WO2020176897A1

WO2020176897A1 - Chimeric antigen receptors and uses thereof

Info

Publication number: WO2020176897A1
Application number: PCT/US2020/020521
Authority: WO
Inventors: Robbie G. MAJZNER; Crystal L. MACKALL; Skyler P. RIETBERG
Original assignee: The Board Of Trustees Of The Leland Stanford Junior University
Priority date: 2019-02-28
Filing date: 2020-02-28
Publication date: 2020-09-03

Abstract

The present disclosure generally relates to novel compositions and methods for improving the efficiency of adoptive cell therapies and enhancing CAR-T cell functionality by simultaneously targeting different epitopes on a same antigen molecule associated with or specific for a target cell, e.g., cancer cell. Some embodiments of the disclosure provides recombinant cells containing one or more chimeric antigen receptors (CARs) directed to different epitopes present on a same target antigen. Also provided are nucleic acids encoding the disclosed CARs, recombinant cells expressing the same, pharmaceutical compositions containing the disclosed nucleic acids and/or recombinant cells, and methods useful for modulating an activity of a target cell, e.g., cancer cell, as well as methods for treating a disease.

Description

CHIMERIC ANTIGEN RECEPTORS AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 62/812,105, filed on February 28, 2020. The disclosure of the above- referenced application is herein expressly incorporated by reference it its entirety, including any drawings.

INCORPORATION OF THE SEQUENCE LISTING

[0002] The material in the accompanying Sequence Listing is hereby incorporated by reference into this application. The accompanying Sequence Listing text file, named 078430- 510001WO_Sequence Listing.txt, was created on February 22, 2020 and is 60 KB.

FIELD

[0003] The present disclosure relates generally to the fields of immuno-therapeutics, and particularly relates to novel polypeptides, e.g., chimeric antigen receptors that bind an antigen of interest and having selectable specificities and activities. The disclosure also provides compositions and methods useful for producing such molecules, as well as methods for the detection and treatment of diseases, such as cancer.

BACKGROUND

[0004] Biopharmaceuticals or the use of pharmaceutical compositions comprising a therapeutic protein for the treatment of diseases or health conditions is a core strategy for a number of pharmaceutical and biotechnology companies. For example, in cancer immunotherapy, the development of agents that activate T cells of the host's immune system to prevent the proliferation of or kill cancer cells has emerged as a promising therapeutic approach to complement existing standards of care. Examples of such immunotherapy approaches include the development of chimeric antigen receptors (CARs) for use in modulating the immune system to inhibit or kill cancer cells. In particular, adoptive transfer of T cells, especially CAR-engineered T cells, has emerged as a promising approach for immunotherapy and made headlines in clinical trials conducted by a number of

pharmaceutical and biotechnology companies.

[0005] However, it has been reported that CAR potency is often limited, particularly in solid tumors. This is often due to low target antigen density and immune suppressive factors in the microenvironment. In particular, low target antigen density is a major impediment to CAR T cells success in solid tumors, where antigens are often expressed at heterogeneous levels. In addition, tumors can relapse in patients after CAR therapy by downregulating the CAR target. In fact, antigen downregulation and antigen escape have emerged as major issues impacting the durability of CAR T-cell therapy. Recent clinical data from CAR T-cell trials in B-cell malignancies demonstrate that a common mechanism of resistance to this novel class of therapeutics is the emergence of tumors with loss or downregulation of the target antigen. Antigen loss or antigen-low escape is likely to emerge as an even greater barrier to success in solid tumors, which manifest greater heterogeneity in target antigen expression.

