctc cacccagcac cgcacctgga ggatccgggg caggggagga gcggatgcct 660

ccatctctgc aggagagagt gccaagggac tgggatccac agcctctggg accacctacc 720

cctggagtgc cagacctggt ggatttccag ccaccccctg agctggtgct gcgggaggca 780

ggagaggagg tgccagacgc aggacctaga gagggcgtga gctttccctg gtccaggcca 840

ccaggacagg gagagttccg cgccctgaac gcccggctgc ccctgaatac agacgcctac 900

ctgtctctgc aggagctgca gggccaggat cctacccacc tggtg 945

<210> 75

<211> 1297

<212> PRT

<213> Artificial sequence

<220>

<223> FRB_RACR_CD19 CAR lentiviral vector construct

<400> 75

Met Glu Met Trp His Glu Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe

1 5 10 15

Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu Pro Leu His

20 25 30

Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn

35 40 45

Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp Cys Arg Lys

50 55 60

Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Leu Gln Ala Trp Asp Leu

65 70 75 80

Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Gly Ser Gly Ala Thr Asn

85 90 95

Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro

100 105 110

Met Pro Leu Gly Leu Leu Trp Leu Gly Leu Ala Leu Leu Gly Ala Leu

115 120 125

His Ala Gln Ala Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly

130 135 140

Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly

145 150 155 160

Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys

165 170 175

Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu

180 185 190

Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile

195 200 205

Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro

210 215 220

Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Gly Glu

225 230 235 240

Gly Ser Asn Thr Ser Lys Glu Asn Pro Phe Leu Phe Ala Leu Glu Ala

245 250 255

Val Val Ile Ser Val Gly Ser Met Gly Leu Ile Ile Ser Leu Leu Cys

260 265 270

Val Tyr Phe Trp Leu Glu Arg Thr Met Pro Arg Ile Pro Thr Leu Lys

275 280 285

Asn Leu Glu Asp Leu Val Thr Glu Tyr His Gly Asn Phe Ser Ala Trp

290 295 300

Ser Gly Val Ser Lys Gly Leu Ala Glu Ser Leu Gln Pro Asp Tyr Ser

305 310 315 320

Glu Arg Leu Cys Leu Val Ser Glu Ile Pro Pro Lys Gly Gly Ala Leu

325 330 335

Gly Glu Gly Pro Gly Ala Ser Pro Cys Asn Gln His Ser Pro Tyr Trp

340 345 350

Ala Pro Pro Cys Tyr Thr Leu Lys Pro Glu Thr Gly Ser Gly Ala Thr

355 360 365

Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly

370 375 380

Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu

385 390 395 400

Leu His Ala Ala Arg Pro Ile Leu Trp His Glu Met Trp His Glu Gly

405 410 415

Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly

420 425 430

Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly Pro

435 440 445

Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu

450 455 460

Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn Val

465 470 475 480

Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg Arg

485 490 495

Ile Ser Lys Gly Lys Asp Thr Ile Pro Trp Leu Gly His Leu Leu Val

500 505 510

Gly Leu Ser Gly Ala Phe Gly Phe Ile Ile Leu Val Tyr Leu Leu Ile

515 520 525

Asn Cys Arg Asn Thr Gly Pro Trp Leu Lys Lys Val Leu Lys Cys Asn

530 535 540

Thr Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Ser Ser Glu His Gly

545 550 555 560

Gly Asp Val Gln Lys Trp Leu Ser Ser Pro Phe Pro Ser Ser Ser Phe

565 570 575

Ser Pro Gly Gly Leu Ala Pro Glu Ile Ser Pro Leu Glu Val Leu Glu

580 585 590

Arg Asp Lys Val Thr Gln Leu Leu Leu Gln Gln Asp Lys Val Pro Glu

595 600 605

Pro Ala Ser Leu Ser Ser Asn His Ser Leu Thr Ser Cys Phe Thr Asn

610 615 620

Gln Gly Tyr Phe Phe Phe His Leu Pro Asp Ala Leu Glu Ile Glu Ala

625 630 635 640

Cys Gln Val Tyr Phe Thr Tyr Asp Pro Tyr Ser Glu Glu Asp Pro Asp

645 650 655

Glu Gly Val Ala Gly Ala Pro Thr Gly Ser Ser Pro Gln Pro Leu Gln

660 665 670

Pro Leu Ser Gly Glu Asp Asp Ala Tyr Cys Thr Phe Pro Ser Arg Asp

675 680 685

Asp Leu Leu Leu Phe Ser Pro Ser Leu Leu Gly Gly Pro Ser Pro Pro

690 695 700

Ser Thr Ala Pro Gly Gly Ser Gly Ala Gly Glu Glu Arg Met Pro Pro

705 710 715 720

Ser Leu Gln Glu Arg Val Pro Arg Asp Trp Asp Pro Gln Pro Leu Gly

725 730 735

Pro Pro Thr Pro Gly Val Pro Asp Leu Val Asp Phe Gln Pro Pro Pro

740 745 750

Glu Leu Val Leu Arg Glu Ala Gly Glu Glu Val Pro Asp Ala Gly Pro

755 760 765

Arg Glu Gly Val Ser Phe Pro Trp Ser Arg Pro Pro Gly Gln Gly Glu

770 775 780

Phe Arg Ala Leu Asn Ala Arg Leu Pro Leu Asn Thr Asp Ala Tyr Leu

785 790 795 800

Ser Leu Gln Glu Leu Gln Gly Gln Asp Pro Thr His Leu Val Gly Ser

805 810 815

Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu

820 825 830

Asn Pro Gly Pro Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu

835 840 845

Leu Pro His Pro Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln

850 855 860

Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser

865 870 875 880

Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln

885 890 895

Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu

900 905 910

His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

915 920 925

Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr

930 935 940

Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr

945 950 955 960

Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser

965 970 975

Gly Glu Gly Ser Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro

980 985 990

Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser

995 1000 1005

Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro

1010 1015 1020

Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu

1025 1030 1035

Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile

1040 1045 1050

Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu

1055 1060 1065

Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr

1070 1075 1080

Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

1085 1090 1095

Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

1100 1105 1110

Cys Pro Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala

1115 1120 1125

Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

1130 1135 1140

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

1145 1150 1155

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys

1160 1165 1170

Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys

1175 1180 1185

Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

1190 1195 1200

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

1205 1210 1215

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

1220 1225 1230

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

1235 1240 1245

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly

1250 1255 1260

Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu

1265 1270 1275

Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

1280 1285 1290

Leu Pro Pro Arg

1295

<210> 76

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Preparation in the lab - Bare FRB_P2A part of the construct

<400> 76

Met Glu Met Trp His Glu Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe

1 5 10 15

Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu Pro Leu His

20 25 30

Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn

35 40 45

Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp Cys Arg Lys

50 55 60

Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Leu Gln Ala Trp Asp Leu

65 70 75 80

Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Gly Ser Gly Ala Thr Asn

85 90 95

Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro

100 105 110

<210> 77

<211> 273

<212> PRT

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - RACRg(FKBP12_IL-2Rγ)_P2A portion of the construct

<400> 77

Met Pro Leu Gly Leu Leu Trp Leu Gly Leu Ala Leu Leu Gly Ala Leu

1 5 10 15

His Ala Gln Ala Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly

20 25 30

Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly

35 40 45

Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys

50 55 60

Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu

65 70 75 80

Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile

85 90 95

Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro

100 105 110

Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Gly Glu

115 120 125

Gly Ser Asn Thr Ser Lys Glu Asn Pro Phe Leu Phe Ala Leu Glu Ala

130 135 140

Val Val Ile Ser Val Gly Ser Met Gly Leu Ile Ile Ser Leu Leu Cys

145 150 155 160

Val Tyr Phe Trp Leu Glu Arg Thr Met Pro Arg Ile Pro Thr Leu Lys

165 170 175

Asn Leu Glu Asp Leu Val Thr Glu Tyr His Gly Asn Phe Ser Ala Trp

180 185 190

Ser Gly Val Ser Lys Gly Leu Ala Glu Ser Leu Gln Pro Asp Tyr Ser

195 200 205

Glu Arg Leu Cys Leu Val Ser Glu Ile Pro Pro Lys Gly Gly Ala Leu

210 215 220

Gly Glu Gly Pro Gly Ala Ser Pro Cys Asn Gln His Ser Pro Tyr Trp

225 230 235 240

Ala Pro Pro Cys Tyr Thr Leu Lys Pro Glu Thr Gly Ser Gly Ala Thr

245 250 255

Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly

260 265 270

Pro

<210> 78

<211> 451

<212> PRT

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - RACRb(FRB_IL-2Rβ)_P2A portion of the construct

<400> 78

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Ile Leu Trp His Glu Met Trp His Glu Gly Leu

20 25 30

Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met

35 40 45

Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly Pro Gln

50 55 60

Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met

65 70 75 80

Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys

85 90 95

Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile

100 105 110

Ser Lys Gly Lys Asp Thr Ile Pro Trp Leu Gly His Leu Leu Val Gly

115 120 125

Leu Ser Gly Ala Phe Gly Phe Ile Ile Leu Val Tyr Leu Leu Ile Asn

130 135 140

Cys Arg Asn Thr Gly Pro Trp Leu Lys Lys Val Leu Lys Cys Asn Thr

145 150 155 160

Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Ser Ser Glu His Gly Gly

165 170 175

Asp Val Gln Lys Trp Leu Ser Ser Pro Phe Pro Ser Ser Ser Phe Ser

180 185 190

Pro Gly Gly Leu Ala Pro Glu Ile Ser Pro Leu Glu Val Leu Glu Arg

195 200 205

Asp Lys Val Thr Gln Leu Leu Leu Gln Gln Asp Lys Val Pro Glu Pro

210 215 220

Ala Ser Leu Ser Ser Asn His Ser Leu Thr Ser Cys Phe Thr Asn Gln

225 230 235 240

Gly Tyr Phe Phe Phe His Leu Pro Asp Ala Leu Glu Ile Glu Ala Cys

245 250 255

Gln Val Tyr Phe Thr Tyr Asp Pro Tyr Ser Glu Glu Asp Pro Asp Glu

260 265 270

Gly Val Ala Gly Ala Pro Thr Gly Ser Ser Pro Gln Pro Leu Gln Pro

275 280 285

Leu Ser Gly Glu Asp Asp Ala Tyr Cys Thr Phe Pro Ser Arg Asp Asp

290 295 300

Leu Leu Leu Phe Ser Pro Ser Leu Leu Gly Gly Pro Ser Pro Pro Ser

305 310 315 320

Thr Ala Pro Gly Gly Ser Gly Ala Gly Glu Glu Arg Met Pro Pro Ser

325 330 335

Leu Gln Glu Arg Val Pro Arg Asp Trp Asp Pro Gln Pro Leu Gly Pro

340 345 350

Pro Thr Pro Gly Val Pro Asp Leu Val Asp Phe Gln Pro Pro Pro Glu

355 360 365

Leu Val Leu Arg Glu Ala Gly Glu Glu Val Pro Asp Ala Gly Pro Arg

370 375 380

Glu Gly Val Ser Phe Pro Trp Ser Arg Pro Pro Gly Gln Gly Glu Phe

385 390 395 400

Arg Ala Leu Asn Ala Arg Leu Pro Leu Asn Thr Asp Ala Tyr Leu Ser

405 410 415

Leu Gln Glu Leu Gln Gly Gln Asp Pro Thr His Leu Val Gly Ser Gly

420 425 430

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

435 440 445

Pro Gly Pro

450

<210> 79

<211> 267

<212> PRT

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - CD19 scFv portion of the construct

<400> 79

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser

20 25 30

Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser

35 40 45

Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly

50 55 60

Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr

85 90 95

Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln

100 105 110

Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

115 120 125

Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser

130 135 140

Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala

145 150 155 160

Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu

165 170 175

Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu

180 185 190

Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser

195 200 205

Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln

210 215 220

Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr

225 230 235 240

Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

245 250 255

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

260 265

<210> 80

<211> 194

<212> PRT

<213> Artificial sequence

<220>

<223> Prepared in the laboratory - IgG4 linker-CD28 TM_4-1BB-CD3ζ portion of the construct

<400> 80

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp Val

1 5 10 15

Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr

20 25 30

Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly Arg Lys Lys Leu Leu

35 40 45

Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu

50 55 60

Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys

65 70 75 80

Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln

85 90 95

Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu

100 105 110

Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly

115 120 125

Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu

130 135 140

Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly

145 150 155 160

Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser

165 170 175

Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro

180 185 190

Pro Arg

<210> 81

<211> 3894

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding the FRB_RACR_CD19 CAR lentiviral vector construct

<400> 81

atggagatgt ggcacgaggg actggagaggag gcaagcagac tgtactttgg cgagaggaac 60

gtgaagggca tgttcgaggt gctggagcca ctgcacgcaa tgatggagag gggaccacag 120

accctgaagg agacatcttt caaccaggca tacggaaggg acctgatgga ggcacaggag 180

tggtgccgga agtatatgaa gagcggcaat gtgaaggacc tgctgcaggc ctgggatctg 240

tactatcacg tgtttcggag aatctccaag ggctctggcg ccaccaactt ctccctgctg 300

aagcaggccg gcgatgtgga ggagaatcct ggaccaatgc cactgggact gctgtggctg 360

ggactggccc tgctgggcgc cctgcacgcc caggccggcg tgcaggtgga gacaatcagc 420

cctggcgacg gacgcacctt tccaaagagg ggacagacat gcgtggtgca ctacaccggc 480

atgctggagg atggcaagaa gttcgacagc tccagagata ggaataagcc ctttaagttc 540

atgctgggca agcaggaagt gatcagggga tgggaggagg gagtggcaca gatgtctgtg 600

ggacagcggg ccaagctgac aatcagccca gactatgcat acggagcaac cggacaccct 660

ggaatcatcc cacctcacgc cacactggtg tttgatgtgg agctgctgaa gctgggcgag 720

ggcagcaaca cctccaagga gaatccattt ctgttcgccc tggaggccgt ggtcatctct 780

gtgggcagca tgggcctgat catctccctg ctgtgcgtgt acttttggct ggagcgcaca 840

atgccacgga tccccaccct gaagaacctg gaggacctgg tgaccgagta ccacggcaat 900

ttctccgcct ggtctggcgt gagcaaggga ctggcagagt ctctgcagcc agattatagc 960

gagcggctgt gcctggtgag cgagatccca cccaagggag gcgccctggg agagggacca 1020

ggagcctccc cttgcaacca gcactctcct tactgggccc ctccatgtta taccctgaag 1080

ccagagacag gcagcggagc tactaacttc tccctgctga agcaagcagg cgacgtggaa 1140

gaaaatcctg gaccaatggc actgccagtg accgccctgc tgctgcctct ggccctgctg 1200

ctgcacgcag ccagacccat cctgtggcac gaaatgtggc atgaaggcct ggaggaggca 1260

agcaggctgt actttggcga gcggaatgtg aaaggaatgt ttgaagtgct ggagcctctg 1320

cacgccatga tggagagggg ccctcagacc ctgaaggaga catcctttaa ccaggcctac 1380

ggcagagacc tgatggaggc ccaggagtgg tgcaggaagt atatgaagtc tggaaatgtg 1440

aaagacctgc tgcaggcctg ggatctgtat tatcacgtgt tcaggcgcat ctctaagggc 1500

aaggatacaa tcccttggct gggacacctg ctggtgggac tgagcggagc ctttggcttc 1560

atcatcctgg tgtatctgct gatcaactgc cgcaatacag gcccatggct gaagaaggtg 1620

ctgaagtgta acacccccga cccttccaag ttcttttctc agctgtctag cgagcacggc 1680

ggcgatgtgc agaagtggct gtcctctcca tttcccagct cctctttcag cccaggagga 1740

ctggcaccag agatctcccc actggaggtg ctggagaggg acaaggtgac ccagctgctg 1800

ctgcagcagg ataaggtgcc tgagccagcc tccctgagct ccaaccactc cctgacctct 1860

tgctttacaa atcagggcta cttctttttc cacctgccag acgcactgga gatcgaggca 1920

tgtcaggtgt atttcacata cgatccctat agcgaggagg accctgatga gggagtggcc 1980

ggcgccccaa ccggatctag cccacagcct ctgcagccac tgagcggaga ggacgatgca 2040

tattgtacat ttccttcccg cgacgatctg ctgctgttct ctccaagcct gctggggagga 2100

ccaagcccac cttccaccgc accaggcggc tccggggcag gggaggagcg gatgccaccc 2160

tctctgcagg agagagtgcc aagggactgg gatccacagc cactgggacc tccaacccct 2220

ggagtgccag acctggtgga tttccagccc cctccagagc tggtgctgag agaggcagga 2280

gaggaggtgc ctgacgcagg accaagagag ggcgtgagct ttccttggtc caggccacct 2340

ggacagggag agttcagagc cctgaacgcc aggctgcccc tgaatacaga cgcctacctg 2400

tctctgcagg agctgcaggg ccaggatcct acacacctgg tcggatctgg cgccaccaac 2460

tttagcctgc tgaagcaggc aggcgacgtg gaagagaacc ctggaccaat gctgctgctg 2520

gtgaccagcc tgctgctgtg cgagctgcca caccctgcct tcctgctgat cccagatatc 2580

cagatgacac agaccacatc ctctctgtcc gcctctctgg gcgacagagt gaccatctct 2640

tgtagggcca gccaggatat ctccaagtac ctgaactggt atcagcagaa gcctgacggc 2700

acagtgaagc tgctgatcta ccacacctct aggctgcaca gcggagtgcc atcccggttc 2760

agcggatccg gatctggaac agactattct ctgaccatca gcaacctgga gcaggaggat 2820

atcgccacat acttttgcca gcagggcaat accctgccat atacattcgg cggaggaacc 2880

aagctggaga tcaccggaag cacatccgga tctggcaagc caggatccgg agagggatct 2940

acaaagggag aggtgaagct gcaggagagc ggaccaggac tggtggcacc cagccagtcc 3000

ctgtctgtga cctgtacagt gtctggcgtg agcctgcccg attacggcgt gtcctggatc 3060

agacagccac caaggaaggg actggagtgg ctgggcgtga tctggggctc tgagaccaca 3120

tactataata gcgccctgaa gtcccggctg accatcatca aggacaacag caagtcccag 3180

gtgtttctga agatgaatag cctgcagacc gacgatacag ccatctacta ttgcgccaag 3240

cactactatt acggcggctc ctacgccatg gattattggg gccagggcac ctccgtgaca 3300

gtgagctccg agtctaagta tggccctcca tgcccccctt gtcctatgtt ctgggtgctg 3360

gtggtggtgg gaggcgtgct ggcctgttac tccctgctgg tgaccgtggc ctttatcatc 3420

ttctgggtga agcgcggccg gaagaagctg ctgtatatct ttaagcagcc cttcatgaga 3480

cctgtgcaga ccacacagga ggaggacggc tgcagctgta ggtttccaga ggaggaggag 3540

ggaggatgcg agctgcgcgt gaagttctct cggagcgccg atgcccctgc ctaccagcag 3600

ggacagaacc agctgtataa cgagctgaat ctgggccgga gagaggagta cgacgtgctg 3660

gataagagga ggggaagaga cccagagatg ggaggcaagc ctcggagaaa gaacccacag 3720

gagggcctgt acaatgagct gcagaaggac aagatggccg aggcctattc cgagatcggc 3780

atgaagggag agaggcgccg gggcaaggga cacgatggcc tgtaccaggg cctgagcacc 3840

gccacaaagg acacctatga tgccctgcac atgcaggccc tgccacccag gtga 3894

<210> 82

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding the naked FRB_P2A portion of the vector construct

<400> 82

atggagatgt ggcacgaggg actggagaggag gcaagcagac tgtactttgg cgagaggaac 60

gtgaagggca tgttcgaggt gctggagcca ctgcacgcaa tgatggagag gggaccacag 120

accctgaagg agacatcttt caaccaggca tacggaaggg acctgatgga ggcacaggag 180

tggtgccgga agtatatgaa gagcggcaat gtgaaggacc tgctgcaggc ctgggatctg 240

tactatcacg tgtttcggag aatctccaag ggctctggcg ccaccaactt ctccctgctg 300

aagcaggccg gcgatgtgga ggagaatcct ggacca 336

<210> 83

<211> 819

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - RACRg(FKBP12_IL-2Rγ)_P2A encoding vector construct

Partial polynucleotide

<400> 83

atgccactgg gactgctgtg gctgggactg gccctgctgg gcgccctgca cgcccaggcc 60

ggcgtgcagg tggagacaat cagccctggc gacggacgca cctttccaaa gaggggacag 120

acatgcgtgg tgcactacac cggcatgctg gaggatggca agaagttcga cagctccaga 180

gataggaata agccctttaa gttcatgctg ggcaagcagg aagtgatcag gggatggggag 240

gagggagtgg cacagatgtc tgtggggacag cgggccaagc tgacaatcag cccagactat 300

gcatacggag caaccggaca ccctggaatc atcccacctc acgccacact ggtgtttgat 360

gtggagctgc tgaagctggg cgagggcagc aacacctcca aggagaatcc atttctgttc 420

gccctggagg ccgtggtcat ctctgtgggc agcatgggcc tgatcatctc cctgctgtgc 480

gtgtactttt ggctggagcg cacaatgcca cggatcccca ccctgaagaa cctggaggac 540

ctggtgaccg agtaccacgg caatttctcc gcctggtctg gcgtgagcaa gggactggca 600

gagtctctgc agccagatta tagcgagcgg ctgtgcctgg tgagcgagat cccacccaag 660

ggaggcgccc tgggagaggg accaggagcc tccccttgca accagcactc tccttatactgg 720

gcccctccat gttataccct gaagccagag acaggcagcg gagctactaa cttctccctg 780

ctgaagcaag caggcgacgt ggaagaaaat cctggacca 819

<210> 84

<211> 1353

<212> DNA

<213> Artificial sequence

<220>

<223> Prepare in the laboratory - Encoding the RACRb (FRB_IL-2Rβ)_P2A portion of the vector construct

