CD19-specific antibody constructs and compositions thereof

Abstract

Disclosed herein are antibodies or antigen-binding fragments thereof that specifically bind to human CD19. Also disclosed herein are chimeric antigen receptors and chimeric antigen receptor transgenes that contain an antigen-binding domain that specifically binds to human CD19. Also described herein are immune cells, viral vectors, and other compositions that contain the antibodies, antigen-binding fragments, chimeric antigen receptors, and / or chimeric antigen receptor transgenes.

Description

[Technical Field]

[0001] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 479,328, filed January 10, 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on January 10, 2024, is named 15147_6006-00000_SL.xml and is 248 kilobytes in size.

[0003] The present disclosure relates to an antibody or antigen-binding fragment thereof that specifically binds to human CD19. Also disclosed are chimeric antigen receptors and chimeric antigen receptor transgenes comprising an antigen-binding domain that specifically binds to human CD19. Also disclosed are immune cells, viral vectors, and other compositions containing the antibody, antibody-binding fragment, chimeric antigen receptor, and / or chimeric antigen receptor transgene. Also disclosed are fusion proteins comprising henipavirus glycoprotein G and a human CD19 antibody or an antigen-binding fragment thereof. Also disclosed are viral vectors and other compositions containing the antibody or antigen-binding fragment, chimeric antigen receptor, and chimeric antigen receptor transgene, as well as the fusion protein. The present disclosure additionally relates to cells expressing chimeric antigen receptors, as well as methods for delivering various antibodies and chimeric antigen receptors and methods for using cells expressing chimeric antigen receptors. [Background technology]

[0004] Introduction Cluster of differentiation antigen 19 (CD19), also known as B lymphocyte antigen CD19, is a transmembrane protein in the immunoglobin (Ig) superfamily expressed on cells of the B cell lineage. CD19 is expressed at all stages of B cell development until terminal differentiation into plasma cells. Notably, CD19 expression is regulated, with mature B cells expressing more CD19 than immature B cells. CD19 expression has been used as a marker in the diagnosis of numerous cancers, including B cell lymphoma, acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL). T lymphocytes are one of the primary targets in gene therapy, and even more so since chimeric antigen receptor (CAR) T cells reached the clinic. Genetically modifying T cells with CAR constructs is the most common approach for generating tumor-specific T cells. The use of modified T cells is an emerging cell therapy approach within the field of adoptive cell transfer (ACT). This approach involves collecting T cells from a patient (autologous) or a healthy donor (allogeneic), genetically modifying or manipulating these T cells, and transferring the modified or engineered T cells into the patient to treat various diseases. While the use of allogeneic T cells has several advantages over the use of autologous T cells, the latter presents challenges, such as the patient having insufficient healthy T cells for harvest and the patient experiencing disease progression, comorbidities, or even death during the time required to produce the engineered T cells. Additionally, CAR-T cells engineered solely with human components may limit host immunogenicity induced by xenogeneic AR. There is a need for methods to efficiently generate effective CAR-T cells, including allogeneic CAR-T cells, that target specific tumor antigens. The present disclosure addresses this need.

[0005] There is a significant unmet need for novel CAR-T cells designed to treat B-cell malignancies, including multiple myeloma. Many recipients of CAR-T therapy fail to respond to treatment or relapse. Furthermore, many recipients of CAR-T therapy develop humoral immunity to available CAR-T therapeutics. For many patients, current manufacturing methods and capabilities pose significant challenges to the availability and access of CAR-T therapeutics, including those targeted to B-cell malignancies. Thus, there is a need for novel CAR-T cells for the treatment of patients with B-cell malignancies, such as multiple myeloma, via targeting human CD19 and other potential secondary antigens. Summary of the Invention

[0006] The present disclosure provides isolated polypeptides that specifically bind to human Cluster of Differentiation 19 (CD19). In some embodiments, the isolated polypeptides comprise a predetermined heavy chain variable region (VH) and / or a predetermined light chain variable region (VL). In some embodiments, the isolated polypeptides comprise a predetermined heavy chain complementarity determining region (HCDR1, HCDR2, and HCDR3) and / or a predetermined light chain complementarity determining region (LCDR1, LCDR2, and LCDR3).

[0007] The present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to human Cluster of Differentiation 19 (CD19). In some embodiments, the antibodies or antigen-binding fragments thereof comprise a predetermined heavy chain variable region (VH) and / or a predetermined light chain variable region (VL). In some embodiments, the antibodies or antigen-binding fragments thereof comprise predetermined heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and / or predetermined light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3). The present disclosure also provides isolated polynucleotides, vectors, and host cells comprising anti-CD19 antibodies or antigen-binding fragments thereof.

[0008] The present disclosure also provides chimeric antigen receptors (CARs) that specifically bind to human cluster of differentiation 19 (CD19). In some embodiments, the CAR comprises at least one of a signal peptide, an extracellular binding domain, a hinge domain, a transmembrane domain, an intracellular costimulatory domain, and / or an intracellular signaling domain. In some embodiments, the CAR extracellular binding domain comprises an antigen-binding domain comprising an antibody or antigen-binding fragment thereof disclosed herein. The present disclosure also provides isolated polynucleotides, vectors, and host cells comprising human anti-CD19 CARs.

[0009] The present disclosure also provides a viral vector that targets immune cells, the vector comprising an antibody or antigen-binding fragment thereof that binds to a cell surface molecule on an immune cell and at least one polynucleotide encoding a chimeric antigen receptor (CAR) disclosed herein. In some embodiments, the antibody or antigen-binding fragment thereof binds to CD4 or CD8. In some embodiments, the vector comprises a henipavirus F protein molecule or a biologically active portion thereof. In some embodiments, the vector comprises a henipavirus envelope glycoprotein G (G protein) or a biologically active portion thereof. In some embodiments, the antibody or antigen-binding fragment thereof that binds to a cell surface molecule is bound to a membrane-associated protein in the viral vector envelope. In some embodiments, the antibody or antigen-binding fragment thereof that binds to a cell surface molecule is bound to a fusogen on the outer surface of the viral vector.

[0010] The present disclosure also provides a fusion protein comprising henipavirus envelope glycoprotein G (G protein), or a biologically active portion thereof, and an anti-CD19 antibody, or antigen-binding fragment thereof, disclosed herein.

[0011] The present disclosure provides a method for selectively modulating the activity of an immune cell, the method comprising delivering to the immune cell an effective amount of a viral vector comprising a polynucleotide encoding a CAR disclosed herein. The present disclosure also provides a method for generating a chimeric antigen receptor (CAR) immune cell, the method comprising delivering to the immune cell an effective amount of a viral vector comprising a polynucleotide encoding a CAR disclosed herein. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a primary T cell. In some embodiments, the polynucleotide encoding a CAR disclosed herein is inserted into a site-specific locus. In some embodiments, the polynucleotide encoding a CAR disclosed herein is inserted by homologous recombination repair. In some embodiments, the immune cell expresses one or more CARs disclosed herein.

[0012] The present disclosure additionally provides engineered cells comprising a CAR disclosed herein and one or more modifications that (i) reduce expression of one or more MHC class I molecules and / or one or more MHC class II molecules, and / or (ii) increase expression of one or more tolerance-inducing factors, wherein the reduced expression of (i) and the increased expression of (ii) are compared to a cell of the same cell type that does not contain the modifications.

[0013] The present disclosure additionally provides a method of administering an effective amount of the CAR cells disclosed herein to a subject in need thereof. The present disclosure also provides a method for treating a disease in a subject. The present disclosure provides a population of immune cells expressing a CAR disclosed herein. The present disclosure provides a composition of immune cells expressing a CAR disclosed herein. The present disclosure also provides a pharmaceutical composition of immune cells expressing a CAR disclosed herein. The present disclosure provides the use of the cells or methods disclosed herein for the treatment of a disease. In some embodiments, the disease is cancer. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the cancer is a solid malignancy. [Brief explanation of the drawings]

[0014] [Figure 1A] We present the in vitro characterization of cluster of differentiation 19 (CD19) chimeric antigen receptor constructs in human T cells. [Figure 1B] We present the in vitro characterization of cluster of differentiation 19 (CD19) chimeric antigen receptor constructs in human T cells. [Figure 2A] 1 shows the effect of cluster of differentiation 19 (CD19) chimeric antigen receptor constructs on NALM-6 tumor cells at various effector:target cell ratios. [Figure 2B] 1 shows the effect of cluster of differentiation 19 (CD19) chimeric antigen receptor constructs on NALM-6 tumor cells at various effector:target cell ratios. [Figure 2C] 1 shows the effect of cluster of differentiation 19 (CD19) chimeric antigen receptor constructs on NALM-6 tumor cells at various effector:target cell ratios. [Figure 2D] 1 shows the effect of cluster of differentiation 19 (CD19) chimeric antigen receptor constructs on NALM-6 tumor cells at various effector:target cell ratios. [Figure 2E] 1 shows the effect of cluster of differentiation 19 (CD19) chimeric antigen receptor constructs on NALM-6 tumor cells at various effector:target cell ratios. [Figure 3A] 1 shows the binding of CD19 binder 1 to recombinant CD19 in an enzyme-linked immunosorbent assay (ELISA). [Figure 3B] Figure 1 shows the binding of CD19 binder 2 to recombinant CD19 in an ELISA. [Figure 3C] 1 shows the binding of CD19 binder 3 to recombinant CD19 in an ELISA. [Figure 4A] Figure 1 shows the binding of CD19 binder 1 to two different cell types. Figure 2 shows the binding of CD19 binder 1 to CD19+ Raji cells. [Figure 4B] 1 shows the binding of CD19 binder 1 to two different cell types. 1 shows the binding of CD19 binder 1 to CD19-293 cells. [Figure 4C] Figure 1 shows the binding of CD19 binder 1 to two different cell types. The EC50 for CD19 binder 1 is shown. [Figure 5A] Figure 1 shows the binding of CD19 binder 2 to two different cell types. Figure 2 shows the binding of CD19 binder 2 to CD19+ Raji cells. [Figure 5B] 1 shows the binding of CD19 binder 2 to two different cell types. 2 shows the binding of CD19 binder 2 to CD19-293 cells. [Figure 5C] Figure 1 shows the binding of CD19 binder 2 to two different cell types. The EC50 for CD19 binder 2 is shown. [Figure 6A] Figure 1 shows the binding of CD19 binder 3 to two different cell types. Figure 2 shows the binding of CD19 binder 3 to CD19+ Raji cells. [Figure 6B] Figure 1 shows the binding of CD19 binder 3 to two different cell types. Figure 2 shows the binding of CD19 binder 2 to CD19-293 cells. [Figure 6C] Figure 1 shows the binding of CD19 binder 3 to two different cell types. The EC50 for CD19 binder 3 is shown. [Figure 7A] Figure 1 shows in vivo tumor growth as measured by flux. [Figure 7B] Shown is the total area under the curve for survival of mice receiving CD19 CAR-T cells after tumor challenge. [Figure 7C] Shown is the survival rate of mice receiving CD19 CAR-T cells after tumor challenge. [Figure 8A] 1 shows the effect of administration of CD8-targeted fusosomes containing fully humanized CAR400 (VL-VH) on tumor growth in vivo. [Figure 8B] Tumor intensity in mice receiving mock or fully humanized CAR400 (VL-VH) is shown. [Figure 9A] Figure 1 shows the transduction rates of activated PBMCs transduced with CD8-retargeting fusogens containing FMC63, CAR400 VLVH, or CAR400 VHVL CARs across different PBMC donors. [Figure 9B] Figure 1 shows the transduction rates of activated PBMCs transduced with CD8-retargeting fusogens containing FMC63, CAR400 VLVH, or CAR400 VHVL CARs across different PBMC donors. [Figure 9C] Figure 1 shows the transduction rates of activated PBMCs transduced with CD8-retargeting fusogens containing FMC63, CAR400 VLVH, or CAR400 VHVL CARs across different PBMC donors. [Figure 9D] Figure 1 shows the transduction rates of activated PBMCs transduced with CD8-retargeting fusogens containing FMC63, CAR400 VLVH, or CAR400 VHVL CARs across different PBMC donors. [Figure 9E] Figure 1 shows the transduction rates of activated PBMCs transduced with CD8-retargeting fusogens containing FMC63, CAR400 VLVH, or CAR400 VHVL CARs across different PBMC donors. [Figure 9F] Figure 1 shows the transduction rates of activated PBMCs transduced with CD8-retargeting fusogens containing FMC63, CAR400 VLVH, or CAR400 VHVL CARs across different PBMC donors. [Figure 10] A-C show the vector copy number (VCN) per target cell genome in activated PBMCs transduced with CD8-retargeting fusogens containing FMC63, CAR400 VLVH, or CAR400 VHVL CARs across different PBMC donors. [Figure 11]Representative flow cytometry showing CAR expression (FMC63, CAR400 VLVH, and CAR400 VHVL) in transduced and activated PBMCs is shown. [Figure 12A] 1 shows CAR-mediated killing of NAML6 cells for three different CD8-retargeted fusogens with different CD19 CARs (FMC63, CAR400 VLVH, and CAR400 VHVL). [Figure 12B] 1 shows CAR-mediated killing of NAML6 cells for three different CD8-retargeted fusogens with different CD19 CARs (FMC63, CAR400 VLVH, and CAR400 VHVL). [Figure 12C] 1 shows CAR-mediated killing of NAML6 cells for three different CD8-retargeted fusogens with different CD19 CARs (FMC63, CAR400 VLVH, and CAR400 VHVL). [Figure 12D] 12A-12C show CAR-mediated killing of NAML6 cells for three different CD8-retargeted fusogens with different CD19 CARs (FMC63, CAR400 VLVH, and CAR400 VHVL). Results from Figures 12A-12C are shown normalized to CAR+ cells. [Figure 12E] 12A-12C show CAR-mediated killing of NAML6 cells for three different CD8-retargeted fusogens with different CD19 CARs (FMC63, CAR400 VLVH, and CAR400 VHVL). Results from Figures 12A-12C are shown normalized to CAR+ cells. [Figure 12F] 12A-12C show CAR-mediated killing of NAML6 cells for three different CD8-retargeted fusogens with different CD19 CARs (FMC63, CAR400 VLVH, and CAR400 VHVL). Results from Figures 12A-12C are shown normalized to CAR+ cells. [Figure 13A]Figure 1 shows the transduction rates of resting PBMCs (e.g., in an ex vivo medication background) transduced with CD8-retargeting fusogens containing FMC63, CAR400 VLVH, or CAR400 VHVL CARs across different PBMC donors. [Figure 13B] Figure 1 shows the transduction rates of resting PBMCs (e.g., in an ex vivo medication background) transduced with CD8-retargeting fusogens containing FMC63, CAR400 VLVH, or CAR400 VHVL CARs across different PBMC donors. [Figure 13C] Figure 1 shows the transduction rates of resting PBMCs (e.g., in an ex vivo medication background) transduced with CD8-retargeting fusogens containing FMC63, CAR400 VLVH, or CAR400 VHVL CARs across different PBMC donors. [Figure 13D] Shown are vector copy numbers (VCN) per target cell genome for resting PBMCs (e.g., in ex vivo medication background) transduced with CD8-retargeting fusogens containing FMC63, CAR400 VLVH, or CAR400 VHVL CARs across different PBMC donors. [Figure 13E] Shown are vector copy numbers (VCN) per target cell genome for resting PBMCs (e.g., in ex vivo medication background) transduced with CD8-retargeting fusogens containing FMC63, CAR400 VLVH, or CAR400 VHVL CARs across different PBMC donors. [Figure 13F] Shown are vector copy numbers (VCN) per target cell genome for resting PBMCs (e.g., in ex vivo medication background) transduced with CD8-retargeting fusogens containing FMC63, CAR400 VLVH, or CAR400 VHVL CARs across different PBMC donors. [Figure 14] Figure 1 shows an additional assessment of transduction rates of resting PBMCs (e.g., in an ex vivo medication background) transduced with CD8-retargeting fusogens containing FMC63 control #1, CAR400 VLVH, CAR400 VHVL CAR, or FMC63 control #2. [Figure 15A]1 shows the results of total flux in a B cell tumor animal model when animals received PBMCs from donor 1603C after administration of Nalm6:Wasabi-ffLuc cells, and various doses of CAR400 VLVH, CAR400 VHVL, and FMC63. [Figure 15B] 1 shows the results of total flux in a B cell tumor animal model when animals received PBMCs from donor 1603C after administration of Nalm6:Wasabi-ffLuc cells, and various doses of CAR400 VLVH, CAR400 VHVL, and FMC63. [Figure 15C] 1 shows the results of total flux in a B cell tumor animal model when animals received PBMCs from donor 1603C after administration of Nalm6:Wasabi-ffLuc cells, and various doses of CAR400 VLVH, CAR400 VHVL, and FMC63. [Figure 15D] 1 shows the results of total flux in a B cell tumor animal model when animals received PBMCs from donor 1603C after administration of Nalm6:Wasabi-ffLuc cells, and various doses of CAR400 VLVH, CAR400 VHVL, and FMC63. [Figure 15E] 1 shows the results of total flux in a B cell tumor animal model when animals received PBMCs from donor 1603C after administration of Nalm6:Wasabi-ffLuc cells, and various doses of CAR400 VLVH, CAR400 VHVL, and FMC63. [Figure 15F] 1 shows the results of total flux in a B cell tumor animal model when animals received PBMCs from donor 1603C after administration of Nalm6:Wasabi-ffLuc cells, and various doses of CAR400 VLVH, CAR400 VHVL, and FMC63. [Figure 15G]1 shows the results of total flux in a B cell tumor animal model when animals received PBMCs from donor 1603C after administration of Nalm6:Wasabi-ffLuc cells, and various doses of CAR400 VLVH, CAR400 VHVL, and FMC63. [Figure 15H] 1 shows the results of total flux in a B cell tumor animal model when animals received PBMCs from donor 1603C after administration of Nalm6:Wasabi-ffLuc cells, and various doses of CAR400 VLVH, CAR400 VHVL, and FMC63. [Figure 15I] 1 shows the results of total flux in a B cell tumor animal model when animals received PBMCs from donor 1603C after administration of Nalm6:Wasabi-ffLuc cells, and various doses of CAR400 VLVH, CAR400 VHVL, and FMC63. [Figure 15J] 1 shows the results of total flux in a B cell tumor animal model when animals received PBMCs from donor 1603C after administration of Nalm6:Wasabi-ffLuc cells, and various doses of CAR400 VLVH, CAR400 VHVL, and FMC63. [Figure 15K] 1 shows the results of total flux in a B cell tumor animal model when animals received PBMCs from donor 1603C after administration of Nalm6:Wasabi-ffLuc cells, and various doses of CAR400 VLVH, CAR400 VHVL, and FMC63. [Figure 15L] 1 shows the results of total flux in a B cell tumor animal model when animals received PBMCs from donor 1603C after administration of Nalm6:Wasabi-ffLuc cells, and various doses of CAR400 VLVH, CAR400 VHVL, and FMC63. [Figure 15M]1 shows the results of total flux in a B cell tumor animal model when animals received PBMCs from donor 1603C after administration of Nalm6:Wasabi-ffLuc cells, and various doses of CAR400 VLVH, CAR400 VHVL, and FMC63. [Figure 15N] 1 shows the results of total flux in a B cell tumor animal model when animals received PBMCs from donor 1603C after administration of Nalm6:Wasabi-ffLuc cells, and various doses of CAR400 VLVH, CAR400 VHVL, and FMC63. [Figure 16A] 1 shows the results of total flux in a B cell tumor animal model in which animals received PBMCs from donor 3001C after administration of Nalm6:Wasabi-ffLuc cells, and various doses of CAR400 VLVH, CAR400 VHVL, and FMC63. [Figure 16B] 1 shows the results of total flux in a B cell tumor animal model in which animals received PBMCs from donor 3001C after administration of Nalm6:Wasabi-ffLuc cells, and various doses of CAR400 VLVH, CAR400 VHVL, and FMC63. [Figure 16C] 1 shows the results of total flux in a B cell tumor animal model in which animals received PBMCs from donor 3001C after administration of Nalm6:Wasabi-ffLuc cells, and various doses of CAR400 VLVH, CAR400 VHVL, and FMC63. [Figure 16D] 1 shows the results of total flux in a B cell tumor animal model in which animals received PBMCs from donor 3001C after administration of Nalm6:Wasabi-ffLuc cells, and various doses of CAR400 VLVH, CAR400 VHVL, and FMC63. [Figure 16E]1 shows the results of total flux in a B cell tumor animal model in which animals received PBMCs from donor 3001C after administration of Nalm6:Wasabi-ffLuc cells, and various doses of CAR400 VLVH, CAR400 VHVL, and FMC63. [Figure 16F] 1 shows the results of total flux in a B cell tumor animal model in which animals received PBMCs from donor 3001C after administration of Nalm6:Wasabi-ffLuc cells, and various doses of CAR400 VLVH, CAR400 VHVL, and FMC63. [Figure 16G] 1 shows the results of total flux in a B cell tumor animal model in which animals received PBMCs from donor 3001C after administration of Nalm6:Wasabi-ffLuc cells, and various doses of CAR400 VLVH, CAR400 VHVL, and FMC63. [Figure 16H] 1 shows the results of total flux in a B cell tumor animal model in which animals received PBMCs from donor 3001C after administration of Nalm6:Wasabi-ffLuc cells, and various doses of CAR400 VLVH, CAR400 VHVL, and FMC63. [Figure 16I] 1 shows the results of total flux in a B cell tumor animal model in which animals received PBMCs from donor 3001C after administration of Nalm6:Wasabi-ffLuc cells, and various doses of CAR400 VLVH, CAR400 VHVL, and FMC63. [Figure 16J] 1 shows the results of total flux in a B cell tumor animal model in which animals received PBMCs from donor 3001C after administration of Nalm6:Wasabi-ffLuc cells, and various doses of CAR400 VLVH, CAR400 VHVL, and FMC63. [Figure 16K]1 shows the results of total flux in a B cell tumor animal model in which animals received PBMCs from donor 3001C after administration of Nalm6:Wasabi-ffLuc cells, and various doses of CAR400 VLVH, CAR400 VHVL, and FMC63. [Figure 16L] 1 shows the results of total flux in a B cell tumor animal model in which animals received PBMCs from donor 3001C after administration of Nalm6:Wasabi-ffLuc cells, and various doses of CAR400 VLVH, CAR400 VHVL, and FMC63. [Figure 16M] 1 shows the results of total flux in a B cell tumor animal model in which animals received PBMCs from donor 3001C after administration of Nalm6:Wasabi-ffLuc cells, and various doses of CAR400 VLVH, CAR400 VHVL, and FMC63. [Figure 16N] 1 shows the results of total flux in a B cell tumor animal model in which animals received PBMCs from donor 3001C after administration of Nalm6:Wasabi-ffLuc cells, and various doses of CAR400 VLVH, CAR400 VHVL, and FMC63. [Figure 17] A and B show the area under the curve (AUC) for tumor burden in animals receiving different CARs (CAR400 VLVH, CAR400 VHVL, and FMC63) throughout the 34-day study. [Figure 18A] The total number and percentage of CAR-positive cells within the peripheral blood CD4+ or CD8+ cell populations are shown. The relative counts of CD4+ cells in animals on day 14 of the study are shown. [Figure 18B] The total number and percentage of CAR-positive cells within the peripheral blood CD4+ or CD8+ cell populations are shown. The relative counts of CD8+ cells in animals on day 14 of the study are shown. [Figure 18C]The total number and percentage of CAR-positive cells within peripheral blood CD4+ or CD8+ cell populations are shown. The percentage of CAR-positive cells among total CD4+ cells is shown. [Figure 18D] The total number and percentage of CAR-positive cells within peripheral blood CD4+ or CD8+ cell populations are shown. The percentage of CAR-positive cells among total CD8+ cells is shown. [Figure 18E] The total number and percentage of CAR-positive cells within the peripheral blood CD4+ or CD8+ cell population are shown. The percentage of tumor cells detected in the peripheral blood of the animals is shown. [Figure 19] A to C show the plasmid maps corresponding to CD47-FMC63, CD47-CAR400 VHVL, and CD47-CAR400 VLVH CAR, respectively. [Figure 20A-1] Figure 1 shows confirmation of CAR transduction in target cells. A representative gating strategy for flow cytometry of CD47-CD19 low immune CAR T cells is shown. [Figure 20A-2] Figure 1 shows confirmation of CAR transduction in target cells. A representative gating strategy for flow cytometry of CD47-CD19 low immune CAR T cells is shown. [Figure 20A-3] Figure 1 shows confirmation of CAR transduction in target cells. A representative gating strategy for flow cytometry of CD47-CD19 low immune CAR T cells is shown. [Figure 20B] Figure 1 shows confirmation of CAR transduction in target cells. Figure 2 shows flow cytometry-based (QIFI) quantification of surface CD47 protein expression on CAR-positive T cells. [Figure 20C] Figure 1 shows confirmation of CAR transduction in target cells. Figure 2 shows the integration of the CAR construct into target cells calculated by digital-droplet PCR. [Figure 21]A-C show the physical and functional titers of the generated CD47-CD19 VSV-g lentiviral vectors as demonstrated by genomic quantification (GQA) (A), functional titer (SupT1 IU / mL) (B), and calculated particle-to-infectivity ratio (GQA / IU) (C). [Figure 22] A and B show the dose titration of the lentiviral vector used to transduce primary T cells. [Figure 23] A and B show digital-droplet PCR confirmation of dose titration by measuring integrated vector copy number (VCN) within a bulk primary T cell pool. B shows delU3 VCN normalized to CAR-positive T cells only. [Figure 24A] Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on NALM-6 and NALM-6 CD19 knockout tumor cells. Figure 2 shows the viability of NALM-6 and NALM-6 CD19 knockout tumor cells, respectively, when tumor cells were cultured with CD47-CD19 hypoimmune CAR T cells at different effector:target cell ratios for 24 hours. [Figure 24B] Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on NALM-6 and NALM-6 CD19 knockout tumor cells. Cytokine levels measured by Meso-Scale Discovery (MSD) after culturing CD47-CD19 hypoimmune CAR T cells with NALM-6 tumor cells are shown. [Figure 24C] Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on NALM-6 and NALM-6 CD19 knockout tumor cells. Cytokine levels measured by Meso-Scale Discovery (MSD) after culturing CD47-CD19 hypoimmune CAR T cells with NALM-6 tumor cells are shown. [Figure 24D]Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on NALM-6 and NALM-6 CD19 knockout tumor cells. Cytokine levels measured by Meso-Scale Discovery (MSD) after culturing CD47-CD19 hypoimmune CAR T cells with NALM-6 tumor cells are shown. [Figure 24E] Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on NALM-6 and NALM-6 CD19 knockout tumor cells. Cytokine levels measured by Meso-Scale Discovery (MSD) after culturing CD47-CD19 hypoimmune CAR T cells with NALM-6 tumor cells are shown. [Figure 24F] Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on NALM-6 and NALM-6 CD19 knockout tumor cells. Figure 2 shows the viability of NALM-6 and NALM-6 CD19 knockout tumor cells, respectively, when tumor cells were cultured with CD47-CD19 hypoimmune CAR T cells at different effector:target cell ratios for 24 hours. [Figure 24G] Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on NALM-6 and NALM-6 CD19 knockout tumor cells. Cytokine levels measured after culturing CD47-CD19 hypoimmune CAR T cells with NALM-6 CD19 knockout tumor cells are shown. [Figure 24H] Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on NALM-6 and NALM-6 CD19 knockout tumor cells. Cytokine levels measured after culturing CD47-CD19 hypoimmune CAR T cells with NALM-6 CD19 knockout tumor cells are shown. [Figure 24I]Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on NALM-6 and NALM-6 CD19 knockout tumor cells. Cytokine levels measured after culturing CD47-CD19 hypoimmune CAR T cells with NALM-6 CD19 knockout tumor cells are shown. [Figure 24J] Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on NALM-6 and NALM-6 CD19 knockout tumor cells. Cytokine levels measured after culturing CD47-CD19 hypoimmune CAR T cells with NALM-6 CD19 knockout tumor cells are shown. [Figure 25A] Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on Raji and Raji CD19 knockout tumor cells. Figure 2 shows the viability of Raji and Raji CD19 knockout tumor cells, respectively, when tumor cells were cultured with CD47-CD19 hypoimmune CAR T cells at different effector:target cell ratios for 24 hours. [Figure 25B] Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on Raji and Raji CD19 knockout tumor cells. Cytokine levels measured by MSD after culturing CD47-CD19 hypoimmune CAR T cells with Raji tumor cells. [Figure 25C] Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on Raji and Raji CD19 knockout tumor cells. Cytokine levels measured by MSD after culturing CD47-CD19 hypoimmune CAR T cells with Raji tumor cells. [Figure 25D] Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on Raji and Raji CD19 knockout tumor cells. Cytokine levels measured by MSD after culturing CD47-CD19 hypoimmune CAR T cells with Raji tumor cells. [Figure 25E]Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on Raji and Raji CD19 knockout tumor cells. Cytokine levels measured by MSD after culturing CD47-CD19 hypoimmune CAR T cells with Raji tumor cells. [Figure 25F] Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on Raji and Raji CD19 knockout tumor cells. Figure 2 shows the viability of Raji and Raji CD19 knockout tumor cells, respectively, when tumor cells were cultured with CD47-CD19 hypoimmune CAR T cells at different effector:target cell ratios for 24 hours. [Figure 25G] Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on Raji and Raji CD19 knockout tumor cells. Cytokine levels measured after culturing CD47-CD19 hypoimmune CAR T cells with Raji CD19 knockout tumor cells. [Figure 25H] Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on Raji and Raji CD19 knockout tumor cells. Cytokine levels measured after culturing CD47-CD19 hypoimmune CAR T cells with Raji CD19 knockout tumor cells. [Figure 25I] Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on Raji and Raji CD19 knockout tumor cells. Cytokine levels measured after culturing CD47-CD19 hypoimmune CAR T cells with Raji CD19 knockout tumor cells. [Figure 25J] Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on Raji and Raji CD19 knockout tumor cells. Cytokine levels measured after culturing CD47-CD19 hypoimmune CAR T cells with Raji CD19 knockout tumor cells. [Figure 26A]Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on K562-CD19+ and parental K562 tumor cells. Figure 2 shows K562 and K562 CD19 knockout tumor cell viability, respectively, when tumor cells are cultured with CD47-CD19 hypoimmune CAR T cells at different effector:target cell ratios for 24 hours. [Figure 26B] Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on K562-CD19+ and parental K562 tumor cells. Cytokine levels measured by MSD after culturing CD47-CD19 hypoimmune CAR T cells with K562 tumor cells are shown. [Figure 26C] Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on K562-CD19+ and parental K562 tumor cells. Cytokine levels measured by MSD after culturing CD47-CD19 hypoimmune CAR T cells with K562 tumor cells are shown. [Figure 26D] Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on K562-CD19+ and parental K562 tumor cells. Cytokine levels measured by MSD after culturing CD47-CD19 hypoimmune CAR T cells with K562 tumor cells are shown. [Figure 26E] Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on K562-CD19+ and parental K562 tumor cells. Cytokine levels measured by MSD after culturing CD47-CD19 hypoimmune CAR T cells with K562 tumor cells are shown. [Figure 26F] Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on K562-CD19+ and parental K562 tumor cells. Figure 2 shows K562 and K562 CD19 knockout tumor cell viability, respectively, when tumor cells are cultured with CD47-CD19 hypoimmune CAR T cells at different effector:target cell ratios for 24 hours. [Figure 26G]Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on K562-CD19+ and parental K562 tumor cells. Cytokine levels measured after culturing CD47-CD19 hypoimmune CAR T cells with K562 CD19 knockout tumor cells. [Figure 26H] Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on K562-CD19+ and parental K562 tumor cells. Cytokine levels measured after culturing CD47-CD19 hypoimmune CAR T cells with K562 CD19 knockout tumor cells. [Figure 26I] Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on K562-CD19+ and parental K562 tumor cells. Cytokine levels measured after culturing CD47-CD19 hypoimmune CAR T cells with K562 CD19 knockout tumor cells. [Figure 26J] Figure 1 shows the cytotoxic effect of hypoimmune CD47-CD19 CAR T cells on K562-CD19+ and parental K562 tumor cells. Cytokine levels measured after culturing CD47-CD19 hypoimmune CAR T cells with K562 CD19 knockout tumor cells. [Figure 27A] Demonstrating the cytotoxicity of hypoimmune CD47-CD19 CAR T cells. Incucyte analysis of NALM-6 iRFP713+ tumor cell growth when cultured with CD19CAR+ or mock untransduced T cells generated from three different donors. [Figure 27B] Demonstrating the cytotoxicity of hypoimmune CD47-CD19 CAR T cells. Incucyte analysis of NALM-6 iRFP713+ tumor cell growth when cultured with CD19CAR+ or mock untransduced T cells generated from three different donors. [Figure 27C] Demonstrating the cytotoxicity of hypoimmune CD47-CD19 CAR T cells. Incucyte analysis of NALM-6 iRFP713+ tumor cell growth when cultured with CD19CAR+ or mock untransduced T cells generated from three different donors. [Figure 27D] Shows cytotoxicity of hypoimmune CD47-CD19 CAR T cells. Shows Incucyte analysis of T cell proliferation over the course of the study with hypoimmune CD47-CD19 CAR T cells generated from three different donors when cultured with NALM-6 iRFP713+ tumor cells. [Figure 27E] Shows cytotoxicity of hypoimmune CD47-CD19 CAR T cells. Shows Incucyte analysis of T cell proliferation over the course of the study with hypoimmune CD47-CD19 CAR T cells generated from three different donors when cultured with NALM-6 iRFP713+ tumor cells. [Figure 27F] Shows cytotoxicity of hypoimmune CD47-CD19 CAR T cells. Shows Incucyte analysis of T cell proliferation over the course of the study with hypoimmune CD47-CD19 CAR T cells generated from three different donors when cultured with NALM-6 iRFP713+ tumor cells. [Figure 28A] Demonstrating the cytotoxicity of hypoimmune CD47-CD19 CAR T cells. Incucyte analysis of NALM-6 CD19 knockout iRFP713+ tumor cell growth when cultured with CD19CAR+ or mock untransduced T cells generated from three different donors. [Figure 28B] Demonstrating the cytotoxicity of hypoimmune CD47-CD19 CAR T cells. Incucyte analysis of NALM-6 CD19 knockout iRFP713+ tumor cell growth when cultured with CD19CAR+ or mock untransduced T cells generated from three different donors. [Figure 28C] Demonstrating the cytotoxicity of hypoimmune CD47-CD19 CAR T cells. Incucyte analysis of NALM-6 CD19 knockout iRFP713+ tumor cell growth when cultured with CD19CAR+ or mock untransduced T cells generated from three different donors. [Figure 28D]Showing cytotoxicity of hypoimmune CD47-CD19 CAR T cells. Shown is an Incucyte analysis of T cell proliferation over the course of the study with hypoimmune CD47-CD19 CAR T cells generated from three different donors when cultured with NALM-6 CD19KO iRFP713+ tumor cells. [Figure 28E] Showing cytotoxicity of hypoimmune CD47-CD19 CAR T cells. Shown is an Incucyte analysis of T cell proliferation over the course of the study with hypoimmune CD47-CD19 CAR T cells generated from three different donors when cultured with NALM-6 CD19KO iRFP713+ tumor cells. [Figure 28F] Showing cytotoxicity of hypoimmune CD47-CD19 CAR T cells. Shown is an Incucyte analysis of T cell proliferation over the course of the study with hypoimmune CD47-CD19 CAR T cells generated from three different donors when cultured with NALM-6 CD19KO iRFP713+ tumor cells. [Figure 29A] Shown is GM-CSF measured by MSD from culture supernatants 24 hours after incubation of hypoimmune CAR T cells with NALM-6 and NALM-6 CD19 knockout cells. [Figure 29B] Shown is IFNγ measured by MSD from culture supernatants 24 hours after incubation of hypoimmune CAR T cells with NALM-6 and NALM-6 CD19 knockout cells. [Figure 29C] Shown is IL-2 measured by MSD from culture supernatants 24 hours after incubation of hypoimmune CAR T cells with NALM-6 and NALM-6 CD19 knockout cells. [Figure 29D] Shown is TNFα measured by MSD from culture supernatants 24 hours after incubation of hypoimmune CAR T cells with NALM-6 and NALM-6 CD19 knockout cells. [Figure 30A]Figure 1 shows the results of total flux in a B-cell tumor animal model when animals received hypoimmune CD47-CD19 CAR T cells generated from three different donors after administration of Nalm6:Wasabi-ffLuc cells. [Figure 30B] Figure 1 shows the results of total flux in a B-cell tumor animal model when animals received hypoimmune CD47-CD19 CAR T cells generated from three different donors after administration of Nalm6:Wasabi-ffLuc cells. [Figure 30C] Figure 1 shows the results of total flux in a B-cell tumor animal model when animals received hypoimmune CD47-CD19 CAR T cells generated from three different donors after administration of Nalm6:Wasabi-ffLuc cells. [Figure 31] AC show the levels of CAR+ cells in the circulating blood at days 13 and 31 in a B cell tumor model. [Figure 32] AC show median fluorescence intensity (MFI) for CD47 measured by flow cytometry from cells in circulating blood on days 13 and 31 in a B cell tumor model. DETAILED DESCRIPTION OF THE INVENTION