[0006] One potential approach would be to treat patients with agents that increase expression of the target antigen. Other approaches to overcome this challenge include engineering CAR T cells to achieve multi-specificity and to respond to lower levels of target antigen and more efficient induction of natural anti-tumor immune responses as a result of CAR induced inflammation. For appropriate targets where the differential expression of target antigen between tumor and normal tissue is high, the efficacy of CAR therapies would be enhanced by engineering CAR T cells to respond to lower antigen densities. For some CARs, it appears that altering the affinity can result in recognition of lower levels of target antigen. However, recent studies reported that increasing the affinity of two different CD22 CARs did not result in enhanced function (Haso W. et al, Blood 2013;121 : 1165-74; and Lynn RC et al., Blood 2015;125:3466-76). Therefore, it remains unclear whether the impact of enhancing scFv activity plateaus.

[0007] Thus, there remains a need for more potent CARs to overcome these obstacles to extend the reach of this new class of therapeutics to more diseases and to treat more patients. The invention described herein provides solutions to address these obstacles and provides additional benefits.

SUMMARY

[0008] The present disclosure relates generally to the development of immuno- therapeutics, such as enhanced polypeptides and chimeric antigen receptors (CARs) and pharmaceutical compositions comprising the same for use in treating diseases such as cancer. In particular, the disclosure provides compositions and methods for improving the efficiency of adoptive cell therapies and enhancing CAR-T cell functionality by simultaneously targeting different epitopes on a same antigen molecule. As will be discussed more thoroughly herein, the low target antigen density is a major impediment to the success of CAR-T cell therapies in solid tumors, where antigens are often expressed at heterogeneous levels. In fact, it has been reported that CAR potency is limited in many tumors, which is often due to low target antigen density. In addition, tumors can relapse in patients after CAR therapy by downregulating the CAR target. Some embodiments of the disclosure provide a new approach to increase the number of engaged CAR molecules per target cell by simultaneously targeting two or more different epitopes on a same antigen associated with or a same antigen specific for a target cell, e.g., cancer cell. This new strategy allows to improving CAR T cell functionality, e.g. , increasing cytokine production and enhancing tumor cell killing, which in turn allows to improving adoptive cell therapies for cancer, such as hematologic malignancies, solid tumors, brain tumors.

[0009] In one aspect, some embodiments of the disclosure relate to a chimeric polypeptide including, in N-terminus to C-terminus: (a) an extracellular domain including a first antigen-binding moiety capable of binding to a first epitope of an antigen, and a second antigen-binding moiety capable of binding to a second epitope of the antigen; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first and the second antigen-binding moieties are in reverse orientation relative to the spatial orientation of their respective epitopes on the antigen.

[0010] Non-limiting exemplary embodiments of the chimeric polypeptide of the disclosure can include one or more of the following features. In some embodiments, the first and the second antigen-binding moieties are operably linked to each other in tandem. In some embodiments, the intracellular signaling domain further includes a 0Ό3z intracellular signaling domain. In some embodiments, the chimeric polypeptide is a chimeric antigen receptor (CAR). In some embodiments, the first and the second epitopes are non-overlapping epitopes on the antigen. In some embodiments, the epitope binding of the first antigen binding moiety does not significantly interfere with epitope binding of the second antigen binding moiety. In some embodiments, epitope binding of the first antigen-binding moiety does not significantly compete with epitope binding of the second antigen-binding moiety. In some embodiments, epitope binding of the first antigen-binding moiety does not compete with epitope binding of the second antigen-binding moiety (e.g., the first epitope and the second epitope are non-competing epitopes). In some embodiments, the first and the second antigen-binding moieties are capable of binding the same antigen expressed by a target cell.

In some embodiments, the antigen is expressed at low density by the target cell. In some embodiments, the target cell is a cancer cell. In some embodiments, the cancer cell is selected from the group consisting of a leukemia cell, an acute myeloma leukemia cell, a lymphoma cell, an anaplastic lymphoma cell, an astrocytoma cell, a B-cell cancer cell, a T-cell cancer, an epithelial type cancer cell, a mesenchymal type cancer cell, a breast cancer cell, a colon cancer cell, an ependymoma cell, an esophageal cancer cell, a glioblastoma cell, a glioma cell, a leiomyosarcoma cell, a liposarcoma cell, a liver cancer cell, a lung cancer cell, a mantle cell lymphoma cell, a melanoma cell, a neuroblastoma cell, a non-small cell lung cancer cell, an oligodendroglioma cell, an ovarian cancer cell, a pancreatic cancer cell, a peripheral T-cell lymphoma cell, a renal cancer cell, a stomach cancer cell, a carcinoma cell, a mesothelioma cell, and a sarcoma cell.

