Fully human chimeric antigen receptors targeting CD19 and uses thereof

Abstract

The present disclosure generally relates to, inter alia, to novel polypeptides, e.g., fully human chimeric antigen receptors that target CD19. The disclosure also provides compositions and methods useful for producing such molecules, as well as methods for the detection and treatment of health conditions, such as diseases (e.g., cancer) associate with CD19.

Description

Attorney Docket: 078430-541001WOFULLY HUMAN CHIMERIC ANTIGEN RECEPTORS TARGETING CD 19AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 754,370; filed on February 5, 2025. The disclosures of the above-referenced application is herein expressly incorporated by reference it its entireties, including any drawings.INCORPORATION OF THE SEQUENCE LISTING

[0002] This application contains a Sequence Listing, which is hereby incorporated herein by reference in its entirety. The accompanying Sequence Listing XML file, named “078430-541001WO_Sequence Listing_ST26.xml,” was created on February 3, 2026, and is115,598 bytes in size.FIELD

[0003] The present disclosure relates generally to the fields of oncology and immunotherapeutics, and particularly relates to novel polypeptides, e.g., fully human chimeric antigen receptors that target CD 19. The disclosure also provides compositions and methods useful for producing such molecules, as well as methods for the detection and treatment of health conditions, such as diseases (e.g., cancer) associate with CD 19.BACKGROUND

[0004] Recent advances in genetic engineering and synthetic biology are enabling the development of novel gene therapies and engineered-cell therapies for the treatment of many diseases and health disorders. For example, chimeric antigen receptors (CARs) have emerged as a promising approach for immunotherapy and made headlines in clinical trials conducted by a number of pharmaceutical and biotechnology companies. CARs are antigen-specific recombinant receptors, which, in a single molecule, redirect the specificity and function of a number of immune cells, including T lymphocytes, natural killer (NK) cells, natural killer T (NKT) cells, and macrophages. For example, in CAR-T cell therapy, the general premise for the use of CAR-T cells in cancer immunotherapy is to rapidly generate tumor-targeted T cells, bypassing the barriers and incremental kinetics of active immunization, and eliminating MHC restriction inAttorney Docket: 078430-541001WOantigen-recognition. Once expressed in T cells, the CAR-modified T cells acquire supra-physiological properties and act as “living drugs” that may exert both immediate and long-term effects. Multiple iterations of CARs have been developed, mainly focusing on antigen-binding moiety and intracellular signaling modules, which are deemed crucial for CAR design. To achieve appropriate costimulatory signals in order to activate effector T cells, improve response, and prolong persistence, many different types of costimulatory receptors can be incorporated, alone, in tandem, or in larger arrays.

[0005] However, the deployment of first-generation CAR-T cell therapies in humans continues to present significant challenges for clinical development. One major potential side effect of first-generation CAR-T cell therapies is the ability of the engineered CAR constructs to induce host immune responses. Immunogenicity risk factors can be associated with the presence of nonhuman or partially human sequences in the engineered CAR constructs, as well as with the presence of residual viral proteins or other non-human origin proteins used as part of the production of the first-generation engineered CAR constructs. These types of immune response have the potential to drive immune rejection of CAR-T cell therapies, for example possibly affecting engineered cell expansion and persistence, and therefore the overall safety and clinically meaningful response of the treatment.

[0006] Consequently, there remains a need for fully human CARs to overcome these obstacles to extend the reach of these therapeutics to more diseases and to treat more patients. The invention described herein provides solutions to address these obstacles and provides additional benefits as well.SUMMARY

[0007] The present disclosure relates generally to the development of immuno-therapeutics, including enhanced polypeptides and chimeric antigen receptors (CARs). In particular, some embodiments of the disclosure relate to include recombinant polypeptides and CARS containing an extracellular domain (ECD) that targets CD 19, wherein the ECD includes a fully human antibody or an antigen-binding fragment thereof. The disclosure also provides compositions and methods useful for producing such molecules, as well as methods for the detection, prevention, and treatment of health conditions, such as diseases (e.g., cancer) associate with CD 19.

[0008] In one aspect, provided herein are various chimeric polypeptides including: (a) anAttorney Docket: 078430-541001WOextracellular domain (ECD) having a binding affinity for human CD 19, wherein the ECD comprises a fully human antibody or an antigen-binding fragment thereof, (b) a hinge domain and a transmembrane (TM) domain from CD28, and (c) an intracellular signaling domain (ICD) comprising one or more costimulatory domains.

[0009] Non-limiting exemplary embodiments of the disclosed chimeric polypeptide of the disclosure include one or more of the following features. In some embodiments, the ICD further includes a CD3^ T cell activation domain. In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, includes all six complementary determining regions (CDRs) of an antibody selected from the group consisting of 21D4, 21D4a, 47G4, 27F3, 3C10, 5G7, 13F1 and 46E8. In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, includes the light chain region (VL) and heavy chain region (VH) of the antibody. In some embodiments, the antigen-binding fragment thereof is or includes a singlechain antibody fragment (scFv), a F(ab) fragment, a F(ab') fragment, a F(ab')2 fragment, Fd fragment, a Fab'-SH fragment, or a Fv fragment.

[0010] In some embodiments, the antigen-binding fragment thereof is or includes a singlechain antibody fragment (scFv) comprising a light chain variable region (VL) and a heavy chain variable region (VH) operably linked to each other via a linker. In some embodiments, the light chain variable region (VL) is N-terminally linked to the heavy chain variable region (VH). In some embodiments, the light chain variable region (VL) is C-terminally linked to the heavy chain variable region (VH). In some embodiments, the linker is a polypeptide linker (peptidal linkage). In some embodiments, the polypeptide linker is or comprises a glycine-serine linker or a Whitlow linker (GSTSGSGKPGSGEGSTKG; SEQ ID NO: 77).

[0011] In some embodiments of the disclosure, the human CD 19 scFv includes, in N-terminal to C-terminal direction: (a) a VL region, a linker, and a VH region; or (b) a VH region, a linker, and a VL region. In some embodiments of the disclosure, the human CD 19 scFv includes, in N-terminal to C-terminal direction: (a) a VL region, a (GGGGS)s linker, and a VH region; (b) a VL region, a Whitlow linker, and a VH region; (c) a VH region, a (GGGGS)s linker (GGGGSGGGGSGGGGS; SEQ ID NO: 78), and a VL region; or (d) a VH region, a Whitlow linker, and a VL region. In some embodiments, the chimeric polypeptide of the disclosure is or includes a chimeric antigen receptor (CAR).

[0012] In some embodiments of the disclosure, the one or more costimulatory domains is fromAttorney Docket: 078430-541001WOa polypeptide selected from the group consisting of a 4- IBB (CD 137), CD27 (TNFRSF7), CD28, 0X40 (CD 134), CD70, LFA-2 (CD2), CD5, ICAM-1 (CD54), LFA-1 (CDlla / CD18), DAP10, DAP12, a co-stimulatory inducible T-cell costimulatory (ICOS) polypeptide sequence, and a combination of any thereof. In some embodiments the one or more costimulatory domains is or includes a costimulatory 4- IBB (CD 137) polypeptide sequence.

[0013] In some embodiments, the chimeric polypeptide of the disclosure includes, in N-terminal to C-terminal direction: (a) a human CD 19 scFv, (b) a hinge domain and a TM domain from CD28, (c) a costimulatory domain from 4-1BB; and (d) a CD3 ICD.

[0014] In some embodiments, the chimeric polypeptide of the disclosure comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

[0015] In another aspect, provided herein are various recombinant nucleic acid molecules including nucleic acid sequences encoding the chimeric polypeptide as disclosed herein. Nonlimiting exemplary embodiments of the recombinant nucleic acid molecules include one or more of the following features. In some embodiments, the chimeric polypeptide is a CAR. In some embodiments, the recombinant nucleic acid molecule includes a nucleic acid sequence that has at least 80% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. In some embodiments, the recombinant nucleic acid molecule is operably linked to a heterologous nucleic acid sequence. In some embodiments, the recombinant nucleic acid molecule is further defined as (e.g., incorporated into) an expression cassette in a vector. In some embodiments, the vector is a plasmid vector or a viral vector. In some embodiments, the viral vector is derived from a lentivirus, an adenovirus, an adeno-associated virus (AAV), a baculovirus, or a retrovirus.

[0016] In another aspect, some embodiments of the disclosure relate to a recombinant cell including: (a) a chimeric polypeptide as disclosed herein; and / or (b) a nucleic acid molecule as disclosed herein. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the recombinant cell is an immune system cell. In some embodiments, the immune system cell is a T cell, B cell, a monocyte, a natural killer (NK) cell, a natural killer T (NKT) cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a monocyte, a hematopoietic stem cell, or a hematopoietic stem cell progenitor. In some embodiments, the cell is an IPSC-derived T cell, a regulatory T cell, a helper T cell (TH), a cytotoxic T cell (TCTL), aAttorney Docket: 078430-541001WOmemory T cell, a gamma delta (y3) T cell, a CD8+ T cytotoxic lymphocyte cell, or a CD4+ T helper lymphocyte cell. In some embodiments, the CD8+ T cytotoxic lymphocyte cell is selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, effector CD8+ T cells, CD8+ stem memory T cells, bulk CD8+ T cells. In some embodiments, the CD4+ T helper lymphocyte cell is selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, effector CD4+ T cells, CD4+ stem memory T cells, and bulk CD4+ T cells. In some embodiments, the T cell is an exhausted T cell or a non-exhausted T cell.

[0017] In another aspect, some embodiments disclosed herein relate to methods for making a recombinant cell, wherein the method includes (a) providing a host cell capable of protein expression; and (b) transducing the provided host cell with a recombinant nucleic acid as disclosed herein to produce a recombinant cell. Accordingly, in a related aspect, also provided herein are recombinant cells produced by the methods of the disclosure. In a further related aspect, some embodiments of the disclosure provide cell cultures that include at least one recombinant cell of the disclosure and a culture medium.

[0018] In another aspect, some embodiments of the disclosure relate to a composition, e.g., a pharmaceutical composition including a pharmaceutically acceptable carrier and one or more of: (a) a chimeric polypeptide as disclosed herein; (b) a nucleic acid molecule as disclosed herein; and / or (c) a recombinant cell as disclosed herein. Non-limiting exemplary embodiments of the composition include one or more of the following features. In some embodiments, the composition includes a recombinant nucleic acid as disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the recombinant nucleic acid is encapsulated in a viral capsid or a lipid nanoparticle. In some embodiments, the composition includes a recombinant cell as disclosed herein and a pharmaceutically acceptable carrier.

[0019] In yet another aspect, some embodiments of the disclosure relate to methods for preventing and / or treating a health condition associated with expression of CD 19 in a subject in need thereof, wherein the methods include administering to the subject a composition including one or more of the following: (a) a chimeric polypeptide as disclosed herein; (b) a nucleic acid molecule as disclosed herein; (c) a recombinant T cell as disclosed herein; and / or (d) a pharmaceutical composition as disclosed herein.

[0020] Exemplary embodiments of the disclosed methods include one or more of the followingAttorney Docket: 078430-541001WOfeatures. In some embodiments, the recombinant T cell is autologous to the subject. In some embodiments, the T cell is obtained by leukapheresis of a sample obtained from a subject. In some embodiments, the health condition is a disease or disorder associate with expression of CD 19. In some embodiments, the disease is a cancer, a malignancy, a precancerous condition, an autoimmune disease (AID), or a non-cancer related indication associated with expression of CD 19. In some embodiments, the autoimmune disease (AID) is systemic lupus erythematosus (SLE), idiopathic inflammatory myositis (IIM), or systemic sclerosis (SSc).

[0021] In some embodiments, the disease is a cancer and CD 19 is expressed at a heterogenous level or a low level in cancer cells. In some embodiments, the disease or disorder is a B-cell malignancy. In some embodiments, the B-cell malignancy is a hematologic malignancy selected from the group consisting B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), acute myelogenous leukemia (AML), acute lymphoid leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B cell pro-lymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, null-acute lymphoblastic leukemia, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, splenic, marginal zone lymphoma, and Waldenstrom macroglobulinemia.

[0022] In some embodiments of the disclosure, the administered composition confers increased production of interferon gamma (IFNy) and / or interleukin-2 (IL-2) in the subject. In some embodiments, the administered composition inhibits or kills malignant B-cells in the subject. In some embodiments, the administered composition inhibits tumor growth or metastasis of the cancer in the subject.

[0023] In some embodiments, the composition is administered to the subject individually as a sole therapy or as a first therapy or in combination with a second therapy. In some embodiments, the second therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, and surgery. In some embodiments, the second therapy includes an additional CAR T cell and / or a bispecific antibody targeting CD3 and a B-cell antigen. In some embodiments, the second therapy includes targeting at least one antigenAttorney Docket: 078430-541001WOexpressed preferentially or exclusively on a B-cell malignancy. In some embodiments, the at least one antigen expressed on a B-cell malignancy is CD22, CD20, CD79a, CD79b, BAFF-R, BCMA, CD72, CD 123, or a combination thereof. In some embodiments, the first therapy and the second therapy are administered concomitantly. In some embodiments, the first therapy is administered at the same time as the second therapy. In some embodiments, the first therapy and the second therapy are administered sequentially. In some embodiments, the first therapy is administered before the second therapy. In some embodiments, the therapy is administered after the second therapy. In some embodiments, the first therapy is administered before and / or after the second therapy. In some embodiments, the first therapy and the second therapy are administered in rotation. In some embodiments, the first therapy and the second therapy are administered together in a single formulation.

[0024] In another aspect, some embodiments of the disclosure provide various kits for the practice of the methods disclosed herein. Some embodiments relate to kits for methods of the diagnosis, prevention, and / or treatment of a condition in a subject in need thereof, wherein the kits include one or more of: a chimeric polypeptide of the disclosure; a recombinant nucleic acid of the disclosure; a recombinant cell of the disclosure, and a pharmaceutical composition of the disclosure.

[0025] In another aspect, provided herein is the use of one or more of: a chimeric polypeptide of the disclosure, a recombinant nucleic acid of the disclosure, a recombinant cell of the disclosure, and a pharmaceutical composition, for the diagnosis, prevention, and / or treatment of a condition. In some embodiments, the condition is a proliferative disease. In some embodiments, the proliferative disease is a cancer.

[0026] In another aspect, provided herein is the use of one or more of the following: a chimeric polypeptide of the disclosure, a recombinant nucleic acid of the disclosure, a recombinant cell of the disclosure, or a pharmaceutical composition of the disclosure, in the manufacture of a medicament for the prevention and / or treatment of a health condition. In some embodiments, the condition is a proliferative disease. In some embodiments, the proliferative disease is a cancer.

[0027] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative embodiments and features described herein, further aspects, embodiments, objects and features of the disclosure will become fully apparent from the drawings and the detailed description and the claims.Attorney Docket: 078430-541001WO

[0028] Each of the aspects and embodiments described herein are capable of being used together, unless excluded either explicitly or clearly from the context of the embodiment or aspect.

[0029] Throughout this specification, various patents, patent applications and other types of publications (e.g., journal articles, electronic database entries, etc.) are referenced. The disclosure of all patents, patent applications, and other publications cited herein are hereby incorporated by reference in their entirety for all purposes.BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIGS. 1A-1B show schematic diagrams of a previously reported CD19-CD8H / TM-CD28-zeta CAR (NCT02659943) as well as four exemplary human CD19 CAR constructs (HuCD19 CAR) of the present disclosure and controls using the murine FMC63 anti-CD19 binder. FIG. 1 A: Hui 9 CAR T cell schematics with corresponding scFv orientations, linkers, transmembrane domains and co-stimulatory signaling molecules. In each scFv, the light chain variable region (VL) and a heavy chain variable region (VH) were linked to each other via a linker. Whitlow linker (solid) was used in the top three constructs, whereas (GGGGS)s linker (dashed) was used in the bottom two constructs. FIG. IB: FMC63-based CAR controls depicting the same as FIG. 1A.

[0031] FIG. 2: Recombinant, Fc- and fluorophore- conjugated CD19 protein used for flow cytometry detection of HuCD19 CAR constructs and mouse FMC63 -based CAR controls described in FIGS. 1A-1B above. In these experiments, Hui 9 CAR T cells and FMC63 controls surface expression were evaluated using CD19-protein for detection via flow cytometry. Mock cells were untransduced, activated T cells from the same donor. All the CAR T cell constructs were incubated with conjugated CD19Fc-DyLight650 protein (R&D Systems) and assayed via flow cytometry for surface CAR expression at D10 post-activation.