[0015] Unless otherwise defined, all terms, notations, and other technical and scientific terms or terminology of the art used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms having commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not be construed as necessarily representing a substantial difference from what is commonly understood in the art.

[0016] Unless otherwise defined, all technical and scientific terms, acronyms, and abbreviations used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Unless otherwise indicated, abbreviations and symbols for chemical and biochemical names follow IUPAC-IUB nomenclature. Unless otherwise indicated, all numerical ranges include the values ​​defining the range and all integer values ​​therebetween.

[0017] As used herein, the articles "a" and "an" refer to one or to more than one, i.e., to at least one, of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0018] As used herein, the term "about" will be understood by those of skill in the art and will vary to some extent depending on the context in which it is used. In some embodiments, the term "about," when referring to a measurable value, e.g., an amount, a time period, is meant to encompass variations accepted in the art based on the standard error in making such measurements. In some embodiments, the term "about," when referring to such a value, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, where such variations are appropriate for performing the disclosed methods.

[0019] As used herein, "CD19" or "cluster of differentiation 19" refers to a transmembrane glycoprotein expressed on cells of the B cell lineage. CD19 is a marker for B cell development.

[0020] As used herein, "CD4" or "cluster of differentiation 4" refers to a transmembrane glycoprotein that is a specific marker for a subclass of T cells (including helper T cells). The CD4 protein acts as a coreceptor together with the T cell receptor (TCR) to recognize antigens presented by MHC class II cells. CD4 plays a role in T cell development and activation of mature T cells.

[0021] As used herein, "CD8" or "cluster of differentiation 8" refers to a transmembrane glycoprotein that is a specific marker for a subclass of T cells (including cytotoxic T cells). CD8 organizes as either a heterodimer of CD8 alpha ("CD8α" or "CD8A") and CD8 beta ("CD8β" or "CD8B") subunits ("CD8αβ" or "CD8AB"), or as a CD8 alpha homodimer ("CD8αα" or "CD8AA"). The assembled dimeric CD8 complex acts as a coreceptor together with the T cell receptor (TCR) to recognize antigens presented by MHC class I cells. CD8 plays a role in T cell development and the activation of mature T cells.

[0022] As used herein, "affinity" refers to the strength of the sum of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The affinity of a molecule for its partner is usually measured by the equilibrium dissociation constant (K D ) (or its reciprocal, the equilibrium association constant, K A Affinity can be measured by common methods known in the art, including those described herein. See, for example, Pope ME, Soste MV, Eyford BA, Anderson NL, Pearson TW, (2009) J. Immunol. Methods. 341(1-2):86-96 and the methods described therein.

[0023] As used herein, "antibody" is meant in a broad sense and includes immunoglobulin molecules including monoclonal antibodies, including murine, human, humanized and chimeric antibodies, antibody fragments, bispecific or multispecific antibodies formed from at least two intact antibodies or antibody fragments, dimeric, tetrameric, or multimeric antibodies, single chain antibodies, and any other modified configuration of an immunoglobulin molecule that contains an antigen recognition site of the required specificity.

[0024] Immunoglobulins can be assigned to five major classes, namely, IgA, IgD, IgE, IgG, and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further subdivided into IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely, kappa (K) and lambda (A), based on the amino acid sequence of their constant domain.

[0025] The term "antigen" refers to an immunogenic molecule that elicits an immune response. This immune response involves antibody production, activation of specific immunocompetent cells, or both. Antigens are, for example, peptides, glycopeptides, polypeptides, glycopolypeptides, polynucleotides, polysaccharides, lipids, etc. It is readily apparent that antigens can be synthetic, recombinantly produced, or derived from biological samples. Exemplary biological samples that may contain one or more antigens include tissue samples, tumor samples, cells, biological fluids, or combinations thereof. Antigens can also be produced by cells that have been modified or genetically engineered to express the antigen.

[0026] As used herein, "antigen-binding fragment" or "antibody fragment" refers to a portion of an immunoglobulin molecule that retains a heavy and / or light chain antigen-binding site, e.g., heavy chain complementarity-determining region (HCDR) 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3), light chain complementarity-determining region (LCDR) 1 (LCDR1), 2 (LCDR2), and 3 (LCDR3), a heavy chain variable region (VH), or a light chain variable region (VL). Antibody fragments include Fab fragments (monovalent fragments consisting of a VL or VH); F(ab)2 fragments (bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region); Fd fragments consisting of the VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single antibody arm; dAb fragments consisting of the VH domain; and variable domains (VHH) from, for example, human or camelid origin. VH and VL domains can be engineered and linked together via synthetic linkers to form various types of single chain antibody designs in which the VH / VL domains pair intramolecularly, or intermolecularly in those embodiments in which the VH and VL domains are expressed as separate single chain antibody constructs, to form a monovalent antigen-binding site, e.g., a single-chain Fv (scFv) or diabody. These antibody fragments are obtained using well-known techniques, and the fragments are characterized in the same manner as intact antibodies.

[0027] Antibody variable regions consist of a "framework" region interrupted by three "antigen-binding sites," which are defined using various terms, including, for example, (i) "complementarity-determining regions" (CDRs), three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3) (Wu and Kabat, J Exp Med 132:211-50, 1970; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), and (ii) "hypervariable regions," "HVRs," or "HVs," three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3) (Chothia and Lesk Mol Biol 196:901-17, 1987). Other terms include "IMGT-CDR" (Lefranc et al., Dev Comparat Immunol 27:55-77, 2003) and "specificity-determining residue usage" (SDRU) (Almagro Mol Recognit, 17:132-43, 2004). The International ImMunoGeneTics (IMGT) database (http: / / www_imgt org) provides standardized numbering and definitions of antigen-binding sites. The correspondence between CDR, HV, and IMGT delineations is described in Lefranc et al., Dev Comparat Immunol 27:55-77, 2003.

[0028] The term "framework," or "FR," or "framework sequence," refers to the remaining sequences of the variable region other than those sequences defined as the antigen-binding site. Because the antigen-binding site can be defined by various terms as described above, the exact amino acid sequence of the framework depends on how the antigen-binding site is defined.

[0029] A "binding domain," also referred to as a "binding region," refers to an antibody or portion thereof that possesses the ability to specifically and non-covalently associate with, integrate with, or combine with a target. Binding domains include any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule, molecular complex, or other target of interest. Exemplary binding domains include receptor ectodomains, ligands, scFvs, disulfide-linked Fvs, sdAbs, VHH antibodies, Fab fragments, Fab' fragments, F(ab')2 fragments, diabodies, or other synthetic polypeptides selected for their specific ability to bind to a biological molecule, molecular complex, or other target of interest.

[0030] The term "CDR" refers to a complementarity determining region defined in at least one particular manner by one skilled in the art.The precise amino acid sequence boundaries of a given CDR or FR can be determined using the "Kabat" numbering scheme in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD; Al-Lazikani et al. (1997) JMB 273, 927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745 ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol. 2003 Jan;27(1):55-77 ("IMGT" numbering scheme); Honegger A and Plueckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol,2001 Jun 8;309(3):657-70, ("Aho" numbering scheme); and Martin et al., "Modeling antibody hypervariable loops: a combined algorithm," PNAS,1989,86(23):9268-9272, ("AbM" numbering scheme).

[0031] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering for both the Kabat and Chothia schemes is based on the length of the most common antibody region sequences, with insertions provided by an insert character, e.g., "30a," and deletions that appear in some antibodies. The two schemes place certain insertions and deletions ("indels") in different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is in many ways similar to the Chothia numbering scheme. The AbM scheme is a compromise between the Kabat and Chothia definitions, based on those used by Oxford Molecular's AbM antibody modeling software.

[0032] In some embodiments, CDRs may be defined according to any of the Chothia numbering scheme, the Kabat numbering scheme, the IMGT numbering scheme, a combination of Kabat, IMGT, and Chothia, the AbM definition, and / or the contact definition. An sdAb variable domain comprises three CDRs designated CDR1, CDR2, and CDR3. Table 1 lists exemplary position boundaries for CDR-H1, CDR-H2, and CDR-H3 as identified by the Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1, residue numbering is listed using both the Kabat and Chothia numbering schemes. FRs are located between the CDRs; for example, FR-H1 precedes CDR-H1, FR-H2 is located between CDR-H1 and CDR-H2, FR-H3 is located between CDR-H2 and CDR-H3, etc. It is noted that the Kabat numbering scheme shown places the insertion at H35A and H35B, so the ends of the Chothia CDR-H1 loop, when numbered using the Kabat numbering convention shown, vary between H32 and H34 depending on the length of the loop.

[0033] Thus, unless otherwise specified, the "CDRs" or "complementarity determining regions" of a given antibody or regions thereof, such as the variable regions thereof, or each identified CDR (e.g., CDR-H1, CDR-H2, CDR-H3), should be understood to encompass one (or a particular) complementarity determining region defined by any of the aforementioned schemes. For example, when a particular CDR (e.g., CDR-H3) is described as containing the amino acid sequence of the corresponding CDR in a given sdAb amino acid sequence, it is understood that such CDR has the sequence of the corresponding CDR (e.g., CDR-H3) in the sdAb defined by any of the aforementioned schemes. It is understood that any antibody, such as an sdAb, includes CDRs, and such are identified according to any of the other aforementioned numbering schemes or other numbering schemes known to those of skill in the art.

[0034] As used herein, "Fv" refers to the minimum antibody fragment containing a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In this configuration, the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three hypervariable regions specific for an antigen) may have the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0035] As used herein, a "single-chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0036] As used herein, "VHH" or "VHH antibody" refers to a single domain antibody consisting of the variable region of the heavy chain of an IgG antibody. For example, the terms "VHH" and "VHH antibody" can refer to the antigen-binding domain of a heavy chain IgG (hcIgG) molecule produced by mammals of the Camelidae family (e.g., llamas, camels, and alpacas).

[0037] As used herein, the term "specifically binds" to a target molecule such as an antigen means that a binding molecule, e.g., a single domain antibody (sdAb), reacts or associates with a particular target molecule more frequently, rapidly, for a longer duration, and / or with higher affinity than alternative molecules. A binding molecule, such as an sdAb or scFv, "specifically binds" to a target molecule if it binds with higher affinity, avidity, ease, and / or duration than it binds to other molecules. It is understood that a binding molecule, e.g., an sdAb or scFv, that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific binding" does not necessarily require (but can include) exclusive binding.

[0038] As used herein, the term "cell surface molecule" refers to a molecule present on the outer surface of a cell. In some embodiments, the cell surface molecule is an antigen, as defined and disclosed herein. In some embodiments, the cell surface molecule is, for example, a non-immunogenic peptide, glycopeptide, polypeptide, glycopolypeptide, polynucleotide, polysaccharide, lipid, etc.

[0039] As used herein, "percent amino acid sequence identity" and "homology" with respect to peptide, polypeptide, or antibody sequences are used interchangeably and are defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in another peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve maximum sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared.

[0040] Amino acid substitutions can include, but are not limited to, replacing one amino acid in a polypeptide with another. Exemplary substitutions are shown in Table 2. Amino acid substitutions are introduced into the antibody of interest and the products are screened for the desired activity, e.g., retained / improved binding.

[0041] Amino acids can be grouped according to common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Tyr, Tyr, Phe.

[0042] Non-conservative substitutions will involve exchanging one member of these classes for another. The term "corresponding to" with respect to a nucleotide or amino acid position of a sequence as shown in the sequence listing refers to a nucleotide or amino acid position identified by alignment with a target sequence based on structural sequence alignment or by using a standard alignment algorithm, such as the GAP algorithm. For example, corresponding residues of similar sequences (e.g., fragments or species variants) can be determined by alignment with a reference sequence by structural alignment methods. By aligning sequences, those skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guides.

[0043] The term "construct" refers to any polynucleotide containing a recombinant nucleic acid molecule. A construct is present in a vector (e.g., a bacterial vector, a viral vector) and is integrated into a genome. A "vector" is a nucleic acid molecule that can introduce a specific nucleic acid sequence into a cell or another nucleic acid sequence, or serve as a means of transporting another nucleic acid molecule. A vector is, for example, a plasmid, a cosmid, a virus, an RNA vector, or a linear or circular DNA or RNA molecule that can include chromosomal, non-chromosomal, semisynthetic, or synthetic nucleic acid molecules. Exemplary vectors are those capable of autonomous replication (episomal vectors), those capable of delivering a polynucleotide into a cellular genome (e.g., viral vectors), or those capable of expressing a linked nucleic acid molecule (expression vectors).

[0044] As used herein, "polypeptide" refers to a polymer comprising amino acids linked together. In some embodiments, a polypeptide is a linear polymer of nucleic acid in a chain. In some embodiments, a polypeptide is a polymer of nucleic acid folded into a structure or shape.

[0045] The terms "hypoimmunogenic," "hypoimmunogenic," "hypoimmunogenic," "hypoimmune," or "hypoimmune" are used interchangeably to refer to cells that have a lower tendency to be immunorejected by a subject into which they are transplanted. For example, compared to unaltered or unmodified wild-type cells, such hypoimmunogenic cells have a reduced tendency to be immunorejected by a subject into which they are transplanted by about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or more. In some examples described herein, genome editing techniques are used to regulate the expression of MHC I and / or MHC II genes, thereby generating hypoimmunogenic cells. In other examples described herein, tolerance-inducing factors, when introduced into and expressed in cells, can regulate or affect the ability of the cells to be recognized by the host immune system, thereby conferring hypoimmunogenicity. The low immunogenicity of cells is determined by assessing the cells' ability to induce adaptive and innate immune responses. Such immune responses can be measured using assays recognized by those skilled in the art, for example, by measuring the effect of hypoimmunogenic cells on T cell proliferation, T cell activation, T cell killing, NK cell proliferation, NK cell activation, and macrophage activity. Hypoimmunogenic cells may undergo reduced killing by T cells and / or NK cells after administration to a subject, or exhibit reduced macrophage engulfment compared to unmodified or wild-type cells. In some embodiments, hypoimmunogenic cells induce a reduced or attenuated immune response in a recipient subject compared to corresponding unmodified wild-type cells. In some embodiments, hypoimmunogenic cells are non-immunogenic or fail to induce an immune response in a recipient subject.

[0046] The term "isolated," as used herein, refers to a molecule that is separated from at least some of the components typically found or produced in nature. For example, a polypeptide is referred to as "isolated" if it is separated from at least some of the components of the cell in which it is produced. If the polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell in which it was produced is considered to "isolate" the polypeptide. Similarly, a polynucleotide is referred to as "isolated" if it is not part of a larger polynucleotide (e.g., in the case of a DNA polynucleotide, genomic DNA, mitochondrial DNA, etc.) in which it is typically found in nature or if it is separated from at least some of the components of the cell in which it was produced. Thus, a DNA polynucleotide contained in a vector within a host cell is referred to as "isolated."

[0047] As used herein, "lipid particle" refers to any biological or synthetic particle containing a bilayer of amphiphilic lipids surrounding a lumen or cavity. Typically, lipid particles do not contain a nucleus. Examples of lipid particles include nanoparticles, virus-derived particles, or cell-derived particles. Such lipid particles include, but are not limited to, virus particles (e.g., lentiviral particles), virus-like particles, viral vectors (e.g., lentiviral vectors), exosomes, enucleated cells, vesicles (e.g., microvesicles, membrane vesicles, extracellular membrane vesicles, plasma membrane vesicles, and giant plasma membrane vesicles), apoptotic bodies, mitoparticles, pyrenocytes, or lysosomes. In some embodiments, the lipid particle is a fusosome. In some embodiments, the lipid particle is not a platelet.

[0048] As used herein, with respect to a protein, e.g., a G protein or an F protein, a "biologically active portion" refers to a portion of a protein that exhibits or retains an activity or property of the full-length protein. For example, a biologically active portion of an F protein, when combined with a G protein, retains fusion activity when each is embedded in a lipid bilayer. A biologically active portion of a G protein, when combined with an F protein, retains fusion activity when each is embedded in a lipid bilayer. In some embodiments, the retained activity includes 10% to 150% or more of the activity of the full-length or wild-type F protein or G protein. Examples of biologically active portions of F and G proteins include truncated forms of the cytoplasmic domain, e.g., truncated forms of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 22, 25, 30, 33, 34, 35, or more consecutive amino acids. See, e.g., Khetawat and Broder 2010 Virology Journal 7:312; Witting et al. 2013 Gene Therapy 20:997-1005; published international patent application WO / 2013 / 148327.

[0049] As used herein, "G protein" refers to the henipavirus envelope-associated glycoprotein G or a biologically active portion thereof. "F protein" refers to the henipavirus fusion protein F or a biologically active portion thereof. In some embodiments, the F and G proteins are from Hendra (HeV) or Nipah (NiV) viruses and are wild-type proteins or variants thereof that exhibit reduced binding to their native binding partners. The F (fusion) and G (association) glycoproteins mediate cell entry of Nipah viruses. The G protein initiates infection by binding to the cell surface receptor ephrin-B2 (EphB2) or EphB3. Subsequent release of the viral genome into the cytoplasm is mediated by the action of the F protein, which induces fusion of the viral envelope with the cell membrane. In some embodiments, the transduction efficiency of targeted lipid particles is improved by engineering hyperfusion mutations in one or both of the F protein (e.g., NiV-F) and G protein (e.g., NiV-G).

[0050] As used herein, "fusosome" refers to a particle containing a bilayer of amphiphilic lipids surrounding a lumen or cavity and a fusogen that interacts with the amphiphilic lipid bilayer. In some embodiments, the fusosome contains nucleic acid. In some embodiments, the fusosome is a membrane-enclosed preparation. In some embodiments, the fusosome is derived from a source cell. As used herein, "fusosome composition" refers to a composition comprising one or more fusosomes.

[0051] As used herein, "fusogen" refers to an agent or molecule that creates an interaction between two membrane-enclosed lumens. In embodiments, the fusogen facilitates membrane fusion. In other embodiments, the fusogen creates a connection, e.g., a pore, between two lumens (e.g., the lumen of the retroviral vector and the cytoplasm of the target cell). In some embodiments, the fusogen comprises a complex of two or more proteins, e.g., none of the proteins alone contains fusogenic activity. In some embodiments, the fusogen comprises a targeting domain.

[0052] As used herein, a "retargeted fusogen" refers to a fusogen that includes a targeting site with a sequence that is not part of the naturally occurring form of the fusogen. In embodiments, the fusogen includes a targeting site that is different from the targeting site in the naturally occurring form of the fusogen. In embodiments, the naturally occurring form of the fusogen lacks a targeting domain, and the retargeted fusogen includes a targeting site that is not present in the naturally occurring form of the fusogen. In embodiments, the fusogen is modified to include a targeting site. In embodiments, the fusogen includes one or more sequence modifications outside the targeting site compared to the naturally occurring form of the fusogen, e.g., in the transmembrane domain, the fusogenically active domain, or the cytoplasmic domain.

[0053] As used herein, a "targeting envelope protein" refers to a polypeptide containing a henipavirus G protein (G protein) linked to a single domain antibody (sdAb) variable domain, e.g., a VL or VH sdAb, scFv, nanobody, camelid VHH domain, shark IgNAR, or fragment thereof, that targets a molecule on a desired cell type. In some such embodiments, the linkage can be direct or indirect via a linker, such as a peptide linker. A "targeting envelope protein" may also be referred to as a "fusion protein" comprising a G protein and an antibody or antigen-binding fragment of the present disclosure, wherein the antibody or antigen-binding fragment is fused to the C-terminus of the G protein, or biologically active portion thereof.

[0054] As used herein, "targeted lipid particle" refers to a lipid particle containing a targeting envelope protein embedded in the lipid bilayer, such as a targeting envelope protein that targets CD4 or CD8. Such targeted lipid particles can be any lipid particle disclosed herein, such as a virus particle, a virus-like particle, a nanoparticle, a vesicle, an exosome, a dendrimer, a lentivirus, a viral vector, an enucleated cell, a microvesicle, a membrane vesicle, an extracellular membrane vesicle, a plasma membrane vesicle, and a giant plasma membrane vesicle, an apoptotic body, a mitoparticle, a pyrenocyte, a lysosome, another membrane-enclosed vesicle, or a lentiviral vector, a virus-based particle, a virus-like particle (VLP), or a cell-derived particle.

[0055] As used herein, "retroviral nucleic acid" refers to a nucleic acid that contains at least the minimum sequence requirements for packaging into a retrovirus or retroviral vector, alone or in combination with a helper cell, helper virus, or helper plasmid. In some embodiments, the retroviral nucleic acid further comprises or encodes an exogenous material, a positive target cell-specific regulatory element, a non-target cell-specific regulatory element, or a negative TCSSE. In some embodiments, the retroviral nucleic acid comprises one or more (e.g., all) of a 5' LTR (e.g., to facilitate integration), a U3 (e.g., to activate viral genome RNA transcription), an R (e.g., a Tat binding region), a U5, a 3' LTR (e.g., to facilitate integration), a packaging site (e.g., psi (Ψ)), and an RRE (e.g., to bind Rev and facilitate nuclear export). Retroviral nucleic acid can comprise RNA (e.g., as part of a virion) or DNA (e.g., when introduced into a source cell or after reverse transcription in a recipient cell). In some embodiments, the retroviral nucleic acid is packaged using a helper cell, helper virus, or helper plasmid containing one or more (eg, all) of gag, pol, and env.

[0056] As used herein, "target cell" refers to a cell type to which it is desired that the targeted lipid particle deliver an exogenous substance. In embodiments, the target cell is a specific tissue type or class of cell, such as an immune effector cell, e.g., a T cell. In some embodiments, the target cell is a diseased cell, e.g., a cancer cell. In some embodiments, the fusogen, e.g., a retargeted fusogen, results in preferential delivery of the exogenous substance to the target cell compared to non-target cells.

[0057] As used herein, "non-target cells" refers to cells of a type to which it is not desired for the targeted lipid particles to deliver exogenous material. In some embodiments, the non-target cells are cells of a particular tissue type or class. In some embodiments, the non-target cells are non-diseased cells, e.g., non-cancer cells. In some embodiments, the fusogen, e.g., the retargeted fusogen, results in lower delivery of exogenous material to non-target cells compared to target cells.

[0058] The term "effective amount," as used herein, refers to an amount of a pharmaceutical composition sufficient to significantly and favorably modify the symptoms and / or condition being treated (e.g., provide a favorable clinical response). The effective amount of the targeted lipid particles of the present disclosure for use in a pharmaceutical composition will vary depending on the particular condition being treated, the severity of the condition, the duration of treatment, the nature of any concomitant therapy, the particular lipid particles being used, the particular pharmaceutically acceptable excipient(s) and / or carrier(s) utilized, and similar factors within the knowledge and expertise of the attending physician.

[0059] "Exogenous material," as used herein with respect to targeted lipid particles, refers to a factor not contained in or encoded by a corresponding wild-type virus or fusogen produced from a corresponding wild-type source cell. In some embodiments, the exogenous material is not naturally occurring, e.g., a protein or nucleic acid having a sequence that is altered (e.g., by insertion, deletion, or substitution) compared to a naturally occurring protein. In some embodiments, the exogenous material is not naturally occurring in the source cell. In some embodiments, the exogenous material is naturally occurring in the source cell but exogenous to the virus. In some embodiments, the exogenous material is not naturally occurring in the recipient cell. In some embodiments, the exogenous material is naturally present in the recipient cell but not at the desired level or for the desired time. In some embodiments, the exogenous material comprises DNA, RNA, or protein.

[0060] As used herein, the term "operably linked" refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other.

[0061] As used herein, "nucleic acid" or "polynucleotide" refers to a polymeric compound comprising covalently linked nucleotides containing naturally occurring subunits (e.g., purine or pyrimidine bases). In some embodiments, the polynucleotide comprises a transgene. Purine bases include adenine and guanine, and pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid molecules include ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) (including cDNA, genomic DNA, and synthetic DNA), either single-stranded or double-stranded. A nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence.

[0062] As used herein, "transgene" refers to genetic material that has been transferred into a cell (e.g., a host cell). A transgene comprises a nucleic acid and, in some embodiments, is incorporated into a cell via any of the methods disclosed herein.

[0063] As used herein, "promoter" refers to a cis-regulatory DNA sequence that induces transcription of a gene when operably linked to a sequence encoding the gene. A promoter may contain one or more transcription factor binding sites. In some embodiments, a promoter works in conjunction with one or more enhancers located distal to the gene.