Polynucleotide

<400> 84

atggcactgc cagtgaccgc cctgctgctg cctctggccc tgctgctgca cgcagccaga 60

cccatcctgt ggcacgaaat gtggcatgaa ggcctggagg aggcaagcag gctgtacttt 120

ggcgagcgga atgtgaaagg aatgtttgaa gtgctggagc ctctgcacgc catgatggag 180

aggggccctc agaccctgaa ggagacatcc tttaaccagg cctacggcag agacctgatg 240

gaggcccagg agtggtgcag gaagtatatg aagtctggaa atgtgaaaga cctgctgcag 300

gcctgggatc tgtattatca cgtgttcagg cgcatctcta agggcaagga tacaatccct 360

tggctgggac acctgctggt gggactgagc ggagcctttg gcttcatcat cctggtgtat 420

ctgctgatca actgccgcaa tacaggccca tggctgaaga aggtgctgaa gtgtaacacc 480

cccgaccctt ccaagttctt ttctcagctg tctagcgagc acggcggcga tgtgcagaag 540

tggctgtcct ctccatttcc cagctcctct ttcagcccag gaggactggc accagagatc 600

tccccactgg aggtgctgga gagggacaag gtgacccagc tgctgctgca gcaggataag 660

gtgcctgagc cagcctccct gagctccaac cactccctga cctcttgctt tacaaatcag 720

ggctacttct ttttccacct gccagacgca ctggagatcg aggcatgtca ggtgtatttc 780

acatacgatccctatagcgaggaggaccctgatgaggggagtggccggcgccccaaccgga 840

tctagcccac agcctctgca gccactgagc ggagaggacg atgcatattg tacatttcct 900

tcccgcgacg atctgctgct gttctctcca agcctgctgg gaggaccaag cccaccttcc 960

accgcaccag gcggctccgg ggcaggggag gagcggatgc caccctctct gcaggagaga 1020

gtgccaaggg actgggatcc acagccactg ggacctccaa cccctggagt gccagacctg 1080

gtggatttcc agccccctcc agagctggtg ctgagagagg caggagagga ggtgcctgac 1140

gcaggaccaa gagagggcgt gagctttcct tggtccaggc cacctggaca gggagagttc 1200

agagccctga acgccaggct gcccctgaat acagacgcct acctgtctct gcaggagctg 1260

cagggccagg atcctacaca cctggtcgga tctggcgcca ccaactttag cctgctgaag 1320

caggcaggcg acgtggaaga gaaccctgga cca 1353

<210> 85

<211> 801

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding the CD19 scFv portion of the vector construct

<400> 85

atgctgctgc tggtgaccag cctgctgctg tgcgagctgc cacaccctgc cttcctgctg 60

atcccagata tccagatgac acagaccaca tcctctctgt ccgcctctct gggcgacaga 120

gtgaccatct cttgtagggc cagccaggat atctccaagt acctgaactg gtatcagcag 180

aagcctgacg gcacagtgaa gctgctgatc taccacacct ctaggctgca cagcggagtg 240

ccatcccggt tcagcggatc cggatctgga acagactatt ctctgaccat cagcaacctg 300

gagcaggagg atatcgccac atacttttgc cagcagggca ataccctgcc atatacattc 360

ggcggaggaa ccaagctgga gatcaccgga agcacatccg gatctggcaa gccaggatcc 420

ggagaggggat ctacaaaggg agaggtgaag ctgcaggaga gcggaccagg actggtggca 480

cccagccagt ccctgtctgt gacctgtaca gtgtctggcg tgagcctgcc cgattacggc 540

gtgtcctgga tcagacagcc accaaggaag ggactggagt ggctgggcgt gatctggggc 600

tctgagacca catactataa tagcgccctg aagtcccggc tgaccatcat caaggacaac 660

agcaagtccc aggtgtttct gaagatgaat agcctgcaga ccgacgatac agccatctac 720

tattgcgcca agcactacta ttacggcggc tcctacgcca tggattattg gggccagggc 780

acctccgtga cagtgagctc c 801

<210> 86

<211> 582

<212> DNA

<213> Artificial sequence

<220>

<223> Prepared in the laboratory - IgG4 linker encoding vector construct-CD28 TM_4-1BB-CD3ζ

Partial polynucleotide

<400> 86

gagtctaagt atggccctcc atgcccccct tgtcctatgt tctgggtgct ggtggtggtg 60

ggaggcgtgc tggcctgtta ctccctgctg gtgaccgtgg cctttatcat cttctgggtg 120

aagcgcggcc ggaagaagct gctgtatatc tttaagcagc ccttcatgag acctgtgcag 180

accacacagg aggaggacgg ctgcagctgt aggtttccag aggaggagga gggaggatgc 240

gagctgcgcg tgaagttctc tcggagcgcc gatgcccctg cctaccagca gggacagaac 300

cagctgtata acgagctgaa tctgggccgg agagaggagt acgacgtgct ggataagagg 360

aggggaagag acccagagat gggaggcaag cctcggagaa agaacccaca ggagggcctg 420

tacaatgagc tgcagaagga caagatggcc gaggcctatt ccgagatcgg catgaaggga 480

gagaggcgcc ggggcaaggg acacgatggc ctgtaccagg gcctgagcac cgccacaaag 540

gacacctatg atgccctgca catgcaggcc ctgccaccca gg 582

<210> 87

<211> 794

<212> PRT

<213> Artificial sequence

<220>

<223> FRB_CD19 CAR_TGFbDN lentiviral vector construct

<400> 87

Met Glu Met Trp His Glu Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe

1 5 10 15

Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu Pro Leu His

20 25 30

Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn

35 40 45

Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp Cys Arg Lys

50 55 60

Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Leu Gln Ala Trp Asp Leu

65 70 75 80

Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Gly Ser Gly Ala Thr Asn

85 90 95

Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro

100 105 110

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

115 120 125

Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser

130 135 140

Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser

145 150 155 160

Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly

165 170 175

Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val

180 185 190

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr

195 200 205

Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln

210 215 220

Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

225 230 235 240

Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser

245 250 255

Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala

260 265 270

Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu

275 280 285

Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu

290 295 300

Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser

305 310 315 320

Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln

325 330 335

Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr

340 345 350

Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

355 360 365

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Glu Ser Lys Tyr Gly

370 375 380

Pro Pro Cys Pro Pro Cys Pro Met Phe Trp Val Leu Val Val Val Gly

385 390 395 400

Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile

405 410 415

Phe Trp Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

420 425 430

Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser

435 440 445

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys

450 455 460

Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln

465 470 475 480

Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu

485 490 495

Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg

500 505 510

Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met

515 520 525

Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly

530 535 540

Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp

545 550 555 560

Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly

565 570 575

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

580 585 590

Pro Gly Pro Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His

595 600 605

Ile Val Leu Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln

610 615 620

Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val

625 630 635 640

Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys

645 650 655

Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys

660 665 670

Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu

675 680 685

Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His

690 695 700

Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu

705 710 715 720

Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp

725 730 735

Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn

740 745 750

Pro Asp Leu Leu Leu Val Ile Phe Gln Val Thr Gly Ile Ser Leu Leu

755 760 765

Pro Pro Leu Gly Val Ala Ile Ser Val Ile Ile Ile Phe Tyr Cys Tyr

770 775 780

Arg Val Asn Arg Gln Gln Lys Arg Arg Arg

785 790

<210> 88

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Preparation in the lab - Bare FRB_P2A part of the construct

<400> 88

Met Glu Met Trp His Glu Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe

1 5 10 15

Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu Pro Leu His

20 25 30

Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn

35 40 45

Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp Cys Arg Lys

50 55 60

Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Leu Gln Ala Trp Asp Leu

65 70 75 80

Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Gly Ser Gly Ala Thr Asn

85 90 95

Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro

100 105 110

<210> 89

<211> 267

<212> PRT

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - CD19 scFv portion of the construct

<400> 89

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser

20 25 30

Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser

35 40 45

Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly

50 55 60

Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr

85 90 95

Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln

100 105 110

Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

115 120 125

Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser

130 135 140

Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala

145 150 155 160

Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu

165 170 175

Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu

180 185 190

Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser

195 200 205

Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln

210 215 220

Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr

225 230 235 240

Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

245 250 255

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

260 265

<210> 90

<211> 216

<212> PRT

<213> Artificial sequence

<220>

<223> Prepared in the laboratory - IgG4 linker-CD28 TM_4-1BB-CD3ζ_P2A portion of the construct

<400> 90

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp Val

1 5 10 15

Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr

20 25 30

Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly Arg Lys Lys Leu Leu

35 40 45

Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu

50 55 60

Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys

65 70 75 80

Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln

85 90 95

Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu

100 105 110

Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly

115 120 125

Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu

130 135 140

Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly

145 150 155 160

Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser

165 170 175

Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro

180 185 190

Pro Arg Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly

195 200 205

Asp Val Glu Glu Asn Pro Gly Pro

210 215

<210> 91

<211> 199

<212> PRT

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - TGFβ DN portion of the construct

<400> 91

Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His Ile Val Leu

1 5 10 15

Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln Lys Ser Val

20 25 30

Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro

35 40 45

Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln

50 55 60

Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro

65 70 75 80

Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr

85 90 95

Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile

100 105 110

Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys

115 120 125

Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn

130 135 140

Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu

145 150 155 160

Leu Leu Val Ile Phe Gln Val Thr Gly Ile Ser Leu Leu Pro Pro Leu

165 170 175

Gly Val Ala Ile Ser Val Ile Ile Ile Phe Tyr Cys Tyr Arg Val Asn

180 185 190

Arg Gln Gln Lys Arg Arg Arg

195

<210> 92

<211> 2385

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding FRB_CD19 CAR_TGFbDN lentiviral vector construct

<400> 92

atggagatgt ggcacgaggg actggaggag gcatccagac tgtacttcgg cgagaggaac 60

gtgaagggca tgtttgaggt gctggagcca ctgcacgcca tgatggagag aggcccccag 120

accctgaagg agacatcttt caaccaggcc tatggaaggg acctgatgga ggcacaggag 180

tggtgccgga agtacatgaa gagcggcaat gtgaaggacc tgctgcaggc ctgggatctg 240

tactatcacg tgttccggag aatcagcaag ggctccggcg ccaccaactt tagcctgctg 300

aagcaggcag gcgacgtgga ggagaatcca ggacctatgc tgctgctggt gacatccctg 360

ctgctgtgcg agctgccaca cccagccttc ctgctgatcc ccgatatcca gatgacccag 420

accacaagct ccctgagcgc ctccctgggc gacagggtga caatctcttg tcgggccagc 480

caggatatct ccaagtatct gaattggtac cagcagaagc ccgacggcac cgtgaagctg 540

ctgatctatc acacatctag actgcacagc ggcgtgcctt ccaggttttc tggcagcggc 600

tccggcaccg actactctct gacaatcagc aacctggagc aggaggatat cgccacctat 660

ttctgccagc agggcaatac cctgccttac acatttggcg gcggcacaaa gctggagatc 720

accggctcta caagcggatc cggcaagcca ggatccggag agggatctac caagggagag 780

gtgaagctgc aggagagcgg acctggactg gtggcaccat ctcagagcct gtccgtgacc 840

tgtacagtgt ctggcgtgag cctgccagat tatggcgtga gctggatcag gcagccacct 900

aggaagggac tggagtggct gggcgtgatc tggggctccg agaccacata ctataacagc 960

gccctgaagt cccgcctgac catcatcaag gacaactcta agagccaggt gttcctgaag 1020

atgaattccc tgcagaccga cgatacagcc atctactatt gcgccaagca ctactattac 1080

ggcggctctt atgccatgga ttactggggc cagggcacca gcgtgacagt gtctagcgag 1140

tccaagtacg gcccaccctg ccctccatgt cccatgtttt gggtgctggt ggtggtggga 1200

ggcgtgctgg cctgttattc cctgctggtg accgtggcct tcatcatctt ttgggtgaag 1260

cgcggccgga agaagctgct gtacatcttc aagcagccct tcatgagacc cgtgcagacc 1320

acacaggagg aggacggctg cagctgtagg ttcccagagg aggaggaggg aggatgcgag 1380

ctgagggtga agttttcccg gtctgccgat gcccctgcct atcagcaggg ccagaatcag 1440

ctgtacaacg agctgaatct gggcaggcgc gaggagtacg acgtgctgga taagaggaga 1500

ggaagggacc ctgagatggg aggcaagcca aggcgcaaga accctcagga gggcctgtat 1560

aatgagctgc agaaggacaa gatggccgag gcctactccg agatcggcat gaagggagag 1620

cggagaaggg gcaagggaca cgatggcctg tatcagggcc tgagcaccgc cacaaaggac 1680

acctacgatg cactgcacat gcaggccctg ccacctagag gatctggagc cacaaacttc 1740

agcctgctga agcaggccgg cgatgtggag gagaatcctg gaccaatggg aagaggactg 1800

ctgaggggac tgtggccact gcacatcgtg ctgtggacca ggatcgcctc tacaatccca 1860

ccccacgtgc agaagagcgt gaacaatgac atgatcgtga ccgataacaa tggcgccgtg 1920

aagtttcccc agctgtgcaa gttctgtgac gtgcgctttt ccacctgtga taaccagaag 1980

tcctgcatgt ctaattgtag catcacatcc atctgcgaga agcctcagga ggtgtgcgtg 2040

gccgtgtggc ggaagaacga cgagaatatc accctggaga cagtgtgcca cgatcccaag 2100

ctgccttatc acgacttcat cctggaggat gccgcctctc ctaagtgtat catgaaggag 2160

aagaagaagc caggcgagac cttctttatg tgcagctgtt cctctgacga gtgcaacgat 2220

aatatcatct tctccgagga gtacaacacc tctaatcctg acctgctgct ggtcatcttt 2280

caggtgacag gcatctccct gctgcctcca ctgggcgtgg ccatctctgt gatcatcatc 2340

ttttattgtt acagagtgaa caggcagcag aagcgccggc gctag 2385

<210> 93

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding the naked FRB_P2A portion of the vector construct

<400> 93

atggagatgt ggcacgaggg actggaggag gcatccagac tgtacttcgg cgagaggaac 60

gtgaagggca tgtttgaggt gctggagcca ctgcacgcca tgatggagag aggcccccag 120

accctgaagg agacatcttt caaccaggcc tatggaaggg acctgatgga ggcacaggag 180

tggtgccgga agtacatgaa gagcggcaat gtgaaggacc tgctgcaggc ctgggatctg 240

tactatcacg tgttccggag aatcagcaag ggctccggcg ccaccaactt tagcctgctg 300

aagcaggcag gcgacgtgga ggagaatcca ggacct 336

<210> 94

<211> 801

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding the CD19 scFv portion of the vector construct

<400> 94

atgctgctgc tggtgacatc cctgctgctg tgcgagctgc cacacccagc cttcctgctg 60

atccccgata tccagatgac ccagaccaca agctccctga gcgcctccct gggcgacagg 120

gtgacaatct cttgtcgggc cagccaggat atctccaagt atctgaattg gtaccagcag 180

aagcccgacg gcaccgtgaa gctgctgatc tatcacacat ctagactgca cagcggcgtg 240

ccttccaggt tttctggcag cggctccggc accgactact ctctgacaat cagcaacctg 300

gagcaggagg atatcgccac ctatttctgc cagcagggca ataccctgcc ttacacattt 360

ggcggcggca caaagctgga gatcaccggc tctacaagcg gatccggcaa gccaggatcc 420

ggagaggggat ctaccaaggg agaggtgaag ctgcaggaga gcggacctgg actggtggca 480

ccatctcaga gcctgtccgt gacctgtaca gtgtctggcg tgagcctgcc agattatggc 540

gtgagctgga tcaggcagcc acctaggaag ggactggagt ggctgggcgt gatctggggc 600

tccgagacca catactataa cagcgccctg aagtcccgcc tgaccatcat caaggacaac 660

tctaagagcc aggtgttcct gaagatgaat tccctgcaga ccgacgatac agccatctac 720

tattgcgcca agcactacta ttacggcggc tctttatgcca tggattactg gggccagggc 780

accagcgtga cagtgtctag c 801

<210> 95

<211> 648

<212> DNA

<213> Artificial sequence

<220>

<223> Prepared in the laboratory - IgG4 linker encoding vector construct-CD28 TM_4-1BB-CD3ζ_P2A

Partial polynucleotide

<400> 95

gagtccaagt acggcccacc ctgccctcca tgtcccatgt tttgggtgct ggtggtggtg 60

ggaggcgtgc tggcctgtta ttccctgctg gtgaccgtgg ccttcatcat cttttgggtg 120

aagcgcggcc ggaagaagct gctgtacatc ttcaagcagc ccttcatgag acccgtgcag 180

accacacagg aggaggacgg ctgcagctgt aggttcccag aggaggagga gggaggatgc 240

gagctgaggg tgaagttttc ccggtctgcc gatgcccctg cctatcagca gggccagaat 300

cagctgtaca acgagctgaa tctgggcagg cgcgaggagt acgacgtgct ggataagagg 360

agaggaaggg accctgagat gggaggcaag ccaaggcgca agaaccctca ggagggcctg 420

tataatgagc tgcagaagga caagatggcc gaggcctact ccgagatcgg catgaaggga 480

gagcggagaa ggggcaaggg acacgatggc ctgtatcagg gcctgagcac cgccacaaag 540

gacacctacg atgcactgca catgcaggcc ctgccaccta gaggatctgg agccacaaac 600

ttcagcctgc tgaagcaggc cggcgatgtg gaggagaatc ctggacca 648

<210> 96

<211> 600

<212> DNA

<213> Artificial sequence

<220>

<223> Preparation in the laboratory - Polynucleotide encoding the TGFβ DN portion of the vector construct