[0032] FIGS. 3A-3B graphically summarize the results of co-culture experiments and illustrating the production of relevant cytokines (IL-2 and IFNy). FIG. 3A: NALM6 clones expressing high (NALM6 WT-CD19hlgh; top panel) and low (NALM6-CD19963; bottom panel) densities of surface CD 19 molecules were cocultured ata 1:1 ratio with Hui 9 CAR T cells and tumor cell killing was measured tracking tumor GFP expression in an Incucyte assay. FIG. 3B:IL-2 and IFNy cytokine production after 24 hr co-culture at 1:1 ratio of Hu 19 CAR Ts withAttorney Docket: 078430-541001WONALM6 clones expressing high (NALM6 WT-CD19hlgh; top panels) (top panels) and low (NALM6-CD19963; bottom panel) densities of CD19, respectively. In these experiments, IL-2 and IFNy were measured via ELISA assay using supernatant from the 24 hr co-culture.

[0033] FIGS. 4A-4C graphically summarize the results of experiments demonstrating effectiveness of the Hui 9 CAR T cells disclosed herein in low density NALM6-CD19 (Nalm6963) model in vivo. FIG. 4A: Mice were inoculated with IxlO6NALM6-CD19963cells at Day -3 and treated with 3xl06of corresponding CAR T cells. Tumor growth was tracked with bioluminescence imaging for 9 days with graphed values for Day 0 and Day 9. FIG. 4B: Mean tumor total flux values of the different treatments in FIG.4A. FIG. 4C: Mice were inoculated with 106NALM6-CD19963cells at Day -3 and treated with 3xl06of corresponding CAR T cells. In these experiments, overall survival was tracked for until the end of the experiment. A log-rank test was used to compare survival curves between groups p-value<0.0001. One-way analysis of variance (ANOVA) was used to compare the mean flux values of the different treatment groups p-value<0.00001.

[0034] FIG. 5 graphically summarize the results of experiments demonstrating effectiveness of the Hul9 CAR T cells disclosed herein in high density / wildtype NALM6-CD19 ( NALM6 WT-CD19hlgh) model in vivo total tumor flux. In these experiments, mice were inoculated with 1 xlO6NALM6_WT-CD19hlghcells at Day -3 and treated with IxlO6of corresponding CAR T cells. Tumor growth was tracked with bioluminescence imaging for 91 days.

[0035] FIG. 6 graphically summarize the results of experiments demonstrating effectiveness of the Hul9 CAR T cells disclosed herein in high density / wildtype NALM6-CD19 ( NALM6 WT-CD19hlgh) model in vivo overall survival. In these experiments, mice were inoculated with IxlO6NALM6(WT)-CD19hlghat Day -3 and treated with 1 xlO6of corresponding CAR T cells. Overall survival was tracked for 91 days.

[0036] FIGS. 7A-7B graphically summarize the results of experiments illustrating in vivo overall persistence of the Hul9 CAR T cells in high density / wildtype NALM6-CD19 (NALM6_WT-CD19hlgh) model. In these experiments, mice were inoculated with IxlO6NALM6_WT-CD19hlghat Day -3 and treated with a curative dose of 6xl06of the corresponding CAR T cells. Experiment was terminated at Day 26 and total human T cells numbers (FIG. 7A) as well as total CAR+T cells (FIG. 7B) in each group were plotted. One-way analysis of variance (ANOVA) was used to compare the mean numbers of T cells and CAR+T cells in theAttorney Docket: 078430-541001WOdifferent treatment groups p-value**<0.01.DETAILED DESCRIPTION OF THE DISCLOSURE

[0037] The present disclosure relates generally to, inter alia, chimeric polypeptides and chimeric antigen receptors (CARs) that include: (a) an extracellular domain (ECD) having a binding affinity for human CD 19, wherein the ECD comprises a fully human antibody or an antigen-binding fragment thereof, (b) a hinge domain and a transmembrane (TM) domain from CD28, and (c) an intracellular signaling domain (ICD) comprising one or more costimulatory domains. The disclosure also provides compositions and methods useful for making such polypeptides and CARs, as well as methods for the detection and treatment of conditions, such as proliferative diseases ( .g., cancers).

[0038] The experimental results presented herein demonstrate that a fully human anti-CD19 antibody or an antigen-binding fragment thereof incorporated in several CAR designs was capable of increasing the antitumor efficacy of the corresponding CAR T cells and, since they are fully humanized, are more amenable to use in humans due to reduced chance of immune mediated rejection. As described in greater detail below, Applicant utilized a fully human antigen-binding molecule having binding affinity for the target antigen CD 19 to generate fully human CD 19 CARs that are capable of recognizing CD 19 low tumors while maintaining persistence in vivo. These had surprising level of efficacy in vivo against tumors with high antigen density as well. These novel constructs dramatically outperformed other previously described constructs utilizing this fully human binder including against CD19-low tumors. In particular, experiments described in the Examples demonstrate that fully human anti-CD19 CAR T cells as engineered resulted in substantially improved CAR T cell persistence in a curative model of B cell leukemia compared to clinical benchmarks and other previously explored constructs, illustrating the unique benefit to the designs employed here.

[0039] In some embodiments of the disclosure, the CAR constructs disclosed herein have the advantage of recognition of low antigen density through the CD28 Hinge-TM domain as well as improved persistence through 41 -BB (both important clinically). Additionally, they demonstrated a surprising level of efficacy in vivo compared to clinical benchmarks. The use of 4- IBB costimulation and a fully human binder both could contribute to improved persistence as 4- IBB signaling drives persistence and the fully human binder greatly reduces the chance ofAttorney Docket: 078430-541001WOimmune mediated rejection.

[0040] In particular, the CAR T cells disclosed herein could be used for treating low or heterogenous CD 19 density hematological malignancies where clinically approved CAR T cells fail. Given their fully human components, they could be used for re-infusion of CAR T cell-infused patients that underwent currently approved CAR T cell therapy.

[0041] These results suggest potential novel strategies in designing more effective chimeric antigen receptors to complement existing immunotherapeutic approaches. In particular, the human CAR T cells described here showcase improved recognition of low antigen hematological tumors while also demonstrating superior in vivo activity and persistence to clinical benchmarks.DEFINITION

[0042] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art.

[0043] The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof. “A and / or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B”.

[0044] The term “about”, as used herein, has its ordinary meaning of approximately. If the degree of approximation is not otherwise clear from the context, “about” means either within plus or minus 10% of the provided value, or rounded to the nearest significant figure, in all cases inclusive of the provided value. Where ranges are provided, they are inclusive of the boundary values.

[0045] As used herein, the term “antibody” refers to a class of proteins that are generally known as immunoglobulins that specifically bind to an antigen molecule. The term antibody includes full-length monoclonal antibodies (mAb), such as IgG2 monoclonal antibodies, whichAttorney Docket: 078430-541001WOinclude immunoglobulin Fc regions. The term antibody also includes bispecific antibodies, diabodies, single-chain antibody fragments (scFv), and antibody fragments such as Fab, F(ab')2, and Fv. In instances where the antibody is a bispecific antibody, the bispecific antibody can be in many different formats. The antibody can be monoclonal or polyclonal and can be prepared by techniques that are well known in the art, such as immunization of a host and collection of sera (polyclonal), or by preparing continuous hybrid cell lines and collecting the secreted protein (monoclonal), or by cloning and expressing nucleotide sequences or mutagenized versions thereof coding at least for the amino acid sequences required for specific binding of natural antibodies. As such, antibodies may include a complete immunoglobulin or fragment thereof, which immunoglobulins include the various classes and isotypes, such as IgA, IgD, IgE, IgGl , IgG2a, IgG2b and IgG3, IgM, etc. Fragments thereof may include Fab, Fv and F(ab’)2, Fab’, and the like. In addition, aggregates, polymers, and conjugates of immunoglobulins or their fragments can be used where appropriate so long as binding affinity for a particular target (e.g., CD 19.

[0046] The terms “cell”, “cell culture”, “cell line” refer not only to the particular subject cell, cell culture, or cell line but also to the progeny or potential progeny of such a cell, cell culture, or cell line, without regard to the number of transfers or passages in culture. It should be understood that not all progeny are exactly identical to the parental cell. This is because certain modifications may occur in succeeding generations due to either mutation (e.g., deliberate or inadvertent mutations) or environmental influences (e.g., methylation or other epigenetic modifications), such that progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein, so long as the progeny retain the same functionality as that of the original cell, cell culture, or cell line.

[0047] As used herein, the term “chimeric antigen receptor” (CAR) refers to a polypeptide construct comprising at least an extracellular antigen-binding domain, a TMD and a cytoplasmic signaling domain (also referred to as “an intracellular signaling domain” or ICD). In some cases, the cytoplasmic signaling domain includes a functional signaling domain derived from a stimulatory molecule. The stimulatory molecule often is the zeta chain associated with the T cell receptor complex. Optionally, the ICD can further include one or more functional signaling domains derived from at least one costimulatory molecule, such as e.g., 4- IBB (i.e., CD 137), CD27, and / or CD28. Optionally, the ICD further comprises a T cell activation domain, such as aAttorney Docket: 078430-541001WOCD3^ T cell activation domain.

[0048] Generally, the CARs of the disclosure include an ectodomain and an endodomain each as defined by the host cell wall. In this regard, the terms “ectodomain” or “extracellular domain” generally refer to the portion of the CAR polypeptide outside of the cell or exterior to the membranous lipid bilayer, which may include the antigen recognition binding domains, an optional hinge domain, and any spacer domains exterior to the amino acid residues physically spanning the membrane. Conversely, the terms “endodomain” or “intracellular domain” generally refer to the portion of the CAR polypeptide inside the cell or interior to the membranous lipid bilayer, which may also include any spacer domains interior to the amino acid residues physically spanning the membrane, as well as the ICD, which comprises one or more costimulatory signaling domains (e.g., IT AM- containing sequences, costimulatory domains, etc.).

[0049] One skilled in the art will understand that the term “derived from” when used in reference to a nucleic acid or polypeptide molecule refers to the origin or source of the molecule, and may include naturally occurring, recombinant, unpurified, or purified molecules. Nucleic acid or polypeptide molecules are considered “derived from” when they include portions or elements assembled in such a way that they produce a functional unit. The portions or elements can be assembled from multiple sources provided that they retain evolutionarily conserved function. In some embodiments, the derivative nucleic acid or polypeptide molecules include substantially the same sequence as the source nucleic acid or polypeptide molecule. For example, the derivative nucleic acid or polypeptide molecules of the present disclosure may have at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the source nucleic acid or polypeptide molecule.

[0050] The terms “nucleic acid molecule” and “polynucleotide” are used interchangeably herein, and refer to both RNA and DNA molecules, including nucleic acid molecules comprising cDNA, genomic DNA, synthetic DNA, and DNA or RNA molecules containing nucleic acid analogs. A nucleic acid molecule can be double-stranded or single-stranded (e.g., a sense strand or an antisense strand). A nucleic acid molecule may contain unconventional or modified nucleotides. The terms "polynucleotide sequence" and "nucleic acid sequence" as used herein interchangeably refer to the sequence of a polynucleotide molecule.

[0051] The term “operably linked”, as used herein, denotes a physical or functional linkageAttorney Docket: 078430-541001WObetween two or more elements, e.g., polypeptide sequences or polynucleotide sequences, which permits them to operate in their intended fashion. Operably linked elements (e.g., domains, segments, portions) of the recombinant polynucleotides and polypeptides of the disclosure may be contiguous or non-contiguous (e.g., linked to one another through a linker).

[0052] The term “percent identity” as used herein in the context of two or more nucleic acids or proteins, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids that are the same (e.g., about 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. See e.g, the NCBI web site at ncbi.nlm.nih.gov / BLAST. Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the complement of a sequence. This definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. Sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al, Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul etal., J Mol Biol 215:403, 1990). Sequence identity can be measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with the default parameters thereof. The amino acid substitution(s) may be a conservative amino acid substitution, for example at a non-essential amino acid residue in the CDR sequence(s). A “conservative amino acid substitution” is understood to be one in which the original amino acid residue is substituted with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are known in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. , threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).Attorney Docket: 078430-541001WO

[0053] The term “recombinant” nucleic acid molecule, polypeptide, and cell as used herein, refers to a nucleic acid molecule, polypeptide, and cell that has been altered through human intervention. As non-limiting examples, a recombinant nucleic acid molecule can be one which: 1) has been synthesized or modified in vitro, for example, using chemical or enzymatic techniques, or recombination of nucleic acid molecules; 2) includes conjoined nucleotide sequences that are not conjoined in nature; 3) has been engineered using molecular cloning techniques such that it lacks one or more nucleotides with respect to the naturally occurring nucleic acid molecule sequence; and / or 4) has been manipulated using molecular cloning techniques such that it has one or more sequence changes or rearrangements with respect to the naturally occurring nucleic acid sequence. A non-limiting example of a recombinant protein is a chimeric antigen receptor as provided herein.

[0054] As used herein, a “subject” or an “individual” includes animals, such as human (e.g., human subjects) and non-human animals. In some embodiments, a “subject” or “individual” is a patient under the care of a physician. Thus, the subject can be a human patient or an individual who has, is at risk of having, or is suspected of having a disease of interest (e.g., cancer) and / or one or more symptoms of the disease. The subject can also be an individual who is diagnosed with a risk of the condition of interest at the time of diagnosis or later. The term "non-human animals" includes all vertebrates, e.g, mammals, e.g, rodents, e.g, mice, and non- mammals, such as non-human primates, e.g., sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0055] As used herein, and unless otherwise specified, a “therapeutically effective amount” or a “therapeutically effective number” of an agent is an amount or number sufficient to provide a therapeutic benefit in the treatment or management of a health condition, such as a disease (e.g., a cancer), or to delay or minimize one or more symptoms associated with the disease. A therapeutically effective amount or number of a compound means an amount or number of therapeutic agent, alone or in combination with other therapeutic agents, which provides a therapeutic benefit in the treatment or management of the disease. The term “therapeutically effective amount” can encompass an amount or number that improves overall therapy of the disease, reduces or avoids symptoms or causes of the disease, or enhances therapeutic efficacy of another therapeutic agent. An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.” A “reduction” of aAttorney Docket: 078430-541001WOsymptom means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s). The exact amount of a composition including a “therapeutically effective amount” will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 2010); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (2016); Pickar, Dosage Calculations (2012); anA Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0056] The term “vector” is used herein to refer to a nucleic acid molecule or sequence capable of transferring or transporting another nucleic acid molecule. For example, a vector can be used as a gene delivery vehicle to transfer a gene into a cell. The transferred nucleic acid molecule is generally linked to, e.g, inserted into, the vector nucleic acid molecule. Generally, a vector is capable of replication when associated with the proper control elements. The term “vector” includes cloning vectors and expression vectors, as well as viral vectors and integrating vectors. An “expression vector” is a vector that includes a regulatory region, thereby capable of expressing DNA sequences and fragments in vitro and / or in vivo. A vector may include sequences that direct autonomous replication in a cell, or may include sequences sufficient to allow integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, e.g., replication defective retroviruses and lentiviruses. In some embodiments, a vector is a gene delivery vector.

[0057] It is understood that aspects and embodiments of the disclosure described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments. As used herein, “comprising” is synonymous with “including”, “containing”, or “characterized by”, and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of’ excludes any elements, steps, or ingredients not specified in the claimed composition or method. As used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any recitation herein of the term “comprising”, particularly in a description of components of a composition or in a description of steps of a method, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or steps.Attorney Docket: 078430-541001WO

[0058] Headings, e.g., (a), (b), (i) etc., are presented merely for ease of reading the specification and claims. The use of headings in the specification or claims does not require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.

[0059] As will be understood by one having ordinary skill in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to”, “at least”, “greater than”, “less than”, and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0060] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0061] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub- combination wasAttorney Docket: 078430-541001WOindividually and explicitly disclosed herein.CD 19

[0062] CD19 (also known as B-lymphocyte antigen CD19, B4, and CVID3) is a cell surface molecule expressed only by B lymphocytes and follicular dendritic cells of the hematopoietic system. It is the earliest of the B-lineage-restricted antigens to be expressed and is present on most pre-B-cells and most non-T-cell acute lymphocytic leukemia cells and B-cell type chronic lymphocytic leukemia cells.

[0063] CD 19 is expressed early in B cell differentiation and continues to be expressed until the B cells are triggered to terminally differentiate. The CD 19 extracellular domain contains two C2-type immunoglobulin (IG)-like domains separated by a smaller potentially disulfide-linked domain. The CD 19 cytoplasmic domain is structurally unique, but highly conserved between human, mouse, and guinea pig. CD 19 is part of a protein complex found on the cell surface of B lymphocytes. The protein complex includes CD19, CD21 (complement receptor, type 2), CD81 (TAPA-1), and CD225 (Leu- 13).