[0011] In some embodiments, the antigen is a tumor associated-antigen (TAA) or a tumor-specific antigen (TSA). In some embodiments, the antigen is associated with or specific for a hematologic malignancy. In some embodiments, the antigen is associated with or specific for a solid tumor. In some embodiments, the antigen is selected from the group consisting of CD22, human epidermal growth factor receptor 2 (HER2/neu/ErbB-2), CD276 (B7-H3), MUC1, PSMA, Glypican 2 (GPC2), IL- 13 -receptor alpha 1, IL- 13-receptor alpha 2, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD123, CD93, CD99, CD 117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), ALK, DLK1, FAP, NY-ESO, WT1, HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo- Dl, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor-1, the dimeric form of the pyruvate kinase isoenzyme type M2 (tumor M2-PK), CD 19, CD20, CD5, CD7, CD3, TRBCl, TRBC2, BCMA, CD38, CD123, CD93, CD34, CDla, SLAMF7/CS1, FLT3, CD33, CD123, TALLA-1, CSPG4, DLL3, IgG Kappa light chain, IgA Lamba light chain, CD16/ FcyRIII, CD64, FITC, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), EGFR and isovariants thereof, TEM-8, sperm protein 17 (Spl7), mesothelin, PAP (prostatic acid phosphatase), prostate stem cell antigen (PSCA), prostein, NKG2D, TARP (T cell receptor gamma alternate reading frame protein), Trp-p8, STEAP1 (six-transmembrane epithelial antigen of the prostate 1), an abnormal ras protein, an abnormal p53 protein, integrin b3 (CD61), galactin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), and Ral-B. In some embodiments, the antigen is selected from the group consisting of CD22, HER2/neu/ErbB-2, and CD276 (B7-H3). In some embodiments, the antigen is CD22, HER2/neu/ErbB-2, or CD276 (B7-H3). In some embodiments, the antigen is CD22. In some embodiments, the antigen is HER2/neu/ErbB-2. In some embodiments, the antigen is CD276 (B7-H3).

[0012] In some embodiments, the first antigen-binding moiety and the second antigen binding moiety are independently selected from the group consisting of an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), a full-length immunoglobulin, a nanobody, a single domain antibody (sdAb), a VNAR domain, and a VHH domain, and a diabody, or a functional fragment of any thereof. In some embodiments, at least one of the first antigen-binding moiety and the second antigen-binding moiety is a scFv or a functional fragment thereof. In some embodiments, the intracellular signaling domain includes one or more costimulatory domains. In some embodiments, the one or more costimulatory domains is selected from the group consisting of a costimulatory 4-1BB (CD137) polypeptide sequence, a costimulatory CD27 polypeptide sequence, a costimulatory 0X40 (CD 134) polypeptide sequence, a costimulatory inducible T-cell costimulatory (ICOS) polypeptide sequence, and a CD2 costimulatory polypeptide sequence. In some embodiments, the transmembrane domain is derived from a CD28 transmembrane domain, a CD8a

transmembrane domain, a Oϋ3z transmembrane domain, a CD4 transmembrane domain, a CTLA4 transmembrane domain, and a PD-1 transmembrane domain. In some embodiments, the transmembrane domain is derived from a CD 8a transmembrane domain.