<400> 96

atgggaagag gactgctgag gggactgtgg ccactgcaca tcgtgctgtg gaccaggatc 60

gcctctacaa tcccacccca cgtgcagaag agcgtgaaca atgacatgat cgtgaccgat 120

aacaatggcg ccgtgaagtt tccccagctg tgcaagttct gtgacgtgcg cttttccacc 180

tgtgataacc agaagtcctg catgtctaat tgtagcatca catccatctg cgagaagcct 240

caggaggtgt gcgtggccgt gtggcggaag aacgacgaga atatcaccct ggagacagtg 300

tgccacgatc ccaagctgcc ttatcacgac ttcatcctgg aggatgccgc ctctcctaag 360

tgtatcatga aggagaagaa gaagccaggc gagaccttct ttatgtgcag ctgttcctct 420

gacgagtgca acgataatat catcttctcc gaggagtaca acacctctaa tcctgacctg 480

ctgctggtca tctttcaggt gacaggcatc tccctgctgc ctccactggg cgtggccatc 540

tctgtgatca tcatctttta ttgttacaga gtgaacaggc agcagaagcg ccggcgctag 600

<210> 97

<211> 67

<212> PRT

<213> Artificial sequence

<220>

<223> Human glycophorin A TM_ CT

<400> 97

Ser Gly His Phe Ser Glu Pro Glu Ile Thr Leu Ile Ile Phe Gly Val

1 5 10 15

Met Ala Gly Val Ile Gly Thr Ile Leu Leu Ile Ser Tyr Gly Ile Arg

20 25 30

Arg Leu Ile Lys Lys Ser Pro Ser Asp Val Lys Pro Leu Pro Ser Pro

35 40 45

Asp Thr Asp Val Pro Leu Ser Ser Val Glu Ile Glu Asn Pro Glu Thr

50 55 60

Ser Asp Gln

65

<210> 98

<211> 201

<212> DNA

<213> Artificial sequence

<220>

<223> 218 linker_human glycophorin A extracellular-TM_HIV Env CT

<400> 98

tccggacact tcagcgagcc tgagatcacc ctgatcatct tcggcgtgat ggccggagtg 60

atcggcacaa tcctgctgat cagctacggc atcagaagac tgattaagaa atccccatct 120

gatgtgaagc ctctgccttc tcctgacacc gacgtccccc tgagcagcgt ggaaatcgag 180

aaccccgaaa ccagcgacca g 201

<210> 99

<211> 500

<212> PRT

<213> Artificial sequence

<220>

<223> gag protein

<400> 99

Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Glu Leu Asp Arg Trp

1 5 10 15

Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Lys Leu Lys

20 25 30

His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro

35 40 45

Gly Leu Leu Glu Thr Ser Glu Gly Cys Arg Gln Ile Leu Gly Gln Leu

50 55 60

Gln Pro Ser Leu Gln Thr Gly Ser Glu Glu Leu Arg Ser Leu Tyr Asn

65 70 75 80

Thr Val Ala Thr Leu Tyr Cys Val His Gln Arg Ile Glu Ile Lys Asp

85 90 95

Thr Lys Glu Ala Leu Asp Lys Ile Glu Glu Glu Gln Asn Lys Ser Lys

100 105 110

Lys Lys Ala Gln Gln Ala Ala Ala Asp Thr Gly His Ser Asn Gln Val

115 120 125

Ser Gln Asn Tyr Pro Ile Val Gln Asn Ile Gln Gly Gln Met Val His

130 135 140

Gln Ala Ile Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Val Glu

145 150 155 160

Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser

165 170 175

Glu Gly Ala Thr Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly

180 185 190

Gly His Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu Glu

195 200 205

Ala Ala Glu Trp Asp Arg Val His Pro Val His Ala Gly Pro Ile Ala

210 215 220

Pro Gly Gln Met Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr

225 230 235 240

Ser Thr Leu Gln Glu Gln Ile Gly Trp Met Thr His Asn Pro Pro Ile

245 250 255

Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys

260 265 270

Ile Val Arg Met Tyr Ser Pro Thr Ser Ile Leu Asp Ile Arg Gln Gly

275 280 285

Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr Lys Thr Leu

290 295 300

Arg Ala Glu Gln Ala Ser Gln Glu Val Lys Asn Trp Met Thr Glu Thr

305 310 315 320

Leu Leu Val Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Lys Ala

325 330 335

Leu Gly Pro Gly Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly

340 345 350

Val Gly Gly Pro Gly His Lys Ala Arg Val Leu Ala Glu Ala Met Ser

355 360 365

Gln Val Thr Asn Pro Ala Thr Ile Met Ile Gln Lys Gly Asn Phe Arg

370 375 380

Asn Gln Arg Lys Thr Val Lys Cys Phe Asn Cys Gly Lys Glu Gly His

385 390 395 400

Ile Ala Lys Asn Cys Arg Ala Pro Arg Lys Lys Gly Cys Trp Lys Cys

405 410 415

Gly Lys Glu Gly His Gln Met Lys Asp Cys Thr Glu Arg Gln Ala Asn

420 425 430

Phe Leu Gly Lys Ile Trp Pro Ser His Lys Gly Arg Pro Gly Asn Phe

435 440 445

Leu Gln Ser Arg Pro Glu Pro Thr Ala Pro Pro Glu Glu Ser Phe Arg

450 455 460

Phe Gly Glu Glu Thr Thr Thr Pro Ser Gln Lys Gln Glu Pro Ile Asp

465 470 475 480

Lys Glu Leu Tyr Pro Leu Ala Ser Leu Arg Ser Leu Phe Gly Ser Asp

485 490 495

Pro Ser Ser Gln

500

<210> 100

<211> 1003

<212> PRT

<213> Artificial sequence

<220>

<223> Pol protein

<400> 100

Phe Phe Arg Glu Asp Leu Ala Phe Pro Gln Gly Lys Ala Arg Glu Phe

1 5 10 15

Ser Ser Glu Gln Thr Arg Ala Asn Ser Pro Thr Arg Arg Glu Leu Gln

20 25 30

Val Trp Gly Arg Asp Asn Asn Ser Leu Ser Glu Ala Gly Ala Asp Arg

35 40 45

Gln Gly Thr Val Ser Phe Ser Phe Pro Gln Ile Thr Leu Trp Gln Arg

50 55 60

Pro Leu Val Thr Ile Lys Ile Gly Gly Gln Leu Lys Glu Ala Leu Leu

65 70 75 80

Asp Thr Gly Ala Asp Asp Thr Val Leu Glu Glu Met Asn Leu Pro Gly

85 90 95

Arg Trp Lys Pro Lys Met Ile Gly Gly Ile Gly Gly Phe Ile Lys Val

100 105 110

Arg Gln Tyr Asp Gln Ile Leu Ile Glu Ile Cys Gly His Lys Ala Ile

115 120 125

Gly Thr Val Leu Val Gly Pro Thr Pro Val Asn Ile Ile Gly Arg Asn

130 135 140

Leu Leu Thr Gln Ile Gly Cys Thr Leu Asn Phe Pro Ile Ser Pro Ile

145 150 155 160

Glu Thr Val Pro Val Lys Leu Lys Pro Gly Met Asp Gly Pro Lys Val

165 170 175

Lys Gln Trp Pro Leu Thr Glu Glu Lys Ile Lys Ala Leu Val Glu Ile

180 185 190

Cys Thr Glu Met Glu Lys Glu Gly Lys Ile Ser Lys Ile Gly Pro Glu

195 200 205

Asn Pro Tyr Asn Thr Pro Val Phe Ala Ile Lys Lys Lys Asp Ser Thr

210 215 220

Lys Trp Arg Lys Leu Val Asp Phe Arg Glu Leu Asn Lys Arg Thr Gln

225 230 235 240

Asp Phe Trp Glu Val Gln Leu Gly Ile Pro His Pro Ala Gly Leu Lys

245 250 255

Gln Lys Lys Ser Val Thr Val Leu Asp Val Gly Asp Ala Tyr Phe Ser

260 265 270

Val Pro Leu Asp Lys Asp Phe Arg Lys Tyr Thr Ala Phe Thr Ile Pro

275 280 285

Ser Ile Asn Asn Glu Thr Pro Gly Ile Arg Tyr Gln Tyr Asn Val Leu

290 295 300

Pro Gln Gly Trp Lys Gly Ser Pro Ala Ile Phe Gln Cys Ser Met Thr

305 310 315 320

Lys Ile Leu Glu Pro Phe Arg Lys Gln Asn Pro Asp Ile Val Ile Tyr

325 330 335

Gln Tyr Met Asp Asp Leu Tyr Val Gly Ser Asp Leu Glu Ile Gly Gln

340 345 350

His Arg Thr Lys Ile Glu Glu Leu Arg Gln His Leu Leu Arg Trp Gly

355 360 365

Phe Thr Thr Pro Asp Lys Lys His Gln Lys Glu Pro Pro Phe Leu Trp

370 375 380

Met Gly Tyr Glu Leu His Pro Asp Lys Trp Thr Val Gln Pro Ile Val

385 390 395 400

Leu Pro Glu Lys Asp Ser Trp Thr Val Asn Asp Ile Gln Lys Leu Val

405 410 415

Gly Lys Leu Asn Trp Ala Ser Gln Ile Tyr Ala Gly Ile Lys Val Arg

420 425 430

Gln Leu Cys Lys Leu Leu Arg Gly Thr Lys Ala Leu Thr Glu Val Val

435 440 445

Pro Leu Thr Glu Glu Ala Glu Leu Glu Leu Ala Glu Asn Arg Glu Ile

450 455 460

Leu Lys Glu Pro Val His Gly Val Tyr Tyr Asp Pro Ser Lys Asp Leu

465 470 475 480

Ile Ala Glu Ile Gln Lys Gln Gly Gln Gly Gln Trp Thr Tyr Gln Ile

485 490 495

Tyr Gln Glu Pro Phe Lys Asn Leu Lys Thr Gly Lys Tyr Ala Arg Met

500 505 510

Lys Gly Ala His Thr Asn Asp Val Lys Gln Leu Thr Glu Ala Val Gln

515 520 525

Lys Ile Ala Thr Glu Ser Ile Val Ile Trp Gly Lys Thr Pro Lys Phe

530 535 540

Lys Leu Pro Ile Gln Lys Glu Thr Trp Glu Ala Trp Trp Thr Glu Tyr

545 550 555 560

Trp Gln Ala Thr Trp Ile Pro Glu Trp Glu Phe Val Asn Thr Pro Pro

565 570 575

Leu Val Lys Leu Trp Tyr Gln Leu Glu Lys Glu Pro Ile Ile Gly Ala

580 585 590

Glu Thr Phe Tyr Val Asp Gly Ala Ala Asn Arg Glu Thr Lys Leu Gly

595 600 605

Lys Ala Gly Tyr Val Thr Asp Arg Gly Arg Gln Lys Val Val Pro Leu

610 615 620

Thr Asp Thr Thr Asn Gln Lys Thr Glu Leu Gln Ala Ile His Leu Ala

625 630 635 640

Leu Gln Asp Ser Gly Leu Glu Val Asn Ile Val Thr Asp Ser Gln Tyr

645 650 655

Ala Leu Gly Ile Ile Gln Ala Gln Pro Asp Lys Ser Glu Ser Glu Leu

660 665 670

Val Ser Gln Ile Ile Glu Gln Leu Ile Lys Lys Glu Lys Val Tyr Leu

675 680 685

Ala Trp Val Pro Ala His Lys Gly Ile Gly Gly Asn Glu Gln Val Asp

690 695 700

Lys Leu Val Ser Ala Gly Ile Arg Lys Val Leu Phe Leu Asp Gly Ile

705 710 715 720

Asp Lys Ala Gln Glu Glu His Glu Lys Tyr His Ser Asn Trp Arg Ala

725 730 735

Met Ala Ser Asp Phe Asn Leu Pro Pro Val Val Ala Lys Glu Ile Val

740 745 750

Ala Ser Cys Asp Lys Cys Gln Leu Lys Gly Glu Ala Met His Gly Gln

755 760 765

Val Asp Cys Ser Pro Gly Ile Trp Gln Leu Asp Cys Thr His Leu Glu

770 775 780

Gly Lys Val Ile Leu Val Ala Val His Val Ala Ser Gly Tyr Ile Glu

785 790 795 800

Ala Glu Val Ile Pro Ala Glu Thr Gly Gln Glu Thr Ala Tyr Phe Leu

805 810 815

Leu Lys Leu Ala Gly Arg Trp Pro Val Lys Thr Val His Thr Asp Asn

820 825 830

Gly Ser Asn Phe Thr Ser Thr Thr Val Lys Ala Ala Cys Trp Trp Ala

835 840 845

Gly Ile Lys Gln Glu Phe Gly Ile Pro Tyr Asn Pro Gln Ser Gln Gly

850 855 860

Val Ile Glu Ser Met Asn Lys Glu Leu Lys Lys Ile Ile Gly Gln Val

865 870 875 880

Arg Asp Gln Ala Glu His Leu Lys Thr Ala Val Gln Met Ala Val Phe

885 890 895

Ile His Asn Phe Lys Arg Lys Gly Gly Ile Gly Gly Tyr Ser Ala Gly

900 905 910

Glu Arg Ile Val Asp Ile Ile Ala Thr Asp Ile Gln Thr Lys Glu Leu

915 920 925

Gln Lys Gln Ile Thr Lys Ile Gln Asn Phe Arg Val Tyr Tyr Arg Asp

930 935 940

Ser Arg Asp Pro Val Trp Lys Gly Pro Ala Lys Leu Leu Trp Lys Gly

945 950 955 960

Glu Gly Ala Val Val Ile Gln Asp Asn Ser Asp Ile Lys Val Val Pro

965 970 975

Arg Arg Lys Ala Lys Ile Ile Arg Asp Tyr Gly Lys Gln Met Ala Gly

980 985 990

Asp Asp Cys Val Ala Ser Arg Gln Asp Glu Asp

995 1000

<210> 101

<211> 4307

<212> DNA

<213> Artificial sequence

<220>

<223> gag-pol nucleic acid

<400> 101

atgggcgccc gcgccagcgt gctttctggc ggcgagctgg acaggtggga gaagattcgc 60

ctgcggcctg gaggaaagaa aaagtacaag ctgaagcaca tcgtgtgggc ttctcgggaa 120

ctggaaagat tcgccgtgaa ccctggactg ctagagacct ccgaaggctg cagacagatc 180

ctgggacagc tgcaacctag cctgcagacc ggcagcgagg agctgagaag cctgtacaac 240

accgtcgcca ccctgtattg tgtgcaccaa agaatcgaga tcaaggacac caaggaggct 300

ctggataaga tcgaggaaga gcagaacaag agcaagaaaa aagcccagca ggccgccgct 360

gataccggcc attctaatca ggtgtcccag aactacccca ttgtgcaaaa tatccagggc 420

cagatggtcc accaggccat cagccctaga accctgaatg cctgggtgaa ggtggtggaa 480

gagaaggccttttctccaga ggtgatccct atgttcagcg ccctgagcga gggcgctacc 540

cctcaggacc tgaacacaat gctgaacacc gtgggcggcc accaggccgc catgcagatg 600

ctgaaagaaa ccatcaacga ggaggccgcc gaatgggacc gggtgcaccc cgttcacgcc 660

ggcccaatcg cccctggcca gatgcgggaa cctagaggca gcgacatcgc cggcacaacc 720

agcacactgc aagagcagat cggatggatg acacacaacc cccccatccc cgtgggcgaa 780

atctacaagc ggtggatcat tctgggactg aacaaaatcg ttagaatgta cagcccctacc 840

agcatcctgg atatcagaca gggcccaaag gagcctttcc gggactacgt ggacagattt 900

tacaagaccc tgagagccga acaggcctcc caagaggtga agaactggat gacagagacc 960

ctgctggtgc agaatgccaa ccctgattgt aagacaatcc tgaaggccct cggacctggc 1020

gctacactgg aagaaatgat gaccgcctgc cagggcgtgg gcggccccgg ccacaaggcc 1080

agagtgctgg ccgaggctat gagccaggtg acaaaccccg ccacaatcat gatccagaag 1140

ggcaacttca gaaaccagcg gaagaccgtg aaatgcttca actgcggcaa ggaaggccac 1200

atcgcaaaga actgcagagc ccctaggaag aaaggctgtt ggaagtgcgg aaaggaagga 1260

caccaaatga aagattgtac tgagagacag gctaattttt tagggaagat ctggccttcc 1320

cacaagggaa ggccagggaa ttttcttcag agcagaccag agccaacagc cccaccagaa 1380

gagagcttca ggtttgggga agagacaaca actccctctc agaagcagga gccgatagac 1440

aaggaactgt atcctttagc ttccctcaga tcactctttg gcagcgaccc ctcgtcacaa 1500

taaagatcgg cggacagctg aaagaggcct tgctggacac cggagccgat gacaccgtgc 1560

tggaagaaat gaacctgcct ggaagatgga aacctaagat gatcggtggc atcggcggat 1620

ttatcaaagt gcgacagtat gaccagatcc tgatcgagat ttgcggccac aaagctatcg 1680

gaacagtgct ggtcggcccg accccccgtga acatcattgg ccgcaacctg ctgacacaga 1740

tcggttgtac actgaacttt cctatcagcc ctatcgaaac cgtgccggtc aagctgaagc 1800

ccggcatgga tggccctaag gtgaagcagt ggcccctgac agaggaaaag atcaaggcac 1860

tggtggaaat ctgcacagaa atggaaaaag agggcaagat ttctaaaatc ggcccagaga 1920

acccctacaa cacccctgtt ttcgccatca agaagaaaga ttccaccaag tggaggaagc 1980

tggtggactt tcgggaactg aacaagcgga cccaggattt ctgggaggtg cagctgggca 2040

tcccccaccc tgccggcctg aaacaaaaaa aaagcgtgac cgtgctggac gtgggcgacg 2100

cctatttcag cgtgcctctg gataaggact tccggaaata caccgccttt accatcccta 2160

gcatcaacaa cgagacccct ggcatccggt accagtacaa cgtgctccca cagggctgga 2220

agggctcacc cgccatcttc cagtgcagca tgaccaagat cctggagcct ttcagaaagc 2280

agaatcctga catcgtgatc taccagtaca tggacgacct gtacgtgggc tctgatctgg 2340

agatcggaca gcacagaaca aagatcgaag agctgagaca gcatctgctg agatggggtt 2400

tcaccaccccc cgacaagaag caccagaagg aacctccttt tctgtggatg ggctacgagc 2460

tgcaccccga taagtggaca gtgcagccca tcgtgctgcc cgagaaggac tcctggaccg 2520

tgaacgacat tcagaagctg gtcggaaagc tgaattgggc ttcccaaatc tacgccggca 2580

tcaaggtgcg gcagctgtgc aagcttctgc gcggcacaaa ggccctgacg gaagtcgtgc 2640

cactgaccga ggaagccgaa ttagagctgg ccgaaaacag agaaattctg aaagaacctg 2700

tgcacggcgt ttactacgac ccttctaagg acctgatcgc cgaaatccag aaacaaggcc 2760

agggccagtg gacttaccaa atctaccagg agcctttcaa aaacctcaag accggcaagt 2820

acgccagaat gaagggagcc catacaaacg acgtgaagca gctgacagag gctgttcaga 2880

agatcgccac agaaagcatc gtgatctggg gcaagacccc aaagttcaag ctgcctatcc 2940

aaaaggaaac ctgggaggcc tggtggaccg agtactggca ggccacctgg attcctgaat 3000

gggagttcgt gaacacacca cctcttgtga agctgtggta ccagctggaa aaggagccaa 3060

tcatcggcgc cgagacattc tacgtggacg gcgccgccaa ccgggagacc aaactgggaa 3120

aggccggata tgtgaccgac agaggcagac aaaaggtggt gcctctgacc gatacaacta 3180

accagaaaac agagctgcag gccattcacc tggccctgca agacagcggc ctggaagtga 3240

atatcgtgac agacagtcag tacgccctgg gcatcatcca ggctcagcct gacaagagcg 3300

agagcgagct ggtgtcccag atcatcgagc agctgatcaa aaaggagaag gtttatctgg 3360

cctgggtgcc cgcccacaag ggcatcggag gcaacgagca ggtggacaag ctggtcagcg 3420

ccggcatccg gaaggtgctg ttcctggacg gcatcgacaa ggcccaggag gaacacgaga 3480

agtaccacag caactggcgg gccatggcca gcgacttcaa cctgccacct gtggttgcta 3540

aggagatcgt cgcctcttgt gataagtgcc agctgaaggg cgaggccatg cacggccaag 3600

tggattgcag ccctgggatc tggcagctag actgtaccca cctggagggc aaggtgatcc 3660

tggtggcagt gcacgtggcc agcggctaca tcgaggctga ggtgatcccc gccgaaacgg 3720

gccaggagac cgcctacttt ctgctgaagc tagccggccg gtggcctgtg aagaccgtgc 3780

acaccgataa cggcagcaat ttcaccagca caaccgtgaa ggctgcctgc tggtgggctg 3840

gaatcaagca ggagttcggc atcccataca atcctcagtc tcagggcgtg atcgagagca 3900

tgaacaagga actgaagaag atcattggtc aggtcagaga tcaggccgag cacctgaaaa 3960

ccgccgttca aatggctgtg ttcatccata acttcaaaag aaaaggcggc atcggcggct 4020

acagcgccgg cgaaagaatc gtggatatca tcgcgaccga catccaaaca aaagagctgc 4080

aaaagcaaat caccaagatc cagaacttca gagtgtacta cagagatagc agagatcctg 4140

tgtggaaggg acctgccaag ctgctgtgga agggcgaggg cgccgtggtg atccaggaca 4200

atagcgacat caaggtcgtg cccagaagaa aggctaaaat cattagagac tacggcaaac 4260

agatggccgg agatgattgc gtggcttcta gacaggacga ggactga 4307

<210> 102

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<223> Rev protein

<400> 102

Met Ala Gly Arg Ser Gly Asp Ser Asp Glu Asp Leu Leu Lys Ala Val

1 5 10 15

Arg Leu Ile Lys Phe Leu Tyr Gln Ser Asn Pro Pro Pro Asn Pro Glu

20 25 30

Gly Thr Arg Gln Ala Arg Arg Asn Arg Arg Arg Arg Trp Arg Glu Arg

35 40 45

Gln Arg Gln Ile His Ser Ile Ser Glu Arg Ile Leu Ser Thr Tyr Leu

50 55 60

Gly Arg Ser Ala Glu Pro Val Pro Leu Gln Leu Pro Pro Leu Glu Arg

65 70 75 80

Leu Thr Leu Asp Cys Asn Glu Asp Cys Gly Thr Ser Gly Thr Gln Gly

85 90 95

Val Gly Ser Pro Gln Ile Leu Val Glu Ser Pro Thr Ile Leu Glu Ser

100 105 110

Gly Ala Lys Glu

115

<210> 103

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> Rev Nucleic Acid

<400> 103

atggccggca gaagcggcga cagcgacgag gatctgctga aagccgtgcg gctgatcaag 60

ttcctgtacc agagcaaccc tcctcctaac cccgagggca ccagacaggc tagacggaac 120

cgcagaagaa ggtggcggga acggcaaaga cagatccact ctatcagcga gagaatcctg 180

agcacctacc tgggaagatc cgccgagcct gtccccctgc agctgcctcc actggaaaga 240

ctgaccctgg attgtaatga ggactgcggc acaagcggaa cccagggcgt gggcagcccc 300

cagattctgg tggaatcccc tacaatcctc gagtctggcg ccaaggaatg a 351

<210> 104

<211> 1539

<212> DNA

<213> Artificial sequence

<220>

<223> Cocal glycoprotein

<400> 104

atgaactttc tgctgctgac cttcatcgtg ctgcctctgt gcagccacgc caagttttcc 60

atcgtgttcc cacagtccca gaagggcaac tggaagaatg tgcccagctc ctaccactat 120

tgtccttcta gctccgacca gaactggcac aatgatctgc tgggcatcac catgaaggtg 180

aagatgccta agacacacaa ggccatccag gcagatggat ggatgtgcca cgcagccaag 240

tggatcacca catgtgactt tcggtggtac ggccccaagt atatcaccca cagcatccac 300

tccatccagc ctacaagcga gcagtgcaag gagtccatca agcagaccaa gcagggcaca 360

tggatgtctc ccggcttccc ccctcagaac tgtggctacg ccaccgtgac agatagcgtg 420

gcagtggtgg tgcaggcaac cccacaccac gtgctggtgg atgagtatac aggcgagtgg 480

atcgacagcc agtttcccaa cggcaagtgc gagaccgagg agtgtgagac agtgcacaat 540

tctaccgtgt ggtacagcga ttataaggtg accggcctgt gcgacgccac actggtggat 600

accgagatca cattcttttc cgaggacggc aagaaggagt ctatcggcaa gcccaacacc 660

ggctacaggt ctaattactt cgcctatgag aagggcgata aggtgtgcaa gatgaattat 720

tgtaagcacg ccggggtgcg gctgccaagc ggcgtgtggt ttgagttcgt ggaccaggac 780

gtgtacgcag cagcaaagct gccagagtgc ccagtgggag caaccatcag cgcccccacc 840

cagacatctg tggacgtgag cctgatcctg gatgtggaga gaatcctgga ctactccctg 900

tgccaggaga catggtccaa gatccgctct aagcagcccg tgagcccagt ggacctgtct 960

tacctggcac caaagaaccc tggaacagga cctgccttta ccatcatcaa tggcacactg 1020

aagtacttcg agacccggta tatcagaatc gacatcgata acccaatcat ctccaagatg 1080

gtgggcaaga tctccggctc tcagaccgag agagagctgt ggacagagtg gttcccatac 1140

gagggcgtgg agatcggccc caatggcatc ctgaagaccc ctacaggcta taagtttcca 1200

ctgttcatga tcggccacgg catgctggac tctgatctgc acaagaccag ccaggccgag 1260

gtgtttgagc acccacacct ggcagaggca ccaaagcagc tgcccgagga ggagaccctg 1320

ttctttggcg atacaggcat ctccaagaac cctgtggagc tgatcgaggg ctggttttct 1380

agctggaagt ctaccgtggt gacattcttt ttcgccatcg gcgtgttcat cctgctgtac 1440

gtggtggcaa ggatcgtgat cgccgtgcgg tacagatatc agggcagcaa caataagaga 1500

atctataatg acatcgagat gtccaggttc cgcaagtga 1539

<210> 105

<211> 65

<212> PRT

<213> Artificial sequence

<220>

<223> Human glycophorin A hinge-TM-CT

<400> 105

His Phe Ser Glu Pro Glu Ile Thr Leu Ile Ile Phe Gly Val Met Ala

1 5 10 15

Gly Val Ile Gly Thr Ile Leu Leu Ile Ser Tyr Gly Ile Arg Arg Leu

20 25 30

Ile Lys Lys Ser Pro Ser Asp Val Lys Pro Leu Pro Ser Pro Asp Thr

35 40 45

Asp Val Pro Leu Ser Ser Val Glu Ile Glu Asn Pro Glu Thr Ser Asp

50 55 60

Gln

65

<210> 106

<211> 195

<212> DNA

<213> Artificial sequence

<220>

<223> Human Glycophorin A Hinge-TM-CT - Nucleic Acid

<400> 106

cacttcagcg agcctgagat caccctgatc atcttcggcg tgatggccgg agtgatcggc 60

acaatcctgc tgatcagcta cggcatcaga agactgatta agaaatcccc atctgatgtg 120

aagcctctgc cttctcctga caccgacgtc cccctgagca gcgtggaaat cgagaacccc 180

gaaaccagcg accag 195

<210> 107

<211> 324

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-CD3scFv_Short Hinge-TM-CT

<400> 107

Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu

1 5 10 15

Ala Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

20 25 30

Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr

35 40 45

Met Asn Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro Lys Arg Trp Ile

50 55 60

Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

65 70 75 80

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

85 90 95

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe

100 105 110

Thr Phe Gly Gln Gly Thr Lys Leu Gln Ile Thr Arg Thr Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln

130 135 140

Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg

145 150 155 160

Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His

165 170 175

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile

180 185 190

Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Val Lys Asp Arg

195 200 205

Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Ala Phe Leu Gln Met

210 215 220

Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys Ala Arg Tyr

225 230 235 240

Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Pro Val

245 250 255

Thr Val Ser Ser Ala Ser Gly Val Glu Leu Ile Glu Gly Trp Phe Ser

260 265 270

Ser Trp Lys Ser Thr Val Val Thr Phe Phe Phe Ala Ile Gly Val Phe

275 280 285

Ile Leu Leu Tyr Val Val Ala Arg Ile Val Ile Ala Val Arg Tyr Arg

290 295 300

Tyr Gln Gly Ser Asn Asn Lys Arg Ile Tyr Asn Asp Ile Glu Met Ser

305 310 315 320

Arg Phe Arg Lys

<210> 108

<211> 972

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-CD3scFv_Short Hinge-TM-CT - Nucleic Acid

<400> 108

atgggcgtga aagtgctgtt cgccctgatc tgcatcgcag ttgctgaagc cgacatccag 60

atgacccagt ctcctagcag cctcagcgct agcgtgggcg atagagtgac catcacatgt 120

agcgccagca gcagcgtgtc ctacatgaac tggtaccagc aaacacctgg aaaggcccct 180

aaaaggtgga tctatgacac atctaagctg gcttctggag tgccatctag attttctggc 240

agcggctccg gcactgatta tacattcacc atcagcagcc tgcagcccga ggatatcgcc 300

acctactact gtcagcagtg gtcctctaat cccttcacct tcggccaggg caccaagctg 360

cagatcacca gaaccagcgg cgggggagga agcggcgggg gaggatctgg cggcggcggc 420

agccaggtgc agctggtgca gagcggcggc ggcgtggtgc aacctggcag aagcctgaga 480

ctgagctgca aggcctctgg ctacaccttc acccggtaca ccatgcattg ggtgcggcag 540

gcccctggca agggcctgga atggattgga tacatcaacc ccagcagagg ctacaccaac 600

tacaaccaga aggtgaagga cagattcaca atttctcggg acaacagcaa gaataccgcc 660

ttcctgcaaa tggactccct gcgcccagaa gataccggcg tgtacttctg cgctagatat 720

tacgacgacc actactgcct ggactactgg ggccagggca cccctgtgac cgtgtccagc 780

gcctccggag tggaactgat cgagggctgg ttcagcagct ggaaaagcac cgtggttaca 840

ttctttttcg ccatcggcgt gttcatcctg ctgtacgtgg tcgccagaat tgtgatcgcc 900

gtgcggtata gataccaggg cagcaacaac aagcggatct acaacgacat cgagatgagc 960

agattcagaa ag 972

<210> 109

<211> 355

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-CD3scFv_Long Hinge_TM_CT

<400> 109

Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu

1 5 10 15

Ala Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

20 25 30

Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr

35 40 45

Met Asn Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro Lys Arg Trp Ile

50 55 60

Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

65 70 75 80

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

85 90 95

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe

100 105 110

Thr Phe Gly Gln Gly Thr Lys Leu Gln Ile Thr Arg Thr Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln

130 135 140

Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg

145 150 155 160

Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His

165 170 175

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile

180 185 190

Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Val Lys Asp Arg

195 200 205

Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Ala Phe Leu Gln Met

210 215 220

Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys Ala Arg Tyr

225 230 235 240

Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Pro Val

245 250 255

Thr Val Ser Ser Ala Ser Ser Gly Phe Glu His Pro His Leu Ala Glu

260 265 270

Ala Pro Lys Gln Leu Pro Glu Glu Glu Thr Leu Phe Phe Gly Asp Thr

275 280 285

Gly Ile Ser Lys Asn Pro Val Glu Leu Ile Glu Gly Trp Phe Ser Ser

290 295 300

Trp Lys Ser Thr Val Val Thr Phe Phe Phe Ala Ile Gly Val Phe Ile

305 310 315 320

Leu Leu Tyr Val Val Ala Arg Ile Val Ile Ala Val Arg Tyr Arg Tyr

325 330 335

Gln Gly Ser Asn Asn Lys Arg Ile Tyr Asn Asp Ile Glu Met Ser Arg

340 345 350

Phe Arg Lys

355

<210> 110

<211> 1062

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-CD3scFv_Long Hinge_TM_CT - Nucleic Acid

<400> 110

atgggcgtga aagtgctgtt cgccctgatc tgcatcgcag ttgctgaagc cgacatccag 60

atgacccagt ctcctagcag cctcagcgct agcgtgggcg atagagtgac catcacatgt 120

agcgccagca gcagcgtgtc ctacatgaac tggtaccagc aaacacctgg aaaggcccct 180

aaaaggtgga tctatgacac atctaagctg gcttctggag tgccatctag attttctggc 240

agcggctccg gcactgatta tacattcacc atcagcagcc tgcagcccga ggatatcgcc 300

acctactact gtcagcagtg gtcctctaat cccttcacct tcggccaggg caccaagctg 360

cagatcacca gaaccagcgg cgggggagga agcggcgggg gaggatctgg cggcggcggc 420

agccaggtgc agctggtgca gagcggcggc ggcgtggtgc aacctggcag aagcctgaga 480

ctgagctgca aggcctctgg ctacaccttc acccggtaca ccatgcattg ggtgcggcag 540

gcccctggca agggcctgga atggattgga tacatcaacc ccagcagagg ctacaccaac 600

tacaaccaga aggtgaagga cagattcaca atttctcggg acaacagcaa gaataccgcc 660

ttcctgcaaa tggactccct gcgcccagaa gataccggcg tgtacttctg cgctagatat 720

tacgacgacc actactgcct ggactactgg ggccagggca cccctgtgac cgtgtccagc 780

gcctccggat tcgagcaccc ccacctggcc gaggccccta agcagctgcc tgaagaagag 840

acactgtttt tcggagatac cggcatcagc aaaaaccccg tggagctgat cgagggctgg 900

ttcagctctt ggaagagcac cgtggtcaca ttctttttcg ccatcggcgt ctttatcctg 960

ctgtacgtgg tagccagaat cgtgatcgcc gtgcggtaca gataccaggg cagcaacaac 1020

aagcggatct acaacgacat cgagatgagc cggttcagaa ag 1062

<210> 111

<211> 374

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-CD3scFv_ hGlycophorin A_TM_HIV Env CT

<400> 111

Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu

1 5 10 15

Ala Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

20 25 30

Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr

35 40 45

Met Asn Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro Lys Arg Trp Ile

50 55 60

Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

65 70 75 80

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

85 90 95

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe

100 105 110

Thr Phe Gly Gln Gly Thr Lys Leu Gln Ile Thr Arg Thr Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln

130 135 140

Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg

145 150 155 160

Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His

165 170 175

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile

180 185 190

Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Val Lys Asp Arg

195 200 205

Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Ala Phe Leu Gln Met

210 215 220

Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys Ala Arg Tyr

225 230 235 240

Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Pro Val

245 250 255

Thr Val Ser Ser Ala Ser Gly Gly Ser Thr Ser Gly Ser Gly Lys Pro

260 265 270

Gly Ser Gly Glu Gly Ser Thr Lys Gly Pro Glu Ile Thr Leu Ile Ile

275 280 285

Phe Gly Val Met Ala Gly Val Ile Gly Thr Ile Leu Leu Ile Ser Tyr

290 295 300

Gly Ile Arg Arg Leu Ala Leu Lys Tyr Trp Trp Asn Leu Leu Gln Tyr

305 310 315 320

Trp Ser Gln Glu Leu Lys Asn Ser Ala Val Ser Leu Leu Asn Ala Thr

325 330 335

Ala Ile Ala Val Ala Glu Gly Thr Asp Arg Val Ile Glu Val Val Gln

340 345 350

Gly Ala Cys Arg Ala Ile Arg His Ile Pro Arg Arg Ile Arg Gln Gly

355 360 365

Leu Glu Arg Ile Leu Leu

370

<210> 112

<211> 1122

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-CD3scFv_ hGlycophorin A_TM_HIV Env CT

<400> 112

atgggcgtga aagtgctgtt cgccctgatc tgcatcgcag ttgctgaagc cgacatccag 60

atgacccagt ctcctagcag cctcagcgct agcgtgggcg atagagtgac catcacatgt 120

agcgccagca gcagcgtgtc ctacatgaac tggtaccagc aaacacctgg aaaggcccct 180

aaaaggtgga tctatgacac atctaagctg gcttctggag tgccatctag attttctggc 240

agcggctccg gcactgatta tacattcacc atcagcagcc tgcagcccga ggatatcgcc 300

acctactact gtcagcagtg gtcctctaat cccttcacct tcggccaggg caccaagctg 360

cagatcacca gaaccagcgg cgggggagga agcggcgggg gaggatctgg cggcggcggc 420

agccaggtgc agctggtgca gagcggcggc ggcgtggtgc aacctggcag aagcctgaga 480

ctgagctgca aggcctctgg ctacaccttc acccggtaca ccatgcattg ggtgcggcag 540

gcccctggca agggcctgga atggattgga tacatcaacc ccagcagagg ctacaccaac 600

tacaaccaga aggtgaagga cagattcaca atttctcggg acaacagcaa gaataccgcc 660

ttcctgcaaa tggactccct gcgcccagaa gataccggcg tgtacttctg cgctagatat 720

tacgacgacc actactgcct ggactactgg ggccagggca cccctgtgac cgtgtccagc 780

gcctccggag gatctacaag cggctctggc aagcctggca gcggagaagg cagcaccaag 840

ggccctgaga tcacactgat catcttcggc gtgatggccg gcgtcatcgg caccatcctg 900

ctgatcagct acggcatcag aagactggct ctgaagtact ggtggaatct gctgcaatac 960

tggagccagg agctgaaaaa cagcgccgtg tccctgctca acgccaccgc catcgccgtg 1020

gccgagggca ccgacagagt gatcgaggtg gtgcagggga cctgcagagc tattcggcac 1080

atccccagac ggatcaggca gggcctggaa agaatcctgc tg 1122

<210> 113

<211> 380

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-CD3scFv_218 linker_HIV Env extracellular-TM-CT

<400> 113

Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu

1 5 10 15

Ala Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

20 25 30

Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr

35 40 45

Met Asn Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro Lys Arg Trp Ile

50 55 60

Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

65 70 75 80

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

85 90 95

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe

100 105 110

Thr Phe Gly Gln Gly Thr Lys Leu Gln Ile Thr Arg Thr Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln

130 135 140

Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg

145 150 155 160

Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His

165 170 175

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile

180 185 190

Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Val Lys Asp Arg

195 200 205

Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Ala Phe Leu Gln Met

210 215 220

Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys Ala Arg Tyr

225 230 235 240

Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Pro Val

245 250 255

Thr Val Ser Ser Ala Ser Gly Gly Ser Thr Ser Gly Ser Gly Lys Pro

260 265 270

Gly Ser Gly Glu Gly Ser Thr Lys Gly Asn Trp Leu Trp Tyr Ile Arg

275 280 285

Ile Phe Ile Ile Ile Val Gly Ser Leu Ile Gly Leu Arg Ile Val Phe

290 295 300

Ala Val Leu Ser Leu Val Asn Arg Gly Trp Glu Ala Leu Lys Tyr Trp

305 310 315 320

Trp Asn Leu Leu Gln Tyr Trp Ser Gln Glu Leu Lys Asn Ser Ala Val

325 330 335

Ser Leu Leu Asn Ala Thr Ala Ile Ala Val Ala Glu Gly Thr Asp Arg

340 345 350

Val Ile Glu Val Val Gln Gly Ala Cys Arg Ala Ile Arg His Ile Pro

355 360 365

Arg Arg Ile Arg Gln Gly Leu Glu Arg Ile Leu Leu

370 375 380

<210> 114

<211> 1140

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-CD3scFv_218 linker_HIV Env extracellular-TM-CT - nucleic acid

<400> 114

atgggcgtga aagtgctgtt cgccctgatc tgcatcgcag ttgctgaagc cgacatccag 60

atgacccagt ctcctagcag cctcagcgct agcgtgggcg atagagtgac catcacatgt 120

agcgccagca gcagcgtgtc ctacatgaac tggtaccagc aaacacctgg aaaggcccct 180

aaaaggtgga tctatgacac atctaagctg gcttctggag tgccatctag attttctggc 240

agcggctccg gcactgatta tacattcacc atcagcagcc tgcagcccga ggatatcgcc 300

acctactact gtcagcagtg gtcctctaat cccttcacct tcggccaggg caccaagctg 360

cagatcacca gaaccagcgg cgggggagga agcggcgggg gaggatctgg cggcggcggc 420

agccaggtgc agctggtgca gagcggcggc ggcgtggtgc aacctggcag aagcctgaga 480

ctgagctgca aggcctctgg ctacaccttc acccggtaca ccatgcattg ggtgcggcag 540

gcccctggca agggcctgga atggattgga tacatcaacc ccagcagagg ctacaccaac 600

tacaaccaga aggtgaagga cagattcaca atttctcggg acaacagcaa gaataccgcc 660

ttcctgcaaa tggactccct gcgcccagaa gataccggcg tgtacttctg cgctagatat 720

tacgacgacc actactgcct ggactactgg ggccagggca cccctgtgac cgtgtccagc 780

gcctccggag gaagcaccag cggctctggc aagcctggca gcggcgaggg ctctaccaag 840

ggcaattggc tgtggtacat cagaatcttc atcatcatcg tgggcagcct gatcggcctg 900

agaatcgtgt tcgccgtgct gagcctggtg aaccggggct gggaagctct gaagtactgg 960

tggaacctgc tgcaatactg gtcccaggag ctgaaaaaca gcgctgtgtc cctgctcaac 1020

gccaccgcca tcgccgtcgc cgagggaaca gacagagtga tcgaggtggt gcagggagcc 1080

tgcagagcca ttcggcacat ccccagacgc atcagacagg gcctggaaag aatcctgctg 1140

<210> 115

<211> 373

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-CD3scFv_ G4S linker_HIV Env extracellular-TM-CT

<400> 115

Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu

1 5 10 15

Ala Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

20 25 30

Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr

35 40 45

Met Asn Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro Lys Arg Trp Ile

50 55 60

Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

65 70 75 80

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

85 90 95

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe

100 105 110

Thr Phe Gly Gln Gly Thr Lys Leu Gln Ile Thr Arg Thr Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln

130 135 140

Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg

145 150 155 160

Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His

165 170 175

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile

180 185 190

Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Val Lys Asp Arg

195 200 205

Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Ala Phe Leu Gln Met

210 215 220

Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys Ala Arg Tyr

225 230 235 240

Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Pro Val

245 250 255

Thr Val Ser Ser Ala Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly

260 265 270

Ser Gly Gly Gly Gly Ser Tyr Ile Arg Ile Phe Ile Ile Ile Val Gly

275 280 285

Ser Leu Ile Gly Leu Arg Ile Val Phe Ala Val Leu Ser Leu Val Asn

290 295 300

Arg Gly Trp Glu Ala Leu Lys Tyr Trp Trp Asn Leu Leu Gln Tyr Trp

305 310 315 320

Ser Gln Glu Leu Lys Asn Ser Ala Val Ser Leu Leu Asn Ala Thr Ala

325 330 335

Ile Ala Val Ala Glu Gly Thr Asp Arg Val Ile Glu Val Val Gln Gly

340 345 350

Ala Cys Arg Ala Ile Arg His Ile Pro Arg Arg Ile Arg Gln Gly Leu

355 360 365

Glu Arg Ile Leu Leu

370

<210> 116

<211> 1119

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-CD3scFv_ G4S linker_HIV Env extracellular-TM-CT - nucleic acid

<400> 116

atgggcgtga aagtgctgtt cgccctgatc tgcatcgcag ttgctgaagc cgacatccag 60

atgacccagt ctcctagcag cctcagcgct agcgtgggcg atagagtgac catcacatgt 120

agcgccagca gcagcgtgtc ctacatgaac tggtaccagc aaacacctgg aaaggcccct 180

aaaaggtgga tctatgacac atctaagctg gcttctggag tgccatctag attttctggc 240

agcggctccg gcactgatta tacattcacc atcagcagcc tgcagcccga ggatatcgcc 300

acctactact gtcagcagtg gtcctctaat cccttcacct tcggccaggg caccaagctg 360

cagatcacca gaaccagcgg cgggggagga agcggcgggg gaggatctgg cggcggcggc 420

agccaggtgc agctggtgca gagcggcggc ggcgtggtgc aacctggcag aagcctgaga 480

ctgagctgca aggcctctgg ctacaccttc acccggtaca ccatgcattg ggtgcggcag 540

gcccctggca agggcctgga atggattgga tacatcaacc ccagcagagg ctacaccaac 600

tacaaccaga aggtgaagga cagattcaca atttctcggg acaacagcaa gaataccgcc 660

ttcctgcaaa tggactccct gcgcccagaa gataccggcg tgtacttctg cgctagatat 720

tacgacgacc actactgcct ggactactgg ggccagggca cccctgtgac cgtgtccagc 780

gcctccggag gcggtggagg ctctggtggc ggagggagcg gtggcggagg cagctacatc 840

agaatcttca tcatcatcgt gggcagcctg atcggcctga gaatcgtgtt cgccgttctg 900

agcctggtga accggggctg ggaagccctg aagtactggt ggaatctgct ccagtactgg 960

tctcaggagc tgaagaacag cgccgtgtcc ctgctgaacg ctacagctat cgccgtcgcc 1020

gagggcaccg acagagtgat cgaggtggtg cagggcgcct gcagagccat ccggcacatc 1080

cctagaagga ttcggcaagg cctggaaaga atcctgctg 1119

<210> 117

<211> 856

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-CD3scFv_Short Hinge_TM_CT_T2A_ Cocal Envelope

<400> 117

Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu

1 5 10 15

Ala Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

20 25 30

Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr

35 40 45

Met Asn Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro Lys Arg Trp Ile

50 55 60

Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

65 70 75 80

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

85 90 95

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe

100 105 110

Thr Phe Gly Gln Gly Thr Lys Leu Gln Ile Thr Arg Thr Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln

130 135 140

Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg

145 150 155 160

Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His

165 170 175

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile

180 185 190

Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Val Lys Asp Arg

195 200 205

Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Ala Phe Leu Gln Met

210 215 220

Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys Ala Arg Tyr

225 230 235 240

Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Pro Val

245 250 255

Thr Val Ser Ser Ala Ser Gly Val Glu Leu Ile Glu Gly Trp Phe Ser

260 265 270

Ser Trp Lys Ser Thr Val Val Thr Phe Phe Phe Ala Ile Gly Val Phe

275 280 285

Ile Leu Leu Tyr Val Val Ala Arg Ile Val Ile Ala Val Arg Tyr Arg

290 295 300

Tyr Gln Gly Ser Asn Asn Lys Arg Ile Tyr Asn Asp Ile Glu Met Ser

305 310 315 320

Arg Phe Arg Lys Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys

325 330 335

Gly Asp Val Glu Glu Asn Pro Gly Pro Asn Phe Leu Leu Leu Thr Phe

340 345 350

Ile Val Leu Pro Leu Cys Ser His Ala Lys Phe Ser Ile Val Phe Pro

355 360 365

Gln Ser Gln Lys Gly Asn Trp Lys Asn Val Pro Ser Ser Tyr His Tyr

370 375 380

Cys Pro Ser Ser Ser Asp Gln Asn Trp His Asn Asp Leu Leu Gly Ile

385 390 395 400

Thr Met Lys Val Lys Met Pro Lys Thr His Lys Ala Ile Gln Ala Asp

405 410 415

Gly Trp Met Cys His Ala Ala Lys Trp Ile Thr Thr Cys Asp Phe Arg

420 425 430

Trp Tyr Gly Pro Lys Tyr Ile Thr His Ser Ile His Ser Ile Gln Pro

435 440 445

Thr Ser Glu Gln Cys Lys Glu Ser Ile Lys Gln Thr Lys Gln Gly Thr

450 455 460

Trp Met Ser Pro Gly Phe Pro Pro Gln Asn Cys Gly Tyr Ala Thr Val

465 470 475 480

Thr Asp Ser Val Ala Val Val Val Gln Ala Thr Pro His His Val Leu

485 490 495

Val Asp Glu Tyr Thr Gly Glu Trp Ile Asp Ser Gln Phe Pro Asn Gly

500 505 510

Lys Cys Glu Thr Glu Glu Cys Glu Thr Val His Asn Ser Thr Val Trp

515 520 525

Tyr Ser Asp Tyr Lys Val Thr Gly Leu Cys Asp Ala Thr Leu Val Asp

530 535 540

Thr Glu Ile Thr Phe Phe Ser Glu Asp Gly Lys Lys Glu Ser Ile Gly

545 550 555 560

Lys Pro Asn Thr Gly Tyr Arg Ser Asn Tyr Phe Ala Tyr Glu Lys Gly

565 570 575

Asp Lys Val Cys Lys Met Asn Tyr Cys Lys His Ala Gly Val Arg Leu

580 585 590

Pro Ser Gly Val Trp Phe Glu Phe Val Asp Gln Asp Val Tyr Ala Ala

595 600 605

Ala Lys Leu Pro Glu Cys Pro Val Gly Ala Thr Ile Ser Ala Pro Thr

610 615 620

Gln Thr Ser Val Asp Val Ser Leu Ile Leu Asp Val Glu Arg Ile Leu

625 630 635 640

Asp Tyr Ser Leu Cys Gln Glu Thr Trp Ser Lys Ile Arg Ser Lys Gln

645 650 655

Pro Val Ser Pro Val Asp Leu Ser Tyr Leu Ala Pro Lys Asn Pro Gly

660 665 670

Thr Gly Pro Ala Phe Thr Ile Ile Asn Gly Thr Leu Lys Tyr Phe Glu

675 680 685

Thr Arg Tyr Ile Arg Ile Asp Ile Asp Asn Pro Ile Ile Ser Lys Met

690 695 700

Val Gly Lys Ile Ser Gly Ser Gln Thr Glu Arg Glu Leu Trp Thr Glu

705 710 715 720

Trp Phe Pro Tyr Glu Gly Val Glu Ile Gly Pro Asn Gly Ile Leu Lys

725 730 735

Thr Pro Thr Gly Tyr Lys Phe Pro Leu Phe Met Ile Gly His Gly Met

740 745 750

Leu Asp Ser Asp Leu His Lys Thr Ser Gln Ala Glu Val Phe Glu His

755 760 765

Pro His Leu Ala Glu Ala Pro Lys Gln Leu Pro Glu Glu Glu Thr Leu

770 775 780

Phe Phe Gly Asp Thr Gly Ile Ser Lys Asn Pro Val Glu Leu Ile Glu

785 790 795 800

Gly Trp Phe Ser Ser Trp Lys Ser Thr Val Val Thr Phe Phe Phe Ala

805 810 815

Ile Gly Val Phe Ile Leu Leu Tyr Val Val Ala Arg Ile Val Ile Ala

820 825 830

Val Arg Tyr Arg Tyr Gln Gly Ser Asn Asn Lys Arg Ile Tyr Asn Asp

835 840 845

Ile Glu Met Ser Arg Phe Arg Lys

850 855

<210> 118

<211> 2568

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-CD3scFv_Short Hinge_TM_CT_T2A_ Cocal Envelope - Nucleic Acid

<400> 118

atgggcgtga aagtgctgtt cgccctgatc tgcatcgcag ttgctgaagc cgacatccag 60

atgacccagt ctcctagcag cctcagcgct agcgtgggcg atagagtgac catcacatgt 120

agcgccagca gcagcgtgtc ctacatgaac tggtaccagc aaacacctgg aaaggcccct 180

aaaaggtgga tctatgacac atctaagctg gcttctggag tgccatctag attttctggc 240

agcggctccg gcactgatta tacattcacc atcagcagcc tgcagcccga ggatatcgcc 300

acctactact gtcagcagtg gtcctctaat cccttcacct tcggccaggg caccaagctg 360

cagatcacca gaaccagcgg cgggggagga agcggcgggg gaggatctgg cggcggcggc 420

agccaggtgc agctggtgca gagcggcggc ggcgtggtgc aacctggcag aagcctgaga 480

ctgagctgca aggcctctgg ctacaccttc acccggtaca ccatgcattg ggtgcggcag 540

gcccctggca agggcctgga atggattgga tacatcaacc ccagcagagg ctacaccaac 600

tacaaccaga aggtgaagga cagattcaca atttctcggg acaacagcaa gaataccgcc 660

ttcctgcaaa tggactccct gcgcccagaa gataccggcg tgtacttctg cgctagatat 720

tacgacgacc actactgcct ggactactgg ggccagggca cccctgtgac cgtgtccagc 780

gcctccggag tggaactgat cgagggctgg ttcagcagct ggaaaagcac cgtggttaca 840

ttctttttcg ccatcggcgt gttcatcctg ctgtacgtgg tcgccagaat tgtgatcgcc 900

gtgcggtata gataccaggg cagcaacaac aagcggatct acaacgacat cgagatgagc 960

agattcagaa agggatctgg agagggaagg ggaagcctgc tgacatgcgg cgacgtggag 1020

gagaacccag gaccaaattt tctgctgctg accttcatcg tgctgcctct gtgcagccac 1080

gccaagtttt ccatcgtgtt cccacagtcc cagaagggca actggaagaa tgtgccctct 1140

agctaccact attgcccttc ctctagcgac cagaactggc acaatgatct gctgggcatc 1200

acaatgaagg tgaagatgcc caagacccac aaggccatcc aggcagatgg atggatgtgc 1260

cacgcagcca agtggatcac aacctgtgac tttcggtggt acggccccaa gtatatcaca 1320

cactccatcc actctatcca gcctacctcc gagcagtgca aggagtctat caagcagaca 1380

aagcagggca cctggatgag ccctggcttc ccaccccaga actgtggcta cgccacagtg 1440

accgactccg tggcagtggt ggtgcaggca acacctcacc acgtgctggt ggatgagtat 1500

accggcgagt ggatcgacag ccagtttcca aacggcaagt gcgagacaga ggagtgtgag 1560

accgtgcaca attctacagt gtggtacagc gattataagg tgacaggcct gtgcgacgcc 1620

accctggtgg atacagagat caccttcttt tctgaggacg gcaagaagga gagcatcggc 1680

aagcccaaca ccggctacag atccaattac ttcgcctatg agaagggcga taaggtgtgc 1740

aagatgaatt attgtaagca cgccggggtg cggctgccta gcggcgtgtg gtttgagttc 1800

gtggaccagg acgtgtacgc agcagcaaag ctgcctgagt gcccagtggg agcaaccatc 1860

tccgccccaa cacagacctc cgtggacgtg tctctgatcc tggatgtgga gcgcatcctg 1920

gactacagcc tgtgccagga gacctggagc aagatccggt ccaagcagcc cgtgtcccct 1980

gtggacctgt cttacctggc accaaagaac ccaggaaccg gaccagcctt tacaatcatc 2040

aatggcaccc tgaagtactt cgagacccgc tatatccgga tcgacatcga taaccctatc 2100

atcagcaaga tggtgggcaa gatctctggc agccagacag agagagagct gtggaccgag 2160

tggttccctt acgagggcgt ggagatcggc ccaaatggca tcctgaagac accaaccggc 2220

tataagtttc ccctgttcat gatcggccac ggcatgctgg acagcgatct gcacaagacc 2280

tcccaggccg aggtgtttga gcacccaacac ctggcagagg caccaaagca gctgcctgag 2340

gaggagacac tgttctttgg cgataccggc atctctaaga accccgtgga gctgatcgag 2400

ggctggtttt cctcttggaa gagcacagtg gtgaccttct ttttcgccat cggcgtgttc 2460

atcctgctgt acgtggtggc cagaatcgtg atcgccgtga gatacaggta tcagggctcc 2520

aacaataaga ggatctataa tgacatcgag atgtctcgct tccggaag 2568

<210> 119

<211> 327

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-CD3 scFv_human glycophorin A hinge-TM-CT

<400> 119

Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu

1 5 10 15

Ala Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

20 25 30

Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr

35 40 45

Met Asn Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro Lys Arg Trp Ile

50 55 60

Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

65 70 75 80

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

85 90 95

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe

100 105 110

Thr Phe Gly Gln Gly Thr Lys Leu Gln Ile Thr Arg Thr Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln

130 135 140

Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg

145 150 155 160

Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His

165 170 175

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile

180 185 190

Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Val Lys Asp Arg

195 200 205

Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Ala Phe Leu Gln Met

210 215 220

Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys Ala Arg Tyr

225 230 235 240

Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Pro Val

245 250 255

Thr Val Ser Ser Ala Ser His Phe Ser Glu Pro Glu Ile Thr Leu Ile

260 265 270

Ile Phe Gly Val Met Ala Gly Val Ile Gly Thr Ile Leu Leu Ile Ser

275 280 285

Tyr Gly Ile Arg Arg Leu Ile Lys Lys Ser Pro Ser Asp Val Lys Pro

290 295 300

Leu Pro Ser Pro Asp Thr Asp Val Pro Leu Ser Ser Val Glu Ile Glu

305 310 315 320

Asn Pro Glu Thr Ser Asp Gln

325

<210> 120

<211> 981

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-CD3 scFv_human glycophorin A hinge-TM-CT - Nucleic acid