[0064] CD 19 is an important regulator of transmembrane signals in B cells. An increase or decrease in the cell surface density of CD 19 affects B cell development and function, resulting in diseases such as autoimmunity or hypogammaglobulinemia. The CD 19 complex potentiates the response of B cells to antigen in vivo through cross-linking of two separate signal transduction complexes found on B cell membranes. The two signal transduction complexes, associated with membrane IgM and CD 19, activate phospholipase C (PLC) by different mechanisms. CD 19 and B cell receptor cross-linking reduces the number of IgM molecules required to activate PLC. Additionally, CD 19 functions as a specialized adapter protein for the amplification of Arc family kinases.

[0065] CD 19 binding has been shown to both enhance and inhibit B-cell activation and proliferation, depending on the amount of cross-linking that occurs. CD 19 is expressed on greater than 90% of B-cell lymphomas and has been predicted to affect growth of lymphomas in vitro and in vivo. Antibodies generated to CD 19 have been murine antibodies. A disadvantage of using a murine antibody in treatment of human subjects is the human anti-mouse (HAMA) response on administration to the patient. Accordingly, the need exists for improved therapeutic antibodies against CD19 which are more effective for treating and / or preventing diseasesAttorney Docket: 078430-541001WOmediated by CD 19.COMPOSITIONS OF THE DISCLOSURE

[0066] As described in greater detail below, one aspect of the present disclosure relates to novel chimeric polypeptides and CARs that include (a) an extracellular domain (ECD) having a binding affinity for human CD 19, wherein the ECD comprises a fully human antibody or an antigen-binding fragment thereof, (b) a hinge domain and a transmembrane (TM) domain from CD28, and (c) an intracellular signaling domain (ICD) comprising one or more costimulatory domains. Also provided are recombinant nucleic acids encoding such chimeric polypeptides, as well as recombinant cells that have been engineered to express a chimeric polypeptide as disclosed herein and are directed against a cell of interest such as an immune system cell or a cancer cell.

[0067] As used herein, the term “fully human antibody” refers to an antibody consisting entirely of human amino acid sequence / human immunoglobulin protein sequences only. A fully human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. For example, a mouse can be engineered to express human immunoglobulins, e.g., immunoglobulins from a human B cell derived library.CHIMERIC POLYPEPTIDES

[0068] In one aspect, some embodiments disclosed herein relate to chimeric polypeptides which include (a) an extracellular domain (ECD) having a binding affinity for human CD 19, wherein the ECD comprises a fully human antibody or an antigen-binding fragment thereof, (b) a hinge domain and a transmembrane (TM) domain from CD28, and (c) an intracellular signaling domain (ICD) comprising one or more costimulatory domains. The binding of the ECD to its target antigen (e.g., CD 19) can be either in a competitive or non-competitive fashion with a natural ligand of the target antigen, such as CD81, CD21, CD225, or PIK3. Accordingly, in some embodiments of the disclosure, the binding of the ECD to its target antigen can be ligandblocking. In some other embodiments, the binding of the ECD to its target antigen does not block binding of at least one of the CD19’s natural ligands.

[0069] In some embodiments, at least two of the polypeptide segments of the chimeric polypeptides and CARs disclosed herein are directly linked to one another. In some embodiments, all of the polypeptide segments are directly linked to one another. In someAttorney Docket: 078430-541001WOembodiments, at least two of the polypeptide segments are directly linked to one another via at least one covalent bond. In some embodiments, at least two of the polypeptide segments are directly linked to one another via at least one peptide bond. In some embodiments, the chimeric polypeptides of the disclosure include one or more linkers which join the two or more polypeptide segments together. In some embodiments, at least two of the polypeptide segments are operably linked to one another via a linker. There is no particular limitation on the linkers that can be used in the chimeric polypeptides described herein. In some embodiments, the linker is a synthetic compound linker such as, for example, a chemical cross-linking agent. Nonlimiting examples of suitable cross-linking agents that are available on the market include N-hydroxysuccinimide (NHS), disuccinimidylsuberate (DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidylpropionate) (DSP), dithiobis(sulfosuccinimidylpropionate) (DTSSP), ethyleneglycol bis(succinimidylsuccinate) (EGS), ethyleneglycol bis(sulfosuccinimidylsuccinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).

[0070] The linker can also be a linker peptide sequence. Accordingly, in some embodiments, at least two of the polypeptide segments are operably linked to one another via a linker peptide sequence. In principle, there are no particular limitations to the length and / or amino acid composition of the linker peptide sequence. In some embodiments, any arbitrary single-chain peptide including about one to 100 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. amino acid residues) can be used as a peptide linker. In some embodiments, the linker peptide sequence includes about 5 to 50, about 10 to 60, about 20 to 70, about 30 to 80, about 40 to 90, about 50 to 100, about 60 to 80, about 70 to 100, about 30 to 60, about 20 to 80, about 30 to 90 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25, about 20 to 40, about 30 to 50, about 40 to 60, about 50 to 70 amino acid residues. In some embodiments, the linker peptide sequence includes about 40 to 70, about 50 to 80, about 60 to 80, about 70 to 90, or about 80 to 100 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25 amino acid residues.CHIMERIC ANTIGEN RECEPTORS (CARS)

[0071] As described above, in some embodiments of the disclosure, the chimeric polypeptidesAttorney Docket: 078430-541001WOdisclosed herein are configured as chimeric antigen receptors (CARs). CARs are recombinant receptor constructs composed of an extracellular antigen-binding moiety derived from an antibody, joined to a hinge domain and a TMD, which can be further linked to the intracellular T cell signaling domains of the T cell receptor. As such, CAR T cells can combine the specificity of an antibody with the cytotoxic and memory functions of T cells. In some embodiments, the CARs of the disclosure do not include a costimulatory domain. In some embodiments, the disclosed CARs include one or more costimulatory domains.

[0072] In some embodiments of the disclosure, the CARs include (a) an extracellular domain (ECD) having a binding affinity for human CD 19, wherein the ECD comprises a fully human antibody or an antigen-binding fragment thereof, (b) a hinge domain and a transmembrane (TM) domain from CD28, and (c) an intracellular signaling domain (ICD) comprising one or more costimulatory domains. In some embodiments, the ICD further includes a CD3 T cell activation domain.Extracellular domains (ECD)

[0073] In some embodiments, the ECD of the chimeric polypeptides or CARs disclosed herein has a binding affinity for CD 19. In some embodiments, the ECD of the chimeric polypeptides or CARs disclosed herein has a binding affinity for human CD 19, wherein the ECD comprises a fully human antibody or a functional antigen-binding fragment thereof.

[0074] One skilled in the art upon reading the present disclosure will readily understand that the term “functional fragment thereof’ or “functional variant thereof’ refers to a molecule having quantitative and / or qualitative biological activity in common with the wild-type molecule from which the fragment or variant was derived. For example, a functional fragment or a functional variant of an antibody is one which retains essentially the same ability to bind to the same epitope as the antibody from which the functional fragment or functional variant was derived. For instance, an antibody capable of binding to an epitope of a cell surface receptor may be truncated at the N-terminus and / or C-terminus, and the retention of its epitope binding activity assessed using assays known to those of skill in the art.

[0075] The ECD can include naturally-occurring amino acid sequences or can be engineered, designed, or modified so as to provide desired and / or improved properties, e.g., binding affinity. Generally, the binding affinity of an antibody or an antigen-binding moiety for a target antigenAttorney Docket: 078430-541001WO(e.g., CD19 antigen) can be calculated by the Scatchard method described by Frankel etal.,Mol. Immunol, 16: 101-106, 1979. In some embodiments, binding affinity can be measured by an antigen / antibody dissociation rate. In some embodiments, a high binding affinity can be measured by a competition radioimmunoassay. In some embodiments, binding affinity can be measured by ELISA. In some embodiments, antibody affinity can be measured by flow cytometry. An antibody that “selectively binds” a target antigen (such as CD 19 or HER2) is an antibody that binds the target antigen with high affinity and does not significantly bind other unrelated antigens but binds the antigen with high affinity, e.g., with an equilibrium constant (KD) of 100 nM or less, such as 60 nM or less, for example, 30 nM or less, such as, 15 nM or less, or 10 nM or less, or 5 nM or less, or 1 nM or less, or 500 pM or less, or 400 pM or less, or 300 pM or less, or 200 pM or less, or 100 pM or less.

[0076] In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, comprises all six CDRs of an antibody selected from the group consisting of 21D4, 21D4a, 47G4, 27F3, 3C10, 5G7, 13F1 and 46E8 (see, e.g., Table 1). More information regarding the antibodies 21D4, 21D4a, 47G4, 27F3, 3C10, 5G7, 13F1 and 46E8, can be found in, for example, PCT Publication Nos. W02007002223A2, W02009054863A2, and WO2015187528A1, all of which are incorporated by reference herein in their entireties. In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, comprises all six CDRs of the antibody 21D4. In some embodiments, the fully human antibody, or an antigenbinding fragment thereof, comprises all six CDRs of the antibody 21D4a. In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, comprises all six CDRs of the antibody 47G4. In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, comprises all six CDRs of the antibody 27F3. In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, comprises all six CDRs of the antibody 3C10. In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, comprises all six CDRs of the antibody 5G7. In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, comprises all six CDRs of the antibody 13F1. In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, comprises all six CDRs of the antibody 46E8.

[0077] In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, comprises the VH and VL of an antibody selected from the group consisting of 21D4,Attorney Docket: 078430-541001WO21D4a, 47G4, 27F3, 3C10, 5G7, 13F1 and 46E8 (see, e.g., Table 1). In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, comprises the VH and VL of antibody 21D4. In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, comprises the VH and VL of antibody 21D4a. In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, comprises the VH and VL of antibody 47G4. In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, comprises the VH and VL of antibody 27F3. In some embodiments, the fully human antibody, or an antigenbinding fragment thereof, comprises the VH and VL of antibody 3C10. In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, the VH and VL of antibody 5G7. In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, comprises the VH and VL of antibody 13F1. In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, the VH and VL of antibody 46E8.TABLE 1 : Exemplary human CD- 19 antibodies of the disclosure. VH and VL correspond to the sequences of heavy chain variable and light chain variable regions, respectively. Exemplary nucleic acid sequences encoding the heavy chain CDRs (HCDRs) and light chain CDRs (LCDRs) are provided in the Sequence Listing.

[0078] In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, includes the light chain region (VL) and heavy chain region (VH) of the antibody. In some embodiments, the antigen-binding fragment thereof is or includes a single-chain antibody fragment (scFv), a F(ab) fragment, a F(ab') fragment, a F(ab')2 fragment, Fd fragment, a Fab'-SH fragment, or a Fv fragment.Attorney Docket: 078430-541001WOLinkers

[0079] In some embodiments, the antigen-binding fragment thereof is or includes a singlechain antibody fragment (scFv) comprising a light chain variable region (VL) and a heavy chain variable region (VH) operably linked to each other via a linker. In some embodiments, the linker is a synthetic compound linker such as, for example, a chemical cross-linking agent. In some embodiments, the VL region and VH region of each scFv are operably linked to one another via a linker polypeptide sequence (e.g., peptidal linkage). In principle, there are no particular limitations to the length and / or amino acid composition of the linker polypeptide sequence. In some embodiments, any arbitrary single-chain peptide comprising about one to 100 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. amino acid residues) can be used as a polypeptide linker. In some embodiments, the polypeptide linker includes about 5 to 50, about 10 to 60, about 20 to 70, about 30 to 80, about 40 to 90, about 50 to 100, about 60 to 80, about 70 to 100, about 30 to 60, about 20 to 80, about 30 to 90 amino acid residues. In some embodiments, the polypeptide linker includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25, about 20 to 40, about 30 to 50, about 40 to 60, about 50 to 70 amino acid residues. In some embodiments, the polypeptide linker includes about 40 to 70, about 50 to 80, about 60 to 80, about 70 to 90, or about 80 to 100 amino acid residues. In some embodiments, the polypeptide linker includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25 amino acid residue.

[0080] In certain embodiments, the polypeptide linker contains only glycine and / or serine residues (e.g., glycine-serine linker). Examples of such polypeptide linkers include: Gly, Ser; Gly Ser; Gly Gly Ser; Ser Gly Gly; Gly Gly Gly Ser; Ser Gly Gly Gly; Gly Gly Gly Gly Ser; Ser Gly Gly Gly Gly; Gly Gly Gly Gly Gly Ser; Ser Gly Gly Gly Gly Gly; Gly Gly Gly Gly Gly Gly Ser; Ser Gly Gly Gly Gly Gly Gly; (Gly Gly Gly Gly Ser)n, wherein n is an integer of one or more; and (Ser Gly Gly Gly Gly)n, wherein n is an integer of one or more. In some embodiments, the polypeptide linker is modified such that the amino acid sequence GSG (that occurs at the junction of traditional Gly / Ser linker peptide repeats) is not present. For example, in some embodiments, the polypeptide linker includes an amino acid sequence selected from the group consisting of: (GGGXX)nGGGGS and GGGGS(XGGGS)n, where X is any amino acid that can be inserted into the sequence and not result in a polypeptide comprising the sequence GSG, and n is 0 to 4. In some embodiments, the sequence of the polypeptide linker isAttorney Docket: 078430-541001WO(GGGXlX2)nGGGGS and XI is P and X2 is S and n is 0 to 4. In some other embodiments, the sequence of the polypeptide linker is (GGGXlX2)nGGGGS and Xi is G and X2 is Q and n is 0 to 4. In some other embodiments, the sequence of the polypeptide linker is (GGGXlX2)nGGGGS and XI is G and X2 is A and n is 0 to 4. In yet some other embodiments, the sequence of the polypeptide linker is GGGGS(XGGGS)n, and X is P and n is 0 to 4. In some embodiments, the polypeptide linker of the disclosure comprises or consists of the amino acid sequence (GGGGA)2GGGGS. In some embodiments, the polypeptide linker comprises or consists of the amino acid sequence (GGGGQ)2GGGGS. In another embodiment, the polypeptide linker comprises or consists of the amino acid sequence (GGGPS)2GGGGS. In another embodiment, the polypeptide linker comprises or consists of the amino acid sequence GGGGS(PGGGS)2. In some embodiments, the polypeptide linker is (GxS)n or (GxS)nGm with G=glycine, S=serine, and (x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5 and m=0, 1, 2 or 3), preferably x=4 and n=2 or 3, more preferably with x=4, n=2. In some embodiments, the polypeptide linker is (GJS)2. In some embodiments, the polypeptide linker is or comprises a glycine- serine linker or a Whitlow linker (GSTSGSGKPGSGEGSTKG; SEQ ID NO: 77). In some embodiments, the polypeptide linker is or comprises a Whitlow linker. In some embodiments, the polypeptide linker is or comprises a (GGGGS)s linker (GGGGSGGGGSGGGGS; SEQ ID NO: 78).

[0081] In some embodiments, the light chain variable region VL is N-terminally linked to the heavy chain variable region VH. In some embodiments, the light chain variable region VL is C-terminally linked to the heavy chain variable region VH.

[0082] In some embodiments of the disclosure, the human CD 19 scFv includes, in N-terminal to C-terminal direction: (a) a VL region, a linker, and a VH region; or (b) a VH region, a linker, and a VL region. In some embodiments of the disclosure, the human CD 19 scFv includes, in N-terminal to C-terminal direction: (a) a VL region, a (4GS)3linker (GGGGSGGGGSGGGGS; SEQ ID NO: 78), and a VH region; (b) a VL region, a Whitlow linker, and a VH region; (c) a VH region, a (GGGGS)3linker, and a VL region; or (d) a VH region, a Whitlow linker, and a VL region. In some embodiments, the human CD 19 scFv includes, in N-terminal to C-terminal direction: a VL region, a (4GS)3linker (GGGGSGGGGSGGGGS; SEQ ID NO: 78), and a VH region. In some embodiments, the human CD 19 scFv includes, in N-terminal to C-terminal direction: a VL region, a Whitlow linker, and a VH region. In some embodiments, the humanAttorney Docket: 078430-541001WOCD 19 scFv includes, in N-terminal to C-terminal direction: a VH region, a (GGGGS)s linker, and a VL region. In some embodiments, the human CD 19 scFv includes, in N-terminal to C-terminal direction: a VH region, a Whitlow linker, and a VL region.