[0013] In some embodiments, the chimeric polypeptide of the disclosure includes, in N- terminus to C-terminus direction: (a) an extracellular domain including a first and a second antigen-binding moieties capable of binding to different epitopes of an antigen selected from the group consisting of CD22, HER2, and CD276; (b) a transmembrane domain derived from CD8a; (c) an intracellular domain including a costimulatory domain from 4-1BB; and d) a CD3z intracellular signaling domain, wherein the first and the second antigen-binding moieties are in reverse orientation relative to the spatial orientation of their respective epitopes on the antigen.

[0014] In some embodiments, the chimeric polypeptide of the disclosure includes an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-4.

[0015] In another aspect, provided herein are recombinant nucleic acids including a nucleotide sequence that encodes a chimeric polypeptide as disclosed herein. In some embodiments, the nucleotide sequence is incorporated into an expression cassette or an expression vector. In some embodiments, the expression vector is a viral vector. In some embodiments, the viral vector is a lentiviral vector, an adenovirus vector, an adeno-associated virus vector, or a retroviral vector.

[0016] In another aspect, provided herein are recombinant cells including (a) a chimeric polypeptide as disclosed herein and/or (b) a recombinant nucleic acid as disclosed herein. In a related aspect, provided herein are recombinant cells that have been engineered to express the following: (a) a first chimeric polypeptide including, from N-terminus to C-terminus: (i) an extracellular domain including a first antigen-binding moiety capable of binding to a first epitope of an antigen; (ii) a transmembrane domain; and (iii) an intracellular signaling domain; and (b) a second chimeric polypeptide including, from N-terminus to C-terminus: (i) an extracellular domain including a second antigen-binding moiety capable of binding to a second epitope of the antigen; (ii) a transmembrane domain; and (iii) an intracellular signaling domain, wherein the first and the second antigen-binding moieties are capable of binding to different epitopes of the antigen. Also provided, in a related aspect, are cell cultures including at least one recombinant cell as disclosed herein and a culture medium.

[0017] In some embodiments, the recombinant cell disclosed herein is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell. In some embodiments, the immune cell is a B cell, a monocyte, a natural killer cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a T lymphocyte, a regulatory T cell, a helper T cell, a cytotoxic T cell, or other T cell. In some embodiments, the immune cell is a T lymphocyte.

[0018] Another aspect relates to methods for making a recombinant cell that includes: (a) providing a cell capable of protein expression; and (b) transducing the cell with a recombinant nucleic acid as disclosed herein.

[0019] In another aspect, provided herein are pharmaceutical compositions including a pharmaceutical acceptable carrier and one or more of the following: (a) a recombinant nucleic acid as disclosed herein, and (b) a recombinant cell as disclosed herein. In some

embodiments, the disclosed pharmaceutical 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. [0020] In one aspect, provided herein are methods for the treatment of a disease in an individual in need thereof, which includes administering to the individual a first therapy including a composition that includes at least one recombinant cell as disclosed herein. In some embodiments, the provided composition treats the disease in the individual.

[0021] Another aspect relates to methods for the treatment of a disease in an individual in need thereof, the method including administering to the individual a first therapy including a composition that includes at least one recombinant cell engineered to express the following: (a) a first chimeric polypeptide including, from N-terminus to C-terminus: (i) an extracellular domain including a first antigen-binding moiety capable of binding to a first epitope of an antigen; (ii) a transmembrane domain; and (iii) an intracellular signaling domain; and (b) a second chimeric polypeptide including, from N-terminus to C-terminus: (i) an extracellular domain including a second antigen-binding moiety capable of binding to a second epitope of the antigen; (ii) a transmembrane domain; and (iii) an intracellular signaling domain, wherein the first and the second antigen-binding moieties are capable of binding to different epitopes of the antigen. In some embodiments, the provided composition treats the disease in the individual.