[0064] The term "safe harbor locus" refers to a genetic locus that allows for the safe expression of a transgene or exogenous gene. A safe harbor or genomic safe harbor is a site in the genome that can provide for the integration of new genetic material in a manner that allows the newly inserted genetic element to (i) function predictably and (ii) not cause modifications to the host genome that pose a risk to the host cell or organism. Exemplary "safe harbor" loci include the CCR5 gene, the CXCR4 gene, the PPP1R12C (also known as AAVS1) gene, the albumin gene, and the Rosa gene.

[0065] The term "safety switch" refers to a system for controlling the expression of a gene or protein of interest that, when down- or up-regulated, results in cellular elimination or death, for example, via recognition by the host immune system. Safety switches are designed to be or include exogenous molecules administered to prevent or mitigate adverse clinical events. Safety switches are operated by controlling expression at the DNA, RNA, and protein levels. Safety switches can include proteins or molecules that allow for the control of cellular activity in response to adverse events. In some embodiments, a safety switch refers to a factor (e.g., a protein, molecule, etc.) that binds to and targets specific cells for cell death or elimination. In some examples, a safety switch is a blocking factor that binds to a target protein on the surface of a cell, thereby eliciting an immune response. In one embodiment, the safety switch is a "kill switch" that is expressed in an inactive state; activation of the switch by a selective, externally provided substance results in the death of the cell expressing the safety switch. In one embodiment, the safety switch gene is cis-acting with respect to the gene of interest in the construct. Activation of the safety switch causes the cell to kill itself alone or itself and neighboring cells through apoptosis or necrosis.

[0066] The term "tolerance-inducing factor," as used herein, includes hypoimmunogenic factors, complement inhibitors, and other factors that modulate or affect (reduce) the ability of cells, upon administration, transplantation, or engraftment, to be recognized by the immune system of a host or recipient subject. Tolerance-inducing factors include, but are not limited to, CD16, CD24, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD200, CCL22, CTLA4-Ig, C1 inhibitory factor, FASL, IDO1, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IL-10, IL-35, PD-L1, Serpinb9, CC121, Mfge8, A20 / TNFAIP3, CCL21, CD16 Fc receptor, CD27, CR1, DUX4, H2-M3 (HLA-G), HLA-F, IL15-RF, MANF, IL-39, and B2M-HLA-E.

[0067] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells, including solutions, suspensions, liquids, powders, pastes, aqueous, non-aqueous, or any combination thereof.

[0068] As used herein, the term "pharmaceutically acceptable" refers to a substance, such as a carrier or diluent, that does not interfere with the biological activity or properties of the therapeutic compound and that is relatively non-toxic, i.e., the substance may be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0069] As used herein, the term "pharmaceutical composition" refers to a mixture of at least one targeted lipid particle of the present disclosure with other chemical components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and / or excipients. The pharmaceutical composition facilitates administration of the targeted lipid particle to an organism. Multiple techniques for administering the targeted lipid particles of the present disclosure exist in the art, including, but not limited to, intravenous, oral, aerosol, parenteral, ocular, pulmonary, and topical administration.

[0070] "Disease" or "disorder," as used herein, refers to a condition for which treatment is necessary and / or desirable.

[0071] As used herein, the terms "treat," "treating," or "treatment" refer to ameliorating a disease or disorder, e.g., slowing or stopping or reducing the onset of a disease or disorder or reducing at least one of its clinical symptoms. For purposes of this disclosure, ameliorating a disease or disorder can include obtaining beneficial or desired clinical results, including, but not limited to, alleviating one or more symptoms, attenuating the extent of the disease, preventing or delaying the spread of the disease (e.g., metastasis, e.g., to the lungs or lymph nodes), preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, inhibiting the disease or disease progression, inhibiting or slowing the disease or its progression, halting its development, and remission (whether partial or total).

[0072] The terms "individual" and "subject" are used interchangeably herein to refer to animals; e.g., mammals. The terms include humans and veterinary animals. In some embodiments, methods are provided for treating animals, including, but not limited to, humans, rodents, monkeys, cats, dogs, horses, cows, pigs, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets. The animals are male or female and of any suitable age, including infants, juveniles, adolescents, adults, and geriatrics. In some examples, an "individual" or "subject" refers to an animal in need of treatment for a disease or disorder. In some embodiments, an animal receiving treatment is a "patient," which refers to the fact that the animal has been identified as having or at sufficient risk of developing a disorder related to the treatment. In some embodiments, the animal is a human, e.g., a human patient.

[0073] The terms "treat," "treating," and "treatment," as used herein in reference to cancer, refer to partially or completely alleviating cancer; preventing cancer; reducing the likelihood of cancer occurrence or recurrence; slowing the progression or development of cancer; eliminating, reducing, or slowing the development of one or more symptoms associated with cancer; or increasing cancer progression or overall survival. For example, "treating" can refer to preventing or slowing an existing cancer from growing larger; preventing or slowing the formation or metastasis of cancer; and / or slowing the onset of certain symptoms of cancer. In some embodiments, the terms "treat," "treating," or "treatment" mean that a subject has a reduced number or size of cancer cells compared to a subject to which the treatment is not administered. In some embodiments, the terms "treat," "treating," or "treatment" mean that one or more symptoms of cancer are alleviated in a subject who receives a treatment disclosed and described herein compared to a subject who does not receive such treatment.

[0074] All publications, patents, and patent applications cited herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Furthermore, each cited publication, patent, or patent application is incorporated by reference herein to disclose and describe the subject matter in connection with which the publication is cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the technology described herein is not entitled to antedate such publication by prior art. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0075] Before the present technology is further described, it is to be understood that the technology is not limited to the particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims. It should also be understood that the headings used herein are not limiting, but are merely intended to provide orientation to the reader, but that the subject matter generally applies to the technology disclosed herein.

[0076] CD19-specific polypeptides Described herein are novel polypeptides that specifically target and bind to human CD19. In some embodiments, the polypeptides can interact with cynomolgus monkey (or "cyno") or M. nemestrina CD19. In some embodiments, the polypeptides are antibodies or antigen-binding fragments thereof. The present disclosure also provides polynucleotides, vectors, and host cells encoding the polypeptides, and methods of using the polypeptides. In some embodiments, for example, the polypeptides are fused to henipavirus glycoprotein G for targeted binding and transduction of cells. In some embodiments, the polypeptides comprise an antigen-binding region that specifically binds to CD19.

[0077] Sequences for exemplary polypeptides of the present disclosure comprising antigen-binding regions using the Kabat numbering scheme are shown in Tables 3-4 below. In some embodiments, the antigen-binding region comprises one or more heavy chain complementarity-determining regions (HCDRs). In some embodiments, the antigen-binding region comprises one or more light chain complementarity-determining regions (LCDRs). In some embodiments, the antigen-binding region comprises a heavy chain variable region (VH). In some embodiments, the antigen-binding region comprises a light chain variable region (VL). Sequences for exemplary HCDRs of the present disclosure are shown in Table 3. Sequences for exemplary LCDRs of the present disclosure are shown in Table 4.

[0078] Sequences for the disclosed VH and VL domains are provided in Tables 5-6. Tables 7-10 provided herein show the CDR sequences of the disclosed polypeptides using both the Chothia and IMGT numbering schemes.

[0079] In some embodiments, a polypeptide capable of binding to CD19 is disclosed. In some embodiments, the polypeptide comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and the light chain variable region comprises three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3). In some embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequence of any one of SEQ ID NOs: set forth in Tables 3, 7, and 9, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequence of any one of SEQ ID NOs: set forth in Tables 4, 8, and 10. In some embodiments, the heavy chain variable region (VH) comprises the amino acid sequence of any one of SEQ ID NOs: 19-21 (Table 5), and the light chain variable region (VL) comprises the amino acid sequence of any one of SEQ ID NOs: 22-24 (Table 6).

[0080] In some embodiments, the polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 19-21.

[0081] In some embodiments, the polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 22-24.

[0082] In some embodiments, the polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 19-21 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 22-24.

[0083] In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NOs: 1, 4, 7, 10, 13, and 16.

[0084] In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NOs: 2, 5, 8, 11, 14, and 17.

[0085] In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NOs: 3, 6, 9, 12, 15, and 18.

[0086] In some embodiments, the polypeptide is an antibody or antigen-binding fragment thereof disclosed herein.

[0087] Polypeptides whose amino acid sequences differ insubstantially from those shown in Tables 3-6 are encompassed within the scope of the present disclosure. Typically, this involves one or more conservative amino acid substitutions with amino acids having similar charge, hydrophobicity, or stereochemical characteristics in the antigen-binding site or framework without adversely altering the properties of the polypeptide. Conservative substitutions can also be made to improve polypeptide properties, such as stability or affinity. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions are made in the amino acid sequence. For example, a "conservative amino acid substitution" can involve the substitution of a native amino acid residue with a non-native residue that has little or no effect on the polarity or charge of the amino acid residue at that position. The desired amino acid substitution can be determined by one of skill in the art at the time such a substitution is desired. For example, amino acid substitutions can be used to identify key residues in a molecular sequence or to increase or decrease the affinity of the molecules described herein. The following eight groups contain amino acids that are conservative amino acid substitutions for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M).

[0088] In some embodiments, the polypeptide binds to human CD 19. In some embodiments, the polypeptide is an antibody or antigen-binding fragment that specifically binds to CD19 disclosed herein.

[0089] In some embodiments, the polypeptide has an affinity constant (K D ) binds to human CD19. In some embodiments, Dis about 5 nM to about 500 nM, about 6 nM to about 10 nM, about 11 nM to about 20 nM, about 25 nM to about 40 nM, about 40 nM to about 60 nM, about 70 nM to about 90 nM, about 100 nM to about 120 nM, about 125 nM to about 140 nM, about 145 nM to about 160 nM, about 170 nM to about 200 nM, about 210 nM to about 250 nM, about 260 nM to about 300 nM, about 310 nM to about 350 nM, about 360 nM to about 400 nM, about 410 nM to about 450 nM, and about 460 nM to about 500 nM. In some embodiments, the polypeptide has an affinity constant (K) of 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, or 10 nM or less. D In some embodiments, the polypeptide binds to human CD19 with a binding affinity (K ) equivalent to human CD19 and cynomolgus monkey, M. mulatta (rhesus monkey), or M. nemestrina CD19. D ) to join them.

[0090] In some embodiments, the polypeptide binds to cynomolgus monkey, M. mulatta (rhesus monkey), or M. nemestrina CD19. In some embodiments, the polypeptide binds to CD19 of mouse, dog, pig, etc. In some embodiments, the polypeptide binds to CD19 of cynomolgus monkey or M. nemestrina CD19. D is about 5 nM to about 500 nM, about 6 nM to about 10 nM, about 11 nM to about 20 nM, about 25 nM to about 40 nM, about 40 nM to about 60 nM, about 70 nM to about 90 nM, about 100 nM to about 120 nM, about 125 nM to about 140 nM, about 145 nM to about 160 nM, about 170 nM to about 200 nM, about 210 nM to about 250 nM, about 260 nM to about 300 nM, about 310 nM to about 350 nM, about 360 nM to about 400 nM, about 410 nM to about 450 nM, and about 460 nM to about 500 nM. In some embodiments, the polypeptide has an affinity constant (K) of 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, or 10 nM or less.D ) binds to cynomolgus monkey or M. nemestrina CD19.

[0091] A polypeptide that specifically binds to CD19 refers to a polypeptide that binds preferentially to CD19 over other antigen targets, respectively. As used herein, the term is interchangeable with "anti-CD19" polypeptide or "polypeptide that binds to CD19." In some embodiments, a polypeptide capable of binding to CD19 may bind to its antigen with higher affinity than others. In some embodiments, a polypeptide capable of binding to CD19 has a binding affinity of at least about 10, as measured, for example, by surface plasmon resonance or other methods known to those skilled in the art. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 or more (or any value in between) K D can bind to its antigen.

[0092] In some embodiments, the polypeptide is bispecific. In some embodiments, the bispecific polypeptide comprises an antigen-binding region that specifically binds CD19 as disclosed herein and an antigen-binding region that specifically binds CD3, 4-1BB, IL-6, NKG2D, Fc-gamma-RIIIA (CD16), APRIL, CD38, TACI, Fc-gamma-RIIIA (CD16) and NKG2D, CD3 and serum albumin, CD47 and TACI, or CD3 and GPRC5D. In some embodiments, the antigen-binding region comprises an antibody or antibody-binding fragment thereof.

[0093] In some embodiments, the polypeptide is conjugated. In some embodiments, the polypeptide is a polypeptide-drug conjugate, and the polypeptide that specifically binds to CD19 disclosed herein is conjugated to a therapeutic or diagnostic agent. In some embodiments, the polypeptide is conjugated to a tag for detection. In some embodiments, the polypeptide is conjugated to a conjugate that improves polypeptide stability. In some embodiments, the polypeptide is conjugated to a cleavable linker, which allows another molecule to be conjugated to the polypeptide. In some embodiments, the polypeptide is conjugated to a nanoparticle.

[0094] Some embodiments of the present disclosure are isolated polynucleotides encoding any of the polypeptides of the present disclosure. While certain exemplary polynucleotides are disclosed herein, other polynucleotides encoding the polypeptides of the present disclosure, taking into account the degeneracy of the genetic code or codon preference in a given expression system, are also within the scope of the present disclosure. The polynucleotide sequence encoding the antigen-binding region of the polypeptide of the present disclosure is operably linked to one or more regulatory elements, such as a promoter and an enhancer, that enable expression of the nucleotide sequence in the intended host cell. In some embodiments, the polynucleotide is a cDNA.

[0095] Some embodiments of the present disclosure are vectors comprising the polynucleotides of the present disclosure. In some embodiments, such vectors are plasmid vectors, viral vectors, vectors for baculovirus expression, transposon-based vectors, or any other vector suitable for introducing the polynucleotides of the present disclosure into a given organism or genetic background by any means. In some embodiments, the vectors are polycistronic. For example, polynucleotides encoding the light and heavy chain variable regions of a polypeptide of the present disclosure, optionally linked to constant regions, are inserted into an expression vector. The light and heavy chains are cloned in the same or different expression vectors. DNA segments encoding immunoglobulin chains are operably linked to control sequences in the expression vector(s) that ensure expression of the immunoglobulin polypeptide. Such control sequences include signal sequences, promoters (e.g., naturally associated or heterologous promoters), enhancer elements, and transcription termination sequences, and are selected to be compatible with the host cell selected to express the polypeptide. Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the polypeptide encoded by the incorporated polynucleotide.

[0096] Suitable expression vectors are typically replicable in the host organism either as episomes or as an integrated part of the host chromosomal DNA. Expression vectors generally contain a selection marker such as ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance to enable detection of those cells transformed with the desired DNA sequence. Suitable vectors, promoters, and enhancer elements are known in the art, and many are commercially available for generating recombinant constructs of interest.

[0097] Some embodiments of the present disclosure are host cells containing the vectors of the present disclosure. The term "host cell" refers to a cell into which a vector has been introduced. It is understood that the term host cell is intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Such host cells include eukaryotic, prokaryotic, plant, or archaeal cells. Escherichia coli, bacilli such as Bacillus subtilis, and other Enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species are examples of prokaryotic host cells. Other microorganisms, such as yeast, are also useful for expression. Saccharomyces (e.g., S. cerevisiae) and Pichia are examples of suitable yeast host cells. Exemplary eukaryotic cells include cells of mammalian, insect, avian, or other animal origin.

[0098] CD19 specific antibody Described herein are novel antibodies and antigen-binding fragments thereof that specifically target and bind to human CD19. In some embodiments, the antibodies or antigen-binding fragments thereof can interact with cynomolgus monkey (or "cyno") or M. nemestrina CD19. In some embodiments, the antibodies or antigen-binding fragments thereof are single-chain variable fragments (scFv) composed of antigen-binding domains derived from the heavy (VH) and light (VL) chains of an IgG molecule and connected via a linker domain. In some embodiments, the antibodies or antigen-binding fragments thereof are single-domain antibodies (sdAbs) composed of antigen-binding domains derived from the heavy (VH) and light (VL) chains of an IgG molecule. In some embodiments, the antibodies or antigen-binding fragments thereof are VHHs corresponding to the VHs of the IgG molecule. The present disclosure also provides polynucleotides, vectors, and host cells encoding the antibodies and fragments thereof, as well as methods of using the antibodies or antigen-binding fragments thereof. In some embodiments, for example, the antibodies or antigen-binding fragments thereof are fused to henipavirus glycoprotein G for targeted binding and transduction of cells.

[0099] Sequences for exemplary antibodies and antigen-binding fragments of the disclosure using the Kabat numbering scheme are shown below in Tables 3-4. Sequences for exemplary HCDRs of the disclosure are shown in Table 3. Sequences for exemplary LCDRs of the disclosure are shown in Table 4.

[0100] Sequences for the disclosed VH and VL domains are provided in Tables 5-6. Tables 7-10 provided herein show the CDR sequences of the disclosed antibodies and antigen-binding fragments thereof using both the Chothia and IMGT numbering schemes. The complete CD19 binder sequences of the variant CD19 scFvs and VHHs of the disclosure are shown in Table 11.

[0101] In some embodiments, disclosed is an antibody or antigen-binding fragment thereof capable of binding to CD19, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and the light chain variable region comprises three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3). In some embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequence of any one of SEQ ID NOs: set forth in Tables 3, 7, and 9, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequence of any one of SEQ ID NOs: set forth in Tables 4, 8, and 10. In some embodiments, the heavy chain variable region (VH) comprises the amino acid sequence of any one of SEQ ID NOs: 19-21 (Table 5), and the light chain variable region (VL) comprises the amino acid sequence of any one of SEQ ID NOs: 22-24 (Table 6).

[0102] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 19-21.

[0103] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VL having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 22-24.

[0104] In some embodiments, the antibody or antigen-binding fragment comprises a VH having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 19-21, and a VL having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 22-24.

[0105] In some embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 1, 4, 7, 10, 13, and 16, respectively.

[0106] In some embodiments, the antibody or antigen-binding fragment thereof comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 2, 5, 8, 11, 14, and 17, respectively.

[0107] In some embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 3, 6, 9, 12, 15, and 18, respectively.

[0108] In some embodiments, the single domain antibody is human or humanized. In some embodiments, the single domain antibody or fragment thereof is naturally occurring. In some embodiments, the single domain antibody or fragment thereof is synthetic.

[0109] In some embodiments, single domain antibodies are antibodies whose complementarity determining regions are part of a single domain polypeptide. In some embodiments, single domain antibodies are antibody variable domains of only the heavy chain. In some embodiments, single domain antibodies do not include light chains.

[0110] In various embodiments, any of the antibodies or antigen-binding fragments described herein may comprise a heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region is an IgG, IgM, IgA, IgD, or IgE isotype, or a derivative or fragment thereof that retains at least one effector function of an intact heavy chain. In some embodiments, the heavy chain constant region is a human IgG isotype. In some embodiments, the heavy chain constant region is a human IgG1 or human IgG4 isotype. In some embodiments, the heavy chain constant region is a human IgG1 isotype. In some embodiments, the light chain constant region is a human kappa or lambda light chain, or a derivative or fragment thereof that retains at least one effector function of an intact light chain. In some embodiments, the light chain constant region is a human kappa light chain.

[0111] In various embodiments, any of the disclosed antibodies or antigen-binding fragments is a rodent antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a CDR-grafted antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof. In some embodiments, any of the disclosed antibodies or antigen-binding fragments comprises human or human-derived heavy and light chain variable regions comprising a human framework or a human framework with one or more backmutations. In various embodiments, any of the disclosed antibodies or antigen-binding fragments is a Fab, Fab', F(ab'), Fd, scFv, (scFv)2, scFv-Fc, VHH, or Fv fragment.

[0112] Antibodies whose heavy chain CDR, light chain CDR, VH, or VL amino acid sequences differ insubstantially from those shown in Tables 3-6 are encompassed within the scope of the present disclosure. Typically, this involves one or more conservative amino acid substitutions with amino acids having similar charge, hydrophobicity, or stereochemical characteristics in the antigen-binding site or framework without adversely altering the antibody's properties. Conservative substitutions may also be made to improve antibody properties, such as stability or affinity. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions are made in the VH or VL sequence. For example, a "conservative amino acid substitution" may involve the replacement of a native amino acid residue with a non-native residue that has little or no effect on the polarity or charge of the amino acid residue at that position. Desired amino acid substitutions are determined by those of skill in the art at the time such substitutions are desired. For example, amino acid substitutions are used to identify key residues in a molecular sequence or to increase or decrease the affinity of the molecules described herein. The following eight groups contain amino acids that are conservative amino acid substitutions for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M).

[0113] In some embodiments, the antibody or antigen-binding fragment thereof binds to human CD19. In some embodiments, the antibody or antigen-binding fragment thereof that binds to CD19 is a single-chain variable fragment (scFv). In embodiments involving a single polypeptide containing both a heavy chain variable region and a light chain variable region, both orientations of these variable regions are contemplated. In some embodiments, the heavy chain variable region is N-terminal to the light chain variable region, meaning that the heavy chain variable region is closer to the N-terminus of the polypeptide. In other embodiments, the light chain variable region is N-terminal to the heavy chain variable region, meaning that the light chain variable region is closer to the N-terminus of the polypeptide than the heavy chain variable region.

[0114] In some embodiments, the scFv binding protein comprises a linker. In some embodiments, the linker is between the heavy chain variable region (VH) and the light chain variable region (VL) (or vice versa). In some embodiments, the linker comprises the amino acid sequence of any one of GS, GGS, GGGS (SEQ ID NO: 227), GGGGS (SEQ ID NO: 147), GGGGGS (SEQ ID NO: 145), SEQ ID NOs: 165-166, and 32-33, or a combination thereof. Substitutions to introduce a new disulfide bond, for example, by making substitutions G44C in VH FR2 and G100C in VL FR4, are also within the scope of the present disclosure.

[0115] In some embodiments, the anti-CD19 antibody or antigen-binding fragment has an affinity constant (K D ) binds to human CD19. In some embodiments, Dis about 5 nM to about 500 nM, about 6 nM to about 10 nM, about 11 nM to about 20 nM, about 25 nM to about 40 nM, about 40 nM to about 60 nM, about 70 nM to about 90 nM, about 100 nM to about 120 nM, about 125 nM to about 140 nM, about 145 nM to about 160 nM, about 170 nM to about 200 nM, about 210 nM to about 250 nM, about 260 nM to about 300 nM, about 310 nM to about 350 nM, about 360 nM to about 400 nM, about 410 nM to about 450 nM, and about 460 nM to about 500 nM. In some embodiments, the anti-CD19 antibody or antigen-binding fragment has an affinity constant (K) of 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, or 10 nM or less. D In some embodiments, the anti-CD19 antibody or antigen-binding fragment binds to human CD19 with comparable binding affinity (K ) to human CD19 and cynomolgus monkey, M. mulatta (rhesus monkey), or M. nemestrina CD19. D ) to join them.

[0116] In some embodiments, the anti-CD19 antibody or antigen-binding fragment binds to cynomolgus monkey, M. mulatta (rhesus monkey), or M. nemestrina CD19. In some embodiments, the anti-CD19 antibody or antigen-binding fragment binds to CD19 of mouse, dog, pig, etc. In some embodiments, the K for cynomolgus monkey or M. nemestrina CD19 Dis about 5 nM to about 500 nM, about 6 nM to about 10 nM, about 11 nM to about 20 nM, about 25 nM to about 40 nM, about 40 nM to about 60 nM, about 70 nM to about 90 nM, about 100 nM to about 120 nM, about 125 nM to about 140 nM, about 145 nM to about 160 nM, about 170 nM to about 200 nM, about 210 nM to about 250 nM, about 260 nM to about 300 nM, about 310 nM to about 350 nM, about 360 nM to about 400 nM, about 410 nM to about 450 nM, and about 460 nM to about 500 nM. In some embodiments, the anti-CD19 antibody or antigen-binding fragment has an affinity constant (K) of 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, or 10 nM or less. D ) binds to cynomolgus monkey or M. nemestrina CD19.

[0117] An antibody or antigen-binding fragment thereof that specifically binds to CD19 refers to an antibody or binding fragment, respectively, that preferentially binds to CD19 over other antigen targets. As used herein, the term is interchangeable with "anti-CD19" antibody or "antibody that binds to CD19." In some embodiments, an antibody or binding fragment capable of binding to CD19 may bind to its antigen with higher affinity than others relative to the pair. In some embodiments, an antibody or binding fragment capable of binding to CD19 has a binding affinity of at least about 10, as measured, for example, by surface plasmon resonance or other methods known to those skilled in the art. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 or more (or any value in between) K D can bind to its antigen.

[0118] In some embodiments, the antibody or antigen-binding fragment thereof is bispecific. In some embodiments, the bispecific antibody or antigen-binding fragment comprises an antibody or antigen-binding fragment thereof that specifically binds to CD19 disclosed herein and an antigen or antibody-binding fragment thereof that specifically binds to CD3, 4-1BB, IL-6, NKG2D, Fc-gamma-RIIIA (CD16), APRIL, CD38, TACI, Fc-gamma-RIIIA (CD16) and NKG2D, CD3 and serum albumin, CD47 and TACI, or CD3 and GPRC5D.

[0119] In some embodiments, the antibody or antigen-binding fragment thereof is conjugated. In some embodiments, the antibody or antigen-binding fragment thereof is an antibody-drug conjugate, and the antibody or antigen-binding fragment thereof that specifically binds to CD19 disclosed herein is conjugated to a therapeutic or diagnostic agent. In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to a tag for detection. In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to a conjugate that improves the stability of the antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to a cleavable linker, which allows another molecule to be conjugated to the antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to a nanoparticle.

[0120] Some embodiments of the present disclosure are isolated polynucleotides encoding either the antibody heavy chain variable region or the antibody light chain variable region of the present disclosure. While certain exemplary polynucleotides are disclosed herein, other polynucleotides encoding the antibodies or antigen-binding fragments thereof of the present disclosure, taking into account the degeneracy of the genetic code or codon preference in a given expression system, are also within the scope of the present disclosure. The polynucleotide sequence encoding the VH or VL of the antibody or antigen-binding fragment thereof of the present disclosure, or a fragment thereof, is operably linked to one or more regulatory elements, such as a promoter and an enhancer, that enable expression of the nucleotide sequence in the intended host cell. In some embodiments, the polynucleotide is a cDNA.

[0121] Some embodiments of the present disclosure are vectors comprising polynucleotides of the present disclosure. In some embodiments, such vectors are plasmid vectors, viral vectors, vectors for baculovirus expression, transposon-based vectors, or any other vector suitable for introducing polynucleotides of the present disclosure into a given organism or genetic background by any means. In some embodiments, the vectors are polycistronic. For example, polynucleotides encoding the light and heavy chain variable regions of an antibody of the present disclosure, optionally linked to constant regions, are inserted into an expression vector. The light and heavy chains are cloned in the same or different expression vectors. DNA segments encoding immunoglobulin chains are operably linked to control sequences in the expression vector(s) that ensure expression of immunoglobulin polypeptides. Such control sequences include signal sequences, promoters (e.g., naturally associated or heterologous promoters), enhancer elements, and transcription termination sequences, and are selected to be compatible with the host cell chosen to express the antibody. In some embodiments, the polycistronic vector comprises one or more tolerance inducers, safety switches, additional antibodies or antigen-binding fragments thereof, or other regulatory elements disclosed herein. Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high level expression of the proteins encoded by the incorporated polynucleotides.

[0122] Suitable expression vectors are typically replicable in the host organism either as episomes or as an integrated part of the host chromosomal DNA. Expression vectors generally contain a selection marker such as ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance to enable detection of those cells transformed with the desired DNA sequence. Suitable vectors, promoters, and enhancer elements are known in the art, and many are commercially available for generating recombinant constructs of interest.

[0123] Some embodiments of the present disclosure are host cells containing the vectors of the present disclosure. The term "host cell" refers to a cell into which a vector has been introduced. It is understood that the term host cell is intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Such host cells include eukaryotic, prokaryotic, plant, or archaeal cells. Escherichia coli, bacilli such as Bacillus subtilis, and other Enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species are examples of prokaryotic host cells. Other microorganisms, such as yeast, are also useful for expression. Saccharomyces (e.g., S. cerevisiae) and Pichia are examples of suitable yeast host cells. Exemplary eukaryotic cells include cells of mammalian, insect, avian, or other animal origin.

[0124] CD19 chimeric antigen receptor In some embodiments, the provided disclosure relates to chimeric receptors, such as chimeric antigen receptors (CARs), that contain one or more domains that combine an antigen- or ligand-binding domain (e.g., an antibody or antigen-binding fragment thereof) that provides specificity for a desired antigen (e.g., a tumor antigen) with an intracellular signaling domain. In some embodiments, the intracellular signaling domain is a stimulatory or activating intracellular domain portion that provides a primary activation signal or primary signal, e.g., a T cell stimulatory or activation domain. In some embodiments, the intracellular signaling domain contains, or additionally contains, a costimulatory signaling domain to facilitate effector function. In some embodiments, the chimeric receptor, when genetically engineered into immune cells, can regulate T cell activity, and in some embodiments, regulate T cell differentiation or homeostasis, thereby resulting in genetically engineered cells with improved longevity, survival, and / or persistence in vivo, for use, for example, in adoptive cell therapy methods.

[0125] In some embodiments, the chimeric antigen receptor comprises an extracellular portion containing an antibody or antigen-binding fragment thereof comprising an antigen-binding domain. In some aspects, the chimeric antigen receptor comprises an extracellular portion containing an antibody or antigen-binding fragment thereof comprising an antigen-binding domain and an intracellular signaling domain. In some embodiments, the antibody or antigen-binding fragment thereof comprises an scFv.

[0126] In some embodiments, the antigen targeted by the antigen-binding domain is CD19. In some aspects, the antigen-binding domain of the recombinant receptor, e.g., CAR, binds to, e.g., specifically binds to, or specifically recognizes CD19, e.g., human CD19. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH and a VL derived from an antibody or antibody fragment specific for CD19 disclosed herein. In some embodiments, the antibody or antigen-binding fragment thereof is a human antibody, e.g., as described in U.S. Patent Publication No. US2016 / 0152723.

[0127] In some embodiments, the CAR is a CD19 CAR ("CD19-CAR"). In some of these embodiments, the polycistronic vector comprises an expression cassette containing a nucleotide sequence encoding a CD19 CAR or another CAR disclosed herein. CD19 is an immunoglobin (Ig) superfamily member expressed on cells of the B-cell lineage. CD19 is a biomarker for B-cell development. CD19 expression is associated with numerous cancers, such as B-cell lymphoma, acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL). In some embodiments, the CD19 CAR may comprise, in tandem, a signal peptide, an extracellular binding domain that specifically binds CD19, a hinge domain, a transmembrane domain, an intracellular costimulatory domain, and / or an intracellular signaling domain.