[0083] In some embodiments, the chimeric polypeptide of the disclosure is or includes a chimeric antigen receptor (CAR).Costimulatory domains

[0084] Generally, the costimulatory domain suitable for the chimeric polypeptides, e.g., CARs disclosed herein can be any one of the costimulatory domains known in the art. Examples of suitable costimulatory domains that can enhance cytokine production and include, but are not limited to, costimulatory polypeptide sequences derived from 4- IBB (CD 137), CD27, CD28, 0X40 (CD134), and costimulatory inducible T-cell costimulatory (ICOS) polypeptide sequences. Accordingly, in some embodiments, the costimulatory domain of the chimeric polypeptides and CARs disclosed herein is selected from the group consisting of a costimulatory 4-1BB (CD137) polypeptide sequence, a costimulatory CD27 polypeptide sequence, a costimulatory CD28 polypeptide sequence, a costimulatory 0X40 (CD 134) polypeptide sequence, and a costimulatory inducible T-cell costimulatory (ICOS) polypeptide sequence. In some embodiments, the chimeric polypeptides and CARs disclosed herein include a costimulatory domain derived from a costimulatory 4- IBB (CD 137) polypeptide sequence. In some embodiments, the chimeric polypeptides and CARs disclosed herein include a costimulatory 4- IBB (CD 137) polypeptide sequence. In some embodiments, the chimeric polypeptides and CARs disclosed herein include a costimulatory domain derived from a costimulatory CD28 polypeptide sequence. In some embodiments, the chimeric polypeptides and CARs disclosed herein include a costimulatory CD28 polypeptide sequence.

[0085] In some embodiments of the disclosure, the ICD of the disclosed CARs includes conserved amino acid motifs that serve as substrates for phosphorylation such as, for example, immunoreceptor tyrosine-based activation motifs (IT AM), and / or immunoreceptor tyrosinebased inhibition motifs (ITIM). In some embodiments, the ICD of the disclosed CARs includes at least 1, at least 2, at least 3, at least 4, or at least 5 specific tyrosine-based motifs selected from ITAM motifs, an ITIM motifs, or related intracellular motifs that serve as a substrate for phosphorylation. In some embodiments of the disclosure, the ICD of the disclosed CARs includes at least 1, at least 2, at least 3, at least 4, or at least 5 IT AMs. Generally, any ICDAttorney Docket: 078430-541001WOincluding an ITAM can be suitably used for the construction of the chimeric polypeptides as described herein. An ITAM generally includes a conserved protein motif that is often present in the tail portion of signaling molecules expressed in many immune cells. The motif may include two repeats of the amino acid sequence YxxL / I separated by 6-8 amino acids, wherein each x is independently any amino acid, producing the conserved motif YxxL / Ix(6-8)YxxL / I. ITAMs within signaling molecules are important for signal transduction within the cell, which is mediated at least in part by phosphorylation of tyrosine residues in the ITAM following activation of the signaling molecule. ITAMs may also function as docking sites for other proteins involved in signaling pathways. In some embodiments, the ICD includes at least 1, at least 2, at least 3, at least 4, or at least 5 ITAMs independently selected from the ITAMs derived from CD3^, FcRy, and combinations thereof. In some embodiments, the ICDs of the disclosed CARs includes a CD3C ICD.

[0086] In some embodiments, the chimeric polypeptide of the disclosure includes, in N-terminal to C-terminal direction: (a) a human CD 19 scFv, (b) a hinge domain and a TM domain from CD28, (c) a costimulatory domain from 4-1BB; and (d) a CD3 ICD.

[0087] In some embodiments, the chimeric polypeptide or CAR of the disclosure includes an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the chimeric polypeptide or CAR includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the chimeric polypeptide or CAR includes an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the chimeric polypeptide or CAR includes an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0088] In some embodiments, the chimeric polypeptide or CAR of the disclosure includes an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the chimeric polypeptide or CAR includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the chimeric polypeptide or CAR includes an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 2. In someAttorney Docket: 078430-541001WOembodiments, the chimeric polypeptide or CAR includes an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0089] In some embodiments, the chimeric polypeptide or CAR of the disclosure includes an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the chimeric polypeptide or CAR includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the chimeric polypeptide or CAR includes an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the chimeric polypeptide or CAR includes an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 4.

[0090] One skilled in the art will appreciate that any chimeric polypeptide or CAR as disclosed herein may be created, for example, by chemical synthesis, or by creating and translating a chimeric polynucleotide in which the polypeptide segments are encoded in the desired relationship. For example, the complete amino acid sequence of a chimeric polypeptide or CAR of the disclosure can be used to construct a back-translated gene. For example, a DNA oligomer containing a nucleotide sequence coding for a given chimeric polypeptide or CAR can be synthesized. For example, several small oligonucleotides coding for portions of the desired CAR or antibody can be synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[0091] In addition to generating desired chimeric polypeptides or CARs via expression of nucleic acid molecules that have been altered by recombinant molecular biological techniques, a subject chimeric polypeptide or CAR in accordance with the present disclosure can be chemically synthesized. Chemically synthesized polypeptides are routinely generated by those of skill in the art.

[0092] Once assembled (by synthesis, recombinant methodologies, site-directed mutagenesis or other suitable techniques), the DNA sequences encoding a chimeric polypeptide or CAR as disclosed herein can be inserted into an expression vector and operably linked to an expression control sequence appropriate for expression of the chimeric polypeptide or CAR in the desired transformed host. Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host. As is known inAttorney Docket: 078430-541001WOthe art, in order to obtain high expression levels of a transfected gene in a host, take should be taken to ensure that the gene is operably linked to transcriptional and translational expression control sequences that are functional in the chosen expression host.NUCLEIC ACID MOLECULES

[0093] In one aspect, provided herein are various nucleic acid molecules including nucleotide sequences encoding a chimeric polypeptide of the disclosure, including expression cassettes, and expression vectors containing these nucleic acid molecules operably linked to heterologous nucleic acid sequences such as, for example, regulator sequences which allow in vivo expression of the chimeric polypeptide in a host cell or ex-vivo cell-free expression system.

[0094] Nucleic acid molecules of the present disclosure can be nucleic acid molecules of any length, including nucleic acid molecules that are generally between about 0.5 Kb and about 50 Kb, for example between about 0.5 Kb and about 20 Kb, between about 1 Kb and about 15 Kb, between about 2 Kb and about 10 Kb, or between about 5 Kb and about 25 Kb, for example between about 10 Kb to 15 Kb, between about 15 Kb and about 20 Kb, between about 5 Kb and about 20 Kb, about 5 Kb and about 10 Kb, or about 10 Kb and about 25 Kb. In some embodiments, the nucleic acid molecules of the disclosure are between about 1.5 Kb and about 50 Kb, between about 5 Kb and about 40 Kb, between about 5 Kb and about 30 Kb, between about 5 Kb and about 20 Kb, or between about 10 Kb and about 50 Kb, for example between about 15 Kb to 30 Kb, between about 20 Kb and about 50 Kb, between about 20 Kb and about 40 Kb, about 5 Kb and about 25 Kb, or about 30 Kb and about 50 Kb.

[0095] In some embodiments, the recombinant nucleic acid includes a nucleic acid sequence encoding a chimeric polypeptide (e.g., a CAR) that includes (a) an ECD having a binding affinity for human CD 19, wherein the ECD comprises a fully human antibody or an antigen-binding fragment thereof; (b) a hinge domain and a transmembrane (TM) domain from CD28; and (c) an intracellular signaling domain (ICD) comprising one or more costimulatory domains.

[0096] In some embodiments, the ICD further comprises a CD3 T cell activation domain. In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, comprises all six CDRs of an antibody selected from the group consisting of 21D4, 21D4a, 47G4, 27F3, 3C10, 5G7, 13F1 and 46E8. In some embodiments, the fully human antibody, or an antigenbinding fragment thereof, comprises all six CDRs of the antibody 21D4. In some embodiments,Attorney Docket: 078430-541001WOthe fully human antibody, or an antigen-binding fragment thereof, comprises all six CDRs of the antibody 21D4a. In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, comprises all six CDRs of the antibody 47G4. In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, comprises all six CDRs of the antibody 27F3. In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, comprises all six CDRs of the antibody 3C10. In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, comprises all six CDRs of the antibody 5G7. In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, comprises all six CDRs of the antibody 13F1. In some embodiments, the fully human antibody, or an antigenbinding fragment thereof, comprises all six CDRs of the antibody 46E8.

[0097] In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, includes the light chain region (VL) and heavy chain region (VH) of the antibody. In some embodiments, the antigen-binding fragment thereof is or includes a single-chain antibody fragment (scFv), a F(ab) fragment, a F(ab') fragment, a F(ab')2 fragment, Fd fragment, a Fab'-SH fragment, or a Fv fragment.

[0098] In some embodiments, the antigen-binding fragment thereof is or includes a singlechain antibody fragment (scFv) comprising a light chain variable region (VL) and a heavy chain variable region (VH) operably linked to each other via a linker. In some embodiments, the light chain variable region (VL) is N-terminally linked to the heavy chain variable region (VH). In some embodiments, the light chain variable region (VL) is C-terminally linked to the heavy chain variable region (VH). In some embodiments, the linker is a polypeptide linker (peptidal linkage). In some embodiments, the polypeptide linker is or comprises a glycine-serine linker or a Whitlow linker.

[0099] In some embodiments of the disclosure, the human CD 19 scFv includes, in N-terminal to C-terminal direction: (a) a VL region, a linker, and a VH region; or (b) a VH region, a linker, and a VL region. In some embodiments of the disclosure, the human CD 19 scFv includes, in N-terminal to C-terminal direction: (a) a VL region, a (GGGGS)s linker, and a VH region; (b) a VL region, a Whitlow linker, and a VH region; (c) a VH region, a (GGGGS)s linker, and a VL region; or (d) a VH region, a Whitlow linker, and a VL region. In some embodiments, the human CD 19 scFv includes, in N-terminal to C-terminal direction: a VL region, a (GGGGS)s linker (GGGGSGGGGSGGGGS; SEQ ID NO: 78), and a VH region. In some embodiments, the humanAttorney Docket: 078430-541001WOCD 19 scFv includes, in N-terminal to C-terminal direction: a VL region, a Whitlow linker, and a VH region. In some embodiments, the human CD 19 scFv includes, in N-terminal to C-terminal direction: a VH region, a (GGGGS)s linker, and a VL region. In some embodiments, the human CD 19 scFv includes, in N-terminal to C-terminal direction: a VH region, a Whitlow linker, and a VL region. In some embodiments, the chimeric polypeptide of the disclosure is or includes a chimeric antigen receptor (CAR).

[0100] In some embodiments of the disclosure, the one or more costimulatory domains is from a polypeptide selected from the group consisting of a 4- IBB (CD 137), CD27 (TNFRSF7), CD28, 0X40 (CD 134), CD70, LFA-2 (CD2), CD5, ICAM-1 (CD54), LFA-1 (CDlla / CD18), DAP10, DAP12, a co-stimulatory inducible T-cell costimulatory (ICOS) polypeptide sequence, and a combination of any thereof. In some embodiments the one or more costimulatory domains is or includes a costimulatory 4- IBB (CD 137) polypeptide sequence.

[0101] In some embodiments, the chimeric polypeptide of the disclosure includes, in N-terminal to C-terminal direction: (a) a human CD 19 scFv, (b) a hinge domain and a TM domain from CD28, (c) a costimulatory domain from 4-1BB; and (d) a CD3^ ICD.

[0102] In some embodiments, the recombinant nucleic acid includes a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. In some embodiments, the recombinant nucleic acid includes a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

[0103] In some embodiments, the recombinant nucleic acid includes a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the recombinant nucleic acid includes a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the recombinant nucleic acid includes a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the recombinant nucleic acid includes a nucleic acid sequence having 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 5.Attorney Docket: 078430-541001WO

[0104] In some embodiments, the recombinant nucleic acid includes a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the recombinant nucleic acid includes a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the recombinant nucleic acid includes a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the recombinant nucleic acid includes a nucleic acid sequence having 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 6.

[0105] In some embodiments, the recombinant nucleic acid includes a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the recombinant nucleic acid includes a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the recombinant nucleic acid includes a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the recombinant nucleic acid includes a nucleic acid sequence having 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 7.

[0106] In some embodiments, the recombinant nucleic acid includes a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the recombinant nucleic acid includes a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the recombinant nucleic acid includes a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the recombinant nucleic acid includes a nucleic acid sequence having 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 8.

[0107] In some embodiments, the recombinant nucleic acid molecule is operably linked to a heterologous nucleic acid sequence. In some embodiments, the heterologous nucleic acid sequences includes sequences that allow in vivo expression of the chimeric polypeptide in a host cell or ex-vivo cell-free expression system (e.g., promoter sequences).Attorney Docket: 078430-541001WO

[0108] In some embodiments, the recombinant nucleic acid molecule is further defined as (e.g., is incorporated into) an expression cassette or a vector. It will be understood that an expression cassette generally includes a construct of genetic material that contains coding sequences and sufficient regulatory information to direct proper transcription and / or translation of the coding sequences in a recipient cell, in vivo and / or ex vivo. Generally, the expression cassette may be inserted into a vector for targeting to a desired host cell and / or into an individual. As such, in some embodiments, an expression cassette of the disclosure include a coding sequence for the chimeric polypeptide as disclosed herein, which is operably linked to expression control elements, such as a promoter, and optionally, any other sequences or a combination of other nucleic acid sequences that affect the transcription or translation of the coding sequence.

[0109] In some embodiments, the nucleotide sequence is incorporated into an expression vector. It will be understood by one skilled in the art that the term “vector” generally refers to a recombinant polynucleotide construct designed for transfer between host cells, and that may be used for the purpose of transformation, e.g., the introduction of heterologous DNA into a host cell. As such, in some embodiments, the vector can be a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. In some embodiments, the expression vector can be an integrating vector.

[0110] In some embodiments, the expression vector can be a viral vector. As will be appreciated by one of skill in the art, the term “viral vector” is widely used to refer either to a nucleic acid molecule (e.g., a transfer plasmid) that includes virus-derived nucleic acid elements that generally facilitate transfer of the nucleic acid molecule or integration into the genome of a cell or to a viral particle that mediates nucleic acid transfer. Viral particles will generally include various viral components and sometimes also host cell components in addition to nucleic acid(s). The term viral vector may refer either to a virus or viral particle capable of transferring a nucleic acid into a cell or to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements that are primarily derived from a virus. In some embodiments, the vector is a vector derived from a lentivirus, an adeno virus, an adeno-associated virus, a baculovirus, or a retrovirus. The term “retroviral vector” refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus. The term “lentiviral vector” refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, includingAttorney Docket: 078430-541001WOLTRs that are primarily derived from a lentivirus, which is a genus of retrovirus.

[0111] In some embodiments, provided herein are nucleic acid molecules encoding a chimeric polypeptide (e.g., a CAR) with an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97, 98%, 99%, or 100% sequence identity to a chimeric polypeptide disclosed herein. In some embodiments, provided herein are nucleic acid molecules encoding a polypeptide with an amino acid sequence having at least about 80% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. In some embodiments, the nucleic acid molecules encode a polypeptide with an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the nucleic acid molecules encode a polypeptide with an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the nucleic acid molecules encode a polypeptide with an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3. In some embodiments, the nucleic acid molecules encode a polypeptide with an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4.

[0112] The nucleic acid sequences encoding the chimeric polypeptides can be optimized for expression in the host cell of interest. For example, the G-C content of the sequence can be adjusted to average levels for a given cellular host, as calculated by reference to known genes expressed in the host cell. Methods for codon usage optimization are known in the art. Codon usages within the coding sequence of the chimeric receptor disclosed herein can be optimized to enhance expression in the host cell, such that about 1%, about 5%, about 10%, about 25%, about 50%, about 75%, or up to 100% of the codons within the coding sequence have been optimized for expression in a particular host cell.