<400> 120

atgggcgtga aagtgctgtt cgccctgatc tgcatcgcag ttgctgaagc cgacatccag 60

atgacccagt ctcctagcag cctcagcgct agcgtgggcg atagagtgac catcacatgt 120

agcgccagca gcagcgtgtc ctacatgaac tggtaccagc aaacacctgg aaaggcccct 180

aaaaggtgga tctatgacac atctaagctg gcttctggag tgccatctag attttctggc 240

agcggctccg gcactgatta tacattcacc atcagcagcc tgcagcccga ggatatcgcc 300

acctactact gtcagcagtg gtcctctaat cccttcacct tcggccaggg caccaagctg 360

cagatcacca gaaccagcgg cgggggagga agcggcgggg gaggatctgg cggcggcggc 420

agccaggtgc agctggtgca gagcggcggc ggcgtggtgc aacctggcag aagcctgaga 480

ctgagctgca aggcctctgg ctacaccttc acccggtaca ccatgcattg ggtgcggcag 540

gcccctggca agggcctgga atggattgga tacatcaacc ccagcagagg ctacaccaac 600

tacaaccaga aggtgaagga cagattcaca atttctcggg acaacagcaa gaataccgcc 660

ttcctgcaaa tggactccct gcgcccagaa gataccggcg tgtacttctg cgctagatat 720

tacgacgacc actactgcct ggactactgg ggccagggca cccctgtgac cgtgtccagc 780

gcctcccact tcagcgagcc tgagatcacc ctgatcatct tcggcgtgat ggccggagtg 840

atcggcacaa tcctgctgat cagctacggc atcagaagac tgattaagaa atccccatct 900

gatgtgaagc ctctgccttc tcctgacacc gacgtccccc tgagcagcgt ggaaatcgag 960

aaccccgaaa ccagcgacca g 981

<210> 121

<211> 7145

<212> DNA

<213> Artificial sequence

<220>

<223> Payload plasmid 142 - includes anti-CD19 CAR, cytoplasmic FRB, RACR

<400> 121

tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg 60

ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc 120

gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc 180

cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt 240

tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc 300

aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta 360

actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa 420

cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc 480

tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac 540

tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat 600

tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa 660

acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga 720

aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc 780

agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat 840

agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa 900

gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca 960

attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac 1020

ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt 1080

tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga 1140

cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg 1200

ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg 1260

caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt 1320

gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat 1380

ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt 1440

acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac 1500

aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt 1560

ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag 1620

tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc 1680

agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg 1740

gagagagaga cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcggttaa 1800

cttttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata gtagacataa 1860

tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt caaaatttta 1920

tcggccgcgg ggtacctagg aacagagaaa caggagaata tgggccaaac aggatatctg 1980

tggtaagcag ttcctgcccc ggctcagggc caagaacagt tggaacagca gaatatgggc 2040

caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga acagatggtc 2100

cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt ccagggtgcc 2160

ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc gcttctcgct 2220

tctgttcgcg cgcttctgct ccccgagctc tatataagca gagctcgttt agtgaaccgt 2280

cagatcgcct ggagacgcca tccacgctgt tttgacttcc atagaagcct gcagggccgc 2340

caccatgctg ctgctggtga cctccctgct gctgtgcgag ctgcctcacc cagcctttct 2400

gctgatcccc gacatccaga tgacacagac cacaagctcc ctgtctgcca gcctgggcga 2460

cagagtgacc atctcctgta gggcctctca ggatatcagc aagtacctga actggtatca 2520

gcagaagcca gatggcacag tgaagctgct gatctaccac acctccaggc tgcactctgg 2580

agtgccaagc cggttctccg gatctggaag cggcaccgac tattccctga caatctctaa 2640

cctggagcag gaggatatcg ccacatactt ttgccagcag ggcaataccc tgccatatac 2700

attcggcgga ggaaccaagc tggagatcac cggatccaca tctggaagcg gcaagccagg 2760

aagcggagag ggatccacaa agggagaggt gaagctgcag gagagcggac caggactggt 2820

ggcaccatcc cagtctctga gcgtgacctg tacagtgtcc ggcgtgtctc tgcctgacta 2880

cggcgtgtcc tggatcaggc agccacctag gaagggactg gagtggctgg gcgtgatctg 2940

gggctctgag accacatact ataattctgc cctgaagagc cgcctgacca tcatcaagga 3000

caactccaag tctcaggtgt ttctgaagat gaatagcctg cagaccgacg atacagccat 3060

ctactattgc gccaagcact actattacgg cggctcctac gccatggatt attggggcca 3120

gggcacctcc gtgacagtgt ctagcgagtc taagtatggc ccaccctgcc ctccatgtcc 3180

aatgttctgg gtgctggtgg tggtgggagg cgtgctggcc tgttatccc tgctggtgac 3240

cgtggccttt atcatcttct gggtgaagag aggcaggaag aagctgctgt atatctttaa 3300

gcagcccttc atgcgccctg tgcagaccac acaggaggag gacggctgca gctgtcggtt 3360

tccagaggag gaggagggag gatgcgagct gcgcgtgaag ttcagccggt ccgccgatgc 3420

ccctgcctac cagcagggcc agaaccagct gtataacgag ctgaatctgg gccggagaga 3480

ggagtacgac gtgctggata agaggagggg aagggaccca gagatgggag gcaagcctcg 3540

gagaaagaac ccacaggagg gcctgtacaa tgagctgcag aaggacaaga tggccgaggc 3600

ctattctgag atcggcatga agggagagag gcgccggggc aagggacacg atggcctgta 3660

ccagggcctg agcaccgcca caaaggacac atatgatgcc ctgcacatgc aggccctgcc 3720

acctagggga tctggagcca ccaactttag cctgctgaag caggcaggcg atgtggagga 3780

gaatccagga cctgagatgt ggcacgaggg actggaggag gcaagcaggc tgtactttgg 3840

cgagcggaat gtgaagggca tgttcgaggt gctggagcca ctgcacgcaa tgatggagag 3900

gggaccacag accctgaagg agacatcctt caaccaggca tacggaaggg acctgatgga 3960

ggcacaggag tggtgccgga agtatatgaa gtctggcaat gtgaaggacc tgctgcaggc 4020

ctgggatctg tattaccacg tgtttagaag gatcagcaag ggctccggcg ccaccaactt 4080

ctccctgctg aagcaggccg gcgatgtgga agaaaatcca ggaccaatgc ctctgggact 4140

gctgtggctg ggactggccc tgctgggcgc cctgcacgcc caggccggcg tgcaggtgga 4200

gacaatcagc cctggcgacg gcagaacctt tccaaagagg ggccagacat gcgtggtgca 4260

ctacaccggc atgctggagg atggcaagaa gttcgactcc tctcgcgatc ggaacaagcc 4320

ctttaagttc atgctgggca agcaggaagt gatcagaggc tgggaggagg gcgtggccca 4380

gatgtctgtg ggccagaggg ccaagctgac aatcagccca gactatgcat acggagcaac 4440

cggacaccct ggaatcatcc caccacacgc cacactggtg ttcgatgtgg agctgctgaa 4500

gctgggcgag ggctctaaca ccagcaagga gaatccattt ctgttcgcac tggaggcagt 4560

ggtcatctcc gtgggctcta tgggcctgat catctccctg ctgtgcgtgt acttttggct 4620

ggagagaaca atgccaagga tccccaccct gaagaacctg gaggacctgg tgaccgagta 4680

ccacggcaat ttcagcgcct ggtccggcgt gtctaaggga ctggcagagt ccctgcagcc 4740

agattattct gagcggctgt gcctggtgag cgagatccct ccaaagggag gcgccctggg 4800

agagggacca ggagccagcc cctgcaacca gcactcccct tactgggccc ccccttgtta 4860

taccctgaag ccagagacag gctctggcgc caccaacttc agcctgctga agcaagccgg 4920

cgacgtggaa gaaaacccag gaccaatggc actgccagtg accgccctgc tgctgcctct 4980

ggccctgctg ctgcacgcag ccagacccat cctgtggcac gaaatgtggc atgaaggcct 5040

ggaggaggca agcagactgt actttggcga gagaaatgtg aaaggaatgt ttgaggtgct 5100

ggagcctctg cacgccatga tggagagggg ccctcagacc ctgaaggaga catcctttaa 5160

ccaggcctac ggcagagacc tgatggaggc ccaggagtgg tgcaggaagt atatgaagag 5220

cggaaatgtg aaagacctgc tgcaggcctg ggatctgtac taccacgtgt tccgccggat 5280

ctctaagggc aaggatacaa tcccttggct gggacacctg ctggtgggac tgagcggagc 5340

ctttggcttc atcatcctgg tgtatctgct gatcaactgc agaaatacag gcccatggct 5400

gaagaaggtg ctgaagtgta acacccctga cccatccaag ttcttttctc agctgagctc 5460

cgagcacggc ggcgatgtgc agaagtggct gtctagcccc tttccttcct ctagcttcag 5520

ccctggagga ctggcacctg agatctcccc actggaggtg ctggagaggg acaaggtgac 5580

ccagctgctg ctgcagcagg ataaggtgcc agagcccgcc tccctgtcct ctaaccacag 5640

cctgacctcc tgctttacaa atcagggcta cttctttttc cacctgccag acgcactgga 5700

gatcgaggca tgtcaggtgt atttcacata cgatccctat agcgaggagg accctgatga 5760

gggagtggcc ggcgccccaa ccggaagctc ccctcagcca ctgcagccac tgagcggaga 5820

ggacgatgca tattgtacat ttccttcccg cgacgatctg ctgctgttct ctccaagcct 5880

gctggggagga ccatctccac ccagcaccgc acctggagga tccggggcag gggaggagcg 5940

gatgcctcca tctctgcagg agagagtgcc aagggactgg gatccacagc ctctgggacc 6000

acctacccct ggagtgccag acctggtgga tttccagcca ccccctgagc tggtgctgcg 6060

ggaggcagga gaggaggtgc cagacgcagg acctagagag ggcgtgagct ttccctggtc 6120

caggccacca ggacagggag agttccgcgc cctgaacgcc cggctgcccc tgaatacaga 6180

cgcctacctg tctctgcagg agctgcaggg ccaggatcct acccacctgg tgtgacgccg 6240

gcgctagtgt cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc 6300

ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg 6360

ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc 6420

tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg 6480

acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg 6540

ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga 6600

caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaag ctgacgtcct 6660

ttccatggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg 6720

tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc 6780

ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc 6840

cgcctgttta agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga 6900

aaagggggga ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg 6960

tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa 7020

cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct 7080

gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc 7140

tagca 7145

<210> 122

<211> 6926

<212> DNA

<213> Artificial sequence

<220>

<223> Payload plasmid -201, including anti-CD19 CAR, cytoplasmic FRB and RACR

<400> 122

gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60

catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120

acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180

ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240

aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300

ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360

tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 420

ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 480

ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 540

tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccggg 600

tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg 660

cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt 720

gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt 780

ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga 840

ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa 900

aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc 960

gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat 1020

ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg 1080

gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc 1140

agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc 1200

atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa 1260

acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat 1320

atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta 1380

ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga 1440

ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca 1500

atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat 1560

ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag 1620

cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg 1680

atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg 1740

agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa 1800

attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa 1860

aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac 1920

ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt 1980

ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca 2040

ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa 2100

gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atctcgacgg 2160

tatcggttaa cttttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata 2220

gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt 2280

caaaatttta tcgattgcct gacgcgtaat gaaagacccc acctgtaggt ttggcaagct 2340

aggatcaagg tcaggaacag agagacagca gaatatgggc caaacaggat atctgtggta 2400

agcagttcct gccccggctc agggccaaga acagttggaa cagcagaata tgggccaaac 2460

aggatatctg tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggtccccag 2520

atgcggtccc gccctcagca gtttctagag aaccatcaga tgtttccagg gtgccccaag 2580

gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt 2640

tcgcgcgctt ctgctccccg agctctatat aagagcccac aacccctcac tcggcgcgtg 2700

aacacaattc tgcagtcgaa ggcgtaccgt cacttacgag tcggtagcct gcagggccgc 2760

caccatgctg ctgctggtga cctccctgct gctgtgcgag ctgcctcacc cagcctttct 2820

gctgatcccc gacatccaga tgacacagac cacaagctcc ctgtctgcca gcctgggcga 2880

cagagtgacc atctcctgta gggcctctca ggatatcagc aagtacctga actggtatca 2940

gcagaagcca gatggcacag tgaagctgct gatctaccac acctccaggc tgcactctgg 3000

agtgccaagc cggttctccg gatctggaag cggcaccgac tattccctga caatctctaa 3060

cctggagcag gaggatatcg ccacatactt ttgccagcag ggcaataccc tgccatatac 3120

attcggcgga ggaaccaagc tggagatcac cggatccaca tctggaagcg gcaagccagg 3180

aagcggagag ggatccacaa agggagaggt gaagctgcag gagagcggac caggactggt 3240

ggcaccatcc cagtctctga gcgtgacctg tacagtgtcc ggcgtgtctc tgcctgacta 3300

cggcgtgtcc tggatcaggc agccacctag gaagggactg gagtggctgg gcgtgatctg 3360

gggctctgag accacatact ataattctgc cctgaagagc cgcctgacca tcatcaagga 3420

caactccaag tctcaggtgt ttctgaagat gaatagcctg cagaccgacg atacagccat 3480

ctactattgc gccaagcact actattacgg cggctcctac gccatggatt attggggcca 3540

gggcacctcc gtgacagtgt ctagcgagtc taagtatggc ccaccctgcc ctccatgtcc 3600

aatgttctgg gtgctggtgg tggtgggagg cgtgctggcc tgttatccc tgctggtgac 3660

cgtggccttt atcatcttct gggtgaagag aggcaggaag aagctgctgt atatctttaa 3720

gcagcccttc atgcgccctg tgcagaccac acaggaggag gacggctgca gctgtcggtt 3780

tccagaggag gaggagggag gatgcgagct gcgcgtgaag ttcagccggt ccgccgatgc 3840

ccctgcctac cagcagggcc agaaccagct gtataacgag ctgaatctgg gccggagaga 3900

ggagtacgac gtgctggata agaggagggg aagggaccca gagatgggag gcaagcctcg 3960

gagaaagaac ccacaggagg gcctgtacaa tgagctgcag aaggacaaga tggccgaggc 4020

ctattctgag atcggcatga agggagag gcgccggggc aagggacacg atggcctgta 4080

ccagggcctg agcaccgcca caaaggacac atatgatgcc ctgcacatgc aggccctgcc 4140

acctagggga tctggagcca ccaactttag cctgctgaag caggcaggcg atgtggagga 4200

gaatccagga cctgagatgt ggcacgaggg actggaggag gcaagcaggc tgtactttgg 4260

cgagcggaat gtgaagggca tgttcgaggt gctggagcca ctgcacgcaa tgatggagag 4320

gggaccacag accctgaagg agacatcctt caaccaggca tacggaaggg acctgatgga 4380

ggcacaggag tggtgccgga agtatatgaa gtctggcaat gtgaaggacc tgctgcaggc 4440

ctgggatctg tattaccacg tgtttagaag gatcagcaag ggctccggcg ccaccaactt 4500

ctccctgctg aagcaggccg gcgatgtgga agaaaatcca ggaccaatgc ctctgggact 4560

gctgtggctg ggactggccc tgctgggcgc cctgcacgcc caggccggcg tgcaggtgga 4620

gacaatcagc cctggcgacg gcagaacctt tccaaagagg ggccagacat gcgtggtgca 4680

ctacaccggc atgctggagg atggcaagaa gttcgactcc tctcgcgatc ggaacaagcc 4740

ctttaagttc atgctgggca agcaggaagt gatcagaggc tgggaggagg gcgtggccca 4800

gatgtctgtg ggccagaggg ccaagctgac aatcagccca gactatgcat acggagcaac 4860

cggacaccct ggaatcatcc caccacacgc cacactggtg ttcgatgtgg agctgctgaa 4920

gctgggcgag ggctctaaca ccagcaagga gaatccattt ctgttcgcac tggaggcagt 4980

ggtcatctcc gtgggctcta tgggcctgat catctccctg ctgtgcgtgt acttttggct 5040

ggagagaaca atgccaagga tccccaccct gaagaacctg gaggacctgg tgaccgagta 5100

ccacggcaat ttcagcgcct ggtccggcgt gtctaaggga ctggcagagt ccctgcagcc 5160

agattattct gagcggctgt gcctggtgag cgagatccct ccaaagggag gcgccctggg 5220

agagggacca ggagccagcc cctgcaacca gcactcccct tactgggccc ccccttgtta 5280

taccctgaag ccagagacag gctctggcgc caccaacttc agcctgctga agcaagccgg 5340

cgacgtggaa gaaaacccag gaccaatggc actgccagtg accgccctgc tgctgcctct 5400

ggccctgctg ctgcacgcag ccagacccat cctgtggcac gaaatgtggc atgaaggcct 5460

ggaggaggca agcagactgt actttggcga gagaaatgtg aaaggaatgt ttgaggtgct 5520

ggagcctctg cacgccatga tggagagggg ccctcagacc ctgaaggaga catcctttaa 5580

ccaggcctac ggcagagacc tgatggaggc ccaggagtgg tgcaggaagt atatgaagag 5640

cggaaatgtg aaagacctgc tgcaggcctg ggatctgtac taccacgtgt tccgccggat 5700

ctctaagggc aaggatacaa tcccttggct gggacacctg ctggtgggac tgagcggagc 5760

ctttggcttc atcatcctgg tgtatctgct gatcaactgc agaaatacag gcccatggct 5820

gaagaaggtg ctgaagtgta acacccctga cccatccaag ttcttttctc agctgagctc 5880

cgagcacggc ggcgatgtgc agaagtggct gtctagcccc tttccttcct ctagcttcag 5940

ccctggagga ctggcacctg agatctcccc actggaggtg ctggagagggg acaaggtgac 6000

ccagctgctg ctgcagcagg ataaggtgcc agagcccgcc tccctgtcct ctaaccacag 6060

cctgacctcc tgctttacaa atcagggcta cttctttttc cacctgccag acgcactgga 6120

gatcgaggca tgtcaggtgt atttcacata cgatccctat agcgaggagg accctgatga 6180

gggagtggcc ggcgccccaa ccggaagctc ccctcagcca ctgcagccac tgagcggaga 6240

ggacgatgca tattgtacat ttccttcccg cgacgatctg ctgctgttct ctccaagcct 6300

gctggggagga ccatctccac ccagcaccgc acctggagga tccggggcag gggaggagcg 6360

gatgcctcca tctctgcagg agagagtgcc aagggactgg gatccacagc ctctgggacc 6420

acctacccct ggagtgccag acctggtgga tttccagcca ccccctgagc tggtgctgcg 6480

ggaggcagga gaggaggtgc cagacgcagg acctagagag ggcgtgagct ttccctggtc 6540

caggccacca ggacagggag agttccgcgc cctgaacgcc cggctgcccc tgaatacaga 6600

cgcctacctg tctctgcagg agctgcaggg ccaggatcct acccacctgg tgtgacgccg 6660

gcgctagtgt cgacgtagtg gtacctttaa gaccaatgac ttacaaggca gctgtagatc 6720

ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac 6780

aagatctgct ttttgcttgt actgggtctc tctggttaga ccagatctga gcctggggagc 6840

tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc 6900

aatgtgtgtg ttggtttttt gtgtgt 6926

<210> 123

<211> 3020

<212> DNA

<213> Artificial sequence

<220>

<223> Cocal Envelope Plasmid - 209

<400> 123

ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc 60

gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat 120

tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc 180

aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 240

caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt 300

acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 360

ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg 420

gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac 480

gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg 540

tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac 600

gccatccacg ctgttttgac ctccatagaa gacaccgggc gagctcggat cctgagaact 660

tcagggtgag tctatgggac ccttgatgtt ttctttcccc ttcttttcta tggttaagtt 720

catgtcatag gaaggggaga agtaacaggg tacacatatt gaccaaatca gggtaatttt 780

gcatttgtaa ttttaaaaaa tgctttcttc ttttaatata cttttttgtt tatcttattt 840

ctaatacttt ccctaatctc tttctttcag ggcaataatg atacaatgta tcatgcctct 900

ttgcaccatt ctaaagaata acagtgataa tttctgggtt aaggcaatag caatatttct 960

gcatataaat atttctgcat ataaattgta actgatgtaa gaggtttcat attgctaata 1020

gcagctacaa tccagctacc attctgcttt tattttatgg ttgggataag gctggattat 1080

tctgagtcca agctaggccc ttttgctaat catgttcata cctctttatct tcctcccaca 1140

gctcctgggc aacgtgctgg tctgtgtgct ggcccatcac tttggcaaag cacgtgagat 1200

ctgtctgaca tcctgcaggg ccgccaccat gaactttctg ctgctgacct tcatcgtgct 1260

gcctctgtgc agccacgcca agttttccat cgtgttccca cagtcccaga agggcaactg 1320

gaagaatgtg cccagctcct accactattg tccttctagc tccgaccaga actggcacaa 1380

tgatctgctg ggcatcacca tgaaggtgaa gatgcctaag acacacaagg ccatccaggc 1440

agatggatgg atgtgccacg cagccaagtg gatcaccaca tgtgactttc ggtggtacgg 1500

ccccaagtat atcacccaca gcatccactc catccagcct acaagcgagc agtgcaagga 1560

gtccatcaag cagaccaagc agggcacatg gatgtctccc ggcttccccc ctcagaactg 1620

tggctacgcc accgtgacag atagcgtggc agtggtggtg caggcaaccc cacaccacgt 1680

gctggtggat gagtatacag gcgagtggat cgacagccag tttcccaacg gcaagtgcga 1740

gaccgaggag tgtgagacag tgcacaattc taccgtgtgg tacagcgatt ataaggtgac 1800

cggcctgtgc gacgccacac tggtggatac cgagatcaca ttcttttccg aggacggcaa 1860

gaaggagtct atcggcaagc ccaacaccgg ctacaggtct aattacttcg cctatgagaa 1920

gggcgataag gtgtgcaaga tgaattattg taagcacgcc ggggtgcggc tgccaagcgg 1980

cgtgtggttt gagttcgtgg accaggacgt gtacgcagca gcaaagctgc cagagtgccc 2040

agtgggagca accatcagcg cccccaccca gacatctgtg gacgtgagcc tgatcctgga 2100

tgtggagaga atcctggact actccctgtg ccaggagaca tggtccaaga tccgctctaa 2160

gcagcccgtg agcccagtgg acctgtctta cctggcacca aagaaccctg gaacaggacc 2220

tgcctttacc atcatcaatg gcacactgaa gtacttcgag acccggtata tcagaatcga 2280

catcgataac ccaatcatct ccaagatggt gggcaagatc tccggctctc agaccgagag 2340

agagctgtgg acagagtggt tcccatacga gggcgtggag atcggcccca atggcatcct 2400

gaagacccct acaggctata agtttccact gttcatgatc ggccacggca tgctggactc 2460

tgatctgcac aagaccagcc aggccgaggt gtttgagcac ccacacctgg cagaggcacc 2520

aaagcagctg cccgaggagg agaccctgtt ctttggcgat acaggcatct ccaagaaccc 2580

tgtggagctg atcgagggct ggttttctag ctggaagtct accgtggtga cattcttttt 2640

cgccatcggc gtgttcatcc tgctgtacgt ggtggcaagg atcgtgatcg ccgtgcggta 2700

cagatatcag ggcagcaaca ataagagaat ctataatgac atcgagatgt ccaggttccg 2760

caagtgacgc cggcgtcgac gctcaacagc ctcgactgtg ccttctagtt gccagccatc 2820

tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct 2880

ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg 2940

gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg 3000

ggatgcggtg ggctctatgg 3020

<210> 124

<211> 5398

<212> DNA

<213> Artificial sequence

<220>

<223> Helper plasmid 300 - gag/pol

<400> 124

gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60

catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120

acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180

ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240

aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300

ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360

tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 420

ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 480

ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 540

tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc 600

agatcactag aagcttagct gcgaagttgg tcgtgaggca ctgggcaggt aagtatcaag 660

gttacaagac aggtttaagg agaccaatag aaactgggct tgtcgagaca gagaagactc 720

ttgcgtttct gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca 780

ggtgtccact cccagttcaa ttacagctct taaggctagg atccgccgcc accatgggcg 840

cccgcgccag cgtgctttct ggcggcgagc tggacaggtg ggagaagatt cgcctgcggc 900

ctggaggaaa gaaaaagtac aagctgaagc acatcgtgtg ggcttctcgg gaactggaaa 960

gattcgccgt gaaccctgga ctgctagaga cctccgaagg ctgcagcag atcctgggac 1020

agctgcaacc tagcctgcag accggcagcg aggagctgag aagcctgtac aacaccgtcg 1080

ccaccctgta ttgtgtgcac caaagaatcg agatcaagga caccaaggag gctctggata 1140

agatcgagga agagcagaac aagagcaaga aaaaagccca gcaggccgcc gctgataccg 1200

gccattctaa tcaggtgtcc cagaactacc ccattgtgca aaatatccag ggccagatgg 1260

tccaccaggc catcagccct agaaccctga atgcctgggt gaaggtggtg gaagagaagg 1320

ccttttctcc agaggtgatc cctatgttca gcgccctgag cgagggcgct acccctcagg 1380

acctgaacac aatgctgaac accgtgggcg gccaccaggc cgccatgcag atgctgaaag 1440

aaaccatcaa cgaggaggcc gccgaatggg accgggtgca ccccgttcac gccggcccaa 1500

tcgcccctgg ccagatgcgg gaacctagag gcagcgacat cgccggcaca accagcacac 1560

tgcaagagca gatcggatgg atgacacaca acccccccat ccccgtgggc gaaatctaca 1620

agcggtggat cattctggga ctgaacaaaa tcgttagaat gtacagccct accagcatcc 1680

tggatatcag acagggccca aaggagcctt tccgggacta cgtggacaga ttttacaaga 1740

ccctgagagc cgaacaggcc tcccaagagg tgaagaactg gatgacagag accctgctgg 1800

tgcagaatgc caaccctgat tgtaagacaa tcctgaaggc cctcggacct ggcgctacac 1860

tggaagaaat gatgaccgcc tgccagggcg tgggcggccc cggccacaag gccagagtgc 1920