[0128] In some embodiments, the CD19-specific CAR includes an antibody or antigen-binding fragment thereof, a transmembrane domain, a costimulatory signaling domain, and a signaling domain. In some embodiments, the antibody or antigen-binding fragment thereof is an anti-CD19 single-chain antibody fragment (scFv) or single-domain antibody fragment (sdAb). Table 11 provides some non-limiting exemplary sequences of full-length CD19 scFv and sdAb sequences. In some embodiments, the CD19-specific CAR includes an anti-CD19 single-chain antibody fragment (scFv) or single-domain antibody fragment (sdAb), a transmembrane domain, such as one derived from human CD8α, a 4-1BB (CD137) costimulatory signaling domain, and a CD3ζ signaling domain. In some embodiments, the CAR is bispecific and is a CAR of human CD19 and CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD70, CD123, CD138, GPRC5D, LeY, NKG2D, WT1, GD2, HER2, EGFR, EGFRvIII, B7H3, PSMA, PSCA, CAIX, CD171, CEA, CSPG4, EPHA2 , FAP, FRα, IL-13Rα, mesothelin, MUC1, MUC16, ROR1, C-Met, CD133, Ep-CAM, GPC3, HPV16, IL13Ra2, MAGEA3, MAGEA4, MART1, NY-ESO, VEGFR2, α-folate, CD24, CD44v7 / 8, EGP-2, EGP-40, erb-B2, erb-B, FBP, fetal acetylcholine receptor, G D2 , G D3In some embodiments, the bispecific CAR specifically binds to another tumor antigen selected from anti-CD19, HMW-MAA, IL-11Rα, KDR, Lewis Y, L1-cell adhesion molecule, MADE-A1, carcinoembryonic antigen (h5T4), TAG-72, CD19 / 22, syndecan-1, or BCMA. scFv, CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD70, CD123, CD138, GPRC5D, LeY, NKG2D, WT1, GD2, HER2, EGFR, EGFRvIII, B7H3, PSMA, PSCA, CAIX, CD171, CEA, CSPG4, EPHA2, FAP, FRα, IL-13Rα, mesothelin, MUC1, MUC16, ROR1, C-Met, CD133, Ep-CAM, GPC3, HPV16, IL13Ra2, MAGEA3, MAGEA4, MART1, NY-ESO, VEGFR2, α-folate, CD24, CD44v7 / 8, EGP-2, EGP-40, erb-B2, erb-B, FBP, fetal acetylcholine receptor, G D2 , G D3 In some embodiments, the bispecific CAR includes an scFv that specifically binds to one of the following: anti-CD19, HMW-MAA, IL-11Rα, KDR, Lewis Y, L1-cell adhesion molecule, MADE-A1, carcinoembryonic antigen (h5T4), TAG-72, CD19 / 22, syndecan-1, or BCMA, a transmembrane domain, a costimulatory signaling domain, and a signaling domain. scFv, CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD70, CD123, CD138, GPRC5D, LeY, NKG2D, WT1, GD2, HER2, EGFR, EGFRvIII, B7H3, PSMA, PSCA, CAIX, CD171, CEA, CSPG4, EPHA2, FAP, FRα, IL-13Rα, mesothelin, MUC1, MUC16, ROR1, C-Met, CD133, Ep-CAM, GPC3, HPV16, IL13Ra2, MAGEA3, MAGEA4, MART1, NY-ESO, VEGFR2, α-folate, CD24, CD44v7 / 8, EGP-2, EGP-40, erb-B2, erb-B, FBP, fetal acetylcholine receptor, G D2 , GD3 , HMW-MAA, IL-11Rα, KDR, Lewis Y, L1-cell adhesion molecule, MADE-A1, carcinoembryonic antigen (h5T4), TAG-72, CD19 / 22, syndecan-1, or BCMA, a transmembrane domain, such as that derived from human CD8α, a 4-1BB (CD137) costimulatory signaling domain, and a CD3ζ signaling domain.

[0129] In some embodiments, the signal peptide of the CD19 CAR comprises a CD8α signal peptide. In some embodiments, the CD8α signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:28 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:28. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the IgK signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:29 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:29. In some embodiments, the signal peptide comprises a GMCSFR-α or CSF2RA signal peptide. In some embodiments, the GMCSFR-α or CSF2RA signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:30 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:30. In some embodiments, the signal peptide comprises an immunoglobulin heavy chain signal peptide. In some embodiments, the immunoglobulin heavy chain signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:31 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:31. Table 12 provides some non-limiting examples of sequences of exemplary signal peptides.

[0130] In some embodiments, the extracellular binding domain of the CD19 CAR is specific for CD19, e.g., human CD19. The extracellular binding domain of the CD19 CAR is codon-optimized for expression in a host cell or to have a variant sequence to increase the function of the extracellular binding domain.

[0131] In some embodiments, the extracellular binding domain comprises an immunologically active portion of an immunoglobulin molecule, e.g., an scFv. In some embodiments, the extracellular binding domain of the CD19 CAR is derived from an antibody specific for CD19, including, for example, any one of the antibodies or antigen-binding fragments thereof disclosed herein, belantamab, erlanatamab, teclistamab, LCAR-B38M, and siltacabtagene. In any of these embodiments, the extracellular binding domain of the CD19 CAR is derived from the V of any of the antibodies or antigen-binding fragments thereof disclosed herein. H , V L , and / or one or more CDRs.

[0132] In some embodiments, the CD19 CAR extracellular binding domain comprises an scFv. An scFv is a heavy chain variable region (V) connected by a (G4S)3 linker or by a Whitlow linker. H ) and the light chain variable region (V L), the amino acid sequences of which are set forth in SEQ ID NOS: 32 and 33, respectively, in Table 13. In some embodiments, the CD19-specific extracellular binding domain comprises or consists of an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NOS: 25, 26, or 27, or the amino acid sequence set forth in SEQ ID NOS: 25-27 or 228-230 in Table 11. In some embodiments, the CD19-specific extracellular binding domain may comprise one or more heavy chain CDRs having the amino acid sequence set forth in Table 3 and one or more light chain CDRs having the amino acid sequence set forth in Table 4. In some embodiments, the CD19-specific extracellular binding domain may comprise a heavy chain having the amino acid sequence set forth in Table 5. In some embodiments, the CD19-specific extracellular binding domain may comprise a light chain having the amino acid sequence set forth in Table 6. In any of these embodiments, the CD19-specific scFv may comprise one or more CDRs comprising a sequence that contains one or more amino acid substitutions or is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any of the sequences specified. In any of these embodiments, the CD19-specific scFv may comprise one or more heavy chains (VH) comprising a sequence that contains one or more amino acid substitutions or is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any of the sequences specified.In any of these embodiments, the CD19-specific scFv may comprise one or more light chains (VL) comprising a sequence that contains one or more amino acid substitutions or is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any of the sequences specified. In some embodiments, the extracellular binding domain of the CD19 CAR comprises or consists of one or more CDRs described herein.

[0133] In some embodiments, the extracellular binding domain of a CD19 CAR comprises a single variable fragment of a heavy chain (VH) capable of binding to an epitope of CD19.

[0134] In some embodiments, the extracellular binding domain of the CD19 CAR comprises a single domain antibody (sdAb). In any of these embodiments, the CD19-specific extracellular binding domain may comprise one or more CDRs that contain one or more amino acid substitutions or that comprise a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any of the identified sequences. In some embodiments, the extracellular binding domain of the CD19 CAR comprises or consists of one or more CDRs described herein.

[0135] In some embodiments, the hinge domain of the CD19 CAR comprises a CD8α hinge domain, e.g., a human CD8α hinge domain. In some embodiments, the CD8α hinge domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 34 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, the hinge domain comprises a CD28 hinge domain, e.g., a human CD28 hinge domain. In some embodiments, the CD28 hinge domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 35. In some embodiments, the hinge domain comprises an IgG4 hinge domain, e.g., a human IgG4 hinge domain. In some embodiments, the IgG4 hinge domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 37 or SEQ ID NO: 38 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 37 or SEQ ID NO: 38. In some embodiments, the hinge domain comprises an IgG4 hinge-Ch2-Ch3 domain, e.g., a human IgG4 hinge-Ch2-Ch3 domain. In some embodiments, the IgG4 hinge-Ch2-Ch3 domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 39 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 39.Non-limiting exemplary sequences of hinge domains are shown in Table 14.

[0136] In some embodiments, the transmembrane domain comprises one selected from the group comprising the transmembrane region of TCRα, TCRβ, TCRζ, CD3ε, CD3γ, CD3δ, CD3ζ, CD4, CD5, CD8α, CD8β, CD9, CD16, CD28, CD45, CD22, CD33, CD34, CD37, CD40, CD40L / CD154, CD45, CD64, CD80, CD86, OX40 / CD134, 4-1BB / CD137, CD154, FcεRIγ, VEGFR2, FAS, FGFR2B, and functional variants thereof.

[0137] In some embodiments, the transmembrane domain of the CD19 CAR comprises a CD8α transmembrane domain, e.g., a human CD8α transmembrane domain. In some embodiments, the CD8α transmembrane domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:40, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:40. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, e.g., a human CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:41, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:41. In some embodiments, the CD28 transmembrane domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 42 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 42. Non-limiting exemplary sequences of transmembrane domains are shown in Table 15.

[0138] In some embodiments, the signaling domain(s) of the CAR comprise a costimulatory domain(s). For example, the signaling domain can contain a costimulatory domain. Or, the signaling domain can contain one or more costimulatory domains. In some embodiments, the signaling domain comprises a costimulatory domain. In other embodiments, the signaling domain comprises a costimulatory domain. In some embodiments, when the CAR comprises two or more costimulatory domains, the two costimulatory domains are not the same. In some embodiments, the costimulatory domain comprises two costimulatory domains that are not the same. In some embodiments, the costimulatory domain improves cytokine production, CAR-T cell proliferation, and / or CAR-T cell persistence during T cell activation. In some embodiments, the costimulatory domain improves cytokine production, CAR-T cell proliferation, and / or CAR-T cell persistence during T cell activation.

[0139] In some embodiments, the intracellular costimulatory domain of the CD19 CAR comprises a 4-1BB costimulatory domain, e.g., a human 4-1BB costimulatory domain. In some embodiments, the 4-1BB costimulatory domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:43 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:43. In some embodiments, the intracellular costimulatory domain comprises a CD28 costimulatory domain, e.g., a human CD28 costimulatory domain. In some embodiments, the CD28 costimulatory domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:44 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:44. In some embodiments, the CD3 zeta signaling domain of SEQ ID NO:46 can have a mutation at amino acid position 14, e.g., a glutamine (Q) to lysine (K) mutation (see SEQ ID NO:61). Non-limiting exemplary sequences of intracellular costimulatory and / or signaling domains are shown in Table 16.

[0140] In some embodiments, the intracellular signaling domain of the CD19 CAR comprises a CD3 zeta (ζ) signaling domain, e.g., a human CD3ζ signaling domain. In some embodiments, the CD3ζ signaling domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:46 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:46.

[0141] In some embodiments, the CD19 CAR has a corresponding amino acid sequence set forth in SEQ ID NO: 232, 234, 236, 238, 240, or 242, or is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 232, 234, 236, 238, 240, or 242. Non-limiting exemplary amino acid sequences of CD19 CARs are shown in Table 35.

[0142] In some embodiments, the CD19 CAR is encoded by the nucleotide sequence set forth in SEQ ID NO: 233, 235, 237, 239, 241, or 243, or is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 233, 235, 237, 239, 241, or 243. Non-limiting exemplary nucleotide sequences of CD19 CARs are shown in Table 35.

[0143] In some embodiments, the polycistronic vector comprises an expression cassette containing a nucleotide sequence encoding a CD19 CAR, including a CD19 CAR comprising, for example, any of the described CD19-specific extracellular binding domains, the CD8α hinge domain of SEQ ID NO: 34, the CD8α transmembrane domain of SEQ ID NO: 40, the 4-1BB costimulatory domain of SEQ ID NO: 43, the CD3ζ signaling domain of SEQ ID NO: 46, and / or variants thereof (i.e., having a sequence that is at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a disclosed sequence). In any of these embodiments, the CD19 CAR may additionally comprise a described signal peptide (e.g., a CD8α signal peptide).

[0144] In some embodiments, the polycistronic vector comprises an expression cassette containing a nucleotide sequence encoding a CD19 CAR, including a CD19 CAR comprising, for example, any of the described CD19-specific extracellular binding domains, the CD8α hinge domain of SEQ ID NO: 34, the CD8α transmembrane domain of SEQ ID NO: 40, the CD28 costimulatory domain of SEQ ID NO: 44, the CD3ζ signaling domain of SEQ ID NO: 46, and / or variants thereof (i.e., having a sequence that is at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a disclosed sequence). In any of these embodiments, the CD19 CAR may additionally comprise a described signal peptide.

[0145] In some embodiments, the recombinant receptor, e.g., the antibody portion of the CAR, further comprises a spacer between the transmembrane domain and the extracellular antigen-binding domain. In some embodiments, the spacer comprises at least a portion of an immunoglobulin constant region, e.g., a hinge region, e.g., an IgG4 hinge region, and / or a CH1 / CL and / or Fc region. In some embodiments, the constant region or portion is that of human IgG, e.g., IgG4 or IgG1. In some aspects, the portion of the constant region functions as a spacer region between the antigen-recognition component, e.g., an scFv, and the transmembrane domain. The spacer is of a length that provides increased cellular reactivity after antigen binding compared to the absence of the spacer. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, WO2014031687, U.S. Patent No. 8,822,647, or published application no. US2014 / 0271635. In some embodiments, the constant region or portion is that of a human IgG, eg, IgG4 or IgG1.

[0146] In some embodiments, the antigen receptor comprises an intracellular domain linked directly or indirectly to an extracellular domain. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises an ITAM. For example, in some aspects, the antigen recognition domain (e.g., the extracellular domain) is typically linked to one or more intracellular signaling components, e.g., signaling components that mimic activation via an antigen receptor complex, such as a TCR complex in the case of a CAR, and / or signaling via another cell surface receptor. In some embodiments, the chimeric receptor comprises a transmembrane domain linked or fused between the extracellular domain (e.g., an scFv) and the intracellular signaling domain. Thus, in some embodiments, the antigen binding component (e.g., an antibody) is linked to one or more transmembrane and intracellular signaling domains.

[0147] In one embodiment, a transmembrane domain naturally associated with one of the domains in a receptor, such as a CAR, is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domain to the transmembrane domain of the same or different surface membrane protein, so as to minimize interaction with other members of the receptor complex.

[0148] The transmembrane domain, in some embodiments, is derived from either natural or synthetic sources. If the source is natural, the domain, in some aspects, is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e., comprising at least the transmembrane region(s) thereof) the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. Alternatively, the transmembrane domain, in some embodiments, is synthetic. In some aspects, synthetic transmembrane domains comprise primarily hydrophobic residues, e.g., leucine and valine. In some aspects, triplets of phenylalanine, tryptophan, and valine will be found at each end of a synthetic transmembrane domain. In some embodiments, the linkage is by a linker, spacer, and / or transmembrane domain(s). In some embodiments, the transmembrane domain comprises the transmembrane portion of CD28.

[0149] In some embodiments, the extracellular domain and the transmembrane domain are linked directly or indirectly. In some embodiments, the extracellular domain and the transmembrane domain are linked by a spacer, e.g., any of those described herein. In some embodiments, the receptor contains the extracellular portion of the molecule from which the transmembrane domain is derived, e.g., the extracellular portion of CD28.

[0150] Intracellular signaling domains include domains that mimic or approximate signaling through a natural antigen receptor, signaling through such a receptor in combination with a costimulatory receptor, and / or signaling through a costimulatory receptor alone. In some embodiments, a short oligo- or polypeptide linker, e.g., a linker 2-10 amino acids in length, e.g., one containing glycine and serine, e.g., a glycine-serine doublet, is present to form the link between the transmembrane domain and the cytoplasmic signaling domain of the CAR.

[0151] T cell activation, in some embodiments, is described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences). In some embodiments, a CAR includes one or both of these signaling components.

[0152] Receptors, e.g., CARs, typically contain at least one intracellular signaling component(s). In some aspects, CARs contain a primary cytoplasmic signaling sequence that controls the primary activation of the TCR complex. The primary cytoplasmic signaling sequence, which acts in a stimulatory manner, may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. Examples of ITAMs that contain primary cytoplasmic signaling sequences include those derived from the CD3 zeta chain, FcR gamma, CD3 gamma, CD3 delta, and CD3 epsilon. In some embodiments, the cytoplasmic signaling molecule(s) in the CAR contain a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta.

[0153] In some embodiments, the receptor includes an intracellular component of the TCR complex, e.g., a TCR CD3 chain, e.g., the CD3-zeta chain, which mediates T cell activation and cytotoxicity. Thus, in some aspects, the antigen-binding moiety is linked to one or more cell signaling modules. In some embodiments, the cell signaling module includes a CD3 transmembrane domain, a CD3 intracellular signaling domain, and / or another CD transmembrane domain. In some embodiments, the intracellular component is or includes a CD3-zeta intracellular signaling domain. In some embodiments, the intracellular component is or includes a signaling domain from an Fc receptor gamma chain. In some embodiments, the receptor, e.g., a CAR, includes an intracellular signaling domain and further includes portions of one or more additional molecules, e.g., portions such as the transmembrane domain and / or hinge portion of CD8, CD4, CD25, or CD16. For example, in some aspects, a CAR or other chimeric receptor is a chimeric molecule of CD3-zeta (CD3-z) or Fc receptor g and a portion of one of CD8, CD4, CD25, or CD16.

[0154] In some embodiments, upon ligation of a CAR or other chimeric receptor, the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of an immune cell, e.g., a T cell engineered to express the CAR. For example, in some contexts, the CAR induces T cell function, e.g., cytolytic activity or T-helper activity, e.g., secretion of cytokines or other factors. In some embodiments, a truncated portion of the intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, e.g., if it transmits an effector function signal. In some embodiments, the intracellular signaling domain(s) include the cytoplasmic sequence of a T cell receptor (TCR), and in some aspects, that of a co-receptor that acts in concert with such receptor to initiate signaling following antigen receptor engagement.

[0155] In the context of natural TCRs, full activation usually requires not only signal transduction via the TCR but also a costimulatory signal. Thus, in some embodiments, the CAR also contains a component for generating a secondary or costimulatory signal to promote full activation. In other embodiments, the CAR does not contain a component for generating a costimulatory signal. In some aspects, an additional CAR is expressed in the same cell and provides a component for generating a secondary or costimulatory signal.

[0156] In some embodiments, the chimeric antigen receptor contains the intracellular domain of a T cell costimulatory molecule. In some embodiments, the CAR includes the signaling domain and / or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, OX40, DAP10, and ICOS. In some aspects, the same CAR includes both an activating component and a costimulatory component. In some embodiments, the chimeric antigen receptor contains an intracellular domain derived from a T cell costimulatory molecule or a functional variant thereof, such as between the transmembrane domain and the intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 41BB.

[0157] In some embodiments, the activation domain is contained within one CAR, while the costimulatory component is provided by another CAR that recognizes a different antigen. In some embodiments, the CAR includes an activating or stimulatory CAR and a costimulatory CAR, both of which are expressed on the same cell (see WO2014 / 055668). In some aspects, the cell includes one or more stimulatory or activating CARs and / or costimulatory CARs. In some embodiments, the cell further includes an inhibitory CAR (iCAR, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013)), e.g., a CAR that recognizes an antigen other than that associated with and / or specific for a disease or condition, such that the activation signal delivered via the disease-targeting CAR is attenuated or inhibited by binding of the inhibitory CAR to its ligand, e.g., reducing off-target effects.

[0158] In some embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain, hi some embodiments, the intracellular signaling domain comprises a chimeric CD28 and CD137 (4-1BB, TNFRSF9) costimulatory domain linked to a CD3 zeta intracellular domain.

[0159] In some embodiments, the CAR comprises one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., a primary activation domain, in the cytoplasmic portion. Exemplary CARs include the intracellular components of CD3-zeta, CD28, and 4-1BB.

[0160] In some embodiments, the intracellular signaling domain comprises a 4-1BB signaling domain and an intracellular component of a CD3-zeta signaling domain, hi some embodiments, the intracellular signaling domain comprises a CD28 signaling domain and an intracellular component of a CD3-zeta signaling domain.

[0161] In some embodiments, the CAR comprises an extracellular antigen-binding domain (e.g., an antibody or antibody fragment, e.g., an scFv) that binds to an antigen (e.g., a tumor antigen), a spacer (e.g., containing a hinge domain, e.g., any described herein), a transmembrane domain (e.g., any described herein), and an intracellular signaling domain (e.g., any intracellular signaling domain, e.g., a primary signaling domain or a costimulatory signaling domain described herein). In some embodiments, the intracellular signaling domain is or comprises a primary cytoplasmic signaling domain. In some embodiments, the intracellular signaling domain additionally comprises an intracellular signaling domain of a costimulatory molecule (e.g., a costimulatory domain). Non-limiting examples of exemplary components of a CAR are set forth in Table 17. In provided aspects, the sequence of each component in the CAR includes any combination listed in Table 17.

[0162] In some embodiments, the antigen receptor further comprises a marker, and / or cells expressing a CAR or other antigen receptor further comprise a surrogate marker, such as a cell surface marker used to confirm transduction or engineering of cells to express the receptor. In some aspects, the marker comprises all or a portion (e.g., a truncated form) of CD34, NGFR, or epidermal growth factor receptor, e.g., a truncated version of such a cell surface receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding a linker sequence, e.g., a cleavable linker sequence, e.g., T2A. For example, the marker, and optionally the linker sequence, can be any of those disclosed in published patent application WO2014031687. For example, the marker is truncated EGFR (tEGFR), optionally linked to a linker sequence, e.g., a T2A cleavable linker sequence.

[0163] In some embodiments, the marker is a molecule that is not naturally found on or on the surface of T cells, e.g., a cell surface protein, or portion thereof. In some embodiments, the molecule is a non-self molecule, e.g., a non-self protein, i.e., a molecule that is not recognized as "self" by the immune system of the host into which the cells will be adoptively transferred.

[0164] In some embodiments, the marker does not serve a therapeutic function and / or produce any effect other than being used as a marker for genetic manipulation, e.g., for selection of successfully manipulated cells. In other embodiments, the marker is a therapeutic molecule or a molecule that otherwise exerts some desired effect, e.g., a ligand that the cells encounter in vivo, e.g., a costimulatory or immune checkpoint molecule to enhance and / or attenuate the response of the cells upon adoptive transfer and encounter of the ligand.

[0165] In some embodiments, a CAR is referred to as a first-generation, second-generation, third-generation, and / or fourth-generation CAR. In some embodiments, a CAR disclosed herein is selected from the group comprising: (a) a first-generation CAR comprising an antigen-binding domain, a transmembrane domain, and a signaling domain; (b) a second-generation CAR comprising an antigen-binding domain, a transmembrane domain, and at least two signaling domains; (c) a third-generation CAR comprising an antigen-binding domain, a transmembrane domain, and at least three signaling domains; and (d) a fourth-generation CAR comprising an antigen-binding domain, a transmembrane domain, three or four signaling domains, and a domain that induces expression of a cytokine gene upon successful signaling of the CAR.

[0166] As described herein, a fourth-generation CAR may contain an antigen-binding domain, a transmembrane domain, three or four signaling domains, and a domain that induces expression of a cytokine gene upon successful signaling of the CAR. In some examples, the cytokine gene is an endogenous or exogenous cytokine gene of the hypoimmunogenic cell. In some embodiments, the cytokine gene encodes a proinflammatory cytokine. In some embodiments, the proinflammatory cytokine is selected from the group including IL-1, IL-2, IL-9, IL-12, IL-18, TNF, IFN-gamma, and functional fragments thereof. In some embodiments, the domain that induces expression of a cytokine gene upon successful signaling of the CAR is or includes a transcription factor or a functional domain or fragment thereof.

[0167] In some embodiments, the CAR contains an antibody disclosed herein, e.g., an antibody fragment, a transmembrane domain that is or contains the transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain that contains the signaling portion of CD28 or a functional variant thereof and the signaling portion of CD3-zeta or a functional variant thereof. In some embodiments, the CAR contains an antibody disclosed herein, e.g., an antibody fragment, a transmembrane domain that is or contains the transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain that contains the signaling portion of 4-IBB or a functional variant thereof and the signaling portion of CD3-zeta or a functional variant thereof. In some such embodiments, the receptor further includes a spacer that contains a portion of an Ig molecule, e.g., a human Ig molecule, e.g., an Ig hinge, e.g., an IgG4 hinge, e.g., a hinge-only spacer.

[0168] In some aspects, the spacer contains only the hinge region of an IgG, e.g., only an IgG4 or IgG hinge. In other embodiments, the spacer is or contains an Ig hinge, e.g., an IgG4-derived hinge, optionally linked to the CH2 and / or CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to the CH2 and CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to the CH3 domain only. In some embodiments, the spacer is or contains a glycine-serine-rich sequence or other flexible linker, e.g., a known flexible linker.

[0169] For example, in some embodiments, a CAR includes an antibody, such as an antibody fragment, including an scFv and sdAb, disclosed herein, a spacer, e.g., a portion of an immunoglobulin molecule, e.g., a spacer containing a hinge region and / or one or more constant regions of a heavy chain molecule, e.g., an Ig-hinge-containing spacer, a transmembrane domain containing all or a portion of a CD28-derived transmembrane domain, a CD28-derived intracellular signaling domain, and a CD3 zeta-derived signaling domain. In some embodiments, a CAR includes an antibody or fragment, such as an scFv or sdAb, disclosed herein, a spacer, e.g., an Ig-hinge-containing spacer, a CD28-derived transmembrane domain, a 4-1BB-derived intracellular signaling domain, and a CD3 zeta-derived signaling domain.

[0170] Recombinant receptors, such as CARs, expressed by cells administered to a subject typically recognize or specifically bind to a molecule expressed in, associated with, and / or specific to the disease or condition being treated or its cells. Upon specific binding to a molecule, e.g., an antigen, the receptor typically delivers an immunostimulatory signal, e.g., an ITAM transduction signal, to the cell, thereby promoting an immune response targeted to the disease or condition. For example, in some embodiments, the cells express a CAR that specifically binds to an antigen expressed by cells or tissues of, or associated with, the disease or condition.

[0171] Some embodiments of the present disclosure are isolated polynucleotides encoding either the CAR or CAR components of the present disclosure. While certain exemplary polynucleotides are disclosed herein, other polynucleotides encoding the antibodies or antigen-binding fragments thereof of the present disclosure, taking into account the degeneracy of the genetic code or codon preference in a given expression system, are also within the scope of the present disclosure. The polynucleotide sequence encoding the CAR or CAR components of the present disclosure is operably linked to one or more regulatory elements, such as promoters and enhancers, that allow for the expression of the nucleotide sequence in the intended host cell. The polynucleotide is a cDNA.

[0172] Some embodiments of the present disclosure are vectors comprising the polynucleotides of the present disclosure. In some embodiments, such vectors are plasmid vectors, viral vectors, vectors for baculovirus expression, transposon-based vectors, or any other vector suitable for introducing the polynucleotides of the present disclosure into a given organism or genetic background by any means. For example, polynucleotides encoding the light and heavy chain variable regions of an antibody of the present disclosure, optionally linked to constant regions, are inserted into an expression vector. The light and heavy chains are cloned in the same or different expression vectors. In some embodiments, DNA segments encoding immunoglobulin chains are operably linked to control sequences in the expression vector(s) that ensure expression of immunoglobulin polypeptides. Such control sequences include signal sequences, promoters (e.g., naturally associated or heterologous promoters), enhancer elements, and transcription termination sequences, and are selected to be compatible with the host cell selected to express the antibody. Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the protein encoded by the incorporated polynucleotide.

[0173] Suitable expression vectors are typically replicable in the host organism either as episomes or as an integrated part of the host chromosomal DNA. Expression vectors generally contain a selection marker such as ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance to enable detection of those cells transformed with the desired DNA sequence. Suitable vectors, promoters, and enhancer elements are known in the art, and many are commercially available for generating recombinant constructs of interest.

[0174] Some embodiments of the present disclosure are methods of producing a CAR, comprising delivering a polynucleotide encoding a CAR described herein or a vector comprising a polynucleotide encoding a CAR described herein to a host cell. In some embodiments, the method of delivery of the polynucleotide or vector is any method for delivery of nucleic acids known to those of skill in the art, including, but not limited to, transfection, transduction, electroporation, and transformation.

[0175] Some embodiments of the present disclosure are host cells containing the vectors of the present disclosure. The term "host cell" refers to a cell into which a vector has been introduced. It is understood that the term host cell is intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations, either due to mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Such host cells include eukaryotic, prokaryotic, plant, or archaeal cells. Escherichia coli, bacilli, e.g., Bacillus subtilis, and other Enterobacteriaceae, e.g., Salmonella, Serratia, and various Pseudomonas species, are examples of prokaryotic host cells. Other microorganisms, such as yeast, are also useful for expression. The genera Saccharomyces (e.g., S. cerevisiae) and Pichia are examples of suitable yeast host cells. Exemplary eukaryotic cells are of mammalian, insect, avian, or other animal origin.

[0176] Vectors for delivering CAR Also provided herein are targeted lipid particles (e.g., vectors) comprising a targeting antibody or antigen-binding fragment thereof and an exogenous substance for delivery of the targeted lipid particle to a target cell. In some embodiments, the targeted lipid particle comprises a henipavirus F protein molecule or a biologically active portion thereof. In some embodiments, the targeted lipid particle comprises a henipavirus G protein molecule or a biologically active portion thereof. In some embodiments, the targeted lipid particle comprises a henipavirus F protein molecule or a biologically active portion thereof and a henipavirus G protein molecule or a biologically active portion thereof.

[0177] In some embodiments, the targeting antibody or antigen-binding fragment thereof is bound to a membrane-binding protein of the targeted lipid particle. In other embodiments, the targeting antibody or antigen-binding fragment thereof is bound to a fusogen on the outer surface of the targeted lipid particle. In some embodiments, the targeting antibody or antigen-binding fragment thereof is bound to a henipavirus G protein or a biologically active portion thereof. In some embodiments, the targeting antibody or antigen-binding fragment thereof is bound to a henipavirus G protein or a biologically active portion thereof, for example, as described in U.S. Patent Publication No. 2022 / 0333134A1 (incorporated herein by reference in its entirety).