[0113] The nucleic acid molecules provided can contain naturally occurring sequences, or sequences that differ from those that occur naturally, but, due to the degeneracy of the genetic code, encode the same polypeptide, e.g, antibody. These nucleic acid molecules can consist of RNA or DNA (for example, genomic DNA, cDNA, or synthetic DNA, such as that produced by phosphoramidite-based synthesis), or combinations or modifications of the nucleotides within these types of nucleic acids. In addition, the nucleic acid molecules can be double-stranded or single-stranded (e.g., either a sense or an antisense strand).Attorney Docket: 078430-541001WO

[0114] The nucleic acid molecules are not limited to sequences that encode polypeptides (e.g., antibodies); some or all of the non-coding sequences that lie upstream or downstream from a coding sequence (e.g., the coding sequence of a chimeric receptor) can also be included. Those of ordinary skill in the art of molecular biology are familiar with routine procedures for isolating nucleic acid molecules. They can, for example, be generated by treatment of genomic DNA with restriction endonucleases, or by performance of the polymerase chain reaction (PCR). In the event the nucleic acid molecule is a ribonucleic acid (RNA), molecules can be produced, for example, by in vitro transcription.RECOMBINANT CELLS AND CELL CULTURES

[0115] The nucleic acid molecules of the present disclosure can be introduced into a cell, such as an immune system cell (e.g., human T cell) or a cancer cell, to produce a recombinant cell containing the nucleic acid molecule. Accordingly, some embodiments of the disclosure relate to methods for making a recombinant cell, including (a) providing a host cell capable of protein expression; and transducing the provided host cell with a recombinant nucleic acid of the disclosure to produce a recombinant cell. Introduction of the nucleic acid molecules of the disclosure into cells can be achieved by methods known to those skilled in the art such as, for example, viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro-injection, nanoparticle-mediated nucleic acid delivery, and the like.

[0116] Accordingly, in some embodiments, the nucleic acid molecules can be introduced into a host cell by viral or non-viral delivery vehicles known in the art to produce an engineered cell (e.g., recombinant cell). For example, the nucleic acid molecule can be stably integrated in the host genome, or can be episomally replicating, or present in the recombinant host cell as a minicircle expression vector for a stable or transient expression. Accordingly, in some embodiments disclosed herein, the nucleic acid molecule is maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the nucleic acid molecule is stably integrated into the genome of the recombinant cell. Stable integration can be completed using classical random genomic recombination techniques or with more precise genome editing techniques such as using zinc-finger proteins (ZNF), guide RNA directed CRISPR / Cas9, DNA-guidedAttorney Docket: 078430-541001WOendonuclease genome editing NgAgo (Natronobacterium gregoryi Argonaute), or TALEN genome editing (transcription activator-like effector nucleases).

[0117] The nucleic acid molecules can be encapsulated in a viral capsid or a lipid nanoparticle, or can be delivered by viral or non-viral delivery means and methods known in the art, such as electroporation. For example, introduction of nucleic acids into cells may be achieved by viral transduction. In a non-limiting example, baculoviral virus or adeno-associated virus (AAV) can be engineered to deliver nucleic acids to target cells via viral transduction. Several AAV serotypes have been described, and all of the known serotypes can infect cells from multiple diverse tissue types. AAV is capable of transducing a wide range of species and tissues in vivo with no evidence of toxicity, and it generates relatively mild innate and adaptive immune responses.

[0118] Retrovirus-derived vector systems, e.g., lentiviral- derived vector systems are also useful for nucleic acid delivery and gene therapy via viral transduction. Lentiviral vectors offer several attractive properties as gene-delivery vehicles, including: (i) sustained gene delivery through stable vector integration into host genome; (ii) the capability of infecting both dividing and non-dividing cells; (iii) broad tissue tropisms, including important gene- and cell-therapy-target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (vi) a potentially safer integration site profile; and (vii) a relatively easy system for vector manipulation and production.

[0119] In some embodiments, host cells can be genetically engineered (e.g., transduced or transformed or transfected) with, for example, a vector construct of the present application that can be, for example, a viral vector or a vector for homologous recombination that includes nucleic acid sequences homologous to a portion of the genome of the host cell, or can be an expression vector for the expression of the chimeric polypeptides of interest. Host cells can be either untransformed cells or cells that have already been transfected with at least one nucleic acid molecule.

[0120] In some embodiments, the recombinant cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the cell is in vivo. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vitro. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the animal cellAttorney Docket: 078430-541001WOis a mouse cell. In some embodiments, the animal cell is a human cell. In some embodiments, the cell is a non-human primate cell. In some embodiments, the recombinant cell is an immune system cell, e.g., a B cell, a monocyte, a NK cell, a natural killer T (NKT) cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a regulatory T cell, a helper T cell (TH), a cytotoxic T cell (TCTL), a memory T cell, a gamma delta (y8) T cell, another T cell, a hematopoietic stem cell, or a hematopoietic stem cell progenitor.

[0121] In some embodiments, the immune system cell is a lymphocyte. In some embodiments, the lymphocyte is a T lymphocyte. In some embodiments, the lymphocyte is a T lymphocyte progenitor. In some embodiments, the cell is an IPSC-derived T cell, a regulatory T cell, a helper T cell (TH), a cytotoxic T cell (TCTL), a memory T cell, a gamma delta (y3) T cell, a CD8+ T cytotoxic lymphocyte cell, or a CD4+ T helper lymphocyte cell. In some embodiments, the T cell is an exhausted T cell or a non-exhausted T cell.

[0122] In some embodiments, the T lymphocyte is a CD4+ T cell or a CD8+ T cell. In some embodiments, the T lymphocyte is a CD8+ T cytotoxic lymphocyte cell. Non-limiting examples of CD8+ T cytotoxic lymphocyte cell suitable for the compositions and methods disclosed herein include naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, effector CD8+ T cells, CD8+ stem memory T cells, and bulk CD8+ T cells. In some embodiments, the T lymphocyte is a CD4+ T helper lymphocyte cell. Suitable CD4+ T helper lymphocyte cells include, but are not limited to, naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, effector CD4+ T cells, CD4+ stem memory T cells, and bulk CD4+ T cells.

[0123] As outlined above, some embodiments of the disclosure relate to various methods for making a recombinant cell, including (a) providing a host cell capable of protein expression; and transducing the provided host cell with a recombinant nucleic acid of the disclosure to produce a recombinant cell. Non-limiting exemplary embodiments of the disclosed methods for making a recombinant cell can further include one or more of the following features. In some embodiments, the host cell is obtained by leukapheresis performed on a sample obtained from a subject, and the cell is transduced ex vivo. In some embodiments, the recombinant nucleic acid is encapsulated in a viral capsid or a lipid nanoparticle. In some embodiments, the methods further include isolating and / or purifying the produced cells. Accordingly, the recombinant cells produced by the methods disclosed herein are also within the scope of the disclosure.Attorney Docket: 078430-541001WO

[0124] Techniques for transforming a wide variety of the above-mentioned host cells and species are known in the art and described in the technical and scientific literature. For example, DNA vectors can be introduced into eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting cells can be found in Sambrook et al. (2012, supra) and other standard molecular biology laboratory manuals, such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, nucleoporation, hydrodynamic shock, and infection. In some embodiments, the nucleic acid molecule is introduced into a host cell by a transduction procedure, electroporation procedure, or a biolistic procedure. Accordingly, cell cultures including at least one recombinant cell as disclosed herein are also within the scope of this application. Methods and systems suitable for generating and maintaining cell cultures are known in the art.

[0125] In one aspect, some embodiments of the disclosure relate to a recombinant cell including: (a) a chimeric polypeptide as described herein; and / or a nucleic acid molecule according as described herein. In some embodiments, the recombinant cell of the disclosure includes a chimeric polypeptide (e.g., a CAR) that includes (a) an ECD having a binding affinity for human CD 19, wherein the ECD comprises a fully human antibody or an antigen-binding fragment thereof; (b) a hinge domain and a transmembrane (TM) domain from CD28; and (c) an intracellular signaling domain (ICD) comprising one or more costimulatory domains.

[0126] In some embodiments, the ICD further comprises a CD3 T cell activation domain. In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, comprises all six CDRs of an antibody selected from the group consisting of 21D4, 21D4a, 47G4, 27F3, 3C10, 5G7, 13F1 and 46E8. In some embodiments, the fully human antibody, or an antigenbinding fragment thereof, comprises all six CDRs of the antibody 21D4. In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, comprises all six CDRs of the antibody 21D4a. In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, comprises all six CDRs of the antibody 47G4. In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, comprises all six CDRs of the antibody 27F3. In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, comprises all six CDRs of the antibody 3C10. In some embodiments, the fully human antibody,Attorney Docket: 078430-541001WOor an antigen-binding fragment thereof, comprises all six CDRs of the antibody 5G7. In some embodiments, the fully human antibody, or an antigen-binding fragment thereof, comprises all six CDRs of the antibody 13F1. In some embodiments, the fully human antibody, or an antigenbinding fragment thereof, comprises all six CDRs of the antibody 46E8.

[0127] In some embodiments, the antigen-binding fragment thereof is or includes a singlechain antibody fragment (scFv) comprising a light chain variable region (VL) and a heavy chain variable region (VH) operably linked to each other via a linker. In some embodiments, the light chain variable region (VL) is N-terminally linked to the heavy chain variable region (VH). In some embodiments, the light chain variable region (VL) is C-terminally linked to the chain variable region (VH). In some embodiments, the linker is a polypeptide linker (peptidal linkage). In some embodiments, the polypeptide linker is or comprises a glycine-serine linker or a Whitlow linker.

[0128] In some embodiments of the disclosure, the human CD 19 scFv includes, in N-terminal to C-terminal direction: (a) a VL region, a linker, and a VH region; or (b) a VH region, a linker, and a VL region. In some embodiments of the disclosure, the human CD 19 scFv includes, in N-terminal to C-terminal direction: (a) a VL region, a (GGGGS)s linker, and a VH region; (b) a VL region, a Whitlow linker, and a VH region; (c) a VH region, a (GGGGS)s linker, and a VL region; or (d) a VH region, a Whitlow linker, and a VL region. In some embodiments, the human CD 19 scFv includes, in N-terminal to C-terminal direction: a VL region, a (GGGGS)s linker (GGGGSGGGGSGGGGS; SEQ ID NO: 78), and a VH region. In some embodiments, the human CD 19 scFv includes, in N-terminal to C-terminal direction: a VL region, a Whitlow linker, and a VH region. In some embodiments, the human CD 19 scFv includes, in N-terminal to C-terminal direction: a VH region, a (GGGGS)s linker, and a VL region. In some embodiments, the human CD 19 scFv includes, in N-terminal to C-terminal direction: a VH region, a Whitlow linker, and a VL region. In some embodiments, the chimeric polypeptide of the disclosure is or includes a chimeric antigen receptor (CAR).

[0129] In some embodiments of the disclosure, the one or more costimulatory domains is from a polypeptide selected from the group consisting of a 4- IBB (CD 137), CD27 (TNFRSF7), CD28, 0X40 (CD 134), CD70, LFA-2 (CD2), CD5, ICAM-1 (CD54), LFA-1 (CDlla / CD18), DAP10, DAP12, a co-stimulatory inducible T-cell costimulatory (ICOS) polypeptide sequence, and a combination of any thereof. In some embodiments the one or more costimulatory domainsAttorney Docket: 078430-541001WOis or includes a costimulatory 4- IBB (CD 137) polypeptide sequence.

[0130] In some embodiments, the chimeric polypeptide of the disclosure includes, in N-terminal to C-terminal direction: (a) a human CD 19 scFv, (b) a hinge domain and a TM domain from CD28, (c) a costimulatory domain from 4-1BB; and (d) a CD3 ICD.

[0131] In some embodiments, the recombinant cell includes a nucleic acid molecule including a nucleic acid sequence encoding a chimeric polypeptide (e.g., a CAR) with an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1. In some embodiments, the recombinant cell includes a nucleic acid molecule including a nucleic acid sequence encoding a chimeric polypeptide (e.g., a CAR) with an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 2. In some embodiments, the recombinant cell includes a nucleic acid molecule including a nucleic acid sequence encoding a chimeric polypeptide (e.g., a CAR) with an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 3. In some embodiments, the recombinant cell includes a nucleic acid molecule including a nucleic acid sequence encoding a chimeric polypeptide (e.g., a CAR) with an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 4.

[0132] In a related aspect, some embodiments of the disclosure relate to cell cultures including at least one recombinant cell as disclosed herein, and a culture medium. Generally, the culture medium can be any one of suitable culture media for the cell cultures described herein. In some embodiments, the recombinant cell expresses a chimeric polypeptide or a CAR described herein. Accordingly, cell cultures including at least one recombinant cell as disclosed herein are also within the scope of this application. Methods and systems suitable for generating and maintaining cell cultures are known in the art.PHARMACEUTICAL COMPOSITIONS

[0133] In some embodiments, the chimeric polypeptides, chimeric antigen receptors (CARs), nucleic acids, recombinant cells, and / or cell cultures of the disclosure can be incorporated into compositions, including pharmaceutical compositions. Such compositions generally include the chimeric polypeptides, CARs, nucleic acids, recombinant cells, and / or cell cultures as describedAttorney Docket: 078430-541001WOherein and a pharmaceutically acceptable carrier. Accordingly, in one aspect, some embodiments of the disclosure relate to pharmaceutical compositions for treating, preventing, ameliorating, reducing or delaying the onset of a health condition, for example a proliferative disease, e.g., a cancer, a malignancy, a precancerous condition, an autoimmune disease (AID), or a non-cancer related indication associated with expression of CD 19.

[0134] Accordingly, one aspect of the present disclosure relates to pharmaceutical compositions that include a pharmaceutically acceptable carrier and one or more of the following: (a) a chimeric polypeptide of the disclosure; (b) a nucleic acid molecule of the disclosure; and / or (c) a recombinant cell of the disclosure. In some embodiments, the composition includes (a) a recombinant nucleic acid of the disclosure and (b) a pharmaceutically acceptable carrier. In some embodiments, the recombinant nucleic acid is encapsulated in a viral capsid or a lipid nanoparticle. In some embodiments, the composition includes (a) a recombinant cell of the disclosure and (b) a pharmaceutically acceptable carrier.

[0135] In certain embodiments, the pharmaceutical compositions in accordance with some embodiments disclosed herein include cell cultures that can be washed, treated, combined, supplemented, or otherwise altered prior to administration to an individual in need thereof. Furthermore, administration can be at varied doses, time intervals or in multiple administrations.

[0136] The pharmaceutical compositions provided herein can be in any form that allows for the composition to be administered to an individual. In some specific embodiments, the pharmaceutical compositions are suitable for human administration. As used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The carrier can be a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, including injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E.W. Martin. In some embodiments, the pharmaceutical composition is sterilely formulated for administration into an individual. In some embodiments, the individual is a human. One ofAttorney Docket: 078430-541001WOordinary skilled in the art will appreciate that the formulation should suit the mode of administration.

[0137] In some embodiments, the pharmaceutical compositions of the present disclosure are formulated to be suitable for the intended route of administration to an individual. For example, the pharmaceutical composition may be formulated to be suitable for parenteral, intraperitoneal, colorectal, intraperitoneal, and intratumoral administration. In some embodiments, the pharmaceutical composition may be formulated for intravenous, oral, intraperitoneal, intratracheal, subcutaneous, intramuscular, topical, or intratumoral administration.

[0138] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™. (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should be fluid to the extent that easy syringeability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants, e.g., sodium dodecyl sulfate. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be generally to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0139] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterileAttorney Docket: 078430-541001WOpowders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.METHODS OF THE DISCLOSURE

[0140] Administration of any one of the therapeutic compositions described herein, e.g., chimeric polypeptides, CARs, nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions, can be used in the diagnosis, prevention, and / or treatment of relevant conditions, such as proliferative diseases (e.g., cancer). In some embodiments, the chimeric polypeptides, CARs, nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions as described herein can be incorporated into therapies and therapeutic agents for use in methods of preventing and / or treating an individual who has, who is suspected of having, or who may be at high risk for developing one or more health conditions, such as proliferative diseases, e.g, cancers, malignancies, precancerous conditions, autoimmune diseases, or non-cancer related indications associated with expression of CD 19. In some embodiments, the individual is a patient under the care of a physician.

[0141] In yet another aspect, some embodiments of the disclosure relate to methods for preventing and / or treating a health condition associated with expression of CD 19 in a subject in need thereof, wherein the methods include administering to the subject a composition including one or more of the following: (a) a chimeric polypeptide as disclosed herein; (b) a nucleic acid molecule as disclosed herein; (c) a recombinant T cell as disclosed herein; and / or (d) a pharmaceutical composition as disclosed herein.

[0142] Exemplary embodiments of the disclosed methods include one or more of the following features. In some embodiments, the recombinant T cell is autologous to the subject. In some embodiments, the T cell is obtained by leukapheresis of a sample obtained from a subject. In some embodiments, the health condition is a disease or disorder associate with expression of CD 19. In some embodiments, the disease is a cancer, a malignancy, a precancerous condition, an autoimmune disease (AID), or a non-cancer related indication associated with expression of CD 19. In some embodiments, the autoimmune disease (AID) is systemic lupus erythematosus (SLE), idiopathic inflammatory myositis (IIM), or systemic sclerosis (SSc).