[0022] Non-limiting exemplary embodiments of the treatment methods disclosed herein can include one or more of the following features. In some embodiments, the antigen is expressed at low density. In some embodiments, the disease is a cancer. In some embodiments, the cancer is a pancreatic cancer, a colon cancer, an ovarian cancer, a prostate cancer, a lung cancer, mesothelioma, a breast cancer, a urothelial cancer, a liver cancer, a head and neck cancer, a sarcoma, a cervical cancer, a stomach cancer, a gastric cancer, a melanoma, a uveal melanoma, a cholangiocarcinoma, multiple myeloma, leukemia, lymphoma, a B-cell cancer, a T-cell cancer, an epithelial type cancer, mesenchymal type cancer, or glioblastoma. In some embodiments, the administered first therapy inhibits tumor growth or metastasis of the cancer in the individual. In some embodiments, the administered first therapy increases cytokine production and/or enhances killing a cancer cell. In some embodiments, the first therapy is administered to the individual in conjunction with administration of 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 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.

[0023] In another aspect, provided herein are methods for modulating an activity of a target cell, the method including: (a) providing a composition including at least one recombinant cell as disclosed herein; and (b) contacting a target cell with the provided composition. In a related aspect, provided herein are methods for modulating an activity of a target cell, the method including providing composition including at least one recombinant cell engineered to express the following: (a) a first chimeric polypeptide including, from N- terminus to C-terminus: (i) an extracellular domain including a first antigen-binding moiety capable of binding to a first epitope of an antigen; (ii) a transmembrane domain; and (iii) an intracellular signaling domain; and (b) a second chimeric polypeptide including, from N- terminus to C-terminus: (i) an extracellular domain including a second antigen-binding moiety capable of binding to a second epitope of the antigen; (ii) a transmembrane domain; and (iii) an intracellular signaling domain, wherein the first and the second antigen-binding moieties are capable of binding to different epitopes of the antigen; and contacting a target cell with the provided composition.

[0024] Non-limiting exemplary embodiments of the disclosed methods of modulating an activity of a target cell can include one or more of the following features. In some embodiments, the provided composition modulates an activity of the target cell. In some embodiments, the activity of the cell to be modulated is selected from the group consisting of: cell growth, proliferation, apoptosis, non-apoptotic death, differentiation, dedifferentiation, migration, secretion of a molecule, cellular adhesion, and cytolytic activity. In some embodiments, the target cell is a cancer cell. In some embodiments, the step of contacting the recombinant cell with the target cell is carried out contacting is carried out in vivo, ex vivo, or in vitro. In some embodiments, the antigen is expressed at low density by the target cell.

[0025] In another aspect, some embodiments of the disclosure relate to systems for modulating an activity of a target cell or for treating a disease in an individual in need thereof, wherein the systems include one or more of: a chimeric polypeptide of the disclosure; a nucleic acid of the disclosure; a recombinant cell of the disclosure; and/or a pharmaceutical composition of the disclosure.

[0026] Yet another aspect of the disclosure is the use of one or more of: a chimeric polypeptide of the disclosure; a polynucleotide of the disclosure; a recombinant cell of the disclosure; and a pharmaceutical composition of the disclosure; for the treatment of a disease. In some embodiments, the disease is cancer.

[0027] Another aspect of the disclosure is the use of one or more of: a chimeric polypeptide of the disclosure; a polynucleotide of the disclosure; a recombinant cell of the disclosure; or a pharmaceutical composition of the disclosure; for the manufacture of a medicament for the treatment of a disease.

[0028] 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.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIGS. 1A-1C depict three examples of how low antigen density represents a major limiting factor for CAR efficacy and antigen downregulation is a mechanism of immune escape from CAR therapy. FIG. 1A is adapted from Walker AJ et al, Mol Ther.