[0178] In some embodiments, the target cell is an immune cell. In some embodiments, the immune cell is an NK cell, a T cell, a macrophage, or a monocyte. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a CD3+ T cell, a CD4+ T cell, a CD5+ T cell, a naive T cell, a regulatory T (Treg) cell, a non-regulatory T cell, a Th1 cell, a Th2 cell, a Th9 cell, a Th17 cell, a T follicular helper (Tfh) cell, a cytotoxic T lymphocyte (CTL), an effector T (Teff) cell, a central memory T cell, an effector memory T cell, an effector memory T cell expressing CD45RA (TEMRA cell), a tissue-resident memory (Trm) cell, a virtual memory T cell, an innate immune memory T cell, a memory stem cell (Tse), or a gamma delta T cell. In some embodiments, the T cell is a cytotoxic T cell, a helper T cell, a memory T cell, a regulatory T cell, or a tumor-infiltrating lymphocyte. In some embodiments, the T cells are CD4+ T cells. In other embodiments, the T cells are CD8+ T cells.

[0179] A. Lipid bilayer In some embodiments, the targeted lipid particle comprises a naturally occurring bilayer of amphipathic lipids surrounding a lumen or cavity. In some embodiments, the targeted lipid particle comprises a lipid bilayer as its outermost surface. In some embodiments, the lipid bilayer surrounds the lumen. In some embodiments, the lumen is aqueous. In some embodiments, the lumen is in contact with hydrophilic head groups on the interior of the lipid bilayer. In some embodiments, the lumen is the cytosol. In some embodiments, the cytosol contains cellular components present in the source cell. In some embodiments, the cytosol does not contain cellular components present in the source cell. In some embodiments, the lumen is a cavity. In some embodiments, the cavity contains an aqueous environment. In some embodiments, the cavity does not contain an aqueous environment.

[0180] In some aspects, the lipid bilayer is derived from a source cell during the process for generating the lipid-containing particle. In some embodiments, the lipid bilayer includes membrane components of the cell from which the lipid bilayer is generated, e.g., phospholipids, membrane proteins, etc. In some embodiments, the lipid bilayer includes cytosol, including components found in the cell from which the lipid bilayer is generated, e.g., solutes, proteins, nucleic acids, etc., but not all of the cellular components, e.g., it lacks a nucleus. In some embodiments, the lipid bilayer is considered exosome-like. The lipid particle can vary in size, in some instances having a diameter ranging from 30 to 300 nm, e.g., 30 to 150 nm, including 40 to 100 nm.

[0181] In some embodiments, the lipid bilayer is a viral envelope. In some embodiments, the viral envelope is obtained from a source cell. In some embodiments, the viral envelope is obtained by a viral capsid from the plasma membrane of the source cell. In some embodiments, the lipid bilayer is obtained from a membrane other than the plasma membrane of the host cell. In some embodiments, the viral envelope lipid bilayer is embedded with viral proteins, including viral glycoproteins.

[0182] In other aspects, the lipid bilayer comprises a synthetic lipid complex. In some embodiments, the synthetic lipid complex is a liposome. In some embodiments, the lipid particle is a vesicular structure characterized by a phospholipid bilayer membrane and an inner aqueous medium. In some embodiments, the lipid bilayer has multiple lipid layers separated by aqueous medium. In some embodiments, the lipid bilayer forms spontaneously when phospholipids are suspended in an excess of aqueous solution. In some instances, the lipid components undergo self-rearrangement before the formation of a contiguous structure, trapping water and dissolved solutes between the lipid bilayers.

[0183] In some embodiments, the targeting envelope protein and fusogen, e.g., any of those mentioned above, including any that are exogenous to or overexpressed in the source cell, are disposed within the lipid bilayer.

[0184] In some embodiments, the targeted lipid particles comprise several different types of lipids. In some embodiments, the lipid is an amphipathic lipid. In some embodiments, the amphipathic lipid is a phospholipid. In some embodiments, the phospholipid comprises phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine. In some embodiments, the lipid comprises a phospholipid, such as phosphocholine and phosphoinositol. In some embodiments, the lipid comprises DMPC, DOPC, and DSPC.

[0185] In some embodiments, the bilayer comprises one or more lipids of the same or different types. In some embodiments, the source cells comprise cells selected from CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC23 cells, PA317 cells, WEHI cells, COS cells, BSC1 cells, BSC40 cells, BMT10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells.

[0186] B. Targeting antibodies In some embodiments, the targeted lipid particle (eg, vector) comprises a targeting antibody or antigen-binding fragment thereof for delivery of the targeted lipid particle to a target cell.

[0187] In some embodiments, the targeting antibody or antigen-binding fragment thereof is bound to a membrane-associated protein of the targeted lipid particle. In other embodiments, the targeting antibody or antigen-binding fragment thereof is bound to a fusogen on the outer surface of the targeted lipid particle. In some embodiments, the targeting antibody or antigen-binding fragment thereof is bound to a henipavirus G protein or a biologically active portion thereof. In some embodiments, the C-terminus of the targeting antibody or antigen-binding fragment thereof is bound to the C-terminus of the G protein or a biologically active portion thereof. In some embodiments, the N-terminus of the targeting antibody or antigen-binding fragment thereof is exposed on the outer surface of the lipid bilayer. In some embodiments, the N-terminus of the targeting antibody or antigen-binding fragment thereof binds to a cell surface molecule of the target cell. In some embodiments, the targeting antibody or antigen-binding fragment thereof specifically binds to a cell surface molecule present on the target cell. In some embodiments, the cell surface molecule is a protein, glycan, lipid, or low molecular weight molecule.

[0188] In some embodiments, the cell surface molecule of the target cell is an antigen or a portion thereof. In some embodiments, the targeting antibody or antigen-binding fragment thereof is an antibody having a single monomer domain antigen binding / recognition domain that can selectively bind to a specific antigen. In some embodiments, the single domain antibody binds to an antigen present on the target cell. In some embodiments, the cell surface molecule is CD4 or CD8.

[0189] Exemplary cells include immune effector cells, peripheral blood mononuclear cells (PBMCs), such as lymphocytes (T cells, B cells, natural killer cells) and monocytes, granulocytes (neutrophils, basophils, eosinophils), macrophages, dendritic cells, cytotoxic T lymphocytes, polymorphonuclear cells (also known as PMN, PML, or PMNL), stem cells, embryonic stem cells, neural stem cells, mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), human myogenic stem cells, muscle-derived stem cells (Mu Stem), embryonic stem cells (ES or ESC), limbic epithelial stem cells, cardiomyogenic stem cells, cardiomyocytes, progenitor cells, allogeneic cells, resident cardiac cells, induced pluripotent stem cells (iPS), adipose-derived or phenotype-modified stem or progenitor cells, CD133+ cells, aldehyde dehydrogenase positive cells (ALDH+), umbilical cord blood (UCB) cells, peripheral blood stem cells (PBSC), neurons, neural progenitor cells, pancreatic beta cells, glial cells, or hepatocytes.

[0190] In some embodiments, the target cell is a cell of a target tissue, hi some embodiments, the target tissue is the liver, lung, heart, spleen, pancreas, gastrointestinal tract, kidney, testis, ovary, brain, reproductive organs, central nervous system, peripheral nervous system, skeletal muscle, endothelium, inner ear, or eye.

[0191] In some embodiments, the target cell is a muscle cell (e.g., a skeletal muscle cell), a kidney cell, a liver cell (e.g., a hepatocyte), or a cardiac cell (e.g., a cardiomyocyte). In some embodiments, the target cell is a cardiac cell, e.g., a cardiomyocyte (e.g., a quiescent cardiomyocyte), a hepatoblast (e.g., a biliary hepatoblast), an epithelial cell, a T cell (e.g., a naive T cell), a macrophage (e.g., a tumor-infiltrating macrophage), or a fibroblast (e.g., a cardiac fibroblast).

[0192] In some embodiments, the target cell is a tumor-infiltrating lymphocyte, a T cell, a neoplastic or tumor cell, a virally infected cell, a stem cell, a central nervous system (CNS) cell, a hematopoietic stem cell (HSC), a liver cell, or a fully differentiated cell. In some embodiments, the target cell is a CD3+ T cell, a CD4+ T cell, a CD8+ T cell, a hepatocyte, a hematopoietic stem cell, a CD34+ hematopoietic stem cell, a CD105+ hematopoietic stem cell, a CD117+ hematopoietic stem cell, a CD105+ endothelial cell, a B cell, a CD20+ B cell, a CD19+ B cell, a cancer cell, a CD133+ cancer cell, an EpCAM+ cancer cell, a CD19+ cancer cell, a Her2 / Neu+ cancer cell, a GluA2+ neuron, a GluA4+ neuron, an NKG2D+ natural killer cell, a SLC1A3+ astrocyte, a SLC7A10+ adipocyte, or a CD30+ lung epithelial cell.

[0193] In some embodiments, the target cell is an antigen-presenting cell, an MHC class II+ cell, a professional antigen-presenting cell, an atypical antigen-presenting cell, a macrophage, a dendritic cell, a myeloid dendritic cell, a plasmacytoid dendritic cell, a CD11c+ cell, a CD11b+ cell, a spleen cell, a B cell, a hepatocyte, an endothelial cell, or a non-cancer cell.

[0194] i.CD4 antibody In some embodiments, a targeting antibody or antigen-binding fragment thereof specifically targets and binds to CD4 for delivery of targeted lipid particles to cells expressing CD4. In some embodiments, the antibody or antigen-binding fragment thereof can interact with cynomolgus monkey (or "cyno") or M. nemestrina CD4. In some embodiments, the antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv) derived from the heavy (VH) and light (VL) chains of an IgG molecule and composed of antigen-binding domains connected via a linker domain. In some embodiments, the antibody or antigen-binding fragment thereof is a VHH corresponding to the VH of an IgG molecule. The present disclosure also provides polynucleotides, vectors, and host cells encoding the antibodies and fragments thereof, as well as methods of using the antibodies or antigen-binding fragments thereof. In some embodiments, for example, the antibody or antigen-binding fragment thereof is fused to henipavirus glycoprotein G for targeted binding and transduction of cells.

[0195] Sequences for exemplary antibodies and antigen-binding fragments of the present disclosure using the Kabat numbering scheme are set forth below in Tables 18-19. Sequences for exemplary HCDRs of the present disclosure are set forth in Table 18. Sequences for exemplary LCDRs of the present disclosure are set forth in Table 19. Additional suitable sequences for antibodies or antigen-binding fragments thereof that specifically bind to CD4 are disclosed, for example, in U.S. Provisional Application No. 63 / 326,269 and U.S. Provisional Application No. 63 / 341,681, which are incorporated by reference in their entireties.

[0196] The sequences for the disclosed VH and VL domains are provided in Tables 20-21.

[0197] In some embodiments, disclosed is an antibody or antigen-binding fragment thereof capable of binding to CD4, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and the light chain variable region comprises three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3). In some embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequence of any one of SEQ ID NOs: set forth in Table 18, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequence of any one of SEQ ID NOs: set forth in Table 19. In some embodiments, the heavy chain variable region (VH) comprises the amino acid sequence of any one of SEQ ID NOs: 71-74 (Table 20), and the light chain variable region (VL) comprises the amino acid sequence of any one of SEQ ID NOs: 75-77 (Table 21).

[0198] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 71-74.

[0199] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VL having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 75-77.

[0200] In some embodiments, the antibody or antigen-binding fragment comprises a VH having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 71-74, and a VL having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 75-77.

[0201] In some embodiments, the antibody or antigen-binding fragment thereof comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 50, 54, 58, 62, 65, and 68, respectively.

[0202] In some embodiments, the antibody or antigen-binding fragment thereof comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 51, 55, 59, 63, 66, and 69, respectively.

[0203] In some embodiments, the antibody or antigen-binding fragment thereof comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 52, 56, 60, 64, 67, and 70, respectively.

[0204] In some embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 53, 57, and 61, respectively.

[0205] In some embodiments, the single domain antibody is human or humanized. In some embodiments, the single domain antibody or fragment thereof is naturally occurring. In some embodiments, the single domain antibody or fragment thereof is synthetic.

[0206] In some embodiments, single domain antibodies are antibodies whose complementarity determining regions are part of a single domain polypeptide. In some embodiments, single domain antibodies are antibody variable domains of only the heavy chain. In some embodiments, single domain antibodies do not include light chains.

[0207] In various embodiments, any of the antibodies or antigen-binding fragments described herein may comprise a heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region is an IgG, IgM, IgA, IgD, or IgE isotype, or a derivative or fragment thereof that retains at least one effector function of an intact heavy chain. In some embodiments, the heavy chain constant region is a human IgG isotype. In some embodiments, the heavy chain constant region is a human IgG1 or human IgG4 isotype. In some embodiments, the heavy chain constant region is a human IgG1 isotype. In some embodiments, the light chain constant region is a human kappa or lambda light chain, or a derivative or fragment thereof that retains at least one effector function of an intact light chain. In some embodiments, the light chain constant region is a human kappa light chain.

[0208] In various embodiments, any of the disclosed antibodies or antigen-binding fragments is a rodent antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a CDR-grafted antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof. In some embodiments, any of the disclosed antibodies or antigen-binding fragments comprises human or human-derived heavy and light chain variable regions comprising a human framework or a human framework with one or more backmutations. In various embodiments, any of the disclosed antibodies or antigen-binding fragments is a Fab, Fab', F(ab'), Fd, scFv, (scFv)2, scFv-Fc, VHH, or Fv fragment.

[0209] Antibodies whose heavy chain CDR, light chain CDR, VH, or VL amino acid sequences differ insubstantially from those shown in Tables 18-21 are encompassed within the scope of the present disclosure. Typically, this involves one or more conservative amino acid substitutions with amino acids having similar charge, hydrophobicity, or stereochemical characteristics in the antigen-binding site or framework without adversely altering the antibody's properties. Conservative substitutions may also be made to improve antibody properties, such as stability or affinity. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions are made in the VH or VL sequence. For example, a "conservative amino acid substitution" may involve the replacement of a native amino acid residue with a non-native residue that has little or no effect on the polarity or charge of the amino acid residue at that position. Desired amino acid substitutions are determined by those of skill in the art at the time such substitutions are desired. For example, amino acid substitutions are used to identify key residues in a molecular sequence or to increase or decrease the affinity of the molecules described herein. The following eight groups contain amino acids that are conservative amino acid substitutions for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M).

[0210] In some embodiments, the antibody or antigen-binding fragment thereof binds to human CD4. In some embodiments, the antibody or antigen-binding fragment thereof that binds to CD4 is a single-chain variable fragment. In embodiments involving a single polypeptide containing both a heavy chain variable region and a light chain variable region, both orientations of these variable regions are contemplated. In some embodiments, the heavy chain variable region is N-terminal to the light chain variable region, meaning that the heavy chain variable region is closer to the N-terminus of the polypeptide. In other embodiments, the light chain variable region is N-terminal to the heavy chain variable region, meaning that the light chain variable region is closer to the N-terminus of the polypeptide than the heavy chain variable region.

[0211] In some embodiments, the scFv binding protein comprises a linker. In some embodiments, the linker is between the heavy chain variable region (VH) and the light chain variable region (VL) (or vice versa). In some embodiments, the linker comprises the amino acid sequence of any one of GS, GGS, GGGS (SEQ ID NO: 227), GGGGS (SEQ ID NO: 147), GGGGGS (SEQ ID NO: 145), SEQ ID NOs: 165-166, and 32-33, or a combination thereof. Substitutions to introduce a new disulfide bond, for example, by making substitutions G44C in VH FR2 and G100C in VL FR4, are also within the scope of the present disclosure.

[0212] In some embodiments, the anti-CD4 antibody or antigen-binding fragment has an affinity constant (K D ) binds to human CD4. In some embodiments, Dis about 5 nM to about 500 nM, about 6 nM to about 10 nM, about 11 nM to about 20 nM, about 25 nM to about 40 nM, about 40 nM to about 60 nM, about 70 nM to about 90 nM, about 100 nM to about 120 nM, about 125 nM to about 140 nM, about 145 nM to about 160 nM, about 170 nM to about 200 nM, about 210 nM to about 250 nM, about 260 nM to about 300 nM, about 310 nM to about 350 nM, about 360 nM to about 400 nM, about 410 nM to about 450 nM, and about 460 nM to about 500 nM. In some embodiments, the anti-CD4 antibody or antigen-binding fragment has an affinity constant (K) of 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 50 nM, 20 nM, or 10 nM or less. D In some embodiments, the anti-CD4 antibody or antigen-binding fragment binds to human CD4 with comparable binding affinity (K ) to human CD4 and to cynomolgus monkey, M. mulatta (rhesus monkey), or M. nemestrina CD4. D ) to join them.

[0213] In some embodiments, the anti-CD4 antibody or antigen-binding fragment binds to cynomolgus monkey, M. mulatta (rhesus monkey), or M. nemestrina CD4. In some embodiments, the anti-CD4 antibody or antigen-binding fragment binds to CD4 of mouse, dog, pig, etc. In some embodiments, the K for cynomolgus monkey or M. nemestrina CD4 D is about 5 nM to about 500 nM, about 6 nM to about 10 nM, about 11 nM to about 20 nM, about 25 nM to about 40 nM, about 40 nM to about 60 nM, about 70 nM to about 90 nM, about 100 nM to about 120 nM, about 125 nM to about 140 nM, about 145 nM to about 160 nM, about 170 nM to about 200 nM, about 210 nM to about 250 nM, about 260 nM to about 300 nM, about 310 nM to about 350 nM, about 360 nM to about 400 nM, about 410 nM to about 450 nM, and about 460 nM to about 500 nM. In some embodiments, the anti-CD4 antibody or antigen-binding fragment has an affinity constant (K) of 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 50 nM, 20 nM, or 10 nM or less. D) binds to cynomolgus monkey or M. nemestrina CD4.

[0214] An antibody or antigen-binding fragment thereof that specifically binds to CD4 refers to an antibody or binding fragment that preferentially binds to CD4 over other antigen targets. As used herein, the term is interchangeable with "anti-CD4" antibody or "antibody that binds to CD4." In some embodiments, an antibody or binding fragment capable of binding to CD4 may bind to its antigen with higher affinity than others relative to the pair. In some embodiments, an antibody or binding fragment capable of binding to CD4 has a binding affinity of at least about 10, as measured, for example, by surface plasmon resonance or other methods known to those skilled in the art. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 or more (or any value in between) K D can bind to its antigen.

[0215] ii.CD8 antibody In some embodiments, a targeting antibody or antigen-binding fragment thereof specifically targets and binds to CD8 for delivery of targeted lipid particles to cells expressing CD8α or CD8β. In some embodiments, the antibody or antigen-binding fragment thereof can interact with cynomolgus monkey (or "cyno") or M. nemestrina CD8. In some embodiments, the antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv) derived from the heavy (VH) and light (VL) chains of an IgG molecule and composed of antigen-binding domains connected via a linker domain. In some embodiments, the antibody or antigen-binding fragment thereof is a VHH corresponding to the VH of an IgG molecule. The present disclosure also provides polynucleotides, vectors, and host cells encoding the antibodies and fragments thereof, as well as methods of using the antibodies or antigen-binding fragments thereof. In some embodiments, for example, the antibody or antigen-binding fragment thereof is fused to henipavirus glycoprotein G for targeted binding and transduction of cells.

[0216] Sequences for exemplary antibodies and antigen-binding fragments of the present disclosure using the Kabat numbering scheme are set forth below in Tables 22-23. Sequences for exemplary HCDRs of the present disclosure are set forth in Table 22. Sequences for exemplary LCDRs of the present disclosure are set forth in Table 23. Additional suitable sequences for antibodies or antigen-binding fragments thereof that specifically bind to CD8 are disclosed, for example, in PCT Application Publication No. WO2022 / 216915, which is incorporated herein by reference in its entirety.

[0217] The sequences for the disclosed VH and VL domains are provided in Tables 24-25.

[0218] In some embodiments, disclosed is an antibody or antigen-binding fragment thereof capable of binding to CD8α or CD8β, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and the light chain variable region comprises three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3). In some embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequence of any one of SEQ ID NOs: 102-105 (Table 24), and the light chain variable region (VL) comprises the amino acid sequence of any one of SEQ ID NOs: 106-109 (Table 25).

[0219] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 102-105.

[0220] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VL having an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 106-109.

[0221] In some embodiments, the antibody or antigen-binding fragment comprises a VH having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 102-105, and a VL having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 106-109.

[0222] In some embodiments, the antibody or antigen-binding fragment thereof comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 78, 82, 86, 90, 94, and 98, respectively.

[0223] In some embodiments, the antibody or antigen-binding fragment thereof comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 79, 83, 87, 91, 95, and 99, respectively.

[0224] In some embodiments, the antibody or antigen-binding fragment thereof comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 80, 84, 88, 92, 96, and 100, respectively.

[0225] In some embodiments, the antibody or antigen-binding fragment thereof comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 81, 85, 89, 93, 97, and 101, respectively.

[0226] In some embodiments, the single domain antibody is human or humanized. In some embodiments, the single domain antibody or fragment thereof is naturally occurring. In some embodiments, the single domain antibody or fragment thereof is synthetic.

[0227] In some embodiments, single domain antibodies are antibodies whose complementarity determining regions are part of a single domain polypeptide. In some embodiments, single domain antibodies are antibody variable domains of only the heavy chain. In some embodiments, single domain antibodies do not include light chains.

[0228] In various embodiments, any of the antibodies or antigen-binding fragments described herein may comprise a heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region is an IgG, IgM, IgA, IgD, or IgE isotype, or a derivative or fragment thereof that retains at least one effector function of an intact heavy chain. In some embodiments, the heavy chain constant region is a human IgG isotype. In some embodiments, the heavy chain constant region is a human IgG1 or human IgG4 isotype. In some embodiments, the heavy chain constant region is a human IgG1 isotype. In some embodiments, the light chain constant region is a human kappa or lambda light chain, or a derivative or fragment thereof that retains at least one effector function of an intact light chain. In some embodiments, the light chain constant region is a human kappa light chain.

[0229] In various embodiments, any of the disclosed antibodies or antigen-binding fragments is a rodent antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a CDR-grafted antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof. In some embodiments, any of the disclosed antibodies or antigen-binding fragments comprises human or human-derived heavy and light chain variable regions comprising a human framework or a human framework with one or more backmutations. In various embodiments, any of the disclosed antibodies or antigen-binding fragments is a Fab, Fab', F(ab'), Fd, scFv, (scFv)2, scFv-Fc, VHH, or Fv fragment.

[0230] Antibodies whose heavy chain CDR, light chain CDR, VH, or VL amino acid sequences differ insubstantially from those shown in Tables 22-25 are encompassed within the scope of the present disclosure. Typically, this involves one or more conservative amino acid substitutions with amino acids having similar charge, hydrophobicity, or stereochemical characteristics in the antigen-binding site or framework without adversely altering the antibody's properties. Conservative substitutions may also be made to improve antibody properties, such as stability or affinity. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions are made in the VH or VL sequence. For example, a "conservative amino acid substitution" may involve the replacement of a native amino acid residue with a non-native residue that has little or no effect on the polarity or charge of the amino acid residue at that position. Desired amino acid substitutions are determined by those of skill in the art at the time such substitutions are desired. For example, amino acid substitutions are used to identify key residues in a molecular sequence or to increase or decrease the affinity of the molecules described herein. The following eight groups contain amino acids that are conservative amino acid substitutions for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M).

[0231] In some embodiments, the antibody or antigen-binding fragment thereof binds to human CD8α or CD8β. In some embodiments, the antibody or antigen-binding fragment thereof binds to a human CD8α homodimer composed of two α chains. In some embodiments, the antibody or antigen-binding fragment thereof binds to a human CD8 heterodimer composed of one α chain and one β chain.

[0232] In some embodiments, the antibody or antigen-binding fragment that binds to CD8 is a single-chain variable fragment. In embodiments involving a single polypeptide containing both a heavy chain variable region and a light chain variable region, both orientations of these variable regions are contemplated. In some embodiments, the heavy chain variable region is N-terminal to the light chain variable region, meaning that the heavy chain variable region is closer to the N-terminus of the polypeptide. In other embodiments, the light chain variable region is N-terminal to the heavy chain variable region, meaning that the light chain variable region is closer to the N-terminus of the polypeptide than the heavy chain variable region.

[0233] In some embodiments, the scFv binding protein comprises a linker. In some embodiments, the linker is between the heavy chain variable region (VH) and the light chain variable region (VL) (or vice versa). In some embodiments, the linker comprises the amino acid sequence of any one of GS, GGS, GGGS (SEQ ID NO: 227), GGGGS (SEQ ID NO: 147), GGGGGS (SEQ ID NO: 145), SEQ ID NOs: 165-166, and 32-33, or a combination thereof. Substitutions to introduce a new disulfide bond, for example, by making substitutions G44C in VH FR2 and G100C in VL FR4, are also within the scope of the present disclosure.

[0234] In some embodiments, the anti-CD8 antibody or antigen-binding fragment has an affinity constant (K D ) binds to human CD8. In some embodiments, the K Dis about 5 nM to about 500 nM, about 6 nM to about 10 nM, about 11 nM to about 20 nM, about 25 nM to about 40 nM, about 40 nM to about 60 nM, about 70 nM to about 90 nM, about 100 nM to about 120 nM, about 125 nM to about 140 nM, about 145 nM to about 160 nM, about 170 nM to about 200 nM, about 210 nM to about 250 nM, about 260 nM to about 300 nM, about 310 nM to about 350 nM, about 360 nM to about 400 nM, about 410 nM to about 450 nM, and about 460 nM to about 500 nM. In some embodiments, the anti-CD8 antibody or antigen-binding fragment has an affinity constant (K) of 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 50 nM, 20 nM, or 10 nM or less. D In some embodiments, the anti-CD8 antibody or antigen-binding fragment binds to human CD8 with comparable binding affinity (K ) to human CD8 and cynomolgus monkey, M. mulatta (rhesus monkey), or M. nemestrina CD8. D ) to join them.

[0235] In some embodiments, the anti-CD8 antibody or antigen-binding fragment binds to cynomolgus monkey, M. mulatta (rhesus monkey), or M. nemestrina CD8. In some embodiments, the anti-CD8 antibody or antigen-binding fragment binds to CD8 from mice, dogs, pigs, etc. In some embodiments, the K for cynomolgus monkey or M. nemestrina CD8 D is about 5 nM to about 500 nM, about 6 nM to about 10 nM, about 11 nM to about 20 nM, about 25 nM to about 40 nM, about 40 nM to about 60 nM, about 70 nM to about 90 nM, about 100 nM to about 120 nM, about 125 nM to about 140 nM, about 145 nM to about 160 nM, about 170 nM to about 200 nM, about 210 nM to about 250 nM, about 260 nM to about 300 nM, about 310 nM to about 350 nM, about 360 nM to about 400 nM, about 410 nM to about 450 nM, and about 460 nM to about 500 nM. In some embodiments, the anti-CD8 antibody or antigen-binding fragment has an affinity constant (K) of 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 50 nM, 20 nM, or 10 nM or less. D) binds to cynomolgus monkey or M. nemestrina CD8.

[0236] An antibody or antigen-binding fragment thereof that specifically binds to CD8α or CD8β refers to an antibody or binding fragment that preferentially binds to CD8α or CD8β, respectively, over other antigen targets. As used herein, the term is interchangeable with "anti-CD8" antibody or "antibody that binds to CD8." In some embodiments, an antibody or binding fragment capable of binding to CD8α or CD8β may bind to that antigen with a higher affinity than others relative to the pair. In some embodiments, an antibody or binding fragment capable of binding to CD8α or CD8β has a binding affinity of at least about 10, as measured, for example, by surface plasmon resonance or other methods known to those skilled in the art. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 or more (or any value in between) K D can bind to its antigen.

[0237] C. Exogenous substances In some embodiments, the targeting vector further comprises a substance (also referred to herein as "cargo" or "payload") exogenous to the source cell. In some embodiments, the exogenous substance is a small molecule, a protein, or a nucleic acid (e.g., DNA, a chromosome (e.g., a human artificial chromosome), RNA, e.g., mRNA or miRNA). In some embodiments, the exogenous substance or cargo encodes a cytoplasmic protein. In some embodiments, the exogenous substance or cargo encodes or comprises a membrane protein. In some embodiments, the exogenous substance or cargo comprises a therapeutic agent. In some embodiments, the therapeutic agent is selected from one or more of a protein, e.g., an enzyme, a transmembrane protein, a receptor, an antibody; a nucleic acid, e.g., DNA, a chromosome (e.g., a human artificial chromosome), RNA, mRNA, siRNA, miRNA; or a small molecule.

[0238] In some embodiments, the exogenous material is present at at least 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies or less. In some embodiments, the targeted lipid particle has an altered, e.g., increased or decreased level, of one or more endogenous molecules, e.g., proteins or nucleic acids (e.g., in some embodiments, endogenous to the source cell, in some embodiments, endogenous to the target cell), e.g., due to treatment of the source cell, e.g., mammalian source cell, with siRNA or a gene editing enzyme. In some embodiments, an endogenous molecule is present at at least 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies or less. In some embodiments, an endogenous molecule (e.g., RNA or protein) is present at at least 1, 2, 3, 4, 5, 10, 20, 50, 100, 500, 10 ... 3 , 5.0×10 3 , 10 4 , 5.0×10 4 , 10 5 , 5.0×10 5 , 10 6 , 5.0×10 6 , 1.0×10 7 , 5.0×10 7 , or 1.0 × 10 8 In some embodiments, the endogenous molecule (e.g., RNA or protein) is present at a concentration at least 1, 2, 3, 4, 5, 10, 20, 50, 100, 500, 1 ... 3 , 5.0×10 3 , 104 , 5.0×10 4 , 10 5 , 5.0×10 5 , 10 6 , 5.0×10 6 , 1.0×10 7 , 5.0×10 7 , or 1.0 × 10 8 Present in low concentrations.

[0239] In some embodiments, the targeted lipid particle (e.g., a targeting vector) delivers at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the cargo (e.g., a therapeutic agent, e.g., an exogenous therapeutic agent) contained by the targeted lipid particle to the target cell. In some embodiments, the targeted lipid particle that fuses with the target cell(s) delivers an average of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the cargo (e.g., a therapeutic agent, e.g., an exogenous therapeutic agent) contained by the targeted lipid particle that fuses with the target cell(s) to the target cell. In some embodiments, the targeted lipid particle composition delivers at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the cargo (e.g., a therapeutic agent, e.g., an exogenous therapeutic agent) contained by the targeted lipid particle composition to the target tissue.