[0143] In some embodiments, the disease is a cancer and CD 19 is expressed at a heterogenousAttorney Docket: 078430-541001WOlevel or a low level in cancer cells. In some embodiments, the disease or disorder is a B-cell malignancy. In some embodiments, the B-cell malignancy is a hematologic malignancy selected from the group consisting B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), acute myelogenous leukemia (AML), acute lymphoid leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B cell pro-lymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, null-acute lymphoblastic leukemia, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, splenic, marginal zone lymphoma, and Waldenstrom macroglobulinemia.

[0144] In some embodiments, the cancer is a multiply drug resistant cancer or a recurrent cancer. It is contemplated that the compositions and methods disclosed here are suitable for both non-metastatic cancers and metastatic cancers. Accordingly, in some embodiments, the cancer is a non-metastatic cancer. In some other embodiments, the cancer is a metastatic cancer. In some embodiments, the composition administered to the subject inhibits metastasis of the cancer in the subject. In some embodiments, the administered composition inhibits tumor growth in the subject.

[0145] In some embodiments, the administered composition inhibits proliferation of a target cancer cell, and / or inhibits tumor growth of the cancer in the subject. For example, the target cell may be inhibited if its proliferation is reduced, if its pathologic or pathogenic behavior is reduced, if it is destroyed or killed, etc. Inhibition includes a reduction of the measured pathologic or pathogenic behavior of at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the methods include administering to the individual an effective number of the recombinant cells disclosed herein, wherein the recombinant cells inhibit the proliferation of the target cell and / or inhibit tumor growth of a target cancer in the subject compared to the proliferation of the target cell and / or tumor growth of the target cancer in subjects who have not been administered with the recombinant cells.Attorney Docket: 078430-541001WO

[0146] In some embodiments, the antigen CD 19 is expressed at low density on target cells, e.g., less than about 6,000 molecules of the target antigen per cell. In some embodiments, the antigen is expressed at a density of less than about 5,000 molecules, less than about 4,000 molecules, less than about 3,000 molecules, less than about 2,000 molecules, less than about 1,000 molecules, or less than about 500 molecules of the target antigen per cell. In some embodiments, the antigen is expressed at a density of less than about 2,000 molecules, such as e.g., less than about 1,800 molecules, less than about 1,600 molecules, less than about 1,400 molecules, less than about 1,200 molecules, less than about 1,000 molecules, less than about 800 molecules, less than about 600 molecules, less than about 400 molecules, less than about 200 molecules, or less than about 100 molecules of the target antigen per cell. In some embodiments, the antigen is expressed at a density of less than about 1,000 molecules, such as e.g., less than about 900 molecules, less than about 800 molecules, less than about 700 molecules, less than about 600 molecules, less than about 500 molecules, less than about 400 molecules, less than about 300 molecules, less than about 200 molecules, or less than about 100 molecules of the target antigen per cell. In some embodiments, the antigen is expressed at a density ranging from about 5,000 to about 100 molecules of the target antigen per cell, such as e.g., from about 5,000 to about 1,000 molecules, from about 4,000 to about 2,000 molecules, from about 3,000 to about 2,000 molecules, from about 4,000 to about 3,000 molecules, from about 3,000 to about 1,000 molecules, from about 2,000 to about 1,000 molecules, from about 1,000 to about 500 molecules, from about 500 to about 100 molecules of the target antigen per cell.

[0147] The terms “administration” and “administering”, as used herein, refer to the delivery of a bioactive composition or formulation by an administration route including, but not limited to, oral, intravenous, intra-arterial, intramuscular, intraperitoneal, subcutaneous, intramuscular, and topical administration, or combinations thereof. The term includes, but is not limited to, administering by a medical professional and self-administering.

[0148] Administration of the compositions described herein, e.g., polypeptides, CARs, nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions, can be used in the stimulation of an immune response. In some embodiments, polypeptides, CARs, nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions as described herein are administered to an individual after induction of remission of cancer with chemotherapy, or after autologous or allogeneic hematopoietic stem cell transplantation.Attorney Docket: 078430-541001WO

[0149] An effective amount of the compositions described herein, e.g., polypeptides, CARs, nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions, is determined based on the intended goal, for example tumor regression. For example, where existing cancer is being treated, the amount of a composition disclosed herein to be administered may be greater than where administration of the composition is for prevention of cancer. One of ordinary skill in the art would be able to determine the amount of a composition to be administered and the frequency of administration in view of this disclosure. The quantity to be administered, both according to number of treatments and dose, also depends on the individual to be treated, the state of the individual, and the protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Frequency of administration could range from 1-2 days, to 2-6 hours, to 6-10 hours, to 1-2 weeks or longer depending on the judgment of the practitioner.

[0150] Longer intervals between administration and lower amounts of compositions may be employed where the goal is prevention. For instance, amounts of compositions administered per dose may be 50% of the dose administered in treatment of active disease, and administration may be at weekly intervals. One of ordinary skill in the art, in light of this disclosure, would be able to determine an effective amount of compositions and frequency of administration. This determination would, in part, be dependent on the particular clinical circumstances that are present (e.g., type of cancer, severity of cancer).

[0151] In certain embodiments, it may be desirable to provide a continuous supply of a composition disclosed herein to the subject to be treated, e.g., a patient. In some embodiments, continuous perfusion of the region of interest (such as the tumor) may be suitable. The time period for perfusion would be selected by the clinician for the particular subject and situation, but times could range from about 1-2 hours, to 2-6 hours, to about 6-10 hours, to about 10-24 hours, to about 1 -2 days, to about 1 -2 weeks or longer. Generally, the dose of the composition via continuous perfusion will be equivalent to that given by single or multiple injections, adjusted for the period of time over which the doses are administered.

[0152] In some embodiments, administration is by bolus injection. In some embodiments, administration is by intravenous infusion. In some embodiments, a composition is administered is administered in a dosage of about 100 ng / kg of body weight per day to about 100 mg / kg of body weight per day. In some embodiments, a composition as disclosed herein is administered inAttorney Docket: 078430-541001WOa dosage of about 0.001 mg / kg to 100 mg / kg of body weight per day. In some embodiments, the therapeutic agents are administered in a single administration. In some embodiments, therapeutic agents are administered in multiple administrations, (e.g, once or more per week for one or more weeks). In some embodiments, doses are administered about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more days. In some embodiments, there are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more total doses. In some embodiments, 4 doses are administered, with a 3 week span between doses.

[0153] One of ordinary skill in the art would be familiar with techniques for administering compositions of the disclosure to an individual. Furthermore, one of ordinary skill in the art would be familiar with techniques and pharmaceutical reagents necessary for preparation of these compositions prior to administration to an individual.

[0154] In certain embodiments of the present disclosure, the composition of the disclosure will be an aqueous composition that includes one or more of the chimeric polypeptides, CARs, nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions as described herein. Aqueous compositions of the present disclosure contain an effective amount of a composition disclosed herein in a pharmaceutically acceptable carrier or aqueous medium. Thus, the “pharmaceutical preparation” or “pharmaceutical composition” of the disclosure can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the recombinant cells disclosed herein, its use in the manufacture of the pharmaceutical compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. For human administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Center for Biologies.

[0155] One of ordinary skill in the art would appreciate that biological materials should be extensively dialyzed to remove undesired small molecular weight molecules and / or lyophilized for more ready formulation into a desired vehicle, where appropriate. The compositions described herein, e.g, polypeptides, CARs, nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions, will then generally be formulated for administration by any known route, such as parenteral administration. Determination of the amount of compositions to be administered will be made by one of skill in the art, and will in part be dependent on the extentAttorney Docket: 078430-541001WOand severity of cancer, and whether the recombinant cells are being administered for treatment of existing cancer or prevention of cancer. The preparation of the compositions containing the chimeric polypeptides, CARs, nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions of the disclosure will be known to those of skill in the art in light of the present disclosure.

[0156] Upon formulation, the compositions of the disclosure will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The compositions can be administered in a variety of dosage forms, such as the type of injectable solutions described above. For parenteral administration, the compositions disclosed herein should be suitably buffered. As discussed in greater detail below, the compositions as described herein may be administered with other therapeutic agents that are part of the therapeutic regiment of the individual, such as other immunotherapy or chemotherapy.

[0157] The chimeric polypeptides, CARs, nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions described herein can be used to inhibit tumor growth or metastasis of a cancer in the treated subject relative to the tumor growth or metastasis in subjects who have not been administered one of the therapeutic compositions disclosed herein. In some embodiments, the antibodies, CARs, nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions described herein can be used to stimulate immune responses against the tumor via inducing the production of interferon gamma (IFNy) and / or interleukin-2 (IL- 2) and other pro-inflammatory cytokines. In some embodiments, the antibodies, CARs, nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions described herein can be used to stimulate proliferation and / or killing capacity of CAR T-cells in the treated subject relative to the production of these molecules in subjects who have not been administered one of the therapeutic compositions disclosed herein. The production of interferon gamma (IFNy) and / or interleukin-2 (IL-2) can be stimulated to produce up to about 20 fold, such as any of about 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold 16 fold, 17 fold, 18 fold, 19 fold, or 20 fold or higher compared to the production of interferon gamma (IFNy) and / or interleukin-2 (IL-2) in subjects who have not been administered one of the therapeutic compositions disclosed herein.Administration of recombinant cells to a subject

[0158] In some embodiments, the methods of the disclosure involve administering an effectiveAttorney Docket: 078430-541001WOamount or number of the recombinants cells provided here to a subject in need thereof. This administering step can be accomplished using any method of implantation delivery in the art. For example, the recombinant cells can be infused directly in the subject’s bloodstream or otherwise administered to the subject.

[0159] In some embodiments, the methods disclosed herein include administering, which term is used interchangeably with the terms “introducing,” implanting,” and “transplanting,” recombinant cells into an individual, by a method or route that results in at least partial localization of the introduced cells at a desired site such that a desired effect(s) is / are produced. The recombinant cells or their differentiated progeny can be administered by any appropriate route that results in delivery to a desired location in the individual where at least a portion of the administered cells or components of the cells remain viable. The period of viability of the cells after administration to a subject can be as short as a few hours, e.g., twenty-four hours, to a few days, to as long as several years, or even the lifetime of the individual, i.e., long-term engraftment.

[0160] When provided prophylactically, the recombinant cells described herein can be administered to a subject in advance of any symptom of a disease or condition to be treated. Accordingly, in some embodiments the prophylactic administration of a recombinant cell population prevents the occurrence of symptoms of the disease or condition.

[0161] When provided therapeutically in some embodiments, recombinant cells are provided at (or after) the onset of a symptom or indication of a disease or condition, e.g., upon the onset of disease or condition.

[0162] For use in the various embodiments described herein, an effective amount of recombinant cells as disclosed herein, can be at least 102cells, at least 5 102cells, at least 103cells, at least 5 103cells, at least 104cells, at least 5 104cells, at least 105cells, at least 2 x 105cells, at least 3 x 105cells, at least 4 x 105cells, at least 5 x 105cells, at least 6 x 105cells, at least 7 x io5cells, at least 8 x 105cells, at least 9 x 105cells, at least 1 x 106cells, at least 2 x 106cells, at least 3 x 106cells, at least 4 x 106cells, at least 5 x 106cells, at least 6 x 106cells, at least 7 x io6cells, at least 8 x 106cells, at least 9 x 106cells, or multiples thereof. The recombinant cells can be derived from one or more donors or can be obtained from an autologous source. In some embodiments, the recombinant cells are expanded in culture prior to administration to a subject in need thereof.Attorney Docket: 078430-541001WO

[0163] In some embodiments, the delivery of a recombinant cell composition (e.g., a composition including a plurality of recombinant cells according to any of the cells described herein) into a subject by a method or route results in at least partial localization of the cell composition at a desired site. A composition including recombinant cells can be administered by any appropriate route that results in effective treatment in the subject, c.g, administration results in delivery to a desired location in the subject where at least a portion of the composition delivered, c.g, at least 1 * 104cells, is delivered to the desired site for a period of time. Modes of administration include injection, infusion, instillation. “Injection” includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In some embodiments, the route is intravenous. For the delivery of cells, delivery by injection or infusion is a standard mode of administration.

[0164] In some embodiments, the recombinant cells are administered systemically, c.g, via infusion or injection. For example, a population of recombinant cells are administered other than directly into a target site, tissue, or organ, such that it enters, the subject’s circulatory system and, thus, is subject to metabolism and other similar biological processes.

[0165] The efficacy of a treatment including any of the compositions provided herein for the prevention or treatment of a disease or condition can be determined by a skilled clinician.However, one skilled in the art will appreciate that a prevention or treatment is considered effective if any one or all of the signs or symptoms or markers of disease are improved or ameliorated. Efficacy can also be measured by failure of a subject to worsen as assessed by decreased hospitalization or need for medical interventions (e.g., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and / or described herein. Treatment includes any treatment of a disease in a subject or an animal (some non-limiting examples include a human, or a mammal) and includes: (1) inhibiting the disease, e.g, arresting, or slowing the progression of symptoms; or (2) relieving the disease, e.g, causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms.

[0166] Measurement of the degree of efficacy is based on parameters selected with regard to the disease being treated and the symptoms experienced. In general, a parameter is selected thatAttorney Docket: 078430-541001WOis known or accepted as correlating with the degree or severity of the disease, such as a parameter accepted or used in the medical community. For example, in the treatment of a solid cancer, suitable parameters can include reduction in the number and / or size of metastases, number of months of progression-free survival, overall survival, stage or grade of the disease, the rate of disease progression, the reduction in diagnostic biomarkers (for example without limitation, a reduction in circulating tumor DNA or RNA, a reduction in circulating cell-free tumor DNA or RNA, and the like), and combinations thereof. It will be understood that the effective dose and the degree of efficacy will generally be determined with relation to a single subject and / or a group or population of subjects. Therapeutic methods of the disclosure reduce symptoms and / or disease severity and / or disease biomarkers by at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100%.

[0167] As discussed above, a therapeutically effective amount includes an amount of a therapeutic composition that is sufficient to promote a particular beneficial effect when administered to a subject, such as one who has, is suspected of having, or is at risk for a disease. In some embodiments, an effective amount includes an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. It is understood that for any given case, an appropriate effective amount can be determined by one of ordinary skill in the art using routine experimentation.Additional therapies

[0168] As discussed above, any one of the compositions as disclosed herein, e.g., chimeric polypeptides, CARs, nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions, can be administered to a subject in need thereof as a sole therapy (e.g., monotherapy). In addition or alternatively, in some embodiments of the disclosure, the chimeric polypeptides, CARs, nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions described herein can be administered to the subject in combination with one or more additional therapies, e.g., at least one, two, three, four, or five additional therapies. Suitable therapies to be administered in combination with the compositions of the disclosure include, but are not limited to chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, targeted therapy, and surgery. Other suitable therapies include therapeutic agents such as chemotherapeutics, anti-cancer agents, and anti-cancer therapies.Attorney Docket: 078430-541001WO

[0169] Administration “in combination with” one or more additional therapies includes simultaneous (concurrent) and consecutive administration in any order. In some embodiments, the one or more additional therapies is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, and surgery. The term chemotherapy as used herein encompasses anti-cancer agents. Various classes of anti-cancer agents can be suitably used for the methods disclosed herein. Non-limiting examples of anticancer agents include: alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, podophyllotoxin, antibodies (e.g., monoclonal or polyclonal), tyrosine kinase inhibitors (e.g., imatinib mesylate (Gleevec® or Glivec®)), hormone treatments, soluble receptors and other antineoplastics.

[0170] Topoisomerase inhibitors are also another class of anti-cancer agents that can be used herein. Topoisomerases are essential enzymes that maintain the topology of DNA. Inhibition of type I or type II topoisomerases interferes with both transcription and replication of DNA by upsetting proper DNA supercoiling. Some type I topoisomerase inhibitors include camptothecins such as irinotecan and topotecan. Examples of type II inhibitors include amsacrine, etoposide, etoposide phosphate, and teniposide. These are semisynthetic derivatives of epipodophyllotoxins, alkaloids naturally occurring in the root of American Mayapple (Podophyllum peltatum).

[0171] Antineoplastics include the immunosuppressant dactinomycin, doxorubicin, epirubicin, bleomycin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide. The antineoplastic compounds generally work by chemically modifying a cell's DNA.

[0172] Alkylating agents can alkylate many nucleophilic functional groups under conditions present in cells. Cisplatin and carboplatin, and oxaliplatin are alkylating agents. They impair cell function by forming covalent bonds with the amino, carboxyl, sulfhydryl, and phosphate groups in biologically important molecules.