tggccgaggc tatgagccag gtgacaaacc ccgccacaat catgatccag aagggcaact 1980

tcagaaacca gcggaagacc gtgaaatgct tcaactgcgg caaggaaggc cacatcgcaa 2040

agaactgcag agcccctagg aagaaaggct gttggaagtg cggaaaggaa ggacaccaaa 2100

tgaaagattg tactgagaga caggctaatt ttttagggaa gatctggcct tcccacaagg 2160

gaaggccagg gaattttctt cagagcagac cagagccaac agccccacca gaagagagct 2220

tcaggtttgg ggaagagaca acaactccct ctcagaagca ggagccgata gacaaggaac 2280

tgtatccttt agcttccctc agatcactct ttggcagcga cccctcgtca caataaagat 2340

cggcggacag ctgaaagagg ccttgctgga caccggagcc gatgacaccg tgctggaaga 2400

aatgaacctg cctggaagat ggaaacctaa gatgatcggt ggcatcggcg gatttatcaa 2460

agtgcgacag tatgaccaga tcctgatcga gatttgcggc cacaaagcta tcggaacagt 2520

gctggtcggc ccgacccccg tgaacatcat tggccgcaac ctgctgacac agatcggttg 2580

tacactgaac tttcctatca gccctatcga aaccgtgccg gtcaagctga agcccggcat 2640

ggatggccct aaggtgaagc agtggcccct gacagaggaa aagatcaagg cactggtgga 2700

aatctgcaca gaaatggaaa aagagggcaa gatttctaaa atcggcccag agaaccccta 2760

caacacccct gttttcgcca tcaagaagaa agattccacc aagtggagga agctggtgga 2820

ctttcgggaa ctgaacaagc ggacccagga tttctgggag gtgcagctgg gcatccccca 2880

ccctgccggc ctgaaacaaa aaaaaagcgt gaccgtgctg gacgtgggcg acgcctattt 2940

cagcgtgcct ctggataagg acttccggaa atacaccgcc tttaccatcc ctagcatcaa 3000

caacgagacc cctggcatcc ggtaccagta caacgtgctc ccacagggct ggaagggctc 3060

acccgccatc ttccagtgca gcatgaccaa gatcctggag cctttcagaa agcagaatcc 3120

tgacatcgtg atctaccagt acatggacga cctgtacgtg ggctctgatc tggagatcgg 3180

acagcacaga acaaagatcg aagagctgag acagcatctg ctgagatggg gtttcaccac 3240

ccccgacaag aagcaccaga aggaacctcc ttttctgtgg atgggctacg agctgcaccc 3300

cgataagtgg acagtgcagc ccatcgtgct gcccgagaag gactcctgga ccgtgaacga 3360

cattcagaag ctggtcggaa agctgaattg ggcttcccaa atctacgccg gcatcaaggt 3420

gcggcagctg tgcaagcttc tgcgcggcac aaaggccctg acggaagtcg tgccactgac 3480

cgaggaagcc gaattagagc tggccgaaaa cagagaaatt ctgaaagaac ctgtgcacgg 3540

cgtttactac gacccttcta aggacctgat cgccgaaatc cagaaacaag gccagggcca 3600

gtggacttac caaatctacc aggagccttt caaaaacctc aagaccggca agtacgccag 3660

aatgaaggga gcccatacaa acgacgtgaa gcagctgaca gaggctgttc agaagatcgc 3720

cacagaaagc atcgtgatct ggggcaagac cccaaagttc aagctgccta tccaaaagga 3780

aacctgggag gcctggtgga ccgagtactg gcaggccacc tggattcctg aatggggagtt 3840

cgtgaacaca ccacctcttg tgaagctgtg gtaccagctg gaaaaggagc caatcatcgg 3900

cgccgagaca ttctacgtgg acggcgccgc caaccgggag accaaactgg gaaaggccgg 3960

atatgtgacc gacagaggca gacaaaaggt ggtgcctctg accgatacaa ctaaccagaa 4020

aacagagctg caggccattc acctggccct gcaagacagc ggcctggaag tgaatatcgt 4080

gacagacagt cagtacgccc tgggcatcat ccaggctcag cctgacaaga gcgagagcga 4140

gctggtgtcc cagatcatcg agcagctgat caaaaaggag aaggtttatc tggcctgggt 4200

gcccgcccac aagggcatcg gaggcaacga gcaggtggac aagctggtca gcgccggcat 4260

ccggaaggtg ctgttcctgg acggcatcga caaggcccag gaggaacacg agaagtacca 4320

cagcaactgg cgggccatgg ccagcgactt caacctgcca cctgtggttg ctaaggagat 4380

cgtcgcctct tgtgataagt gccagctgaa gggcgaggcc atgcacggcc aagtggattg 4440

cagccctggg atctggcagc tagactgtac ccacctggag ggcaaggtga tcctggtggc 4500

agtgcacgtg gccagcggct acatcgaggc tgaggtgatc cccgccgaaa cgggccagga 4560

gaccgcctac tttctgctga agctagccgg ccggtggcct gtgaagaccg tgcacaccga 4620

taacggcagc aatttcacca gcacaaccgt gaaggctgcc tgctggtggg ctggaatcaa 4680

gcaggagttc ggcatcccat acaatcctca gtctcagggc gtgatcgaga gcatgaacaa 4740

ggaactgaag aagatcattg gtcaggtcag agatcaggcc gagcacctga aaaccgccgt 4800

tcaaatggct gtgttcatcc ataacttcaa aagaaaaggc ggcatcggcg gctacagcgc 4860

cggcgaaaga atcgtggata tcatcgcgac cgacatccaa acaaaagagc tgcaaaagca 4920

aatcaccaag atccagaact tcagagtgta ctacagagat agcagagatc ctgtgtggaa 4980

gggacctgcc aagctgctgt ggaagggcga gggcgccgtg gtgatccagg acaatagcga 5040

catcaaggtc gtgcccagaa gaaaggctaa aatcattaga gactacggca aacagatggc 5100

cggagatgat tgcgtggctt ctagacagga cgaggactga taagaattcc atgtcgacgc 5160

tcaacagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg 5220

ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt 5280

gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc 5340

aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatgg 5398

<210> 125

<211> 1153

<212> DNA

<213> Artificial sequence

<220>

<223> Helper plasmid 246 - Rev

<400> 125

gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60

catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120

acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180

ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240

aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300

ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360

tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 420

ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 480

ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 540

tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc 600

agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggccacca 660

tggccggcag aagcggcgac agcgacgagg atctgctgaa agccgtgcgg ctgatcaagt 720

tcctgtacca gagcaaccct cctcctaacc ccgagggcac cagacaggct agacggaacc 780

gcagaagaag gtggcgggaa cggcaaagac agatccactc tatcagcgag agaatcctga 840

gcacctacct gggaagatcc gccgagcctg tccccctgca gctgcctcca ctggaaagac 900

tgaccctgga ttgtaatgag gactgcggca caagcggaac ccagggcgtg ggcagccccc 960

agattctggt ggaatcccct acaatcctcg agtctggcgc caaggaatga taactagcac 1020

ctgctttatttgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa 1080

taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg 1140

ggaggttttt taa 1153

<210> 126

<211> 2454

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-CD3 plasmid 224

<400> 126

ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc 60

gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat 120

tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc 180

aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 240

caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt 300

acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 360

ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg 420

gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac 480

gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg 540

tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac 600

gccatccacg ctgttttgac ctccatagaa gacaccgggc gagctcggat cctgagaact 660

tcagggtgag tctatgggac ccttgatgtt ttctttcccc ttcttttcta tggttaagtt 720

catgtcatag gaaggggaga agtaacaggg tacacatatt gaccaaatca gggtaatttt 780

gcatttgtaa ttttaaaaaa tgctttcttc ttttaatata cttttttgtt tatcttattt 840

ctaatacttt ccctaatctc tttctttcag ggcaataatg atacaatgta tcatgcctct 900

ttgcaccatt ctaaagaata acagtgataa tttctgggtt aaggcaatag caatatttct 960

gcatataaat atttctgcat ataaattgta actgatgtaa gaggtttcat attgctaata 1020

gcagctacaa tccagctacc attctgcttt tattttatgg ttgggataag gctggattat 1080

tctgagtcca agctaggccc ttttgctaat catgttcata cctctttatct tcctcccaca 1140

gctcctgggc aacgtgctgg tctgtgtgct ggcccatcac tttggcaaag cacgtgagat 1200

ctgccaccat gggcgtgaaa gtgctgttcg ccctgatctg catcgcagtt gctgaagccg 1260

acatccagat gacccagtct cctagcagcc tcagcgctag cgtgggcgat agagtgacca 1320

tcacatgtag cgccagcagc agcgtgtcct acatgaactg gtaccagcaa acacctggaa 1380

aggcccctaa aaggtggatc tatgacacat ctaagctggc ttctggagtg ccatctagat 1440

tttctggcag cggctccggc actgattata cattcaccat cagcagcctg cagcccgagg 1500

atatcgccac ctactactgt cagcagtggt cctctaatcc cttcaccttc ggccagggca 1560

ccaagctgca gatcaccaga accagcggcg ggggaggaag cggcggggga ggatctggcg 1620

gcggcggcag ccaggtgcag ctggtgcaga gcggcggcgg cgtggtgcaa cctggcagaa 1680

gcctgagact gagctgcaag gcctctggct acaccttcac ccggtacacc atgcattggg 1740

tgcggcaggc ccctggcaag ggcctggaat ggattggata catcaacccc agcagaggct 1800

acaccaacta caaccagaag gtgaaggaca gattcacaat ttctcgggac aacagcaaga 1860

ataccgcctt cctgcaaatg gactccctgc gcccagaaga taccggcgtg tacttctgcg 1920

ctagatatta cgacgaccac tactgcctgg actactgggg ccagggcacc cctgtgaccg 1980

tgtccagcgc ctccggagtg gaactgatcg agggctggtt cagcagctgg aaaagcaccg 2040

tggttacatt ctttttcgcc atcggcgtgt tcatcctgct gtacgtggtc gccagaattg 2100

tgatcgccgt gcggtataga taccagggca gcaacaacaa gcggatctac aacgacatcg 2160

agatgagcag attcagaaag taatgactcg aggtgaattc gcgccggcgt cgacgctcaa 2220

cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt 2280

ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat 2340

cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg 2400

gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atgg 2454

<210> 127

<211> 2452

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-CD3 Plasmid - 232

<400> 127

ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc 60

gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat 120

tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc 180

aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 240

caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt 300

acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 360

ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg 420

gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac 480

gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg 540

tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac 600

gccatccacg ctgttttgac ctccatagaa gacaccgggc gagctcggat cctgagaact 660

tcagggtgag tctatgggac ccttgatgtt ttctttcccc ttcttttcta tggttaagtt 720

catgtcatag gaaggggaga agtaacaggg tacacatatt gaccaaatca gggtaatttt 780

gcatttgtaa ttttaaaaaa tgctttcttc ttttaatata cttttttgtt tatcttattt 840

ctaatacttt ccctaatctc tttctttcag ggcaataatg atacaatgta tcatgcctct 900

ttgcaccatt ctaaagaata acagtgataa tttctgggtt aaggcaatag caatatttct 960

gcatataaat atttctgcat ataaattgta actgatgtaa gaggtttcat attgctaata 1020

gcagctacaa tccagctacc attctgcttt tattttatgg ttgggataag gctggattat 1080

tctgagtcca agctaggccc ttttgctaat catgttcata cctctttatct tcctcccaca 1140

gctcctgggc aacgtgctgg tctgtgtgct ggcccatcac tttggcaaag cacgtgagat 1200

ctgccaccat gggcgtgaaa gtgctgttcg ccctgatctg catcgcagtt gctgaagccg 1260

acatccagat gacccagtct cctagcagcc tcagcgctag cgtgggcgat agagtgacca 1320

tcacatgtag cgccagcagc agcgtgtcct acatgaactg gtaccagcaa acacctggaa 1380

aggcccctaa aaggtggatc tatgacacat ctaagctggc ttctggagtg ccatctagat 1440

tttctggcag cggctccggc actgattata cattcaccat cagcagcctg cagcccgagg 1500

atatcgccac ctactactgt cagcagtggt cctctaatcc cttcaccttc ggccagggca 1560

ccaagctgca gatcaccaga accagcggcg ggggaggaag cggcggggga ggatctggcg 1620

gcggcggcag ccaggtgcag ctggtgcaga gcggcggcgg cgtggtgcaa cctggcagaa 1680

gcctgagact gagctgcaag gcctctggct acaccttcac ccggtacacc atgcattggg 1740

tgcggcaggc ccctggcaag ggcctggaat ggattggata catcaacccc agcagaggct 1800

acaccaacta caaccagaag gtgaaggaca gattcacaat ttctcgggac aacagcaaga 1860

ataccgcctt cctgcaaatg gactccctgc gcccagaaga taccggcgtg tacttctgcg 1920

ctagatatta cgacgaccac tactgcctgg actactgggg ccagggcacc cctgtgaccg 1980

tgtccagcgc ctccggacac ttcagcgagc ctgagatcac cctgatcatc ttcggcgtga 2040

tggccggagt gatcggcaca atcctgctga tcagctacgg catcagaaga ctgattaaga 2100

aatccccatc tgatgtgaag cctctgcctt ctcctgacac cgacgtcccc ctgagcagcg 2160

tggaaatcga gaaccccgaa accagcgacc agtgataaga attctagtcg acgctcaaca 2220

gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 2280

ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 2340

cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 2400

gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat gg 2452

<210> 128

<211> 3871

<212> DNA

<213> Artificial sequence

<220>

<223> KanR_p57M-MND-(anti)medCD3-Cocal-wPREw-BGHpA-001

<400> 128

gaacagagaa acaggagaat atgggccaaa caggatatct gtggtaagca gttcctgccc 60

cggctcaggg ccaagaacag ttggaacagc agaatatggg ccaaacagga tatctgtggt 120

aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc ggtcccgccc 180

tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc tgaaatgacc 240

ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc gcgcttctgc 300

tccccgagct ctatataagc agagctcgtt tagtgaaccg tcagatcgcc tggagacgcc 360

atccacgctg ttttgacttc catagaagcc tgcagggccg ccaccatggc actgcctgtg 420

acagccctgc tgctgccact ggccctgctg ctgcacgcag cacgcccaga tatccagatg 480

acccagtccc caagctccct gagcgcctcc gtgggcgacc gggtgacaat cacctgcagc 540

gcctctagct ccgtgtccta catgaactgg tatcagcaga cacctggcaa ggccccaaag 600

agatggatct acgataccag caagctggcc tccggcgtgc cttctaggtt ttctggcagc 660

ggctccggca cagattatac attcaccatc tctagcctgc agccagagga catcgccacc 720

tactattgcc agcagtggtc ctctaatccc tttacattcg gccagggcac caagctgcag 780

atcacaagaa cctctggagg aggaggaagc ggaggaggag gatccggcgg cggcggctct 840

caggtgcagc tggtgcagag cggaggagga gtggtgcagc caggcagaag cctgaggctg 900

tcctgtaagg cctctggcta cacattcacc agatatacaa tgcactgggt gaggcaggca 960

ccaggcaagg gactggagtg gatcggctac atcaacccct ccaggggcta caccaactat 1020

aatcagaagg tgaaggatcg gttcaccatc agcagggaca actccaagaa taccgccttc 1080

ctgcagatgg acagcctgag gccagaggat accggcgtgt acttttgcgc ccggtactat 1140

gacgatcact actgtctgga ttatggggc cagggaacac cagtgaccgt gagctccgcc 1200

gcagcaaagc ctaccacaac ccctgcccca aggccaccta cacccgcccc taccatcgcc 1260

tctcagccac tgagcctgag gccagaggca tccaggcctg ccgcaggggg ggccgtgcac 1320

acccggggcc tggactttgc ctctgatatg gcactgatcg tgctgggagg agtggcagga 1380

ctgctgctgt tcatcggact gggcatcttc ttttgcgtgc gctgtaggca ccggagaagg 1440

cagggatctg gagagggaag gggaagcctg ctgacatgcg gcgacgtgga ggagaaccca 1500

ggaccaaatt ttctgctgct gaccttcatc gtgctgcctc tgtgcagcca cgccaagttt 1560

tccatcgtgt tcccacagtc ccagaagggc aactggaaga atgtgccctc tagctaccac 1620

tattgccctt cctctagcga ccagaactgg cacaatgatc tgctgggcat cacaatgaag 1680

gtgaagatgc ccaagaccca caaggccatc caggcagatg gatggatgtg ccacgcagcc 1740

aagtggatca caacctgtga ctttcggtgg tacggcccca agtatatcac acactccatc 1800

cactctatcc agcctacctc cgagcagtgc aaggagtcta tcaagcagac aaagcagggc 1860

acctggatga gccctggctt cccaccccag aactgtggct acgccacagt gaccgactcc 1920

gtggcagtgg tggtgcaggc aacacctcac cacgtgctgg tggatgagta taccggcgag 1980

tggatcgaca gccagtttcc aaacggcaag tgcgagacag aggagtgtga gaccgtgcac 2040

aattctacag tgtggtacag cgattataag gtgacaggcc tgtgcgacgc caccctggtg 2100

gatacagaga tcaccttctt ttctgaggac ggcaagaagg agagcatcgg caagcccaac 2160

accggctaca gatccaatta cttcgcctat gagaagggcg ataaggtgtg caagatgaat 2220

tattgtaagc acgccggggt gcggctgcct agcggcgtgt ggtttgagtt cgtggaccag 2280

gacgtgtacg cagcagcaaa gctgcctgag tgcccagtgg gagcaaccat ctccgcccca 2340

acacagacct ccgtggacgt gtctctgatc ctggatgtgg agcgcatcct ggactacagc 2400

ctgtgccagg agacctggag caagatccgg tccaagcagc ccgtgtcccc tgtggacctg 2460

tcttacctgg caccaaagaa cccaggaacc ggaccagcct ttacaatcat caatggcacc 2520

ctgaagtact tcgagacccg ctatatccgg atcgacatcg ataaccctat catcagcaag 2580

atggtgggca agatctctgg cagccagaca gagagagagc tgtggaccga gtggttccct 2640

tacgagggcg tggagatcgg cccaaatggc atcctgaaga caccaaccgg ctataagttt 2700

cccctgttca tgatcggcca cggcatgctg gacagcgatc tgcacaagac ctcccaggcc 2760

gaggtgtttg agcacccaca cctggcagag gcaccaaagc agctgcctga ggaggagaca 2820

ctgttctttg gcgataccgg catctctaag aaccccgtgg agctgatcga gggctggttt 2880

tcctcttgga agagcacagt ggtgaccttc tttttcgcca tcggcgtgtt catcctgctg 2940

tacgtggtgg ccagaatcgt gatcgccgtg agatacaggt atcagggctc caacaataag 3000

aggatctata atgacatcga gatgtctcgc ttccggaagt gacgccggcg tcgacaatca 3060

acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt 3120

tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc 3180

tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc 3240

cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg 3300

gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc 3360

cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg 3420

cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc tgctcgcctg 3480

tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc 3540

agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct 3600

tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcctgtgt gccttctagt 3660

tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact 3720

cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat 3780

tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc 3840

aggcatgctg gggatgcggt gggctctatg g 3871

<210> 129

<211> 4525

<212> DNA

<213> Artificial sequence

<220>

<223> p57M-CMV-BGi-(anti)-med-hCD3-Cocal-BG-polyA

<400> 129

gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60

catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120

acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180

ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240

aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300

ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360

tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 420

ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 480

ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 540

tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc 600

agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat 660

ccagcctccc ctcgaagctt acatgtggta ccgagctcgg atcctgagaa cttcagggtg 720

agtctatggg acccttgatg ttttctttcc ccttcttttc tatggttaag ttcatgtcat 780

aggaagggga gaagtaacag ggtacacata ttgaccaaat cagggtaatt ttgcatttgt 840

aattttaaaa aatgctttct tcttttaata tacttttttg tttatcttat ttctaatact 900

ttccctaatc tctttctttc agggcaataa tgatacaatg tatcatgcct ctttgcacca 960

ttctaaagaa taacagtgat aatttctggg ttaaggcaat agcaatattt ctgcatataa 1020

atatttctgc atataaattg taactgatgt aagaggtttc atattgctaa tagcagctac 1080

aatccagcta ccattctgct tttattttat ggttggggata aggctggatt attctgagtc 1140

caagctaggc ccttttgcta atcatgttca tacctcttat cttcctccca cagctcctgg 1200

gcaacgtgct ggtctgtgtg ctggcccatc actttggcaa agcacgtgag atctgaattc 1260

tgacactcct gcagggccgc caccatggca ctgcctgtga cagccctgct gctgccactg 1320

gccctgctgc tgcacgcagc acgcccagat atccagatga cccagtcccc aagctccctg 1380

agcgcctccg tgggcgaccg ggtgacaatc acctgcagcg cctctagctc cgtgtcctac 1440

atgaactggt atcagcagac acctggcaag gccccaaaga gatggatcta cgataccagc 1500

aagctggcct ccggcgtgcc ttctaggttt tctggcagcg gctccggcac agattataca 1560

ttcaccatct ctagcctgca gccagaggac atcgccacct actattgcca gcagtggtcc 1620

tctaatccct ttacattcgg ccagggcacc aagctgcaga tcacaagaac ctctggagga 1680

ggaggaagcg gaggaggagg atccggcggc ggcggctctc aggtgcagct ggtgcagagc 1740

ggaggaggag tggtgcagcc aggcagaagc ctgaggctgt cctgtaaggc ctctggctac 1800

acattcacca gatatacaat gcactgggtg aggcaggcac caggcaaggg actggagtgg 1860

atcggctaca tcaacccctc caggggctac accaactata atcagaaggt gaaggatcgg 1920

ttcaccatca gcagggacaa ctccaagaat accgccttcc tgcagatgga cagcctgagg 1980

ccagaggata ccggcgtgta cttttgcgcc cggtactatg acgatcacta ctgtctggat 2040

tattggggcc agggaacacc agtgaccgtg agctccgccg cagcaaagcc taccacaacc 2100

cctgccccaa ggccacctac acccgcccct accatcgcct ctcagccact gagcctgagg 2160

ccagaggcat ccaggcctgc cgcagggggg gccgtgcaca cccggggcct ggactttgcc 2220

tctgatatgg cactgatcgt gctggggagga gtggcaggac tgctgctgtt catcggactg 2280

ggcatcttct tttgcgtgcg ctgtaggcac cggagaaggc agggatctgg agagggaagg 2340

ggaagcctgc tgacatgcgg cgacgtggag gagaacccag gaccaaattt tctgctgctg 2400

accttcatcg tgctgcctct gtgcagccac gccaagtttt ccatcgtgtt cccacagtcc 2460

cagaagggca actggaagaa tgtgccctct agctaccact attgcccttc ctctagcgac 2520

cagaactggc acaatgatct gctgggcatc acaatgaagg tgaagatgcc caagacccac 2580

aaggccatcc aggcagatgg atggatgtgc cacgcagcca agtggatcac aacctgtgac 2640

tttcggtggt acggccccaa gtatatcaca cactccatcc actctatcca gcctacctcc 2700

gagcagtgca aggagtctat caagcagaca aagcagggca cctggatgag ccctggcttc 2760

ccaccccaga actgtggcta cgccacagtg accgactccg tggcagtggt ggtgcaggca 2820

acacctcacc acgtgctggt ggatgagtat accggcgagt ggatcgacag ccagtttcca 2880

aacggcaagt gcgagacaga ggagtgtgag accgtgcaca attctacagt gtggtacagc 2940

gattataagg tgacaggcct gtgcgacgcc accctggtgg atacagagat caccttcttt 3000

tctgaggacg gcaagaagga gagcatcggc aagcccaaca ccggctacag atccaattac 3060

ttcgcctatg agaagggcga taaggtgtgc aagatgaatt attgtaagca cgccggggtg 3120

cggctgccta gcggcgtgtg gtttgagttc gtggaccagg acgtgtacgc agcagcaaag 3180

ctgcctgagt gcccagtggg agcaaccatc tccgccccaa cacagacctc cgtggacgtg 3240

tctctgatcc tggatgtgga gcgcatcctg gactacagcc tgtgccagga gacctggagc 3300

aagatccggt ccaagcagcc cgtgtcccct gtggacctgt cttacctggc accaaagaac 3360

ccaggaaccg gaccagcctt tacaatcatc aatggcaccc tgaagtactt cgagacccgc 3420

tatatccgga tcgacatcga taaccctatc atcagcaaga tggtgggcaa gatctctggc 3480

agccagacag agagagagct gtggaccgag tggttccctt acgagggcgt ggagatcggc 3540

ccaaatggca tcctgaagac accaaccggc tataagtttc ccctgttcat gatcggccac 3600

ggcatgctgg acagcgatct gcacaagacc tcccaggccg aggtgtttga gcacccacac 3660

ctggcagagg caccaaagca gctgcctgag gaggagacac tgttctttgg cgataccggc 3720

atctctaaga accccgtgga gctgatcgag ggctggtttt cctcttggaa gagcacagtg 3780

gtgaccttct ttttcgccat cggcgtgttc atcctgctgt acgtggtggc cagaatcgtg 3840

atcgccgtga gatacaggta tcagggctcc aacaataaga ggatctataa tgacatcgag 3900

atgtctcgct tccggaagtg acgccggcgc tcaaatcctg cacaacagat tcttcatgtt 3960

tggaccaaat caacttgtga taccatgctc aaagaggcct caattatatt tgagttttta 4020

atttttatga aaaaaaaaaa aaaaaacgga attcacccca ccagtgcagg ctgcctatca 4080

gaaagtggtg gctggtgtgg ctaatgccct ggcccacaag tatcactaag ctcgctttct 4140

tgctgtccaa tttctattaa aggttccttt gttccctaag tccaactact aaactggggg 4200

atattatgaa gggccttgag catctggatt ctgcctaata aaaaacattt attttcattg 4260

caatgatgta tttaaattat ttctgaatat tttaaaaa agggaatgtg ggaggtcagt 4320

gcatttaaaa cataaagaaa tgaagagcta gttcaaacct tgggaaaata cactatatct 4380

taaactccat gaaagaaggt gaggctgcaa acagctaatg cacattggca acagcccctg 4440

atgcctatgc cttattcatc cctcagaaaa ggattcaagt agaggcttga tttggaggtt 4500

aaagttttgc taatgctgta tttta 4525

<210> 130

<211> 2773

<212> DNA

<213> Artificial sequence

<220>

<223> Cocal Envelope Plasmid - 143

<400> 130

gaacagagaa acaggagaat atgggccaaa caggatatct gtggtaagca gttcctgccc 60

cggctcaggg ccaagaacag ttggaacagc agaatatggg ccaaacagga tatctgtggt 120

aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc ggtcccgccc 180

tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc tgaaatgacc 240

ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc gcgcttctgc 300

tccccgagct ctatataagc agagctcgtt tagtgaaccg tcagatcgcc tggagacgcc 360

atccacgctg ttttgacttc catagaagcc tgcagggccg ccaccatgaa ctttctgctg 420

ctgaccttca tcgtgctgcc tctgtgcagc cacgccaagt tttccatcgt gttccccacag 480

tcccagaagg gcaactggaa gaatgtgccc agctcctacc actattgtcc ttctagctcc 540

gaccagaact ggcacaatga tctgctgggc atcaccatga aggtgaagat gcctaagaca 600

cacaaggcca tccaggcaga tggatggatg tgccacgcag ccaagtggat caccacatgt 660

gactttcggt ggtacggccc caagtatatc acccacagca tccactccat ccagcctaca 720

agcgagcagt gcaaggagtc catcaagcag accaagcagg gcacatggat gtctcccggc 780

ttcccccctc agaactgtgg ctacgccacc gtgacagata gcgtggcagt ggtggtgcag 840

gcaaccccac accacgtgct ggtggatgag tatacaggcg agtggatcga cagccagttt 900

cccaacggca agtgcgagac cgaggagtgt gagacagtgc acaattctac cgtgtggtac 960

agcgattata aggtgaccgg cctgtgcgac gccacactgg tggataccga gatcacattc 1020

ttttccgagg acggcaagaa ggagtctatc ggcaagccca acaccggcta caggtctaat 1080

tacttcgcct atgagaaggg cgataaggtg tgcaagatga attattgtaa gcacgccggg 1140

gtgcggctgc caagcggcgt gtggtttgag ttcgtggacc aggacgtgta cgcagcagca 1200

aagctgccag agtgcccagt gggagcaacc atcagcgccc ccacccagac atctgtggac 1260

gtgagcctga tcctggatgt ggagagaatc ctggactact ccctgtgcca ggagacatgg 1320

tccaagatcc gctctaagca gcccgtgagc ccagtggacc tgtcttacct ggcaccaaag 1380

aaccctggaa caggacctgc ctttaccatc atcaatggca cactgaagta cttcgagacc 1440

cggtatatca gaatcgacat cgataaccca atcatctcca agatggtggg caagatctcc 1500

ggctctcaga ccgagagaga gctgtggaca gagtggttcc catacgaggg cgtggagatc 1560

ggccccaatg gcatcctgaa gacccctaca ggctataagt ttccactgtt catgatcggc 1620

cacggcatgc tggactctga tctgcacaag accagccagg ccgaggtgtt tgagcaccca 1680