[0240] In some embodiments, the exogenous substance or cargo is not naturally expressed in the cell from which the targeted lipid particle was derived. In some embodiments, the exogenous substance or cargo is naturally expressed in the cell from which the vector was derived. In some embodiments, the exogenous substance or cargo is loaded into the targeted lipid particle via expression in the cell from which the viral vector was derived (e.g., expression from DNA or mRNA introduced via transfection, transduction, or electroporation). In some embodiments, the exogenous substance or cargo is expressed from DNA integrated into the genome or maintained episomally. In some embodiments, expression of the exogenous substance or cargo is constitutive. In some embodiments, expression of the exogenous substance or cargo is induced. In some embodiments, the exogenous substance or cargo is induced immediately prior to generating the targeted lipid particle. In some embodiments, expression of the exogenous substance or cargo is induced simultaneously with expression of a fusogen.

[0241] In some embodiments, exogenous substances or cargoes are loaded onto targeted lipid particles via electroporation into the targeted lipid particles themselves or into the cells from which the targeted lipid particles originate. In some embodiments, exogenous substances or cargoes are loaded onto targeted lipid particles via transfection (e.g., of DNA or mRNA encoding the cargo) into the targeted lipid particles themselves or into the cells from which the targeted lipid particles originate.

[0242] In some embodiments, the exogenous material or cargo may include one or more nucleic acid sequences, one or more polypeptides, combinations of nucleic acid sequences and / or polypeptides, one or more organelles, and any combination thereof. In some embodiments, the exogenous material or cargo may include one or more cellular components. In some embodiments, the exogenous material or cargo includes one or more cytoplasmic and / or nuclear components.

[0243] In some embodiments, the exogenous material or cargo includes a nucleic acid, e.g., DNA, nDNA (nuclear DNA), mtDNA (mitochondrial DNA), protein-encoding DNA, a gene, a transgene, an operon, a chromosome, a genome, a transposon, a retrotransposon, a viral genome, a vector, a polycistronic vector, an intron, an exon, modified DNA, mRNA (messenger RNA), tRNA (transfer RNA), modified RNA, microRNA, siRNA (small interfering RNA), tmRNA (transfer messenger RNA), rRNA (ribosomal RNA), mtRNA (mitochondrial RNA), snRNA (small nuclear RNA), small nucleolar RNA (snoRNA), SmYRNA (mRNA trans-splicing RNA), gRNA (guide RNA), TERC (telomerase RNA component), aRNA (antisense RNA), cis-NAT (cis-naturally occurring antisense transcript), CRISPR RNA (crRNA), IncRNA (long non-coding RNA), piRNA (piwi-interacting RNA), shRNA (short hairpin RNA), tasiRNA (trans-acting siRNA), eRNA (enhancer RNA), satellite RNA, pcRNA (protein-coding RNA), dsRNA (double-stranded RNA), RNAi (interfering RNA), circRNA (circular RNA), reprogramming RNA, aptamers, and any combination thereof. In some embodiments, the nucleic acid is a wild-type nucleic acid. In some embodiments, the nucleic acid is a mutant nucleic acid. In some embodiments, the nucleic acid is a fusion or chimera of multiple nucleic acid sequences.

[0244] In some embodiments, the exogenous substance or cargo may include a nucleic acid. For example, the exogenous substance or cargo may include RNA for improving the expression of an endogenous protein, or siRNA or miRNA for inhibiting the expression of an endogenous protein. For example, the endogenous protein may regulate the structure or function in a target cell. In some embodiments, the cargo may include a nucleic acid encoding an engineered protein that regulates the structure or function in a target cell. In some embodiments, the exogenous substance or cargo is a nucleic acid that targets a transcriptional activator that regulates the structure or function in a target cell.

[0245] In some embodiments, the exogenous substance or cargo includes a polypeptide, such as an enzyme, a structural polypeptide, a signaling polypeptide, a regulatory polypeptide, a transport polypeptide, a sensory polypeptide, a motor polypeptide, a defensive polypeptide, a conservation polypeptide, a transcription factor, an antibody, a cytokine, a hormone, a catabolic polypeptide, an anabolic polypeptide, a proteolytic polypeptide, a metabolic polypeptide, a kinase, a transferase, a hydrolase, a lyase, an isomerase, a ligase, an enzyme regulator polypeptide, a protein-binding polypeptide, a lipid-binding polypeptide, a membrane fusion polypeptide, a cell differentiation polypeptide, an epigenetic polypeptide, a cell death polypeptide, a nuclear transport polypeptide, a nucleic acid-binding polypeptide, a reprogramming polypeptide, a DNA editing polypeptide, a DNA repair polypeptide, a DNA recombination polypeptide, a transposase polypeptide, a DNA integration polypeptide, a targeting endonuclease (e.g., zinc finger nuclease, transcription activator-like nuclease (TALEN), cas9 and its homologs), a recombinase, and any combination thereof. In some embodiments, the protein targets a protein in a cell for degradation. In some embodiments, the protein targets a protein in a cell for degradation by localizing the protein to the proteasome. In some embodiments, the protein is a wild-type protein. In some embodiments, the protein is a mutant protein. In some embodiments, the protein is a fusion or chimeric protein.

[0246] In some embodiments, the exogenous substance or cargo includes a small molecule, e.g., an ion (e.g., Ca 2+ , C1-, Fe 2+), carbohydrates, lipids, reactive oxygen species, reactive nitrogen species, isoprenoids, signaling molecules, heme, polypeptide cofactors, electron-withdrawing compounds, electron-donating compounds, metabolites, ligands, and any combination thereof. In some embodiments, the small molecule is an agent that interacts with a target in a cell. In some embodiments, the small molecule targets a protein in a cell for degradation. In some embodiments, the small molecule targets a protein in a cell for degradation by localizing the protein to the proteasome. In some embodiments, the small molecule is a proteolysis-targeting chimeric molecule (PROTAC).

[0247] In some embodiments, the exogenous substance or cargo includes a mixture of proteins, nucleic acids, or metabolites, such as multiple polypeptides, multiple nucleic acids, multiple small molecules; combinations of nucleic acids, polypeptides, and small molecules; ribonucleoprotein complexes (e.g., Cas9-gRNA complexes); multiple transcription factors, multiple epigenetic factors, reprogramming factors (e.g., Oct4, Sox2, cMyc, and Klf4); multiple regulatory RNAs; and any combination thereof.

[0248] In some embodiments, the exogenous material or cargo includes one or more organelles, such as chondrisomes, mitochondria, lysosomes, nuclei, cell membranes, cytoplasm, endoplasmic reticulum, ribosomes, vacuoles, endosomes, spliceosomes, polymerases, capsids, acrosomes, autophagosomes, centrioles, glycosomes, glyoxysomes, hydrogenosomes, melanosomes, mitosomes, myofibers, cnidocysts, peroxisomes, proteasomes, vesicles, stress granules, organelle networks, and any combination thereof.

[0249] In some embodiments, the exogenous material encodes a therapeutic or diagnostic agent. In some embodiments, the therapeutic agent is a chimeric antigen receptor (CAR). In some embodiments, the CAR specifically binds to CD19 (e.g., the CAR comprises any of the antibodies or antigen-binding fragments described herein). In some embodiments, the CAR is bispecific and specifically binds to CD19 and also binds to CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD70, CD123, CD138, GPRC5D, LeY, NKG2D, WT1, GD2, HER2, EGFR, EGFRvIII, B7H3, PSMA, PSCA, CAIX, CD171, CEA, CSPG4, EPH A2, FAP, FRα, IL-13Rα, mesothelin, MUC1, MUC16, ROR1, C-Met, CD133, Ep-CAM, GPC3, HPV16, IL13Ra2, MAGEA3, MAGEA4, MART1, NY-ESO, VEGFR2, α-folate, CD24, CD44v7 / 8, EGP-2, EGP-40, erb-B2, erb-B, FBP, fetal acetylcholine receptor, G D2 , G D3 , HMW-MAA, IL-11Rα, KDR, Lewis Y, L1-cell adhesion molecule, MADE-A1, carcinoembryonic antigen (h5T4), TAG-72, CD19 / 22, syndecan-1, or BCMA. In some embodiments, the CAR is engineered to include the intracellular signaling domain of the T cell antigen receptor complex zeta chain (e.g., CD3 zeta). In some embodiments, the intracellular domain is selected from a CD137 (4-1BB) signaling domain, a CD28 signaling domain, and a CD3 zeta signaling domain.

[0250] DG proteins Also provided herein are fusion proteins comprising envelope glycoprotein G, H, and / or F proteins of the Paramyxoviridae family and a targeting antibody or antigen-binding fragment thereof disclosed herein exposed on the surface of a lipid particle or viral vector. In some embodiments, the targeting antibody or antigen-binding fragment thereof disclosed herein is fused to envelope glycoprotein G, H, and / or F proteins of the Paramyxoviridae family. In some embodiments, the fusogen contains Nipah virus protein F, measles virus F protein, tree shrew paramyxovirus F protein, paramyxovirus F protein, Hendra virus F protein, henipavirus F protein, morbillivirus F protein, respirovirus F protein, Sendai virus F protein, rubulavirus F protein, or abulavirus F protein. In some embodiments, the lipid particle contains henipavirus envelope-associated glycoprotein G (G protein) or a biologically active portion thereof and / or henipavirus envelope fusion glycoprotein F (F protein) or a biologically active portion thereof.

[0251] In some embodiments, the fusogen is baculovirus glycoprotein GP64, or glycoprotein GP64 variant E45K / T259A.

[0252] In some embodiments, the fusogen is a hemagglutinin-neuraminidase (HN) and / or fusion (F) protein (F / HN) from a respiratory paramyxovirus. In some embodiments, the respiratory paramyxovirus is Sendai virus. The HN and F glycoproteins of Sendai virus function to bind sialic acid via the HN protein and to mediate cell fusion for cell entry via the F protein. In some embodiments, the fusogen is the F and / or HN protein from murine parainfluenza virus type 1 (see, e.g., U.S. Patent No. 10,704,061).

[0253] In some embodiments, the lipid particle (eg, a viral vector) is pseudotyped with a viral glycoprotein described herein, eg, NiV-F and / or NiV-G protein.

[0254] In some embodiments, the viral vector further comprises a vector surface targeting moiety that specifically binds to a target ligand. In some embodiments, the vector surface targeting moiety is a polypeptide. In some embodiments, a nucleic acid encoding a paramyxovirus envelope protein (e.g., G protein) is modified with a targeting moiety to specifically bind to a target molecule on a target cell. In some embodiments, the targeting moiety is any targeting protein, including, but not necessarily limited to, antibodies and antigen-binding fragments thereof disclosed herein.

[0255] Henipavirus F proteins from various species have been reported to be compatible with G proteins from other species to induce fusion (Brandel-Tretheway et al. Journal of Virology. 2019. 93(13):e00577-19). In some aspects of the provided lipid particles (e.g., lentiviral vectors), the F protein is heterologous to the G protein, i.e., the F and G proteins, or biologically active portions thereof, are from different Henipavirus species. For example, in some embodiments, the G protein is from Hendra virus and the F protein is NiV-F as described. In other aspects, the F and / or G proteins are chimeric F and / or G proteins containing regions of F and / or G proteins from different Henipavirus species. In some embodiments, replacing a portion of the F protein with amino acids from a heterologous sequence in a Henipavirus results in fusion to a G protein with the heterologous sequence (Brandel-Tretheway et al. 2019). In some embodiments, the chimeric F and / or G proteins contain an extracellular domain from one Henipavirus species and a transmembrane and / or cytoplasmic domain from a different Henipavirus species, for example, in some embodiments, the F protein contains a Hendra virus extracellular domain and a Nipah virus transmembrane / cytoplasmic domain.

[0256] In some embodiments, the fusion protein contains a henipavirus envelope-associated glycoprotein G (G protein), or a biologically active portion thereof, and a single-domain antibody (sdAb) variable domain or single-chain variable fragment (scFv). In some embodiments, the sdAb variable domain or scFv is linked directly or indirectly to the G protein. In some embodiments, the sdAb variable domain or scFv is linked to the C-terminus (C-terminal amino acid) of the G protein, or a biologically active portion thereof. In some embodiments, the linkage is via a peptide linker, e.g., a flexible peptide linker. Table 26 provides a non-limiting list of examples of G proteins.

[0257] In some embodiments, the G protein is a Henipavirus G protein or a biologically active portion thereof. In some embodiments, the Henipavirus G protein is a Hendra (HeV) virus G protein, a Nipah (NiV) virus G protein (NiV-G), a Cedar (CedPV) virus G protein, a Mojiang virus G protein, a bat paramyxovirus G protein, or a biologically active portion thereof. Non-limiting examples of G proteins include those corresponding to SEQ ID NOs: 129, 138, 139, 140, and 141.

[0258] In some embodiments, the G-binding protein is a type II transmembrane glycoprotein containing an N-terminal cytoplasmic tail (e.g., corresponding to amino acids 1-49 of SEQ ID NO: 120), a transmembrane domain (e.g., corresponding to amino acids 50-70 of SEQ ID NO: 120), an extracellular domain containing an extracellular stalk (e.g., corresponding to amino acids 71-187 of SEQ ID NO: 120), and a globular head (e.g., corresponding to amino acids 188-602 of SEQ ID NO: 120). In such embodiments, the N-terminal cytoplasmic domain is located within the lumen of the lipid bilayer, and the C-terminal portion is the extracellular domain exposed outside the lipid bilayer. The stalk region in the C-terminal region (e.g., corresponding to amino acids 159-167 of NiV-G) has been shown to be involved in interaction with the F protein and inducing F protein fusion (Liu et al. 2015 J of Virology 89:1838). In wild-type G proteins, the globular head mediates receptor binding to the henipavirus entry receptors ephrinB2 and ephrinB3 but is dispensable for membrane fusion (Brandel-Tretheway et al. Journal of Virology. 2019. 93(13)e00577-19). In some embodiments herein, the targeting of the G protein is modified by linking the G protein or a biologically active fragment thereof (e.g., cytoplasmic truncation) to an sdAb variable domain. Binding of the G protein to a binding partner can trigger fusion mediated by a compatible F protein or a biologically active portion thereof. The G protein sequences disclosed herein are primarily disclosed as expression sequences containing an N-terminal methionine required for translation initiation. Because such N-terminal methionines are generally cleaved co- or post-translationally, the mature protein sequences for all G protein sequences disclosed herein are also contemplated as lacking an N-terminal methionine.

[0259] The G glycoprotein is highly conserved among Henipavirus species. For example, the G proteins of NiV and HeV viruses share 79% amino acid identity. Studies have shown that the G protein is highly compatible with the F proteins of different species, as demonstrated by different types of fusion activation (Brandel-Tretheway et al., Journal of Virology, 2019). As further described below, in some embodiments, targeted lipid particles contain heterologous G and F proteins from different species.

[0260] In some embodiments, the G protein has a sequence set forth in any of SEQ ID NOs: 120, 129, 138, 139, 140, 141, 148, 156, or 158-160, or is at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, or at least about 86% identical to any one of SEQ ID NOs: 120, 129, 138, 139, 140, 141, 148, 156, or 158-160. or a functionally active variant or biologically active portion thereof having a sequence that is about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% identical. In some embodiments, the G protein or functionally active variant or biologically active portion thereof is a henipavirus F protein, e.g., a protein that retains fusion activity in conjunction with an F protein (e.g., NiV-F or HeV-F). Fusogenic activity includes the activity of the G protein, in conjunction with the Henipavirus F protein, to promote or facilitate fusion of two membrane spaces (e.g., the space within a targeted lipid particle having Henipavirus F and G proteins embedded in its lipid bilayer) and the cytoplasm of a target cell (e.g., a cell containing a surface receptor or molecule recognized or bound by the targeted lipid particle). In some embodiments, the F protein and the G protein are from the same Henipavirus species (e.g., NiV-G and NiV-F). In some embodiments, the F protein and the G protein are from different Henipavirus species (e.g., NiV-G and HeV-F).

[0261] In some embodiments, the G protein has a sequence of amino acids set forth in SEQ ID NO: 120, 129, 138, 139, 140, 141, 148, 156, or 158-160, or a functionally active variant thereof or a biologically active portion thereof that retains fusion activity. In some embodiments, a functionally active variant comprises an amino acid sequence having at least or about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 120, 129, 138, 139, 140, 141, 148, 156, or 158-160, and retains fusion activity in conjunction with a Henipavirus F protein (e.g., NiV-F or HeV-F). In some embodiments, the biologically active portion has an amino acid sequence having at least or about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 120, 129, 138, 139, 140, 141, 148, 156, or 158-160, and retains fusion activity in conjunction with a Henipavirus F protein (e.g., NiV-F or HeV-F).

[0262] Reference to retaining fusion activity includes 10% or about 10% to 150% or about 150% or more of the level or degree of binding of the corresponding wild-type G protein as set forth in any one of SEQ ID NOs: 120, 129, 138, 139, 140, 141, 148, 156, or 158-160, e.g., at least 10% or at least about 10% of the level or degree of fusion activity of the corresponding wild-type G protein, e.g., at least 15% or at least about 15% of the level or degree of fusion activity of the corresponding wild-type G protein, e.g., For example, at least 20% or at least about 20% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least 25% or at least about 25% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least 30% or at least about 30% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least 35% or at least about 35% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least 40% or at least about 40%, for example, at least 45% or at least about 45% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least 50% or at least about 50% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least 55% or at least about 55% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least 60% or at least about 60% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least 65% or at least about 65% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least 70% or at least about 70% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least 75% or at least about 75% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least 80% or at least about 80% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least 85% or at least about 85% of the level or degree of fusion activity of the corresponding wild-type G protein, for example,These include activities that are at least 90% or at least about 90% of the level or degree of fusion activity of the corresponding wild-type G protein, e.g., at least 95% or at least about 95% of the level or degree of fusion activity of the corresponding wild-type G protein, e.g., at least 100% or at least about 100% of the level or degree of fusion activity of the corresponding wild-type G protein, or, e.g., at least 120% or at least about 120% (in combination with a Henipavirus F protein) of the level or degree of fusion activity of the corresponding wild-type G protein.

[0263] In some embodiments, the G protein is a mutant G protein that is a functionally active variant or biologically active portion thereof containing one or more amino acid mutations, e.g., one or more amino acid insertions, deletions, substitutions, or truncations. In some embodiments, the mutations described herein relate to amino acid insertions, deletions, substitutions, or truncations relative to a reference G protein sequence. In some embodiments, the reference G protein sequence is the wild-type sequence of a G protein or a biologically active portion thereof. In some embodiments, the functionally active variant or biologically active portion thereof is a mutant of the wild-type Hendra (HeV) virus G protein, the wild-type Nipah (NiV) virus G protein (NiV-G), the wild-type Cedar (CedPV) virus G protein, the wild-type Mojiang virus G protein, the wild-type bat paramyxovirus G protein, or a biologically active portion thereof. In some embodiments, the wild-type G protein has a sequence set forth in any one of SEQ ID NOs: 120, 129, 138, 139, 140, 141, 148, 156, or 158-160.

[0264] In some embodiments, the G protein is a mutant G protein that is a biologically active portion that is an N-terminally and / or C-terminally truncated fragment of the wild-type Hendra (HeV) virus G protein, the wild-type Nipah (NiV) virus G protein (NiV-G), the wild-type Cedar (CedPV) virus G protein, the wild-type Mojiang virus G protein, or the wild-type bat paramyxovirus G protein. In some embodiments, the truncation is an N-terminal truncation of all or part of the cytoplasmic domain. In some embodiments, the mutant G protein is a truncated and biologically active portion that lacks up to 49 consecutive amino acid residues at or near the N-terminus of a wild-type G protein, such as the wild-type G protein set forth in any one of SEQ ID NOs: 120, 129, 138, 139, 140, 141, 148, 156, or 158-160. In some embodiments, the mutant G protein is truncated and lacks up to 49 consecutive amino acids at the N-terminus of the wild-type G protein, for example, up to 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 30, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 consecutive amino acid(s).

[0265] In some embodiments, the G protein is a wild-type Nipah virus G (NiV-G) protein or a Hendra virus G protein, or a functionally active variant or biologically active portion thereof. In some embodiments, the G protein is a NiV-G protein having a sequence set forth in SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148, or a variant thereof that is at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, at least 87% or about 87%, at least 88% or more identical to SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148. or a functional variant or biologically active portion thereof having an amino acid sequence with about 88%, at least or about 89%, at least or about 90%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity.

[0266] In some embodiments, the G protein is a mutant NiV-G protein that is a biologically active portion of wild-type NiV-G. In some embodiments, the biologically active portion is an N-terminally truncated fragment. In some embodiments, the mutant NiV-G protein is truncated and contains up to 5 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), up to 6 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), up to 10 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), up to 12 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), up to 14 ... up to 7 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), up to 8 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), up to 9 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), up to 10 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), up to 11 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), up to 12 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), up to 13 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), up to 14 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), up to 15 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), up to 16 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148),or SEQ ID NO: 148), up to 17 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), up to 18 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), up to 19 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), up to 20 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148). up to 21 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), up to 22 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), up to 23 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), or SEQ ID NO: 148), up to 25 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), up to 26 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), up to 27 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148). up to 28 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), up to 29 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), up to 30 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148),or SEQ ID NO:148), up to 32 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:120, SEQ ID NO:138, or SEQ ID NO:148), up to 33 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:120, SEQ ID NO:138, or SEQ ID NO:148), up to 34 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:120, SEQ ID NO:138, or SEQ ID NO:148). up to 35 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), up to 36 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), up to 37 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), up to 38 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148); up to 39 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148); up to 40 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148); up to 41 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148). residues, up to 42 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), up to 43 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), up to 44 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148),or lacking up to 45 consecutive amino acid residues at or near the N-terminus of SEQ ID NO: 148).

[0267] In some embodiments, the NiV-G protein is a biologically active portion that does not contain a cytoplasmic domain. In some embodiments, the NiV-G protein that does not have a cytoplasmic domain is encoded by SEQ ID NO: 142.

[0268] In some embodiments, the mutant NiV-G protein comprises a sequence set forth in any of SEQ ID NOs: 121-126, 149-154, 132, 142, or 157, or is at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, or at least 87% or more identical to SEQ ID NOs: 121-126, 149-154, 132, 142, or 157. or at least about 87%, at least 88%, or at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or about 99% sequence identity.

[0269] In some embodiments, the mutant NiV-G protein is at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, at least 87% or about 87%, at least 88% or about 88%, at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, or functional variants thereof having at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO: 121 or SEQ ID NO: 149, or at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or is about 83%, at least 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, at least 87% or about 87%, at least 88% or about 88%, at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least a functional variant thereof having 98% or about 98%, or at least 99% or about 99% sequence identity, or at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, at least 87% or about 87%, at least 88% or about 88%, at least 89% or about 89%, at least 90% or about 90% to SEQ ID NO: 149;and functional variants thereof having at least or about 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98%, or at least 99% or about 99% sequence identity, or a 5 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148).

[0270] In some embodiments, the mutant NiV-G protein is as set forth in SEQ ID NO: 122 or a functional variant thereof having at least or about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 122, or to SEQ ID NO: 150 or a functional variant thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 150. and at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, at least 87% or about 87%, at least 88% or about 88%, at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about and functional variants thereof having 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity, and having a 10 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148).

[0271] In some embodiments, the mutant NiV-G protein is as set forth in SEQ ID NO: 123 or a functional variant thereof having an amino acid sequence having at least or about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 123, or to SEQ ID NO: 151 or a functional variant thereof. 151 and at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, at least 87% or about 87%, at least 88% or about 88%, at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92% and functional variants thereof having at least 2%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity.

[0272] In some embodiments, the mutant NiV-G protein is as set forth in SEQ ID NO: 124, or a functional variant thereof having at least or about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 124, or at least to SEQ ID NO: 152. at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least or about 84%, at least 85% or about 85%, at least 86% or about 86%, at least 87% or about 87%, at least 88% or about 88%, at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, and functional variants thereof having at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity.

[0273] In some embodiments, the mutant NiV-G protein is as set forth in SEQ ID NO: 125 or a functional variant thereof having at least or about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 153 or a functional variant thereof. at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, at least 87% or about 87%, at least 88% or about 88%, at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, and functional variants thereof having at least or about 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 98%, or at least 99% or about 99% sequence identity, and having a 25 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148).

[0274] In some embodiments, the mutant NiV-G protein has an identical sequence to SEQ ID NO: 126 or SEQ ID NO: 126 at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, at least 87% or about 87%, at least 88% or about 88%, at least 89% or about 89%, or at least or a functional variant thereof having at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO: 154 or SEQ ID NO: 154. at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, at least 87% or about 87%, at least 88% or about 88%, at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92% , a 30 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), as indicated by functional variants thereof having at least or about 93%, at least 94% or about 94%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity.

[0275] In some embodiments, the mutant NiV-G protein has an identical sequence to SEQ ID NO:132 or SEQ ID NO:132 by at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, at least 87% or about 87%, at least 88% or about 88%, at least 89% or about 89%, or at least or a functional variant thereof having at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO: 155 or SEQ ID NO: 155. at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, at least 87% or about 87%, at least 88% or about 88%, at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92% , a 33 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), as indicated by functional variants thereof having at least or about 93%, at least 94% or about 94%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity.

[0276] In some embodiments, the mutant NiV-G protein has an identical sequence to SEQ ID NO:132 or SEQ ID NO:132 by at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, at least 87% or about 87%, at least 88% or about 88%, at least 89% or about 89%, or at least or a functional variant thereof having at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO: 155 or SEQ ID NO: 155. at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, at least 87% or about 87%, at least 88% or about 88%, at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92% , a 34 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148), as indicated by functional variants thereof having at least or about 93%, at least 94% or about 94%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity.

[0277] In some embodiments, the NiV-G protein has a 34 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148) and one or more amino acid substitutions corresponding to amino acid substitutions selected from E501A, W504A, Q530A, and E533A with respect to the numbering shown in SEQ ID NO: 138.

[0278] In some embodiments, the mutant NiV-G protein is at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, at least 87% or about 87%, at least 88% or about 88%, at least 89% or about 89%, at least 90% or about 90%, or at least The wild-type NiV-G protein (SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148) lacks the N-terminal cytoplasmic domain of the wild-type NiV-G protein, as indicated by functional variants thereof having at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity.

[0279] In some embodiments, the mutant G protein is a mutant HeV-G protein having a sequence set forth in SEQ ID NO: 129 or 156, or having at least or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, at least 87% or about 87%, at least 88% or more sequence identity to SEQ ID NO: 129 or 156. or a functional variant or biologically active portion thereof having an amino acid sequence having at least about 88%, at least or at about 89%, at least or at about 90%, at least or at about 90%, at least or at about 91%, at least or at about 92%, at least or at about 92%, at least or at about 93%, at least or at about 94%, at least or at about 94%, at least 95% or at about 95%, at least or at about 96%, at least or at about 97%, at least or at about 98%, or at least or at about 99% of the amino acid sequence.

[0280] In some embodiments, the G protein is a mutant HeV-G protein that is a biologically active portion of wild-type HeV-G. In some embodiments, the biologically active portion is an N-terminally truncated fragment. In some embodiments, the mutant HeV-G protein is truncated and contains up to 5 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 129 or 156), up to 6 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 129 or 156), up to 7 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 129 or 156), up to 8 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 129 or 156), up to 9 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 129 or 156), up to 10 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 129 or 156), up to 11 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 129 or 156), up to 12 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 129 or 156), up to 13 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 129 or 156), up to 14 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 129 or 156), up to 15 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 129 or 156), up to 16 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 129 or 156), up to 17 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 129 or 156), up to 18 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 129 or 156), up to 19 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 129 or 156),A maximum of 20 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 129 or 156), a maximum of 21 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 129 or 156), a maximum of 22 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 129 or 156), a maximum of 23 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 129 or 156), up to 24 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 129 or 156), up to 25 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 129 or 156), up to 26 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 129 or 156), up to 27 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 129 or 156), wild-type HeV-G protein (SEQ ID NO: 129 or 156) up to 28 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 129 or 156), up to 29 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 129 or 156), up to 30 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 129 or 156), up to 31 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 129 or 156), a maximum of 32 consecutive amino acid residues, a maximum of 33 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 129 or 156), a maximum of 34 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 129 or 156), a maximum of 35 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 129 or 156), a maximum of 36 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 129 or 156),up to 37 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 129 or 156), up to 38 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 129 or 156), up to 39 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 129 or 156), up to 40 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 129 or 156), The wild-type HeV-G protein (SEQ ID NO: 129 or 156) lacks up to 41 consecutive amino acid residues at or near the end, up to 42 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 129 or 156), up to 43 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 129 or 156), up to 44 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 129 or 156), or up to 45 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 129 or 156).

[0281] In some embodiments, the HeV-G protein is a biologically active portion thereof that does not contain a cytoplasmic domain. In some embodiments, the mutant HeV-G protein has a similar sequence to SEQ ID NO: 143 or at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, or at least 91% of SEQ ID NO: 143. The wild-type HeV-G protein (SEQ ID NO: 129 or 156) lacks the N-terminal cytoplasmic domain, as indicated by a functional variant thereof having 0%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity.

[0282] In some embodiments, the G protein, or functionally active variant or biologically active portion thereof, binds to EphrinB2 or EphrinB3. In some aspects, the G protein has a sequence of amino acids set forth in any one of SEQ ID NO: 120, SEQ ID NO: 129, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 148, SEQ ID NO: 140, or SEQ ID NO: 141, or is a functionally active variant or biologically active portion thereof that is capable of binding to EphrinB2 or EphrinB3. In some embodiments, a functionally active variant or biologically active portion has an amino acid sequence having at least or about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:120, SEQ ID NO:129, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:148, SEQ ID NO:140, or SEQ ID NO:141, or a functionally active variant or biologically active portion thereof, and retains binding to ephrin B2 or B3.