[0173] Vinca alkaloids bind to specific sites on tubulin, inhibiting the assembly of tubulin into microtubules (M phase of the cell cycle). The vinca alkaloids include: vincristine, vinblastine, vinorelbine, and vindesine.

[0174] Anti-metabolites resemble purines (azathioprine, mercaptopurine) or pyrimidine and prevent these substances from becoming incorporated in to DNA during the "S" phase of the cell cycle, stopping normal development and division. Anti-metabolites also affect RNA synthesis.

[0175] Plant alkaloids and terpenoids are obtained from plants and block cell division byAttorney Docket: 078430-541001WOpreventing microtubule function. Since microtubules are vital for cell division, without them, cell division cannot occur. The main examples are vinca alkaloids and taxanes.

[0176] Podophyllotoxin is a plant-derived compound which has been reported to help with digestion as well as used to produce two other cytostatic drugs, etoposide and teniposide. They prevent the cell from entering the G1 phase (the start of DNA replication) and the replication of DNA (the S phase).

[0177] Taxanes as a group includes paclitaxel and docetaxel. Paclitaxel is a natural product, originally known as Taxol and first derived from the bark of the Pacific Yew tree. Docetaxel is a semi-synthetic analogue of paclitaxel. Taxanes enhance stability of microtubules, preventing the separation of chromosomes during anaphase.

[0178] In some embodiments, the anti-cancer agents can be selected from remicade, docetaxel, celecoxib, melphalan, dexamethasone (Decadron®), steroids, gemcitabine, cisplatinum, temozolomide, etoposide, cyclophosphamide, temodar, carboplatin, procarbazine, gliadel, tamoxifen, topotecan, methotrexate, gefitinib (Iressa®), taxol, taxotere, fluorouracil, leucovorin, irinotecan, xeloda, CPT-11, interferon alpha, pegylated interferon alpha (e.g., PEGINTRON-A), capecitabine, cisplatin, thiotepa, fludarabine, carboplatin, liposomal daunorubicin, cytarabine, doxetaxol, pacilitaxel, vinblastine, IL-2, GM-CSF, dacarbazine, vinorelbine, zoledronic acid, palmitr onate, biaxin, busulphan, prednisone, bortezomib (Velcade®), bisphosphonate, arsenic trioxide, vincristine, doxorubicin (Doxil®), paclitaxel, ganciclovir, adriamycin, estrainustine sodium phosphate (Emcyt®), sulindac, etoposide, and combinations of any thereof.

[0179] In other embodiments, the anti-cancer agent can be selected from bortezomib, cyclophosphamide, dexamethasone, doxorubicin, interferon-alpha, lenalidomide, melphalan, pegylated interferon-alpha, prednisone, thalidomide, or vincristine.

[0180] In some embodiments, the methods of prevention and / or treatment as described herein further include an immunotherapy. In some embodiments, the immunotherapy includes administration of one or more checkpoint inhibitors. Accordingly, some embodiments of the methods of treatment described herein include further administration of a compound that inhibits one or more immune checkpoint molecules. Non-limiting examples of immune checkpoint molecules include CTLA4, PD-1, PD-L1, A2AR, B7-H3, B7-H4, TIM3, and combinations of any thereof. In some embodiments, the compound that inhibits the one or more immune checkpoint molecules includes an antagonistic antibody. Examples of antagonistic antibodiesAttorney Docket: 078430-541001WOsuitable for the compositions and methods disclosed herein include, but are not limited to, ipilimumab, nivolumab, pembrolizumab, durvalumab, atezolizumab, tremelimumab, and avelumab.

[0181] In some embodiments, the one or more anti-cancer therapy is radiation therapy. In some embodiments, the radiation therapy can include the administration of radiation to kill cancerous cells. Radiation interacts with molecules in the cell such as DNA to induce cell death. Radiation can also damage the cellular and nuclear membranes and other organelles. Depending on the radiation type, the mechanism of DNA damage may vary as does the relative biologic effectiveness. For example, heavy particles (i.e. protons, neutrons) damage DNA directly and have a greater relative biologic effectiveness. Electromagnetic radiation results in indirect ionization acting through short-lived, hydroxyl free radicals produced primarily by the ionization of cellular water. Clinical applications of radiation consist of external beam radiation (from an outside source) and brachytherapy (using a source of radiation implanted or inserted into the patient). External beam radiation consists of X-rays and / or gamma rays, while brachytherapy employs radioactive nuclei that decay and emit alpha particles, or beta particles along with a gamma ray. Radiation also contemplated herein includes, for example, the directed delivery of radioisotopes to cancer cells. Other forms of DNA damaging factors are also contemplated herein such as microwaves and UV irradiation.

[0182] Radiation may be given in a single dose or in a series of small doses in a dose-fractionated schedule. The amount of radiation contemplated herein ranges from about 1 to about 100 Gy, including, for example, about 5 to about 80, about 10 to about 50 Gy, or about 10 Gy. The total dose may be applied in a fractioned regime. For example, the regime may include fractionated individual doses of 2 Gy. Dosage ranges for radioisotopes vary widely, and depends on the half-life of the isotope and the strength and type of radiation emitted. When the radiation includes use of radioactive isotopes, the isotope may be conjugated to a targeting agent, such as a therapeutic antibody, which carries the radionucleotide to the target tissue (e.g., tumor tissue).

[0183] Surgery described herein includes resection in which all or part of a cancerous tissue is physically removed, exercised, and / or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs surgery). Removal of pre-cancers or normal tissues is also contemplated herein.Attorney Docket: 078430-541001WO

[0184] Accordingly, in some embodiments, the methods of the disclosure include administration of a composition disclosed herein to a subject individually as a sole therapy (e.g., monotherapy). In some embodiments, a composition of the disclosure is administered to a subject as a first therapy in combination with a second therapy. In some embodiments, the second therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, and surgery. In some embodiments, the first therapy and the second therapy are administered concomitantly. In some embodiments, the first therapy is administered at the same time as the second therapy. In some embodiments, the first therapy and the second therapy are administered sequentially. In some embodiments, the first therapy is administered before the second therapy. In some embodiments, the first therapy is administered after the second therapy. In some embodiments, the first therapy is administered before and / or after the second therapy. In some embodiments, the first therapy and the second therapy are administered in rotation. In some embodiments, the first therapy and the second therapy are administered together in a single formulation.KITS

[0185] Also provided herein are various kits for the practice of a method described herein. In particular, some embodiments of the disclosure provide kits for the diagnosis of a health condition in a subject. Some other embodiments relate to kits for the prevention of a health condition in a subject in need thereof. Some other embodiments relate to kits for methods of treating a health condition in a subject in need thereof. For example, provided herein, in some embodiments, are kits that include one or more of the chimeric polypeptides (e.g., CARS), recombinant nucleic acids, recombinant cells, or pharmaceutical compositions as provided and described herein, as well as written instructions for making and using the same.

[0186] In some embodiments, the kits of the disclosure further include one or more means useful for the administration of any one of the provided chimeric polypeptides (e.g., CARS), recombinant nucleic acids, recombinant cells, or pharmaceutical compositions to an individual. For example, in some embodiments, the kits of the disclosure further include one or more syringes (including pre-filled syringes) and / or catheters (including pre-filled syringes) used to administer any one of the provided chimeric polypeptides (e.g., CARS), recombinant nucleic acids, recombinant cells, or pharmaceutical compositions to an individual. In some embodiments, a kit can have one or more additional therapeutic agents that can be administeredAttorney Docket: 078430-541001WOsimultaneously or sequentially with the other kit components for a desired purpose, e.g., for diagnosing, preventing, or treating a condition in a subject in need thereof.

[0187] Any of the above-described kits can further include one or more additional reagents, where such additional reagents can be selected from: dilution buffers; reconstitution solutions, wash buffers, control reagents, control expression vectors, negative control polypeptides, positive control polypeptides, reagents suitable for in vitro production of the chimeric polypeptides.

[0188] In some embodiments, the components of a kit can be in separate containers. In some other embodiments, the components of a kit can be combined in a single container.

[0189] In some embodiments, a kit can further include instructions for using the components of the kit to practice the methods disclosed herein. The instructions for practicing the methods are generally recorded on a suitable recording medium. For example, the instructions can be printed on a substrate, such as paper or plastic, etc. The instructions can be present in the kit as a package insert, in the labeling of the container of the kit or components thereof (e.g., associated with the packaging or sub-packaging), etc. The instructions can be present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc. In some instances, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the internet), can be provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate.

[0190] No admission is made that any reference cited herein constitutes prior art. The discussion of the references states what their authors assert, and the inventors reserve the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of information sources, including scientific journal articles, patent documents, and textbooks, are referred to herein; this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0191] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and alternatives will be apparent to those of skill in the art upon review of this disclosure, and are to be included within the spirit and purview of this application.Attorney Docket: 078430-541001WOEXAMPLES

[0192] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.EXAMPLE 1General experimental procedures

[0193] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature, such as Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements through 2014); Bollag, D. M. etal. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. etal. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K. B., Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press;Beaucage, S. L. etal. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements through 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and / or parameters unless otherwise noted.EXAMPLE 2Attorney Docket: 078430-541001WOCell lines

[0194] NAML6 cells expressing GFP and Luciferase with different levels of truncated CD 19 expression were used for in vitro and in vivo studies as previously described (Majzner et al. Cancer Discov. 10, 702-723, 2020). The HEK-293GP retroviral packaging cell line was obtained from the Surgery Branch (National Cancer Institute, National Institutes of Health) and was used to make retroviral-packaged CAR constructs. Peripheral blood mononuclear cells (PBMCs) for CAR T cells were obtained from the Stanford Blood Center under an IRB-exempt protocol. They were isolated using Ficoll-plaque Plus density gradient centrifugation(GE Healthcare). Cell aliquots were frozen in CryoStor CS10 media (Sigma- Aldrich) until thawing for use.EXAMPLE 3CAR T plasmid constructs

[0195] 47G4 scFv-containing Hul9-CD8H / TM-CD28-CD3z CAR sequence was previously described (Alabanza, L. et al. Mol. Ther. 25, 2452-2465, 2017) and sequence was obtained from U.S. Patent Publication No. US20190202913A1 (corresponding to PCT Publication No.WO2015187528A1). Novel CAR constructs were made using restriction enzyme and In-Fusion HD Cloning (Takara Bio) using gene blocks purchased from Integrated DNA Technologies. All genes cloned were codon-optimized for human expression and their amino acid sequences were confirmed using UniProt database. All CARs were cloned into MSGV1 retroviral vectors for T cell integration and verified via sanger sequencing before transfection (Primordium Labs). FIGS.1A-1B shows schematic diagrams of four exemplary human CD19 CAR constructs (HuCD19 CAR) of the present disclosure and murine anti-CD19 binder FMC63 controls. FIG. 1A: Hui 9 CAR T cell schematics with corresponding scFv orientations, linkers, transmembrane domains and co-stimulatory signaling molecules. Whitlow linker (solid), (4GS)s linker (dashed). FIG. IB:FMC63 CAR controls depicting the same as FIG. 1 A.EXAMPLE 4Retroviral supernatant production

[0196] 6.75 x 106HEK293GP cells were added to 100mm poly-D-lysine-precoated plates in DMEM media supplemented with 10% FBS, lOmM HEPES, 100U mL-1 penicillin and lOOpg / mL streptomycin (Gibco). Following overnight incubation, cells were transfected using lipofectamine 2000 at 2: 1 ratio (9pg:4.5pg) of vector to packaging plasmid. Viral supernatantsAttorney Docket: 078430-541001WOwere recovered at 48 and 72h post transfection and used for T cell transduction.EXAMPLE 5CAR T cell transduction

[0197] Thawed PBMCs were thawed on day 0 and cultured with human T- Activator anti-CD3 / anti-CD28 Dynabeads (Gibco) at 3:1 bead: cell ratio in AIM-V media (Gibco) supplemented with 5% FBS, 2mM GlutaMAX, 100U mL-1 penicillin and lOOug mL-1 streptomycin, lOmMHEPES and 100IU mL-1 recombinant human Interleukin-2 (Prepotech). Retroviral transductions were done on days 3 and 4 using 25 pg mL-1 retronectin- coated (Takara), non-tissue culture 12-well plates. Wells were briefly blocked with 2% BSA in PBS before adding the retroviral supernatant onto the plates. One mL of thawed viral supernatant per CAR construct was used per well followed by centrifugation at 3000 rpm for 2 h at 32°C.0.5 x 106T cells were added to each well in 1 ml of complete AIM-V medium and after the second transduction on day 4, viral supernatant was discarded, beads magnetically removed and CAR T cells maintained in AIM-V medium at a density of 0.3 x 106cells / mL. CAR T cells were used for flow cytometry analysis of expression levels as well as for all in vitro and in vivo experiments at day 10 post-bead activation.EXAMPLE 6Flow cytometry

[0198] CAR T cells were washed and stained with 2% FBS in PBS (FACS buffer). Staining was done for 20 min at 4 °C followed by one wash with FACS buffer and analyzed on a NovoCyte Quanteon cytometer (Agilent). FlowJo software (BD) was used for data analysis. CD19-targeting CARs were detected using human CD19-Fc recombinant protein (R&D Systems), fluorophore- using DyLight 650 Microscale Antibody Labeling Kits (Invitrogen) stained at 1:400 dilution.EXAMPLE 7Cytotoxicity assay

[0199] Day 10 CD- 19 targeted CAR T cells were co-cultured with 50,000 tumor cells (NALM6 WT or NALM6963) at 1 : 1 ratio in RPMI medium supplemented with 10 % FBS, 2mM GlutaMAX, 100U mL-1 penicillin and lOOug mL-1 streptomycin. Co-cultures were seeded on 96-well flat-bottom plates and incubated at 37 °C in an Incucyte S3 Live-Cell Analysis SystemAttorney Docket: 078430-541001WO(Sartorius) for approximately 72 hours. The basic analyzer feature on the Incucyte S3 software was used to quantify killing of GFP+ tumor cells by measuring the Total Green Object Integrated Intensity over duration of experiment. Cytotoxicity index was calculated as the percentage of Total Green Object Integrated Intensity at a specific time point divided by the Total Green Object Integrated Intensity at time 0.EXAMPLE 8Cytokine assay

[0200] A total of 1 x 105day 10 CD 19 CAR T cells were co-cultured with tumor cells in a 1:1 ratio in complete RPMI medium and incubated at 37 °C for 24 hours. The supernatants were collected via centrifugation of the co-cultures and IL-2 as well as IFNy were measured by ELISA following the manufacturer’s protocol (BioLegend). Absorbances were measured with a Synergy Hl Hybrid Multi-Mode Reader with Gen5 software (BioTek).EXAMPLE 9Animal studies

[0201] Mice studies were carried out according to approved protocols by Stanford Institutional Animal Care and Use Committee. Immunodeficient NOD-scid IL2Rgnull (NSG, NOD.Cg-PrkdcscidI12rgtml Wjl / SzJl) mice were purchased from The Jackson Laboratory or bred in house. Six-to-ten- week-old male or female mice were intravenously inoculated with 1 x 106CD19+NALM6-WT or CD19+NALM6963cells 3 days before T cell injection in 200 pl PBS. Mice were randomized to ensure even tumor burden between experimental and control groups before treatment.