cacctggcag aggcaccaaa gcagctgccc gaggaggaga ccctgttctt tggcgataca 1740

ggcatctcca agaaccctgt ggagctgatc gagggctggt tttctagctg gaagtctacc 1800

gtggtgacat tctttttcgc catcggcgtg ttcatcctgc tgtacgtggt ggcaaggatc 1860

gtgatcgccg tgcggtacag atatcagggc agcaacaata agagaatcta taatgacatc 1920

gagatgtcca ggttccgcaa gtgacgccgg cgtcgacaat caacctctgg attacaaaat 1980

ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc 2040

tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt 2100

gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg 2160

cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg 2220

tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc 2280

cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt 2340

gttgtcgggg aagctgacgt cctttccatg gctgctcgcc tgtgttgcca cctggattct 2400

gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg 2460

cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg 2520

gatctccctt tgggccgcct ccccgcctgt gtgccttcta gttgccagcc atctgttgtt 2580

tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa 2640

taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg 2700

gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg 2760

gtgggctcta tgg 2773

<210> 131

<211> 6468

<212> DNA

<213> Artificial sequence

<220>

<223> Helper plasmid gag/pol-89

<400> 131

ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag 60

cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc 120

caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg 180

gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca 240

tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc 300

ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt 360

attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata 420

gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt 480

ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca 540

aatgggcggt aggcgtgtac ggtggggaggt ctatataagc agagctcgtt tagtgaaccg 600

tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagac accgggaccg 660

atccagcctc ccctcgaagc ttacatgtgg taccgagctc ggatcctgag aacttcaggg 720

tgagtctatg ggacccttga tgttttcttt ccccttcttt tctatggtta agttcatgtc 780

ataggaaggg gagaagtaac agggtacaca tattgaccaa atcagggtaa ttttgcattt 840

gtaattttaa aaaatgcttt cttcttttaa tatacttttt tgtttatctt atttctaata 900

ctttccctaa tctctttctt tcagggcaat aatgatacaa tgtatcatgc ctctttgcac 960

cattctaaag aataacagtg ataatttctg ggttaaggca atagcaatat ttctgcatat 1020

aaatatttct gcatataaat tgtaactgat gtaagaggtt tcatattgct aatagcagct 1080

acaatccagc taccattctg cttttatttt atggttggga taaggctgga ttatctgag 1140

tccaagctag gcccttttgc taatcatgtt catacctctt atcttcctcc cacagctcct 1200

gggcaacgtg ctggtctgtg tgctggccca tcactttggc aaagcacgtg agatctgaat 1260

tcgagatctg ccgccgccat gggtgcgaga gcgtcagtat taagcggggg agaattagat 1320

cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt aaaacatata 1380

gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt agaaacatca 1440

gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg atcagaagaa 1500

cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag gatagagata 1560

aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag taagaaaaaa 1620

gcacagcaag cagcagctga cacaggacac agcaatcagg tcagccaaaa ttaccctata 1680

gtgcagaaca tccaggggca aatggtacat caggccatat cacctagaac tttaaatgca 1740

tgggtaaaag tagtagaaga gaaggctttc agcccagaag tgatacccat gttttcagca 1800

ttatcagaag gagccacccc acaagattta aacaccatgc taaacacagt ggggggacat 1860

caagcagcca tgcaaatgtt aaaagagacc atcaatgagg aagctgcaga atgggataga 1920

gtgcatccag tgcatgcagg gcctattgca ccaggccaga tgagagaacc aaggggaagt 1980

gacatagcag gaactactag tacccttcag gaacaaatag gatggatgac acataatcca 2040

cctatcccag taggagaaat ctataaaaga tggataatcc tgggattaaa taaaatagta 2100

agaatgtata gccctaccag cattctggac ataagacaag gaccaaagga accctttaga 2160

gactatgtag accgattcta taaaactcta agagccgagc aagcttcaca agaggtaaaa 2220

aattggatga cagaaacctt gttggtccaa aatgcgaacc cagattgtaa gactatttta 2280

aaagcattgg gaccaggagc gacactagaa gaaatgatga cagcatgtca gggagtgggg 2340

ggacccggcc ataaagcaag agttttggct gaagcaatga gccaagtaac aaatccagct 2400

accataatga tacagaaagg caattttagg aaccaaagaa agactgttaa gtgtttcaat 2460

tgtggcaaag aagggcacat agccaaaaat tgcagggccc ctaggaaaaa gggctgttgg 2520

aaatgtggaa aggaaggaca ccaaatgaaa gattgtactg agagacaggc taatttttta 2580

gggaagatct ggccttccca caagggaagg ccagggaatt ttcttcagag cagaccagag 2640

ccaacagccc caccagaaga gagcttcagg tttggggaag agacaacaac tccctctcag 2700

aagcaggagc cgatagacaa ggaactgtat cctttagctt ccctcagatc actctttggc 2760

agcgacccct cgtcacaata aagatagggg ggcaattaaa ggaagctcta ttagatacag 2820

gagcagatga tacagtatta gaagaaatga atttgccagg aagatggaaa ccaaaaatga 2880

tagggggaat tggaggtttt atcaaagtaa gacagtatga tcagatactc atagaaatct 2940

gcggacataa agctataggt acagtattag taggacctac acctgtcaac ataattggaa 3000

gaaatctgtt gactcagatt ggctgcactt taaattttcc cattagtcct attgagactg 3060

taccagtaaa attaaagcca ggaatggatg gcccaaaagt taaacaatgg ccattgacag 3120

aagaaaaaat aaaagcatta gtagaaattt gtacagaaat ggaaaaggaa ggaaaaattt 3180

caaaaattgg gcctgaaaat ccatacaata ctccagtatt tgccataaag aaaaaagaca 3240

gtactaaatg gagaaaatta gtagatttca gagaacttaa taagagaact caagatttct 3300

gggaagttca attaggaata ccacatcctg cagggttaaa acagaaaaaa tcagtaacag 3360

tactggatgt gggcgatgca tatttttcag ttcccttaga taaagacttc aggaagtata 3420

ctgcatttac catacctagt ataaacaatg agacaccagg gattagatat cagtacaatg 3480

tgcttccaca gggatggaaa ggatcaccag caatattcca gtgtagcatg acaaaaatct 3540

tagagccttt tagaaaacaa aatccagaca tagtcatcta tcaatacatg gatgatttgt 3600

atgtaggatc tgacttagaa atagggcagc atagaacaaa aatagaggaa ctgagacaac 3660

atctgttgag gtggggattt accacaccag acaaaaaaca tcagaaagaa cctccattcc 3720

tttggatggg ttatgaactc catcctgata aatggacagt acagcctata gtgctgccag 3780

aaaaggacag ctggactgtc aatgacatac agaaattagt gggaaaattg aattgggcaa 3840

gtcagattta tgcagggatt aaagtaaggc aattatgtaa acttcttagg ggaaccaaag 3900

cactaacaga agtagtacca ctaacagaag aagcagagct agaactggca gaaaacaggg 3960

agattctaaa agaaccggta catggagtgt attatgaccc atcaaaagac ttaatagcag 4020

aaatacagaa gcaggggcaa ggccaatgga catatcaaat ttatcaagag ccatttaaaa 4080

atctgaaaac aggaaagtat gcaagaatga agggtgccca cactaatgat gtgaaacaat 4140

taacagaggc agtacaaaaa atagccacag aaagcatagt aatatgggga aagactccta 4200

aatttaaatt acccatacaa aaggaaacat gggaagcatg gtggacagag tattggcaag 4260

ccacctggat tcctgagtgg gagtttgtca atacccctcc cttagtgaag ttatggtacc 4320

agttagagaa agaacccata ataggagcag aaactttcta tgtagatggg gcagccaata 4380

gggaaactaa attaggaaaa gcaggatatg taactgacag aggaagacaa aaagttgtcc 4440

ccctaacgga cacaacaaat cagaagactg agttacaagc aattcatcta gctttgcagg 4500

attcgggatt agaagtaaac atagtgacag actcacaata tgcattggga atcattcaag 4560

cacaaccaga taagagtgaa tcagagttag tcagtcaaat aatagagcag ttaataaaaa 4620

aggaaaaagt ctacctggca tgggtaccag cacacaaagg aattggagga aatgaacaag 4680

tagataaatt ggtcagtgct ggaatcagga aagtactatt tttagatgga atagataagg 4740

cccaagaaga acatgagaaa tatcacagta attggagagc aatggctagt gattttaacc 4800

taccacctgt agtagcaaaa gaaatagtag ccagctgtga taaatgtcag ctaaaagggg 4860

aagccatgca tggacaagta gactgtagcc caggaatatg gcagctagat tgtacacatt 4920

tagaaggaaa agttatcttg gtagcagttc atgtagccag tggatatata gaagcagaag 4980

taattccagc agagacaggg caagaaacag catacttcct cttaaaatta gcaggaagat 5040

ggccagtaaa aacagtacat acagacaatg gcagcaattt caccagtact acagttaagg 5100

ccgcctgttg gtgggcgggg atcaagcagg aatttggcat tccctacaat ccccaaagtc 5160

aaggagtaat agaatctatg aataaagaat taaagaaaat tataggacag gtaagagatc 5220

aggctgaaca tcttaagaca gcagtacaaa tggcagtatt catccacaat tttaaaagaa 5280

aagggggggat tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca 5340

tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca 5400

gggacagcag agatccagtt tggaaaggac cagcaaagct cctctggaaa ggtgaagggg 5460

cagtagtaat acaagataat agtgacataa aagtagtgcc aagaagaaaa gcaaagatca 5520

tcagggatta tggaaaacag atggcaggtg atgattgtgt ggcaagtaga caggatgagg 5580

attaacacat ggaattccgg agcggccgca ggagctttgt tccttgggtt cttgggagca 5640

gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg 5700

tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg 5760

ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga 5820

tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc 5880

actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac 5940

acgacctgga tggagtggga cagagaaatt aacaattaca caagcttccg cggaattcac 6000

cccaccagtg caggctgcct atcagaaagt ggtggctggt gtggctaatg ccctggccca 6060

caagtatcac taagctcgct ttcttgctgt ccaatttcta ttaaaggttc ctttgttccc 6120

taagtccaac tactaaactg ggggatatta tgaagggcct tgagcatctg gattctgcct 6180

aataaaaaac atttattttc attgcaatga tgtatttaaa ttatttctga atattttact 6240

aaaaagggaa tgtgggaggt cagtgcattt aaaacataaa gaaatgaaga gctagttcaa 6300

accttgggaa aatacactat atcttaaact ccatgaaaga aggtgaggct gcaaacagct 6360

aatgcacatt ggcaacagcc cctgatgcct atgccttatt catccctcag aaaaggattc 6420

aagtagaggc ttgatttgga ggttaaagtt ttgctatgct gtatttta 6468

<210> 132

<211> 972

<212> DNA

<213> Artificial sequence

<220>

<223> Helper plasmid 84 - Rev

<400> 132

aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca 60

tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga 120

tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt 180

ccgcattgca gagatattgt atttaagtgc ctagctcgat acaataaacg ccatttgacc 240

attcaccaca ttggtgtgca cctccaagct cgagctcgtt tagtgaaccg tcagatcgcc 300

tggagacgcc atccacgctg ttttgacctc catagaagac accgggaccg atccagcctc 360

ccctcgaagc tagtcgatta ggcatctcct atggcaggaa gaagcggaga cagcgacgaa 420

gacctcctca aggcagtcag actcatcaag tttctctatc aaagcaaccc acctcccaat 480

cccgagggga cccgacaggc ccgaaggaat agaagaagaa ggtggagaga gagacagaga 540

cagatccatt cgattagtga acggatcctt agcacttatc tgggacgatc tgcggagcct 600

gtgcctcttc agctaccacc gcttgagaga cttactcttg attgtaacga ggattgtgga 660

acttctggga cgcagggggt gggaagccct caaatattgg tggaatctcc tacaatattg 720

gagtcaggag ctaaagaata gtgctgttag cttgctcaat gccacagcta tagcagtagc 780

tgaggggaca gatagggtta tagaagtagt acaagaagct tggcactggc cgtcgtttta 840

caacgtcgtg atctgagcct gggagatctc tggctaacta gggaacccac tgcttaagcc 900

tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 960

taactagaga tc 972

<210> 133

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-CD3- CDR-L1

<400> 133

Ser Ala Ser Ser Ser Val Ser Tyr Met Asn

1 5 10

<210> 134

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-CD3 - CDR-L2

<400> 134

Asp Thr Ser Lys Leu Ala Ser Gly

1 5

<210> 135

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-CD3 - CDR-L3

<400> 135

Gln Gln Trp Ser Ser Asn Pro Phe Thr

1 5

<210> 136

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-CD3 - CDR-H2

<400> 136

Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Val Lys

1 5 10 15

Asp

<210> 137

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-CD3 - CDR-H3

<400> 137

Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr

1 5 10

<210> 138

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-CD19 - CDR-L1

<400> 138

Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn

1 5 10

<210> 139

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-CD19 - CDR-L2

<400> 139

His Thr Ser Arg Leu His Ser

1 5

<210> 140

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-CD19 - CDR-L3

<400> 140

Gln Gln Gly Asn Thr Leu Pro Tyr Thr

1 5

<210> 141

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-CD19 - CDR-H1

<400> 141

Asp Tyr Gly Val

1

<210> 142

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-CD19 - CDR-H2

<400> 142

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Tyr Asn Ser Ala Leu Lys Ser

1 5 10 15

<210> 143

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-CD19 - CDR-H3

<400> 143

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

1 5 10

<210> 144

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-CD3 - CDR-H1

<400> 144

Arg Tyr Thr Met His

1 5

Claims (65)

1. A viral particle comprising a vector genome comprising a polynucleotide sequence encoding an anti-CD 19 chimeric antigen receptor, wherein the viral particle transduces an immune cell in vivo.

2. The particle of claim 1, wherein the viral particle is a lentiviral particle.

3. The particle of claim 1, wherein the immune cell is a T cell.

4. The particle of claim 1, wherein the vector genome comprises a polynucleotide sequence encoding a multi-zoned cell surface receptor.

5. The particle of claim 4, wherein the multi-zoned cell surface receptor is a chemically inducible cell surface receptor.

6. The particle of claim 4, wherein the vector genome comprises a polynucleotide sequence encoding a multi-partitioned cell surface receptor comprising an FKBP-rapamycin complex binding domain (FRB domain) or a functional variant thereof; and the polynucleotide includes a polynucleotide sequence encoding an FK506 binding protein domain (FKBP) or a functional variant thereof.

7. The particle of claim 5, wherein the multi-zoned cell surface receptor is a rapamycin activated cell surface receptor.

8. The particle of claim 1, wherein the vector genome comprises a polynucleotide sequence that confers resistance to an immunosuppressant.

9. The particle of claim 8, wherein the vector genomic sequence conferring resistance to an immunosuppressant encodes a polypeptide that binds rapamycin, wherein optionally the polypeptide is FRB.

10. The particle of claim 4, wherein the vector genome comprises, in 5 'to 3' order on a polycistronic transcript: said polynucleotide sequence encoding said multi-zoned cell surface receptor and said polynucleotide sequence encoding said anti-CD 19 chimeric antigen receptor.

11. The particle of claim 4, wherein the vector genome comprises, in 5 'to 3' order on a polycistronic transcript: said polynucleotide sequence encoding said anti-CD 19 chimeric antigen receptor and said polynucleotide sequence encoding said multi-zoned cell surface receptor, and/or wherein said anti-CD 19 chimeric antigen receptor shares at least 80%, 90%, 95% or 100% identity with SEQ ID No. 51, 79, 89, 121 or 122.

12. The particle of claim 1, wherein the polynucleotide encoding the anti-CD 19 chimeric antigen receptor and/or the polynucleotide encoding the multi-zoned cell surface receptor is operably linked to one or more promoters.

13. The particle of claim 12, wherein at least one of the one or more promoters is an inducible promoter.

14. The particle of claim 1, wherein the viral particle comprises a viral envelope comprising one or more immune cell activating proteins exposed on and/or conjugated to a surface of the viral envelope.

15. The particle of claim 14, wherein the viral envelope comprises an anti-CD 3 single-chain variable fragment exposed on and/or conjugated to a surface of the viral envelope.

16. The particle of claim 14, wherein the viral envelope comprises a Cocal glycoprotein exposed on and/or conjugated to a surface of the viral envelope, optionally wherein the Cocal glycoprotein comprises an R354Q mutation compared to a reference sequence according to SEQ ID No. 5.

17. The particle of claim 14, wherein the viral envelope comprises an anti-CD 3 single chain variable fragment and a Cocal glycoprotein exposed on and/or conjugated to a surface of the viral envelope.

18. The particle of claim 14, wherein the viral envelope comprises an anti-CD 3 single-chain variable fragment sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID No. 2 or 12.

19. The particle of claim 14, wherein the viral envelope comprises a Cocal glycoprotein sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID No. 5, 13, 19, 123, 128, 129 or 130.

20. The particle of claim 13, wherein the promoter is an MND promoter.

21. The particle of claim 14, wherein the vector genome shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID No. 49.

22. The particle of claim 14, wherein the vector genome shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID No. 75.

23. The particle of claim 14, wherein the vector genome shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID No. 87.

24. A method of treating a disease or disorder associated with malignant cd19+ cells, transducing immune cells in vivo, and/or generating immune cells expressing an anti-CD 19 chimeric antigen receptor in a subject in need thereof, the method comprising administering to the subject the viral particle of any one of claims 1 to 23.

25. The method of claim 24, wherein the viral particles are administered by intraperitoneal, subcutaneous, or intranodal injection.

26. A method of treating a disease or disorder associated with malignant cd19+ cells in a subject in need thereof, the method comprising administering to the subject an immune cell transduced with the viral particle of any one of claims 1-23.

27. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a viral particle by intraperitoneal, subcutaneous, or intranodal injection, wherein the viral particle transduces immune cells in vivo.

28. The method of claim 27, wherein the viral particles are administered by intranodal injection through inguinal lymph nodes.

29. The method of claim 27, wherein the viral particles are administered by intraperitoneal injection.

30. A viral particle for transducing an immune cell in vivo, the viral particle comprising a polynucleotide sequence encoding a chimeric antigen receptor.

31. The particle of any one of claims 1 to 23 or claim 30, wherein the viral particle further comprises a polynucleotide sequence encoding a dominant negative tgfβ receptor.

32. The particle of any one of claims 1 or 30 wherein expression of the chimeric antigen receptor is modulated by an FRB-degrader fusion polypeptide, and wherein inhibition of the FRB-degrader fusion polypeptide is inducible by ligand chemistry.

33. The particle of claim 32, wherein the ligand is rapamycin.

34. The particle of any one of claims 1 or 30, wherein expression of the chimeric antigen receptor is modulated by a degradative sub-fusion polypeptide, and wherein inhibition of the degradative sub-fusion polypeptide is inducible by ligand chemistry.

35. The method of any one of claims 24 or 27, wherein the disease or disorder comprises a B-cell malignancy, a relapsed/refractory CD 19-expressing malignancy, diffuse large B-cell lymphoma (DLBCL), burkitt's B-cellymphoma, B-LBL), follicular Lymphoma (FL), chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), mantle Cell Lymphoma (MCL), 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 myelogenous leukemia, glioblastoma, neuroblastoma, medulloblastoma, sarcoma, and any combination thereof.

36. The method of any one of claims 24 or 27, wherein the disease or disorder comprises diffuse large B-cell lymphoma (DLBCL).

37. The method of any one of claims 24 or 27, wherein the disease or disorder comprises burkitt's type large B cell lymphoma (B-LBL).

38. The method of any one of claims 24 or 27, wherein the disease or disorder comprises Follicular Lymphoma (FL).

39. The method of any one of claims 24 or 27, wherein the disease or disorder comprises Chronic Lymphocytic Leukemia (CLL).

40. The method of any one of claims 24 or 27, wherein the disease or disorder comprises Acute Lymphoblastic Leukemia (ALL).

41. The method of any one of claims 24 or 27, wherein the disease or disorder comprises Mantle Cell Lymphoma (MCL).

42. A pharmaceutical composition comprising the viral particle according to any one of claims 1 to 23.

43. A kit comprising a pharmaceutical composition according to claim 42 and optionally a composition comprising a ligand, optionally rapamycin.

44. A viral particle for use in the method of any one of claims 24 to 29 or 35 to 41.

45. Use of a viral particle in a method according to any one of claims 24 to 29 or 35 to 41.

46. The method of claim 24, wherein cd19+ B cells in the subject are depleted by at least 80%, at least 85%, at least 90%, or at least 95% compared to a subject not utilizing viral particles.

47. The method of claim 46, wherein the cd19+ B cells in the peripheral blood of the subject are depleted.

48. The method of claim 46, wherein B cell depletion is maintained in the subject for at least 7 days, at least 10 days, at least 20 days, at least 30 days, at least 40 days, at least 50 days, at least 60 days, at least 70 days, or at least 80 days after administration of the viral particles.

49. The method of claim 24, wherein at least 200, at least 400, at least 600, at least 800, or at least 1000 ten thousand transduction units of viral particles are administered to the subject.

50. The method of claim 32, wherein contacting the immune cells with the ligand increases the number of immune cells in the subject that express an anti-CD 19 chimeric antigen receptor by at least 10-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, or at least 1000-fold.

51. A polypeptide comprising a single chain variable fragment that specifically binds to CD3 (anti-CD 3 scFv) and a glycophorin a transmembrane fragment.

52. The polypeptide of claim 51, wherein the glycophorin a transmembrane fragment shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with HFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETS DQ (SEQ ID NO: 105).

53. The polypeptide of claim 51, wherein the anti-CD 3 scFv shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID No. 2 or 12.

54. The polypeptide of any one of claims 51 to 53, wherein the polypeptide shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID No. 119.

55. The polypeptide of claim 51, wherein the transmembrane fragment shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID No. 13, 19, 25, 31, 37, 43 or 105.

56. A surface engineered lentiviral particle comprising the polypeptide of any one of claims 51 to 54 displayed on the surface of the lentiviral particle.

57. A method of transducing a cell, the method comprising contacting the viral particle of any one of claims 1 to 23 with an immune cell in vivo.

58. A polynucleotide comprising an anti-CD 3 scFv and a glycophorin a transmembrane fragment.

59. The polynucleotide of claim 58, wherein said glycophorin A transmembrane fragment shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO 106.

60. The polynucleotide of claim 58, wherein said anti-CD 3 scFv shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID No. 7 or 15.

61. The polynucleotide of any one of claims 58 to 60, wherein said polypeptide shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID No. 120.

62. The polynucleotide of claim 58, wherein said transmembrane fragment shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID No. 16, 22, 25, 28, 34, 40, 47 or 106.

63. A method of making a viral particle, the method comprising:

a) Providing cells in a culture medium; and

b) Transfecting the cells simultaneously or sequentially with the vector genome, transfer plasmid and packaging plasmid of any one of claims 1 to 23; whereby the cells express surface engineered viral particles.

64. A method of treating a disease or disorder associated with malignant cd19+ cells, the method comprising: transducing immune cells in vivo, and/or producing a viral particle expressing an anti-CD 3 single-chain variable fragment exposed on and/or conjugated to the surface of a viral envelope, said anti-CD 3 single-chain variable fragment sharing at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID No. 2 or 12; and administering the viral particles to a subject.

65. The method of claim 64, wherein the viral particles are administered by intraperitoneal, subcutaneous, or intranodal injection.