[0283] Reference to retaining binding to ephrin B2 or B3 includes at least 5% or at least about 5% of the level or degree of binding of the corresponding wild-type G protein as set forth in SEQ ID NO: 120, SEQ ID NO: 129, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 148, SEQ ID NO: 140, or SEQ ID NO: 141, or a functionally active variant or biologically active portion thereof, of the corresponding wild-type G protein as set forth in SEQ ID NO: 120, SEQ ID NO: 129, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 148, SEQ ID NO: 140, or SEQ ID NO: 141. 10% of the level or degree of binding of the G protein, or a functionally active variant or biologically active portion thereof, as set forth in SEQ ID NO:120, SEQ ID NO:129, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:148, SEQ ID NO:140, or SEQ ID NO:141; 15% of the level or degree of binding of the corresponding wild-type G protein, or a functionally active variant or biologically active portion thereof, as set forth in SEQ ID NO:120, SEQ ID NO:129, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:148, SEQ ID NO:140, or SEQ ID NO:141; or 20% of the level or degree of binding of a corresponding wild-type G protein, or a functionally active variant or biologically active portion thereof, as set forth in SEQ ID NO:120, SEQ ID NO:129, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:148, SEQ ID NO:140, or SEQ ID NO:141, or 25% of the level or degree of binding of a corresponding wild-type G protein, or a functionally active variant or biologically active portion thereof, as set forth in SEQ ID NO:120, SEQ ID NO:129, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:148, SEQ ID NO:140, or SEQ ID NO:141, or 30% of the level or degree of binding of a corresponding wild-type G protein as set forth in SEQ ID NO:1, or a functionally active variant or biologically active portion thereof, as set forth in SEQ ID NO:120, SEQ ID NO:129, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:148, SEQ ID NO:140, or SEQ ID NO:141, 35% of the level or degree of binding of a corresponding wild-type G protein as set forth in SEQ ID NO:120, SEQ ID NO:129, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:148, SEQ ID NO:140,or 40% of the level or degree of binding of the corresponding wild-type G protein as set forth in SEQ ID NO: 141, or a functionally active variant or biologically active portion thereof, SEQ ID NO: 120, SEQ ID NO: 129, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 148, SEQ ID NO: 140, or 45% of the level or degree of binding of the corresponding wild-type G protein as set forth in SEQ ID NO: 141, or a functionally active variant or biologically active portion thereof, SEQ ID NO: 120, SEQ ID NO: 129, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 148, SEQ ID NO: 140 50% of the level or degree of binding of the corresponding wild-type G protein as set forth in SEQ ID NO: 140, or SEQ ID NO: 141, or a functionally active variant or biologically active portion thereof, SEQ ID NO: 120, SEQ ID NO: 129, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 148, ... 60% of the level or degree of binding of a corresponding wild-type G protein as set forth in SEQ ID NO: 148, SEQ ID NO: 140, or SEQ ID NO: 141, or a functionally active variant or biologically active portion thereof; 65% of the level or degree of binding of a corresponding wild-type G protein as set forth in SEQ ID NO: 120, SEQ ID NO: 129, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 148, SEQ ID NO: 140, or SEQ ID NO: 141, or a functionally active variant or biologically active portion thereof; 9, 70% of the level or degree of binding of a corresponding wild-type G protein as set forth in SEQ ID NO: 148, SEQ ID NO: 140, or SEQ ID NO: 141, or a functionally active variant or biologically active portion thereof, e.g., at least 75% or at least about 75% of the level or degree of binding of a corresponding wild-type G protein as set forth in SEQ ID NO: 120, SEQ ID NO: 129, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 148, SEQ ID NO: 140, or SEQ ID NO: 141, or a functionally active variant or biologically active portion thereof, e.g.,At least 80% or at least about 80% of the level or degree of binding of the corresponding wild-type G protein as set forth in SEQ ID NO: 120, SEQ ID NO: 129, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 148, SEQ ID NO: 140, or SEQ ID NO: 141, or a functionally active variant or biologically active portion thereof, for example, at least 85% or at least about 85% of the level or degree of binding of the corresponding wild-type G protein as set forth in SEQ ID NO: 120, SEQ ID NO: 129, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 148, SEQ ID NO: 140, or SEQ ID NO: 141, or a functionally active variant or biologically active portion thereof, for example, For example, binding that is at least 90% or at least about 90% of the level or degree of binding of a corresponding wild-type G protein, or a functionally active variant or biologically active portion thereof, as set forth in SEQ ID NO: 120, SEQ ID NO: 129, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 148, SEQ ID NO: 140, or SEQ ID NO: 141, or at least 95% or at least about 95% of the level or degree of binding of a corresponding wild-type protein, or a functionally active variant or biologically active portion thereof, as set forth in SEQ ID NO: 120, SEQ ID NO: 129, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 148, SEQ ID NO: 140, or SEQ ID NO: 141.

[0284] In some embodiments, the G protein is NiV-G, or a functionally active variant or biologically active portion thereof, and binds to ephrin B2 or ephrin B3. In some aspects, the NiV-G has the sequence of amino acids set forth in SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148, or is a functionally active variant or biologically active portion thereof that is capable of binding to ephrin B2 or ephrin B3. In some embodiments, a functionally active variant or biologically active portion has an amino acid sequence having at least or about 80%, at least 85% or about 85%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148, and retains binding to ephrin B2 or B3. Exemplary biologically active portions include all or part of the cytoplasmic domain, e.g., an N-terminally truncated variant lacking one or more, e.g., 1 to 49 consecutive N-terminal amino acid residues, e.g., as set forth in any one of SEQ ID NOs: 121-126, 142, and 149-154.

[0285] References to retaining binding to ephrin B2 or B3 include at least 5% or at least about 5% of the level or extent of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148, 10% of the level or extent of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148, 15% of the level or extent of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148, 20% of the level or extent of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148; 25% of the level or extent of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148; 30% of the level or extent of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148; 35% of the level or extent of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148; 40% of the level or extent of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148; 45% of the level or extent of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148; 50% of the level or extent of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148 55% of the level or extent of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148, 60% of the level or extent of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148, 65% of the level or extent of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148, 70% of the level or extent of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148, for example, SEQ ID NO: 120, SEQ ID NO: 138,or at least 75% or at least about 75% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 148, e.g., at least 80% or at least about 80% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148, e.g., at least 85% or at least about 85% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148, e.g., at least 90% or at least about 90% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148, or at least 95% or at least about 95% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO: 148.

[0286] In some embodiments, the G protein is HeV-G or a functionally active variant or biologically active portion thereof, and binds to ephrin B2 or ephrin B3. In some aspects, the HeV-G has the amino acid sequence set forth in SEQ ID NO: 129 or 156, or is a functionally active variant or biologically active portion thereof capable of binding to ephrin B2 or ephrin B3. In some embodiments, a functionally active variant or biologically active portion has an amino acid sequence having at least or about 80%, at least 85% or about 85%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO: 129 or 156 and retains binding to ephrin B2 or B3. Exemplary biologically active portions include all or a portion of the cytoplasmic domain, e.g., an N-terminally truncated variant lacking one or more, e.g., 1-49 consecutive N-terminal amino acid residues, e.g., as set forth in any one of SEQ ID NOs: 143.

[0287] References to retaining binding to ephrin B2 or B3 include at least 5% or at least about 5% of the level or extent of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 129 or 156, 10% of the level or extent of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 129 or 156, 15% of the level or extent of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 129 or 156, 20% of the level or extent of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 129 or 156, 25% of the level or extent of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 129 or 156, 30% of the level or extent of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 129 or 156, 35% of the level or extent of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 129 or 156, 40% of the level or extent of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 129 or 156 45% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 129 or 156, 50% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 129 or 156, 55% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 129 or 156, 60% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 129 or 156, 65% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 129 or 156 70% of the level or degree of binding of wild-type HeV-G, for example, at least 75% or at least about 75% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 129 or 156, for example, at least 80% or at least about 80% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 129 or 156, for example, at least 85% or at least about 85% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 129 or 156, for exampleBinding that is at least 90% or at least about 90% of the level or extent of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 129 or 156, or at least 95% or at least about 95% of the level or extent of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 129 or 156, for example.

[0288] In some embodiments, the G protein or biologically active portion thereof is a mutant G protein that exhibits reduced binding to the native binding partner of the wild-type G protein. In some embodiments, the mutant G protein or biologically active portion thereof is a mutant of wild-type Niv-G and exhibits reduced binding to one or both of the native binding partners, ephrin B2 or ephrin B3. In some embodiments, the mutant G protein or biologically active portion thereof, e.g., mutant NiV-G protein, exhibits reduced binding to the native binding partner. In some embodiments, the reduced binding to EphrinB2 or EphrinB3 is reduced by at or about 5%, 10% or about 10%, 15% or about 15%, 20% or about 20%, 25% or about 25%, 30% or about 30%, 40% or about 40%, 50% or about 50%, 60% or about 60%, 70% or about 70%, 80% or about 80%, 90% or about 90%, or more than at or about 100%.

[0289] In some embodiments, the mutations described herein can improve transduction efficiency. In some embodiments, the mutations described herein enable specific targeting of desired cell types other than EphrinB2 or EphrinB3. In some embodiments, the mutations described herein at least partially disable binding to at least one native receptor, e.g., to reduce binding to at least one of EphrinB2 or EphrinB3. In some embodiments, the mutations described herein disrupt native receptor recognition.

[0290] In some embodiments, the mutant NiV-G protein or biologically active portion thereof is truncated and contains up to 5 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 6 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 7 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), or 8 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138). 9 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 10 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 11 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 12 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 13 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 14 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 15 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 16 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 17 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 18 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 19 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 20 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 21 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 22 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 23 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138),24 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 25 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 26 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 27 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 28 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 29 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 30 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 31 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), The wild-type NiV-G protein (SEQ ID NO: 138) lacks 32 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 33 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 34 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 35 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 36 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 37 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 38 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), 39 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138), or 40 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 138).

[0291] In some embodiments, the G protein contains one or more amino acid substitutions at residues involved in interactions with one or both of EphrinB2 and EphrinB3, hi some embodiments, the amino acid substitutions correspond to mutations E501A, W504A, Q530A, and E533A with respect to the numbering set forth in SEQ ID NO: 138.

[0292] In some embodiments, the G protein is a mutant G protein containing one or more amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with respect to the numbering set forth in SEQ ID NO: 138. In some embodiments, the G protein is a mutant G protein containing one or more amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with respect to SEQ ID NO: 138, or a biologically active portion thereof containing an N-terminal truncation. In some embodiments, the G protein is a mutant G protein containing one or more amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A in combination with any one of the N-terminal truncations disclosed above with respect to SEQ ID NO: 138, or a biologically active portion thereof. In some embodiments, any of the above-described mutant G proteins contains one, two, three, or all four amino acids selected from the group consisting of E501A, W504A, Q530A, and E533A, in all pairwise and triplet combinations thereof, with respect to the numbering set forth in SEQ ID NO: 138.

[0293] In some embodiments, the mutant NiV-G protein has an amino acid sequence set forth in SEQ ID NO: 127 or 155, or an amino acid sequence having at least or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO: 127 or 155. In some embodiments, the G protein has an amino acid sequence set forth in SEQ ID NO: 127 or 155.

[0294] In some embodiments, the targeting envelope protein comprises a G protein or a functionally active variant or biologically active portion thereof and a targeting antibody or antigen-binding fragment thereof, wherein the targeting envelope protein exhibits increased binding to another molecule that is different from the native binding partner of the wild-type G protein. In some embodiments, the targeting antibody or antigen-binding fragment thereof is a single-domain antibody (sdAb) or scFv. In some embodiments, the other molecule is a protein expressed on the surface of a desired target cell. In some embodiments, the increased binding to the other molecule is greater than or equal to 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, the binding confers retargeted binding compared to that of the wild-type G protein, conferring new or different binding activity.

[0295] In some embodiments, the C-terminus of the targeting antibody or antigen-binding fragment thereof is linked to the C-terminus of a G protein or biologically active portion thereof. In some embodiments, the N-terminus of the targeting antibody or antigen-binding fragment thereof is exposed on the outer surface of the lipid bilayer. In some embodiments, the N-terminus of the targeting antibody or antigen-binding fragment thereof binds to a cell surface molecule of a target cell. In some embodiments, the targeting antibody or antigen-binding fragment thereof specifically binds to a cell surface molecule present on a target cell. In some embodiments, the cell surface molecule is a protein, glycan, lipid, or low-molecular-weight molecule.

[0296] In some embodiments, the cell surface molecule of the target cell is an antigen or a portion thereof. In some embodiments, the targeting antibody or antigen-binding fragment thereof is an antibody having a single monomer domain antigen-binding / recognition domain that can selectively bind to a specific antigen. In some embodiments, the single domain antibody binds to an antigen present on the target cell.

[0297] Exemplary cells include immune effector cells, peripheral blood mononuclear cells (PBMCs), such as lymphocytes (T cells, B cells, natural killer cells) and monocytes, granulocytes (neutrophils, basophils, eosinophils), macrophages, dendritic cells, cytotoxic T lymphocytes, polymorphonuclear cells (also known as PMN, PML, or PMNL), stem cells, embryonic stem cells, neural stem cells, mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), human myogenic stem cells, muscle-derived stem cells (Mu Stem), embryonic stem cells (ES or ESC), limbic epithelial stem cells, cardiomyogenic stem cells, cardiomyocytes, progenitor cells, allogeneic cells, resident cardiac cells, induced pluripotent stem cells (iPS), adipose-derived or phenotype-modified stem or progenitor cells, CD133+ cells, aldehyde dehydrogenase positive cells (ALDH+), umbilical cord blood (UCB) cells, peripheral blood stem cells (PBSC), neurons, neural progenitor cells, pancreatic beta cells, glial cells, or hepatocytes.

[0298] In some embodiments, the target cell is a cell of a target tissue, hi some embodiments, the target tissue is the liver, lung, heart, spleen, pancreas, gastrointestinal tract, kidney, testis, ovary, brain, reproductive organs, central nervous system, peripheral nervous system, skeletal muscle, endothelium, inner ear, or eye.

[0299] In some embodiments, the target cell is a muscle cell (e.g., a skeletal muscle cell), a kidney cell, a liver cell (e.g., a hepatocyte), or a cardiac cell (e.g., a cardiomyocyte). In some embodiments, the target cell is a cardiac cell, e.g., a cardiomyocyte (e.g., a quiescent cardiomyocyte), a hepatoblast (e.g., a biliary hepatoblast), an epithelial cell, a T cell (e.g., a naive T cell), a macrophage (e.g., a tumor-infiltrating macrophage), or a fibroblast (e.g., a cardiac fibroblast).

[0300] In some embodiments, the target cell is a tumor-infiltrating lymphocyte, a T cell, a neoplastic or tumor cell, a virally infected cell, a stem cell, a central nervous system (CNS) cell, a hematopoietic stem cell (HSC), a liver cell, or a fully differentiated cell. In some embodiments, the target cell is a CD3+ T cell, a CD4+ T cell, a CD8+ T cell, a hepatocyte, a hematopoietic stem cell, a CD34+ hematopoietic stem cell, a CD105+ hematopoietic stem cell, a CD117+ hematopoietic stem cell, a CD105+ endothelial cell, a B cell, a CD20+ B cell, a CD19+ B cell, a cancer cell, a CD133+ cancer cell, an EpCAM+ cancer cell, a CD19+ cancer cell, a Her2 / Neu+ cancer cell, a GluA2+ neuron, a GluA4+ neuron, an NKG2D+ natural killer cell, a SLC1A3+ astrocyte, a SLC7A10+ adipocyte, or a CD30+ lung epithelial cell.

[0301] In some embodiments, the target cell is an antigen-presenting cell, an MHC class II+ cell, a professional antigen-presenting cell, an atypical antigen-presenting cell, a macrophage, a dendritic cell, a myeloid dendritic cell, a plasmacytoid dendritic cell, a CD11c+ cell, a CD11b+ cell, a spleen cell, a B cell, a hepatocyte, an endothelial cell, or a non-cancer cell. In some embodiments, the cell surface molecule is any one of CD8.

[0302] In some embodiments, the G protein, or a functionally active variant or biologically active portion thereof, is directly linked to an sdAb variable domain (e.g., VHH) or scFv. In some embodiments, the targeting envelope protein is a fusion protein having the following structure: (N'-single domain antibody-C')-(C'-G protein-N'). In some embodiments, the targeting envelope protein is a fusion protein having the following structure: (N'-scFv-C')-(C'-G protein-N').

[0303] In some embodiments, the G protein or functionally active variant or biologically active portion thereof is indirectly linked to the sdAb variable domain or scFv via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a chemical linker.

[0304] In some embodiments, the linker is a peptide linker and the targeting envelope protein is a fusion protein containing a G protein, or a functionally active variant or biologically active portion thereof, linked to an sdAb variable domain or scFv via the peptide linker. In some embodiments, the targeting envelope protein is a fusion protein having the following structure: (N'-single domain antibody-C')-linker-(C'-G protein-N'). In some embodiments, the targeting envelope protein is a fusion protein having the following structure: (N'-scFv-C')-linker-(C'-G protein-N'). In some embodiments, the peptide linker is at most 65 amino acids in length. In some embodiments, the peptide linker is between about 2 and 65 amino acids, 2 and 60 amino acids, 2 and 56 amino acids, 2 and 52 amino acids, 2 and 48 amino acids, 2 and 44 amino acids, 2 and 40 amino acids, 2 and 36 amino acids, 2 and 32 amino acids, 2 and 28 amino acids, 2 and 24 amino acids, 2 and 20 amino acids, 2 and 18 amino acids, 2 and 14 amino acids, 2 and 12 amino acids, 2 and 10 amino acids, 2 and 8 amino acids, 2 and 6 amino acids, 6 and 65 amino acids, 6 and 60 amino acids, 6 and 56 amino acids, 6 and 52 amino acids, 6 and 48 amino acids, 6 and 44 amino acids, 6 and 40 amino acids, 6 and 36 amino acids, 6 and 32 amino acids, 6 and 28 amino acids, amino acids, 6-24 amino acids, 6-20 amino acids, 6-18 amino acids, 6-14 amino acids, 6-12 amino acids, 6-10 amino acids, 6-8 amino acids, 8-65 amino acids, 8-60 amino acids, 8-56 amino acids, 8-52 amino acids, 8-48 amino acids, 8-44 amino acids, 8-40 amino acids, 8-36 amino acids, 8-32 amino acids, 8-28 amino acids, 8-24 amino acids, 8-20 amino acids, 8-18 amino acids, 8-14 amino acids, 8-12 amino acids, 8-10 amino acids, 10-65 amino acids, 10-60 amino acids, 10-56 amino acids, 10-52 amino acids, 10-48 amino acids, 10-44 amino acids,10-40 amino acids, 10-36 amino acids, 10-32 amino acids, 10-28 amino acids, 10-24 amino acids, 10-20 amino acids, 10-18 amino acids, 10-14 amino acids, 10-12 amino acids, 12-65 amino acids, 12-60 amino acids, 12-56 amino acids, 12-52 amino acids, 12-48 amino acids, 12-44 amino acids, 12-40 amino acids, 12-36 amino acids, 12-32 amino acids, 12-28 amino acids, 12-24 amino acids, 12-20 amino acids Acids, 12-18 amino acids, 12-14 amino acids, 14-65 amino acids, 14-60 amino acids, 14-56 amino acids, 14-52 amino acids, 14-48 amino acids, 14-44 amino acids, 14-40 amino acids, 14-36 amino acids, 14-32 amino acids, 14-28 amino acids, 14-24 amino acids, 14-20 amino acids, 14-18 amino acids, 18-65 amino acids, 18-60 amino acids, 18-56 amino acids, 18-52 amino acids, 18-48 amino acids, 18-44 amino acids, 18-40 amino acids, 18-36 amino acids, 18-32 amino acids, 18-28 amino acids, 18-24 amino acids, 18-20 amino acids, 20-65 amino acids, 20-60 amino acids, 20-56 amino acids, 20-52 amino acids, 20-48 amino acids, 20-44 amino acids, 20-40 amino acids, 20-36 amino acids, 20-32 amino acids, 20-28 amino acids, 20-26 amino acids, 20-24 amino acids, 24-65 amino acids, 24-60 amino acids, 24- 56 amino acids, 24-52 amino acids, 24-48 amino acids, 24-44 amino acids, 24-40 amino acids, 24-36 amino acids, 24-32 amino acids, 24-30 amino acids, 24-28 amino acids, 28-65 amino acids, 28-60 amino acids, 28-56 amino acids, 28-52 amino acids, 28-48 amino acids, 28-44 amino acids, 28-40 amino acids, 28-36 amino acids, 28-34 amino acids, 28-32 amino acids, 32-65 amino acids, 32-60 amino acids,32-56 amino acids, 32-52 amino acids, 32-48 amino acids, 32-44 amino acids, 32-40 amino acids, 32-38 amino acids, 32-36 amino acids, 36-65 amino acids, 36-60 amino acids, 36-56 amino acids, 36-52 amino acids, 36-48 amino acids, 36-44 amino acids, 36-40 amino acids, 40-65 amino acids, 40-60 amino acids, 40-56 amino acids, 40-52 amino acids, 40-48 amino acids, 40-44 amino acids amino acids, 44-65 amino acids, 44-60 amino acids, 44-56 amino acids, 44-52 amino acids, 44-48 amino acids, 48-65 amino acids, 48-60 amino acids, 48-56 amino acids, 48-52 amino acids, 50-65 amino acids, 50-60 amino acids, 50-56 amino acids, 50-52 amino acids, 54-65 amino acids, 54-60 amino acids, 54-56 amino acids, 58-65 amino acids, 58-60 amino acids, or 60-65 amino acids. In some embodiments, the peptide linker is a polypeptide that is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 amino acids in length.

[0305] In some embodiments, the linker is a flexible peptide linker. In some such embodiments, the linker is 1 to 20 amino acids, e.g., 1 to 20 amino acids including glycine. In some embodiments, the linker is 1 to 20 amino acids, e.g., 1 to 20 amino acids including glycine and serine. In some embodiments, the linker is a flexible peptide linker containing the amino acids glycine and serine, referred to as a GS-linker. In some embodiments, the peptide linker includes the sequence GS, GGS, GGGGS (SEQ ID NO: 147), GGGGGS (SEQ ID NO: 145), or a combination thereof. In some embodiments, the polypeptide linker is the sequence (GGS)n (SEQ ID NO: 231), where n is 1 to 10. In some embodiments, the polypeptide linker is the sequence (GGGGS)n (SEQ ID NO: 146), where n is 1 to 10. In some embodiments, the polypeptide linker is the sequence (GGGGGS)n (SEQ ID NO: 137), where n is 1 to 6.

[0306] Also provided herein are polynucleotides comprising a nucleic acid sequence encoding a targeting envelope protein. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a G protein, or a biologically active portion thereof. In some embodiments, the polynucleotide further comprises a nucleic acid sequence encoding a single domain antibody (sdAb) variable domain or an scFv, or a biologically active portion thereof. The polynucleotide may comprise a sequence of nucleotides encoding any of the targeting envelope proteins described above. In some embodiments, the polynucleotide is a synthetic nucleic acid. Also provided are expression vectors containing any of the provided polynucleotides.

[0307] In some embodiments, expression of natural or synthetic nucleic acids is achieved by operably linking a nucleic acid encoding a gene of interest to a promoter and incorporating the construct into an expression vector. In some embodiments, the vector is suitable for replication and integration in eukaryotes. In some embodiments, the cloning vector contains transcription and translation terminators, initiation sequences, and a promoter useful for expression of the desired nucleic acid sequence. In some embodiments, the plasmid contains a promoter suitable for expression in a cell.

[0308] In some embodiments, the polynucleotide contains at least one promoter operably linked to control expression of a targeting envelope protein containing a G protein and a single domain antibody (sdAb) variable domain or scFv. For expression of the targeting envelope protein, at least one module in each promoter functions to position a start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters that lack a TATA box (such as promoters for mammalian terminal deoxynucleotidyl transferase genes and promoters for the SV40 gene), separate elements overlapping the start site itself help to fix the location of initiation.

[0309] In some embodiments, additional promoter elements, e.g., enhancers, control the frequency of transcription initiation. In some embodiments, the additional promoter elements are located 30-110 bp upstream of the start site, although many promoters have been shown to contain functional elements downstream of the start site as well. In some embodiments, the spacing between promoter elements is frequently flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In some embodiments, such as in the case of the thymidine kinase (tk) promoter, the spacing between promoter elements is increased to 50 bp apart before activity begins to decline. In some embodiments, depending on the promoter, individual elements can function cooperatively or independently to activate transcription.

[0310] In some embodiments, a promoter is one naturally associated with a gene or polynucleotide sequence, such as obtained by isolating the 5' non-coding sequences located upstream of the coding segment and / or exons. In some embodiments, such a promoter is referred to as "endogenous." In some embodiments, an enhancer is one naturally associated with a polynucleotide sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages may be obtained by placing a coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter not normally associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, as well as promoters or enhancers that are not "naturally occurring," i.e., contain different elements of different transcriptional regulatory regions and / or expression-altering mutations. In addition to producing promoter and enhancer nucleic acid sequences synthetically, sequences can be produced using nucleic acid amplification techniques, including recombinant cloning and / or PCR, in connection with the compositions disclosed herein.

[0311] In some embodiments, a suitable promoter is an immediate-early cytomegalovirus (CMV) promoter sequence. In some embodiments, the promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked thereto. In some embodiments, a suitable promoter is elongation growth factor-la (EF-la). In some embodiments, other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate-early promoter, Rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter.

[0312] In some embodiments, the promoter is an inducible promoter. In some embodiments, an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when such expression is desired, or turn off expression when expression is not desired. In some embodiments, inducible promoters include metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0313] In some embodiments, exogenously controlled inducible promoters are used to control expression of G proteins and single domain antibody (sdAb) variable domains or scFvs. For example, radiation-inducible, heat-inducible, and / or drug-inducible promoters are used to selectively induce transgene expression, e.g., in targeted regions. In such embodiments, the location, duration, and level of transgene expression are controlled by administration of an exogenous inducer.

[0314] In some embodiments, expression of a targeting envelope protein containing a G protein and a single domain antibody (sdAb) variable domain or scFv is controlled using a drug-inducible promoter. For example, in some embodiments, the promoter, enhancer, or transactivator comprises a Lac operator sequence, a tetracycline operator sequence, a galactose operator sequence, a doxycycline operator sequence, a rapamycin operator sequence, a tamoxifen operator sequence, or a hormone-responsive operator sequence, or analogs thereof. In some examples, the inducible promoter comprises a tetracycline response element (TRE). In some embodiments, the inducible promoter comprises an estrogen response element (ERE) that can activate gene expression in the presence of tamoxifen. In some examples, a drug-inducible element, e.g., a TRE, is combined with a promoter selected for enhanced transcription in the presence of a drug, e.g., doxycycline. In some embodiments, the drug-inducible promoter is a small molecule-inducible promoter.

[0315] In some embodiments, any of the provided polynucleotides are modified to remove CpG motifs and / or to optimize codons for translation in a particular species, such as human, canine, feline, equine, ovine, bovine, etc. In some embodiments, the polynucleotides are optimized for human codon usage (i.e., human codon-optimized). In some embodiments, the polynucleotides are modified to remove CpG motifs. In other embodiments, the provided polynucleotides are modified to remove CpG motifs and are codon-optimized, e.g., human codon-optimized. Methods for codon optimization and CpG motif detection and modification are well known. Typically, polynucleotide optimization improves transgene expression, increases transgene stability, and preserves the amino acid sequence of the encoded polypeptide.

[0316] To assess expression of the targeted envelope protein, the expression vector introduced into the cell may also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing particles, e.g., viral particles. In other embodiments, the selectable marker is carried on a separate piece of DNA and used in a co-transfection procedure. In some embodiments, both the selectable marker and the reporter gene are flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers are known in the art and include, for example, antibiotic resistance genes, such as neo.

[0317] Reporter genes are used to identify potentially transfected cells and evaluate the functionality of regulatory sequences.Reporter genes that encode easily assayable proteins are well known in the art.Usually, reporter genes are genes that encode proteins that are not present in or expressed by recipient organisms or tissues, and whose expression is revealed by some easily detectable property, such as enzymatic activity.The expression of the reporter gene is assayed at a suitable time after the DNA is introduced into the recipient cells.

[0318] Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (see, e.g., Ui-Tei et al., 2000, FEBS Lett. 479:79-82). Suitable expression systems are well known and can be prepared using well-known techniques or obtained commercially. In some embodiments, internal deletion constructs are generated using unique internal restriction sites or by partial digestion of non-unique restriction sites. The constructs can then be transfected into cells that exhibit high levels of desired polynucleotide and / or polypeptide expression. Typically, the construct with the smallest 5' flanking region that exhibits the highest level of expression of the reporter gene is identified as the promoter. In some embodiments, such promoter regions are linked to a reporter gene and used to evaluate drugs for their ability to modulate promoter-driven transcription.

[0319] i. Mutated paramyxovirus G protein In some embodiments, the paramyxovirus G protein is a mutant paramyxovirus G glycoprotein (e.g., a variant paramyxovirus G glycoprotein) that includes one or more amino acid mutations (i.e., substitutions) that result in reduced glycosylation of the protein. The one or more amino acid mutations, also referred to as deglycosylation mutations, can be one or more amino acid substitutions (also referred to as mutations).

[0320] In some embodiments, the mutant paramyxovirus G glycoprotein comprises an amino acid substitution at one or more amino acid positions that reduces glycosylation of the G glycoprotein. In some embodiments, the one or more amino acid substitutions disrupt an N-linked glycosylation site. In some embodiments, the one or more amino acid substitutions disrupt an O-linked glycosylation site.

[0321] In some embodiments, the mutant paramyxovirus G glycoprotein is derived from a morbillivirus (e.g., measles virus (MeV), canine distemper virus, cetacean morbillivirus, peste des petit ruminants virus, phocine distemper virus, rinderpest virus), henipavirus (e.g., Hendra (HeV), Nipah (NiV), Cedar (CedPV), Mojiang virus, Lanya virus, or bat paramyxovirus). In some embodiments, the mutant paramyxovirus G glycoprotein is a mutant of a paramyxovirus G glycoprotein derived from a Nipah virus or a measles virus. In some embodiments, the mutant paramyxovirus G protein is a mutant of a paramyxovirus G protein selected from the group consisting of SEQ ID NOs: 127, 138, and 155, or a modified paramyxovirus G glycoprotein derived from any one of SEQ ID NOs: 127, 138, and 155 that contains an altered cytoplasmic tail. In some embodiments, the mutant paramyxovirus G protein has a sequence of amino acids at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% identical to any one of SEQ ID NOs: 127, 138, and 155, and contains an acid substitution at one or more amino acid positions that reduces glycosylation of the G glycoprotein provided herein.

[0322] The location of predicted glycosylation sites can be determined using the sequence of the protein. For example, N-glycosylation often occurs at sites of the sequence NXS / T (where "X" is any amino acid except P). Various algorithms and tools for predicting both N- and O-linked glycosylation are available, including SprintGly (http: / / sparks-lab.org / server / sprint-gly / ), NetNGlyc (https: / / services.healthtech.dtu.dk / service.php?NetNGlyc-1.0), NetOGlyc (https: / / services.healthtech.dtu.dk / service.php?NetOGlyc-4.0), and GlycoMinestruct (http: / / glycomine.erc.monash.edu / Lab / GlycoMine_Struct / ), as well as the methods described in Pitti et al., Sci. Reports, 9:15975 (2019) and Pakhrin et al., Molecules 26:7314 (2021). Any predicted glycosylation sites can be substituted as described herein.