2017 Sep 6;25(9):2189-2201, where low ALK levels expressed on neuroblastomas ALK^low NALM-6 were shown to be insufficient to induce maximal CAR functionality. FIG. IB is adapted from Majzner RG et al, Clin Cancer Res. 2019 Apr 15;25(8):2560-2574. B7-H3 was lentivirally expressed on the surface of NALM-6, a B cell leukemia known to be susceptible to CAR T cells and obtained single clones expressing variable amounts of B7-H3 on their surface. FIG. 1C is adapted from Fry TJ et al, Nat Med. 2018 Jan;24(l):20-28, showing that relapse is associated with diminished CD22 site density, and a pattern of acquired resistance to CD22-CAR associated with diminished CD22 site density assessed by flow cytometry.

[0030] FIG. 2 is a schematic illustrating conceptually how targeting two different epitopes of the same target antigen can increase the number of CAR receptors at the immune synapse, increasing CAR T cell reactivity at low antigen density.

[0031] FIG. 3 schematically illustrates a non-limiting example of the application of two CAR constructs each containing a single chain antibody fragments (scFv) targeting different epitopes on the antigen CD22. In these experiments, two CARs were constructed with two single chain variable fragments (scFv) m971 and HA22 which bind two distinct locations on CD22. CAR T cells were then transduced with these CARs.

[0032] FIGS. 4A-4C schematically summarize the results from experiments performed to isolate CAR-T cells containing one or both CAR constructs described in FIG. 3. the CAR- T cells were FACS sorted based on fluorescence to achieve a 100% pure single-positive CAR T cells (i.e. , expressing one CAR construct) or double-positive CAR T cell populations (i.e.. expressing both CAR constructs).

[0033] FIGS. 5A-5B schematically summarize the results of experiments performed to illustrate that engineered human T cells expressing both anti-CD22 single-targeting CAR constructs described in FIG. 3 demonstrate enhanced killing of tumor cells.

[0034] FIGS. 6A-6B schematically summarize the results of experiments performed to illustrate that engineered human T cells expressing both anti-CD22 single-targeting CAR constructs described in FIG. 3 generate more cytokine secretion against CD22^low leukemia than control T cells expressing either individual CAR.

[0035] FIGS. 7A-7B schematically summarize the results of experiments performed with engineered human T cells expressing two CAR constructs each containing a scFv targeting different epitopes on the antigen HER2. In these experiments, two CARs were constructed with two scFv 4D5 and FRP5 which bind two distinct locations on HER2. 4D5 targets proximal epitope and FRP5 target distal epitope. As shown in FIGS. 7A-7B, CAR T cells transduced with both anti-HER2 single-targeting CARs generated more cytokine than control T cells expressing either individual CARs, especially against low antigen density tumors.

[0036] FIG. 8 is a schematic illustrating how a tandem dual -targeting CAR would target an antigen. Both scFv’s are connected in a single CAR molecule in the correct spatial orientation based on the location of their target epitopes.

[0037] FIGS. 9A-9B schematically summarize the results of experiments performed to illustrate that a tandem dual -targeting CAR design with m971 and HA22 scFv’s linked to each other in“normal orientation” does not enhance activity against CD22^low tumor cells.

[0038] FIG. 10 is a schematic illustrating how a reverse oriented tandem dual -targeting CAR would target an antigen. A tandem dual-targeting CAR molecule was constructed with m971 and HA22 scFv’s linked to each other in the orientation opposite to epitope spatial location. In this design, the orientation m971 and HA22 scFv’s was reversed to force engagement of more CAR molecules per T cell.

[0039] Both scFv’s are connected in a single CAR molecule in the reverse spatial orientation based on the location of their target epitopes. Theoretically, this would result in the recruitment of more CAR molecules to the synapse (as each CAR molecule can bind only one epitope due to reverse orientation) and greater activation.

[0040] FIGS. 11A-11B schematically summarize the results of experiments performed to illustrate that a tandem dual -targeting CAR design with m971 and HA22 scFv’s linked to each other in“reverse orientation” demonstrate enhance activity against CD22^low tumor cells.