[0202] CAR T cells were injected intravenously on day 10 after activation: CD19+NALM6963bearing mice received a dose of 3 x 106CAR+ T cells for efficacy assessment while CD19+NALM6-WT bearing mice received 1 * 106CAR+ T cells. Leukemia mouse models were monitored for disease progression once a week using BLI with an IVIS imaging system (Perkin Elmer) and Livingimage software (Perkin Elmer). Mice were humanely euthanized when they showed signs of morbidity as per approved animal protocols.EXAMPLE 10Expression of HuCD19 CAR constructs

[0203] This Example describes experiments performed to illustrate the expression of HuCD19Attorney Docket: 078430-541001WOCAR constructs and murine FMC63 binder controls described in Example 3 and FIGS. 1A-1B above. Surface CAR T cell expression was evaluated using an Agilent NovoCyte Quanteon flow cytometer and FlowJo software (BD) for data analysis. CD19 CAR Ts were detected using a CD19-Fc recombinant protein (R&D Systems). Recombinant proteins were fluor ophore-conjugated using DyLight 650 Microscale Antibody Labeling Kits (Invitrogen) following manufacturer’s instructions. As shown in FIG. 2, Hul9 CAR T cell and FMC63-based CAR controls surface expression were evaluated using CD19-protein for detection via flow cytometry. Mock were untransduced, activated T cells. All the CAR T cell constructs were incubated with conjugated CD19Fc-DyLight650 protein and assayed via flow cytometry for surface CAR expression at DIO post-activation. CAR T cell expression was shown to be homogeneous with comparable expression on the surface of T cells for all CD 19 CAR T cells tested.EXAMPLE 11Effectiveness of Hui 9 CAR T cells in low density NALM6-CD19 (Naim963) and high density NALM6-CD19 (NALM6-WT) models in vitro

[0204] This Example describes experiments performed to illustrate the effectiveness of the Hui 9 CAR T cells disclosed herein in low density NALM6-CD19 (Nalm6963) high density NALM6-CD19 (NALM6-WT) models in vitro. CAR T cells (either the Hui 9 CAR T cells disclosed herein, a previously described huCD19-CD8H / TM-CD28-zeta, or murine FMC63 based controls) were cocultured as described in Examples 7 and 8 above with Nalm6 cells expressing either low or high amounts of CD 19. The Hui 9 CAR T cells disclosed herein demonstrated a distinct advantage to the previously described huCD19-CD8H / TM-CD28-zeta (NCT02659943) in being able to kill CD19lowexpressing tumor cells (FIG. 3A, bottom) while maintaining good killing and cytokine production in response to CD19hlghtumor cells (FIG.3 A, top and FIG.3B).EXAMPLE 12Effectiveness of Hui 9 CAR T cells in low density NALM6-CD19 (Naim963) model in vivo

[0205] This Example describes experiments performed to illustrate the effectiveness of the Hui 9 CAR T cells disclosed herein in low density NALM6-CD19 (Nalm6963) model in vivo. NSG mice were inoculated with 1 106Nalm6 CD19-low cells and then treated three days later with 3 / | 06of the indicated CAR T cell. Tumor burden was tracked over time by bioluminescentAttorney Docket: 078430-541001WOimaging (FIGS.4A-B) as was survival (FIG.4C) These data show the potency and efficacy of Hui 9 CAR T cells reported herein in detecting and controlling low-antigen cancer cells in a xenograft model as compared to the previously reported Hul9-CD8H / TM-CD28-zeta CAR and compared to clinical benchmarks (FMC63-CD28H / TM-CD28-zeta is the CAR structure utilized in FDA approved axicabtagene ciloleucel and FMC63-CD28H / TM-CD28-zeta is the CAR structure utilized in FDA approved tisagenlecleucel. This demonstrates the ability of the uniquely engineered CARs to mediate anti -tumor efficacy against CD 19 low tumor cells, overcoming a major mechanism of immune escape through antigen downregulation.EXAMPLE 13Effectiveness of Hui 9 CAR T cells in high density NALM6-CD19 (NALM6-WT) model in vivo

[0206] This Example describes experiments performed to illustrate the effectiveness of Hu 19 CAR T cells disclosed in some embodiments of the disclosure in high density NALM6-CD19 (NALM6-WT_CD19hlgh) model in vivo total tumor flux. In these experiments, as shown in FIG.5, mice were inoculated with 1 106CD19 WT clone NALM6 at Day -3 and treated with 1 106of corresponding CAR T cells. Tumor growth was tracked with bioluminescence imaging for 91 days and mice were followed for survival. The data in FIG. 5 show the improved efficacy and tumor control by Hui 9 CAR T cells reported herein as compared to both the previously reported Hul9-CD8H / TM-CD28-zeta CAR and compared to a clinical benchmarks (FMC63-CD28H / TM-CD28-zeta is the CAR structure utilized in FDA approved Yescarta. The Hui 9 CAR T cells reported herein also resulted in improved survival compared to controls (FIG. 6).EXAMPLE 14High density NALM6-CD19 (NALM6-WT) model in vivo overall persistence

[0207] This Example describes experiments performed to illustrate in vivo overall persistence of Hui 9 CAR T cells disclosed in some embodiments of the disclosure in high density NALM6-CD19 (NALM6-WT CD 19hlgh) model. In these experiments, as shown in FIGS. 7A-7B, mice were inoculated with 1 106NALM6_WT-CD19hlghat Day -3 and treated with a curative dose of 6 106of the corresponding CAR T cells. Experiment was terminated at Day 26 and total human T cells numbers (FIG. 7A) as well as total CAR+T cells (FIG. 7B) in each group were plotted. One-way analysis of variance (ANOVA) was used to compare the mean numbers of T cells and CAR+T cells in the different treatment groups p-value**<0.01.Attorney Docket: 078430-541001WO

[0208] The experiments described in this Example demonstrate that Hui 9 CAR T cells as engineered resulted in substantially improved CAR T cell persistence in a curative model of B cell leukemia compared to clinical benchmarks and other previously explored constructs, illustrating the unique benefit to the designs employed here.

[0209] While particular alternatives of the present disclosure have been disclosed, it is to be understood that various modifications and combinations are possible and are contemplated within the true spirit and scope of the appended claims. There is no intention, therefore, of limitations to the exact abstract and disclosure herein presented.

Claims

Attorney Docket: 078430-541001WOCLAIMS WHAT IS CLAIMED IS:

1. A recombinant polypeptide comprising:(a) an extracellular domain (ECD) having a binding affinity for human CD 19, wherein the ECD comprises a fully human antibody or an antigen-binding fragment thereof; (b) a hinge domain and a transmembrane (TM) domain from CD28; and(c) an intracellular signaling domain (ICD) comprising one or more costimulatory domains.

2. The chimeric polypeptide of claim 1, wherein the ICD further comprises a CD3 T cell activation domain.

3. The chimeric polypeptide of any one of claims 1 to 2, wherein the fully human antibody, or an antigen-binding fragment thereof, comprises all six complementary determining regions (CDRs) of an antibody selected from the group consisting of 21D4, 21D4a, 47G4, 27F3, 3C10, 5G7, 13F1 and46E8.

4. The chimeric polypeptide of claim 3, wherein the fully human antibody, or an antigenbinding fragment thereof, comprises the light chain region (VL) and heavy chain region (VH) of the antibody.

5. The chimeric polypeptide of any one of claims 1 to 4, wherein the antigen-binding fragment thereof is or comprises a single-chain antibody fragment (scFv), a F(ab) fragment, a F(ab') fragment, a F(ab')2 fragment, Fd fragment, a Fab'-SH fragment, or a Fv fragment.

6. The chimeric polypeptide of claim 5, wherein the antigen-binding fragment thereof is or comprises a single-chain antibody fragment (scFv) comprising a light chain variable region (VL) and a heavy chain variable region (VH) operably linked to each other via a linker.

7. The chimeric polypeptide of claim 6, wherein a light chain variable region (VL) is N-terminally linked to the heavy chain variable region (VH).

8. The chimeric polypeptide of claim 6, wherein a light chain variable region (VL) is C-terminally linked to the heavy chain variable region (VH).Attorney Docket: 078430-541001WO9. The chimeric polypeptide of any one of claims 6 to 8, wherein the linker is a polypeptide linker (peptidal linkage).

10. The chimeric polypeptide of claim 9, wherein the polypeptide linker is or comprises a glycine-serine linker or a Whitlow linker.

11. The chimeric polypeptide of any one of claims 6 to 10, wherein the human CD 19 scFv comprises, in N-terminal to C-terminal direction:(a) a VL region, a linker, and a VH region; or(b) a VH region, a linker, and a VL region.

12. The chimeric polypeptide of any one of claims 6 to 11, wherein the human CD 19 scFv comprises, in N-terminal to C-terminal direction:(a) a VL region, a (4GS)s linker, and a VH region;(b) a VL region, a Whitlow linker, and a VH region;(c) a VH region, a (4GS)s linker, and a VL region; or(d) a VH region, a Whitlow linker, and a VL region.

13. The chimeric polypeptide of any one of claims 1 to 12, wherein the chimeric polypeptide is a chimeric antigen receptor (CAR).

14. The chimeric polypeptide of any one of claims 1 to 13, wherein the one or more costimulatory domains is from a polypeptide selected from the group consisting of a 4- IBB (CD137), CD27 (TNFRSF7), CD28, 0X40 (CD134), CD70, LFA-2 (CD2), CD5, ICAM-1 (CD54), LFA-1 (CD1 la / CD18), DAP10, DAP12, a co-stimulatory inducible T-cell costimulatory (ICOS) polypeptide sequence, and a combination of any thereof.

15. The chimeric polypeptide of claim 14, wherein the one or more costimulatory domains comprises a costimulatory 4- IBB (CD 137) polypeptide sequence.

16. The chimeric polypeptide of any one of claims 1 to 15, wherein the chimeric polypeptide comprises, in N-terminal to C-terminal direction:(a) a human CD 19 scFv,(b) a hinge domain and a TM domain from CD28,Attorney Docket: 078430-541001WO(c) a costimulatory domain from 4-1BB; and(d) a CD3C ICD.

17. The chimeric polypeptide of any one of claims 1 to 16, wherein the chimeric polypeptide comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

18. A recombinant nucleic acid molecule comprising a nucleic acid sequence that encodes a chimeric polypeptide according to of any one of claims 1 to 17.

19. The recombinant nucleic acid molecule of claim 18, wherein the nucleic acid sequence has at least 80% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

20. The recombinant nucleic acid molecule of any one of claims 18 to 19, wherein the recombinant nucleic acid molecule is operably linked to a heterologous nucleic acid sequence.

21. The recombinant nucleic acid molecule of any one of claims 18 to 20, wherein the recombinant nucleic acid molecule is further defined as an expression cassette in a vector.

22. The nucleic acid molecule of claim 21 , wherein the vector is a plasmid vector or a viral vector.

23. The nucleic acid molecule of claim 22, wherein the viral vector is derived from a lentivirus, an adenovirus, an adeno-associated virus (AAV), a baculovirus, or a retrovirus.

24. A recombinant cell comprising:(a) a chimeric polypeptide according to any one of claims 1 to 17; and / or(b) a nucleic acid molecule according to any one of claims 18 to 23.

25. The recombinant cell of claim 24, wherein the recombinant cell is a eukaryotic cell.

26. The recombinant cell of any one of claim 24 to 25, wherein the recombinant cell is an immune system cell.Attorney Docket: 078430-541001WO27. The recombinant cell of claim 26, wherein the immune system cell is a T cell, B cell, a monocyte, a natural killer (NK) cell, a natural killer T (NKT) cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a monocyte, a hematopoietic stem cell, or a hematopoietic stem cell progenitor.

28. The recombinant cell of claim 27, wherein the T cell is an IPSC-derived T cell, a regulatory T cell, a helper T cell (TH), a cytotoxic T cell (TCTL), a memory T cell, a gamma delta (y6) T cell, a CD8+ T cytotoxic lymphocyte cell, or a CD4+ T helper lymphocyte cell.

29. The recombinant cell of claim 28, wherein the CD8+ T cytotoxic lymphocyte cell is selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, effector CD8+ T cells, CD8+ stem memory T cells, bulk CD8+ T cells.

30. The recombinant cell of claim 28, wherein the CD4+ T helper lymphocyte cell is selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, effector CD4+ T cells, CD4+ stem memory T cells, and bulk CD4+ T cells.

31. The recombinant cell of any one of claims 27 to 30, wherein the T cell is an exhausted T cell or a non-exhausted T cell.

32. A method for making a recombinant cell, comprising:a) providing a host cell capable of protein expression; andb) transducing the provided host cell with a recombinant nucleic acid according to any one of claims 18 to 23 to produce a recombinant cell.

33. A recombinant cell produced by a method according to claim 32.

34. A cell culture comprising at least one recombinant cell according to any one of claims 24 to 31 and a culture medium.

35. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and:a) a chimeric polypeptide according to any one of claims 1 to 17;b) a nucleic acid molecule according to any one of claims 18 to 23; and / orc) a recombinant cell according to any one of claims 24 to 31.Attorney Docket: 078430-541001WO36. The pharmaceutical composition of claim 35, wherein the composition comprises a recombinant nucleic acid according to any one of claims 18 to 23, a pharmaceutically acceptable carrier.

37. The pharmaceutical composition of claim 36, wherein the recombinant nucleic acid is encapsulated in a viral capsid or a lipid nanoparticle.

38. The pharmaceutical composition of claim 35, wherein the composition comprises a recombinant cell according to any one of claims 24 to 41, a pharmaceutically acceptable carrier.

39. A method for treating a health condition associated with expression of CD 19 in a subject in need thereof, comprising administering to the subject a composition comprising:a) a chimeric polypeptide according to any one of claims 1 to 17;b) a nucleic acid molecule according to any one of claims 18 to 23;c) a recombinant T cell according to any one of claims 27 to 41; and / ord) a pharmaceutical composition according to any one of claims 35 to 38.

40. The method of claim 39, wherein the recombinant T cell is autologous to the subject.

41. The method of any one of claims 39 to 40, wherein the T cell is obtained by leukapheresis of a sample obtained from a subject.

42. The method of any one of claims 39 to 41, wherein the health condition is a disease or disorder associate with expression of CD 19.

43. The method of claim 40, wherein the disease is a cancer, a malignancy, a precancerous condition, an autoimmune disease (AID), or a non-cancer related indication associated with expression of CD 19.

44. The method of any one of claims 39 to 43, wherein the autoimmune disease (AID) is systemic lupus erythematosus (SLE), idiopathic inflammatory myositis (IIM), or systemic sclerosis (SSc).

45. The method of any one of claims 39 to 44, wherein CD 19 is expressed at a heterogenous level or a low level in cancer cells.

46. The method of any one of claims 39 to 45, wherein the disease or disorder is a B-cell malignancy.Attorney Docket: 078430-541001WO47. The method of any one of claims 39 to 46, wherein the B-cell malignancy is a hematologic malignancy selected from the group consisting B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), acute myelogenous leukemia (AML), acute lymphoid leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B cell pro-lymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell-or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, null-acute lymphoblastic leukemia, nonHodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, splenic, marginal zone lymphoma, and Waldenstrom macroglobulinemia.

48. The method of any one of claims 39 to 47, wherein the administered composition confers increased production of interferon gamma (IFNy) and / or interleukin-2 (IL-2) in the subject.

49. The method of any one of claims 39 to 48, wherein the administered composition inhibits or kills malignant B-cells in the subject.

50. The method of any one of claims 39 to 48, wherein the administered composition inhibits tumor growth or metastasis of the cancer in the subject.

51. The method of any one of claims 39 to 50, wherein the composition is administered to the subject individually as a sole therapy or as a first therapy in combination with a second therapy.

52. The method of claim 51, wherein the second therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, and surgery.

53. The method of any one of claims 51 to 52, wherein the second therapy comprises an additional CAR T cell and / or a bispecific antibody targeting CD3 and a B-cell antigen.

54. The method of any one of claims 51 to 53, wherein the second therapy comprises targeting at least one antigen expressed on a B-cell malignancy.

55. The method of claim 54, wherein the at least one antigen expressed on a B-cell malignancy is CD22, CD20, CD79a, CD79b, BAFF-R, BCMA, CD72, CD 123, or a combination thereof.

56. The method of any one of claims 51 to 52, wherein the first therapy and the second therapy are administered concomitantly.Attorney Docket: 078430-541001WO57. The method of any one of claims 51 to 56, wherein the first therapy is administered at the same time as the second therapy.

58. The method of any one of claims 51 to 52, wherein the first therapy and the second therapy are administered sequentially.

59. The method of claim 58, wherein the first therapy is administered before the second therapy.

60. The method of claim 58, wherein the first therapy is administered after the second therapy.

61. The method of any one of claims 51 to 52, wherein the first therapy is administered before and / or after the second therapy.

62. The method of any one of claims 51 to 52, wherein the first therapy and the second therapy are administered in rotation.

63. The method of any one of claims 51 to 52, wherein the first therapy and the second therapy are administered together in a single formulation.

64. A kit for the diagnosis, prevention, and / or treatment a health condition associated with expression of CD 19 in a subject in need thereof, the kit comprising:a) a chimeric polypeptide according to any one of claims 1 to 17;b) a nucleic acid molecule according to any one of claims 18 to 23;c) a recombinant T cell according to any one of claims 27 to 31; and / ord) a pharmaceutical composition according to any one of claims 35 to 38.

65. Use of:a) a chimeric polypeptide according to any one of claims 1 to 17;b) a nucleic acid molecule according to any one of claims 18 to 23;c) a recombinant T cell according to any one of claims 27 to 31; and / or(d) a pharmaceutical composition according to any one of claims 35 to 38.for the diagnosis, prevention, and / or treatment a health condition associated with expression of CD 19 in a subject in need thereof.

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