[0323] In some embodiments, the paramyxovirus G glycoprotein to which deglycosylation mutations are made is NiV-G set forth in SEQ ID NO: 138 or a modified Nipah G glycoprotein (NiV-G) having an altered cytoplasmic tail compared to native NiV-G (e.g., SEQ ID NO: 138). In some embodiments, the variant paramyxovirus G protein has a sequence of amino acids at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% identical to SEQ ID NO: 138 and contains an acid substitution at one or more amino acid positions that reduces glycosylation of the G glycoprotein provided herein. In some embodiments, the paramyxovirus G glycoprotein to which deglycosylation mutations are made is NiV-G set forth in SEQ ID NO: 127 or a modified Nipah G glycoprotein (NiV-G) having an altered cytoplasmic tail compared to native NiV-G (e.g., SEQ ID NO: 127). In some embodiments, the variant paramyxovirus G protein has a sequence of amino acids at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% identical to SEQ ID NO: 127 and contains an acid substitution at one or more amino acid positions that reduces glycosylation of the G glycoprotein provided herein.

[0324] Exemplary modified NiV-G proteins having altered cytoplasmic tails in which one or more amino acid substitutions can be incorporated to reduce glycosylation are as described herein, see, e.g., Table 26.

[0325] Amino acid positions for substitution are described herein with positions "corresponding" to positions in a reference sequence. It is understood that amino acid substitutions can be made not only in a reference sequence, but also in a similar sequence by aligning the reference position or identifying corresponding residues thereto. For example, a position "corresponding" to a protein position in a reference sequence can be identified by aligning the similar sequence with the reference sequence based on structural sequence alignment or using a standard alignment algorit...

Claims

1. An isolated polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 19-21.

2. An isolated polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to a sequence selected from SEQ ID NOs: 19-21.

3. An isolated polypeptide comprising an amino acid sequence selected from SEQ ID NOs:22-24.

4. An isolated polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to a sequence selected from SEQ ID NOs: 22-24.

5. 10. An isolated protein comprising the isolated polypeptide of claim 1 and the isolated polypeptide of claim 3.

6. 10. An isolated protein comprising the isolated polypeptide of claim 2 and the isolated polypeptide of claim 4.

7. a) SEQ ID NOs: 1, 4, and 7; b) SEQ ID NOs: 2, 5, and 8; and c) SEQ ID NOs: 3, 6, and 9 An isolated polypeptide comprising an amino acid sequence selected from:

8. a) SEQ ID NOs: 1, 4, and 7; b) SEQ ID NOs: 2, 4, and 8; or c) SEQ ID NOs: 3, 6, and 9 An isolated polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to

9. a) SEQ ID NOs: 10, 13, and 16; b) SEQ ID NOs: 11, 14, and 17; or c) SEQ ID NOs: 12, 15, and 18 An isolated polypeptide comprising an amino acid sequence selected from:

10. a) SEQ ID NOs: 10, 13, and 16; b) SEQ ID NOs: 11, 14, and 17; or c) An isolated polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOs: 12, 15, and 18.

11. 10. An isolated protein comprising the isolated polypeptide of claim 7 and the isolated polypeptide of claim 9.

12. 11. An isolated protein comprising the isolated polypeptide of claim 8 and the isolated polypeptide of claim 10.

13. The isolated polypeptide or protein of any one of claims 1 to 12, wherein the isolated polypeptide or protein is an antibody or an antigen-binding fragment thereof.

14. An antibody or antigen-binding fragment thereof that specifically binds to human Cluster of Differentiation 19 (CD19), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from SEQ ID NOs: 28 to 32.

15. An antibody or antigen-binding fragment thereof that specifically binds to human Cluster of Differentiation 19 (CD19), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to a sequence selected from SEQ ID NOs: 19 to 21.

16. An antibody or antigen-binding fragment thereof that specifically binds to human Cluster of Differentiation 19 (CD19), wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising an amino acid sequence selected from SEQ ID NOs: 22 to 24.

17. An antibody or antigen-binding fragment thereof that specifically binds to human Cluster of Differentiation 19 (CD19), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VL) comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to a sequence selected from SEQ ID NOs: 22 to 24.

18. An antibody or antigen-binding fragment thereof that specifically binds to human Cluster of Differentiation 19 (CD19), comprising: i) a heavy chain variable region (VH) of claim 14; and ii) a light chain variable region (VL) of claim 16.

19. An antibody or antigen-binding fragment thereof that specifically binds to human Cluster of Differentiation 19 (CD19), comprising: i) a heavy chain variable region (VH) of claim 15; and ii) a light chain variable region (VL) of claim 17.

20. An antibody or antigen-binding fragment thereof that specifically binds to human Cluster of Differentiation 19 (CD19), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 19 and a light chain variable region (VL) comprising the sequence of SEQ ID NO:

22.

21. An antibody or antigen-binding fragment thereof that specifically binds to human Cluster of Differentiation 19 (CD19), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 20 and a light chain variable region (VL) comprising the sequence of SEQ ID NO:

23.

22. An antibody or antigen-binding fragment thereof that specifically binds to human Cluster of Differentiation 19 (CD19), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 21 and a light chain variable region (VL) comprising the sequence of SEQ ID NO:

24.

23. An antibody or antigen-binding fragment thereof that specifically binds to human cluster of differentiation 19 (CD19), comprising three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 1, 4, 7, 10, 13, and 16, respectively.

24. An antibody or antigen-binding fragment thereof that specifically binds to human cluster of differentiation 19 (CD19), comprising three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 2, 5, 8, 11, 14, and 17, respectively.

25. An antibody or antigen-binding fragment thereof that specifically binds to human cluster of differentiation 19 (CD19), comprising three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 3, 6, 9, 12, 15, and 18, respectively.

26. The antibody or antigen-binding fragment thereof may be Fab, Fab', F(ab')2, Fd, scFv, (scFv) ２ 26. The antibody or antigen-binding fragment thereof according to any one of claims 14 to 25, which is an scFv-Fc, sdAb, VHH, or Fv fragment.

27. The antibody or antigen-binding fragment thereof according to any one of claims 14 to 26, wherein the antibody or antigen-binding fragment thereof is an scFv.

28. The antibody or antigen-binding fragment thereof according to any one of claims 14 to 27, wherein the VH is N-terminal to the VL.

29. The antibody or antigen-binding fragment thereof according to any one of claims 14 to 27, wherein the VL is N-terminal to the VH.

30. The antibody or antigen-binding fragment thereof according to any one of claims 26 to 29, wherein the scFv comprises a linker connecting the VH and the VL.

31. The antibody or antigen-binding fragment thereof of claim 30, wherein the linker connecting the VH and the VL is a Whitlow linker.

32. The antibody or antigen-binding fragment thereof of claim 30, wherein the linker connecting the VH and the VL is a (G4S)3 linker (SEQ ID NO: 32).

33. The antibody or antigen-binding fragment of any one of claims 30 to 32, wherein the linker comprises an amino acid sequence selected from SEQ ID NOs: 32-33, 145-147, and 165-166.

34. The antibody or antigen-binding fragment thereof according to any one of claims 14 to 33, wherein the antibody or antigen-binding fragment thereof comprises a CD8α hinge domain.

35. The antibody or antigen-binding fragment thereof according to any one of claims 14 to 34, wherein the antibody or antigen-binding fragment comprises the CD8α transmembrane domain.

36. The antibody or antigen-binding fragment thereof has an EC ５０ The antibody or antigen-binding fragment thereof according to any one of claims 14 to 35, which binds to human CD19 at the

37. A bispecific antibody or antigen-binding fragment thereof comprising the antibody or antigen-binding fragment thereof of any one of claims 14 to 36 and an antibody or antigen-binding fragment thereof that specifically binds to at least one additional cell surface molecule.

38. 38. The bispecific antibody or antigen-binding fragment thereof of claim 37, wherein the at least one additional cell surface molecule comprises CD3, 4-1BB, IL-6, NKG2D, Fc-gamma-RIIIA (CD16), APRIL, CD38, TACI, Fc-gamma-RIIIA (CD16) and NKG2D, CD3 and serum albumin, CD47 and TACI, or CD3 and GPRC5D.

39. The antibody or antigen-binding fragment thereof of any one of claims 14 to 36 or the bispecific antibody of claim 37 or 38, wherein the antibody or antigen-binding fragment comprises a conjugate.

40. 40. The antibody or antigen-binding fragment of claim 39, wherein the conjugate is a therapeutic agent, a tag for detection, a conjugate that improves antibody stability, a nucleic acid, a cleavable linker, or a nanoparticle.

41. The antibody or antigen-binding fragment thereof of any one of claims 14 to 36 or 39 to 40 or the bispecific antibody of claim 37 or 38, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody.

42. 42. The antibody or antigen-binding fragment of any one of claims 14 to 36 or 39 to 41 or the bispecific antibody of claim 37 or 38, wherein the antibody or antigen-binding fragment is humanized.

43. An isolated polynucleotide encoding an antibody or antigen-binding fragment thereof according to any of claims 14 to 36 or 39 to 42, or a bispecific antibody according to claim 37 or 38.

44. 44. An isolated vector comprising the polynucleotide of claim 43.

45. 45. The isolated vector of claim 44, wherein the vector is a polycistronic vector.

46. 46. ​​The isolated vector of claim 44 or 45, wherein the vector comprises nucleic acid encoding one or more additional molecules.

47. 47. The isolated vector of claim 46, wherein the one or more additional molecules are selected from a tolerance inducer, a suicide switch, a regulatory element, or an antibody or antigen-binding fragment thereof.

48. 48. The isolated vector of claim 46 or 47, wherein the one or more additional molecules comprise a tolerance inducer.

49. 49. The isolated vector of any one of claims 46 to 48, wherein the one or more additional molecules comprises a suicide switch.

50. 50. The isolated vector of any one of claims 46 to 49, wherein the one or more additional molecules comprise a regulatory element.

51. An isolated host cell comprising a polynucleotide according to claim 43 and / or a vector according to any one of claims 44 to 50.

52. 42. A chimeric antigen receptor (CAR) comprising an extracellular binding domain that specifically binds to human cluster of differentiation 19 (CD19), wherein the extracellular binding domain comprises an antigen-binding domain constituting the antibody or antigen-binding fragment of any one of claims 14 to 36 or 39 to 42, or the bispecific antibody of claim 37 or 38.

53. 53. The CAR of claim 52, wherein the CAR comprises one or more of a signal peptide, an extracellular binding domain, and a signaling domain.

54. 54. The CAR of claim 52 or 53, wherein the CAR comprises one or more of a signal peptide, an extracellular binding domain, a hinge domain, a transmembrane domain, an intracellular costimulatory domain, and an intracellular signaling domain.

55. The CAR according to any one of claims 52 to 54, wherein the CAR comprises a CD8α signal peptide.

56. The CAR according to any one of claims 52 to 55, wherein the CAR comprises one or more intracellular domains selected from a CD137 (4-1BB) signaling domain, a CD28 signaling domain, and a CD3 zeta signaling domain.

57. The CARs include CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD70, CD123, CD138, GPRC5D, LeY, NKG2D, WT1, GD2, HER2, EGFR, EGFRvIII, B7H3, PSMA, PSCA, CAIX, CD171, CEA, CSPG4, EPHA2, FAP, FRα, IL-1 3Rα, mesothelin, MUC1, MUC16, ROR1, c-Met, CD133, Ep-CAM, GPC3, HPV16, IL13Ra2, MAGEA3, MAGEA4, MART1, NY-ESO, VEGFR2, α-folate, CD24, CD44v7 / 8, EGP-2, EGP-40, erb-B2, erb-B, FBP, fetal acetylcholine receptor, G Ｄ２ , G Ｄ３ , HMW-MAA, IL-11Rα, KDR, Lewis Y, L1-cell adhesion molecule, MADE-A1, carcinoembryonic antigen (h5T4), TAG-72, CD19 / 22, syndecan 1, or BCMA.

58. An isolated polynucleotide encoding the CAR according to any one of claims 52 to 57.

59. 59. An isolated vector comprising the polynucleotide of claim 58.

60. 60. The isolated vector of claim 59, wherein the vector is a polycistronic vector.

61. 61. The isolated vector of claim 59 or 60, wherein the vector comprises nucleic acid encoding one or more additional molecules.

62. 62. The isolated vector of claim 61 , wherein the one or more additional molecules are selected from a tolerance inducer, a suicide switch, a regulatory element, an antibody or antigen-binding fragment thereof, or a CAR.

63. 63. The isolated vector of claim 61 or 62, wherein the one or more additional molecules comprise a tolerance inducer.

64. 64. The isolated vector of any one of claims 61 to 63, wherein the one or more additional molecules comprises a suicide switch.

65. 65. The isolated vector of any one of claims 61 to 64, wherein the one or more additional molecules comprise a regulatory element.

66. 66. The isolated vector of any one of claims 61 to 65, wherein the one or more additional molecules comprise an antibody or antigen-binding fragment thereof.

67. 67. The isolated vector of any one of claims 61 to 66, wherein the one or more additional molecules comprises a CAR.

68. A method for producing the CAR of any one of claims 52 to 57, the method comprising delivering the polynucleotide of claim 58 or the vector of any one of claims 59 to 67 to a host cell.

69. 68. An isolated host cell comprising a polynucleotide according to claim 58 and / or a vector according to any one of claims 59 to 67.

70. A viral vector that targets immune cells, the viral vector comprising: a) an antibody or antigen-binding fragment thereof that binds to a cell surface molecule on the immune cell, wherein the antibody or antigen-binding fragment thereof is bound to a membrane-associated protein in the envelope of the viral vector or to a fusogen on the outer surface of the viral vector; and b) at least one polynucleotide encoding the chimeric antigen receptor (CAR) of any one of claims 52 to 69. The viral vector comprising:

71. 71. The viral vector of claim 70, wherein the immune cell is a T cell, a B cell, a natural killer cell, a macrophage, or a monocyte.

72. 72. The viral vector of claim 70 or 71, wherein the immune cell is a T cell.

73. The viral vector of any one of claims 70 to 72, wherein the antibody or antigen-binding fragment thereof binds to CD3, CD4, CD7, or CD8.

74. 74. The viral vector of any one of claims 70 to 73, wherein the viral vector comprises Henipavirus envelope glycoprotein G (G protein) or a biologically active portion thereof.

75. 75. The viral vector of any one of claims 70 to 74, wherein the viral vector comprises a Henipavirus F protein molecule or a biologically active portion thereof.

76. 76. The viral vector of any one of claims 70 to 75, wherein the viral vector comprises Henipavirus envelope glycoprotein G (G protein) or a biologically active portion thereof bound to the antibody or antigen-binding fragment thereof.

77. The antibody or antigen-binding fragment thereof binds to CD8 and comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are, respectively: a) SEQ ID NOs: 78, 82, 86, 90, 94, and 98; b) SEQ ID NOs: 79, 83, 87, 91, 95, and 99; c) SEQ ID NOs: 80, 84, 88, 92, 96, and 100; or d) SEQ ID NOs: 81, 85, 89, 93, 97, and 101 The viral vector according to any one of claims 70 to 76, comprising:

78. 78. The viral vector of any one of claims 70 to 77, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) having an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 102 to 105.

79. 79. The viral vector of any one of claims 70 to 78, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) having an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 106 to 109.

80. The viral vector of any one of claims 70 to 79, wherein the antibody or antigen-binding fragment thereof comprises a VH having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 102 to 105, and a VL having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 106 to 109.

81. The antibody or antigen-binding fragment thereof binds to CD4 and comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are, respectively: a) SEQ ID NOs: 50, 54, 58, 62, 65, and 68; b) SEQ ID NOs: 51, 55, 59, 63, 66, and 69; or c) SEQ ID NOs: 52, 56, 60, 64, 67, and 70; or wherein said HCDR1, HCDR2, and HCDR3 each comprise: d) SEQ ID NOs: 53, 57, and 61 The viral vector according to any one of claims 70 to 76, comprising:

82. 82. The viral vector of any one of claims 70-76 or 81, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) having an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 71-74.

83. 83. The viral vector of any one of claims 70-76 or 81-82, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) having an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 75-77.

84. The viral vector of any one of claims 70 to 76 or 81 to 83, wherein the antibody or antigen-binding fragment thereof comprises a VH having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 71 to 74, and a VL having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 75 to 77.

85. 85. The viral vector of any one of claims 70 to 84, wherein the G protein is wild-type Nipah virus G glycoprotein (NiV-G) or a functionally active variant or biologically active portion thereof.

86. 86. The viral vector of claim 85, wherein the NiV-G variant or biologically active portion thereof comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:120, SEQ ID NO:138, or SEQ ID NO:

148.

87. 87. The viral vector of claim 85 or 86, wherein the NiV-G variant or biologically active portion thereof comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from E501A, W504A, Q530A, and E533A with respect to the numbering set forth in SEQ ID NO:

138.

88. The viral vector of any one of claims 85 to 87, wherein the NiV-G variant comprises SEQ ID NO: 127 or 155.

89. 89. The viral vector of any one of claims 71 to 88, wherein the F protein is a wild-type Nipah virus F (NiV-F) protein or a functionally active variant or biologically active portion thereof.

90. 90. The viral vector of claim 89, wherein the NiV-F comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:118, or SEQ ID NO:

119.

91. A fusion protein comprising henipavirus envelope glycoprotein G (G protein) or a biologically active portion thereof and an antibody or antigen-binding fragment thereof according to any one of claims 14 to 42.

92. 92. The fusion protein of claim 91, wherein the antibody or antigen-binding fragment thereof is fused to the G protein via a peptide linker.

93. 93. The fusion protein of claim 91 or 92, wherein the peptide linker comprises (GGGGS)n, where n is 3 (SEQ ID NO: 32).

94. The fusion protein of any one of claims 91 to 93, wherein the antigen-binding fragment is an scFv.

95. 95. The fusion protein of any one of claims 91 to 94, wherein the G protein or biologically active portion thereof is wild-type Nipah virus G glycoprotein (NiV-G) or a functionally active variant or biologically active portion thereof.

96. 96. The fusion protein of claim 95, wherein the NiV-G variant or biologically active portion thereof comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 120, SEQ ID NO: 138, or SEQ ID NO:

148.

97. 97. The fusion protein of claim 95 or 96, wherein the NiV-G variant or biologically active portion thereof comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from E501A, W504A, Q530A and E533A with respect to the numbering set forth in SEQ ID NO:

138.

98. The fusion protein of any one of claims 95 to 97, wherein the NiV-G variant comprises SEQ ID NO: 127 or 155.

99. The fusion protein of any one of claims 91 to 98, wherein the protein is pseudotyped into a lentiviral particle.

100. 91. A method for selectively modulating the activity of an immune cell, comprising delivering to the immune cell an effective amount of a viral vector comprising a polynucleotide encoding a chimeric antigen receptor (CAR), wherein the viral vector is the viral vector of any one of claims 70 to 90.

101. 91. A method for generating a CAR immune cell, comprising delivering to the immune cell an effective amount of a viral vector comprising a polynucleotide encoding a chimeric antigen receptor (CAR), wherein the viral vector is the viral vector of any one of claims 70 to 90.

102. 102. The method of claim 100 or 101, wherein the immune cell is a T cell, a B cell, a natural killer cell, a macrophage, or a monocyte.

103. The method of any one of claims 100 to 102, wherein the immune cells are T cells.

104. 104. The method of claim 102 or 103, wherein the T cells are CD3+ T cells, CD4+ T cells, CDS+ T cells, naive T cells, regulatory T (Treg) cells, non-regulatory T cells, Th1 cells, Th2 cells, Th9 cells, Th17 cells, T follicular helper (Tfh) cells, cytotoxic T lymphocytes (CTLs), effector T (Teff) cells, central memory T cells, effector memory T cells, effector memory T cells expressing CD45RA (TEMRA cells), tissue-resident memory (Trm) cells, virtual memory T cells, innate immune memory T cells, memory stem cells (Tse), or γδ T cells.

105. The method of any one of claims 102 to 104, wherein the T cells are cytotoxic T cells, helper T cells, memory T cells, regulatory T cells, or tumor-infiltrating lymphocytes.

106. The method of any one of claims 102 to 105, wherein the T cells are human T cells.

107. The method of any one of claims 102 to 106, wherein the T cells are autologous T cells.

108. The method of any one of claims 102 to 107, wherein the T cells are allogeneic T cells.

109. 109. The method of claim 108, wherein the allogeneic T cells are primary T cells.

110. 110. The method of claim 109, wherein the primary T cells are collected from a sample comprising cells from a single donor.

111. 110. The method of claim 109, wherein the primary T cells are collected from a sample comprising cells from multiple donors.

112. 112. The method of any one of claims 108 to 111, wherein the allogeneic T cells are differentiated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

113. 113. The method of any one of claims 100 to 112, wherein after delivery to the immune cell, the polynucleotide encoding the CAR is inserted into a site-specific locus.

114. 114. The method of claim 113, wherein the site-specific locus is a safe harbor locus.

115. 115. The method of claim 114, wherein the site-specific locus is selected from TRAC, TRBC1, TRBC2, B2M, CIITA, MICA, MICB, AAVS1, ABO, CCR5, CLYBL, CXCR4, F3, FUT1, HMGB1, KDM5D, LRP1, RHD, ROSA26, or SHS23.

116. 116. The method of any one of claims 113 to 115, wherein the polynucleotide encoding the CAR is inserted by homology directed repair (HDR).

117. 117. The method of Claim 116, wherein the polynucleotide encoding the CAR is inserted by programmable addition via a CRISPR-associated transposase, prime editing, a TnpB polypeptide, or a site-specific targeting element (PASTE).

118. 118. The method of Claim 116 or 117, wherein the polynucleotide encoding the CAR is inserted by a site-specific nuclease.

119. 119. The method of claim 118, wherein the site-specific nuclease is selected from a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), and a CRISPR-Cas combination.

120. The site-specific nuclease may be any of Cas3, Cas4, Cas5, Cas8a, Cas8b, Cas8c, Cas9, Cas10, Cas12, Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f (C2c10), Cas12g, Cas12h, Cas12i, Cas12k (C2c5), Cas13, Cas13a (C2c2), Cas13b, and Cas12c (C2c3).

120. The method of claim 118 or 119, wherein the polypeptide is selected from the group consisting of s13c, Cas13d, C2c4, C2c8, C2c9, Cmr5, Cse1, Cse2, Csf1, Csm2, Csn2, Csx10, Csx11, Csy1, Csy2, Csy3, Mad7, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, CRISPR-associated transposases, and TnpB polypeptides.

121. 121. The method of any one of claims 116 to 120, wherein the polynucleotide encoding the CAR is inserted using a guide RNA (gRNA) and a CRISPR-associated (Cas) nuclease.

122. 122. The method of Claim 121, wherein the gRNA comprises a complementary region, the complementary region comprising a nucleic acid sequence that is complementary to a target nucleic acid sequence within the locus, and the target nucleic acid sequence comprises an insertion site.

123. An immune cell comprising the CAR according to any one of claims 52 to 57.

124. The immune cells include CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD70, CD123, CD138, GPRC5D, LeY, NKG2D, WT1, GD2, HER2, EGFR, EGFRvIII, B7H3, PSMA, PSCA, CAIX, CD171, CEA, CSPG4, EPHA2, FAP, FRα, IL-1 3Rα, mesothelin, MUC1, MUC16, ROR1, c-Met, CD133, Ep-CAM, GPC3, HPV16, IL13Ra2, MAGEA3, MAGEA4, MART1, NY-ESO, VEGFR2, α-folate, CD24, CD44v7 / 8, EGP-2, EGP-40, erb-B2, erb-B, FBP, fetal acetylcholine receptor, G Ｄ２ , G Ｄ３ , HMW-MAA, IL-11Rα, KDR, Lewis Y, L1-cell adhesion molecule, MADE-A1, carcinoembryonic antigen (h5T4), TAG-72, CD19 / 22, syndecan-1, or BCMA.

125. 125. The cell of claim 123 or 124, wherein the cell is a T cell, a B cell, a natural killer cell, a macrophage, or a monocyte.

126. The cell of claim 125, wherein the cell is a T cell.

127. The cell of claim 125 or 126, wherein the T cell is a CD3+ T cell, a CD4+ T cell, a CDS+ T cell, a naive T cell, a regulatory T (Treg) cell, a non-regulatory T cell, a Th1 cell, a Th2 cell, a Th9 cell, a Th17 cell, a T follicular helper (Tfh) cell, a cytotoxic T lymphocyte (CTL), an effector T (Teff) cell, a central memory T cell, an effector memory T cell, an effector memory T cell expressing CD45RA (TEMRA cell), a tissue-resident memory (Trm) cell, a virtual memory T cell, an innate immune memory T cell, a memory stem cell (Tse), or a γδ T cell.

128. The cell of any one of claims 125 to 127, wherein the T cell is a cytotoxic T cell, a helper T cell, a memory T cell, a regulatory T cell, or a tumor-infiltrating lymphocyte.

129. The cell of any one of claims 125 to 128, wherein the T cell is a human T cell.

130. The cell of any one of claims 125 to 129, wherein the T cell is an autologous T cell.

131. The cell of any one of claims 125 to 129, wherein the T cell is an allogeneic T cell.

132. The cell of claim 131 , wherein the allogeneic T cell is a primary T cell.

133. 133. The cell of claim 132, wherein the primary T cells are collected from a sample containing cells from a single donor.

134. 133. The cell of claim 132, wherein the primary T cells are collected from a sample comprising cells from multiple donors.

135. 135. The cell of any one of claims 131 to 134, wherein the allogeneic T cell is differentiated from an embryonic stem cell (ESC) or an induced pluripotent stem cell (iPSC).

136. 1. An engineered cell, comprising: a) the CAR according to any one of claims 52 to 57; and b) one or more modifications that (i) reduce the expression of one or more MHC class I molecules and / or one or more MHC class II molecules, and / or (ii) increase the expression of one or more tolerance-inducing factors, wherein the reduced expression of (i) and the increased expression of (ii) are compared to cells of the same cell type that do not contain the modification(s). The engineered cell comprising:

137. 137. The engineered cell of claim 136, wherein the one or more modifications that increase expression comprise increased cell surface expression, and / or the one or more modifications that decrease expression comprise decreased cell surface expression.

138. The one or more modifications in (i) above are a) one or more MHC class I molecules; b) one or more MHC class II molecules; or c) one or more MHC class I molecules and one or more MHC class II molecules 138. The engineered cell of claim 136 or 137, wherein the engineered cell reduces expression of:

139. 139. The engineered cell of any one of Claims 136-138, wherein the one or more modifications in (i) reduce expression of one or more molecules selected from B2M, TAP I, NLRC5, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, HLA-DM, HLA-DO, RFX5, RFXANK, RFXAP, NFY-A, NFY-B, and NFY-C.

140. 140. The engineered cell of claim 139, wherein the engineered cell does not express one or more molecules selected from B2M, TAP I, NLRC5, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, HLA-DM, HLA-DO, RFX5, RFXANK, RFXAP, NFY-A, NFY-B, and NFY-C.

141. 141. The engineered cell of any one of claims 136-140, wherein said reduced expression comprises inactivation, disruption, or knockout of one or both alleles of genes encoding or regulating expression of said one or more MHC class I molecules and / or said one or more MHC class II molecules.

142. 142. The engineered cell of any one of Claims 136-141, wherein the one or more tolerance inducers comprise one or more tolerance inducers selected from A20 / TNFAIP3, C1-inhibitory factor, CCL21, CCL22, CD16, CD16 Fc receptor, CD24, CD27, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD200, CR1, CTLA4-Ig, DUX4, FasL, H2-M3, HLA-C, HLA-E, HLA-E heavy chain, HLA-F, HLA-G, IDO1, IL-10, IL-15RF, IL-35, MANF, Mfge8, PD-L1, and Serpinb9.

143. 143. The engineered cell of any one of claims 136-142, wherein the increased expression comprises a modification that increases the activity of a gene that encodes or controls the expression of the one or more tolerance-inducing factors.

144. A method comprising administering an effective amount of the CAR cell of any one of claims 123 to 143 to a subject in need thereof.

145. The method of claim 1244, wherein the method is for treating a disease in the subject.

146. 144. A population of immune cells expressing the CAR of any one of claims 52 to 57 or the cells of any one of claims 123 to 143 for use in treating a disease in a subject.

147. 144. A composition of immune cells expressing the CAR of any one of claims 52 to 57 or the cells of any one of claims 123 to 143 for use in treating a disease in a subject.

148. 144. A pharmaceutical composition of an immune cell expressing the CAR of any one of claims 52 to 57 or the cell of any one of claims 123 to 143 for use in treating a disease in a subject.

149. 149. Use of the population of cells described in claim 146, the composition of cells described in claim 147, or the pharmaceutical composition described in claim 148 for use in treating a disease in a subject.

150. Use of the population of cells described in claim 146, the composition of cells described in claim 147, or the pharmaceutical composition described in claim 148 in the manufacture of a medicament for the treatment of a disease.

151. 150. The method of claim 144 or 145, the population of cells of claim 146, the composition of claim 147, the pharmaceutical composition of claim 148, or the use of claim 149 or 150, wherein the disease is cancer.

152. The cancer may be characterized by the expression of CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD70, kappa, lambda, B-cell maturation factor (BCMA), G protein-coupled receptor family C group 5 member D (GPRC5D), CD123, LeY, NKG2D ligand, WT1, GD2, HER2, EGFR, EGFRvIII, B7H3, PSMA, PSCA, CAIX, CD171, CEA, CSPG4, EPHA2 , FAP, FRα, IL-13Rα, mesothelin, MUC1, MUC16, ROR1, c-Met, CD133, Ep-CAM, GPC3, HPV16-E6, IL13Ra2, MAGEA3, MAGEA4, MART1, NY-ESO-1, VEGFR2, α-folate receptor, CD24, CD44v7 / 8, EGP-2, EGP-40, erb-B2, erb-B2, 3, 4, FBP, fetal acetylcholine receptor, G Ｄ２ , G Ｄ３ , HMW-MAA, IL-11Rα, KDR, Lewis Y, L1-cell adhesion molecule, MAGE-A1, carcinoembryonic antigen (h5T4), and / or TAG-72 expression.

153. 153. The method, population of cells, composition, pharmaceutical composition or use of any of claims 144-152, wherein said cancer is a hematological malignancy.

154. 154. The method, population of cells, composition, pharmaceutical composition, or use of any one of claims 144 to 153, wherein the hematological malignancy is selected from myeloid neoplasms, myelodysplastic syndromes (MDS), myeloproliferative / myelodysplastic syndromes, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), multiple myeloma (MM), blast crisis chronic myelogenous leukemia (bcCML), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), T-cell lymphoma, and B-cell lymphoma.

155. 155. The method, population of cells, composition, pharmaceutical composition or use of any one of claims 144 to 154, wherein the cancer is a solid malignant tumor.

156. 156. The method, population of cells, composition, pharmaceutical composition or use of any one of claims 144 to 155, wherein the solid malignant tumor is selected from breast cancer, ovarian cancer, colon cancer, prostate cancer, epithelial cancer, renal cell carcinoma, pancreatic adenocarcinoma, cervical cancer, colorectal cancer, glioblastoma, rhabdomyosarcoma, neuroblastoma, melanoma, Ewing's sarcoma, osteosarcoma, mesothelioma, and adenocarcinoma.

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