[0041] FIGS. 12A-12C schematically summarize the results of experiments performed to illustrate that a tandem dual -targeting CAR design with 4D5 and FRP5 scFv’s linked to each other in“normal orientation” does not enhance activity against HER2^low tumor cells.

[0042] FIGS. 13A-13C schematically summarize the results of experiments performed to illustrate that a tandem dual -targeting CAR design with 4D5 and FRP5 scFv’s linked to each other in“reverse orientation” (e.g. , in the orientation opposite to epitope spatial location) demonstrate enhance activity against HER2^low tumor cells.

[0043] FIG. 14 schematically summarizes the results of experiments performed to illustrate that human T cells transduced with both anti-CD276 single-targeting CARs generated more cytokine than control T cells expressing either individual CARs.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0044] The present disclosure relates generally to the development of immuno- therapeutics, such as enhanced polypeptides and chimeric antigen receptors (CARs) and pharmaceutical compositions comprising the same for use in treating diseases such as cancer. In particular, the disclosure provides compositions and methods for improving the efficiency of adoptive cell therapies and enhancing CAR-T cell functionality by simultaneously targeting different epitopes on a same antigen molecule. As will be discussed more thoroughly herein, the low target antigen density is a major impediment to the success of CAR-T cell therapies in solid tumors, where antigens are often expressed at heterogeneous levels. As illustrated in FIGS. 1A-1C, it has been reported that there is a threshold number of activated CAR molecules required for an effective CAR T cell response. When antigen density is limiting, the number of activated CAR molecules falls below a threshold needed for anti-tumor activity.

[0045] In fact, it has been reported that CAR potency is limited in many tumors, which is often due to low target antigen density. In addition, tumors can relapse in patients after CAR therapy by downregulating the CAR target. Some embodiments of the disclosure provide a new approach to increase the number of engaged CAR molecules per target cell by simultaneously targeting two or more different epitopes on a same antigen associated with or a same antigen specific for a target cell, e.g., cancer cell. This new strategy allows to improving adoptive cell therapy for cancer, such as hematologic malignancies, solid tumors, brain tumors, by improving CAR T cell functionality, e.g., increasing cytokine production and enhancing tumor cell killing. As demonstrated by the experimental data presented in the Examples below, simultaneously targeting different epitopes on the same antigen allows for efficient anti-tumor activity against cancers that express low levels of the target antigen.

[0046] In particular, several exemplary CARs were generated for three different target antigens: CD22, Her2, and B7-H3. For each target, two antigen-binding moieties, e.g., single chain variable fragments (scFv’s), targeting different epitopes present on two different locations on the target antigen (e.g., one distal and one proximal) were selected.

Subsequently, dual -targeting CAR T cells were generated by using two approaches. In one approach, referred to as“cotransduced dual -targeting CARs”, a number of dual-targeting CAR T cells were genetically engineered to express two individual CAR constructs each containing a single chain antibody fragments (scFv) targeting different epitopes on the antigen (see, e.g., FIG. 3 and Examples 1-3). In a second approach, referred to as“tandem dual-targeting CAR”, a number of dual -targeting CAR T cells were genetically engineered to express a single CAR construct containing two scFv’s operably linked in tandem (see, e.g., FIGS. 8 and 10 and Examples 4-5).

[0047] In experiments where the tandem dual-targeting CAR approach was employed, tandem CARs were generated in two orientations: one that would align the antigen-binding domains spatially with their target epitopes and one that had the opposite orientation. Human T cells were then transduced with single CARs recognizing individual epitopes, e.g., either “cotransduced” constructs, or“tandem” constructs. T cells transduced with individual “cotransduced” constructs had similar CAR expression levels and were flow sorted to a pure population (see, e.g., FIG. 4). Traditional single-targeting CAR T cells and dual -targeting CAR T cells were assayed in vitro for cytotoxicity and cytokine secretion in response to tumor lines with a range of target antigen densities (see, e.g., FIGS. 5-6, 11, and 13).

[0048] It was observed that“dual -targeting” CAR T cells outperformed traditional CAR T cells with single CARs recognizing single epitopes in both tumor cell killing and cytokine secretion (see, e.g., FIGS. 5-6 and 8-13). This was especially noticeable against tumor lines engineered to express low levels of antigen. Unexpectedly, it was observed that the“tandem dual-targeting” CARs of the present disclosure outperformed traditional CARs only when the scFv’s are linked to each other in tandem and in reverse orientation relative to the spatial orientation of their respective epitopes on the antigen (see, e.g., Examples 3-4).

[0049] Accordingly, the generation of dual-targeting CAR T cells capable of recognizing two different epitopes on the same antigen has been demonstrated herein a powerful strategy to increase CAR-T cell efficacy at low tumor antigen density. Without being bound to any theory, it is believe that the mechanism for increased efficacy may involve engagement of more CAR molecules at the immune synapse.

[0050] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols generally identify similar components, unless context dictates otherwise. The illustrative alternatives described in the detailed description, drawings, and claims are not meant to be limiting. Other alternatives may be used and other changes may be made without departing from the spirit or scope of the subject matter presented here. It will be readily understood that the aspects, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are explicitly contemplated and make part of this application.

[0051] 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 application 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.

A DEFINITIONS

[0052] 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.”

[0053] The terms“administration” and“administering”, as used herein, refer to the delivery of a composition or formulation as disclosed herein by an administration route including, but not limited to, intravenous, intra-arterial, intracranial, intramuscular, intraperitoneal, subcutaneous, intramuscular, or combinations thereof. The term includes, but is not limited to, administration by a medical professional and self-administration.

[0054] One skilled in the art will understand that the term“antigen” generally refers to a molecule or a portion of a molecule capable of being bound by a selective antigen binding agent, such as an antigen binding moiety, an antigen binding protein (e.g., an antibody), and optionally capable of being used in an animal to produce antibodies capable of binding to that antigen. An antigen may possess one or more epitopes that are capable of interacting with different antigen binding agents, e.g., antigen binding moieties and antibodies.

[0055] The term“epitope” refers to an antigenic determinant of a molecule that is bound by an antigen-binding agent, such as an antigen-binding moiety or antigen-binding protein (e.g., antibody). A single antigen may have more than one epitope. Strategies, techniques, and materials useful for determining of the spatial orientation and/or distribution of epitopes on an antigen are known in art. Examples of such methods include competitive binding assays described by Choudhary A. et al. (J. Immunol. 2018 May 1; 200(9): 3053- 3066) and Unfer RC (Iowa State University PhD Dissertation, 1991). In addition, spatial epitope prediction can be performed by using web-based tools such as SEPPA 2.0 described by Qi et al. (2014 Nucl. Acids Res., 2014, Vol. 42, Web Server issue W59-W63), which is hereby incorporated by reference in its entirety. Different antigen-binding moieties may bind to different areas (e.g., epitopes) on an antigen and may have different biological effects.

Most often, epitopes reside on proteins, but in some instances may reside on other kinds of molecules, such as nucleic acids. Epitope determinants may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three dimensional structural characteristics, and/or specific charge characteristics. An epitope can be contiguous (i.e., linear), or non-contiguous

(discontinuous) (e.g., in a polypeptide, amino acid residues that are not contiguous to one another in the polypeptide sequence but that within in context of the molecule are bound by the antigen binding protein). An epitope can be conformational epitope, which is an epitope that exists within the conformation of an active protein but is not present in a denatured protein. A conformational epitope is generally produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. Generally, an epitope includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-cons ecutive amino acids in a unique spatial conformation. See, e.g. , Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996). As discussed above, encompassed by the term “epitope” are simple epitopes comprising only a few contiguous amino acid residues as well as complex epitopes that encompass discontinuous amino acids. In some cases, complex epitopes comprise amino acids separated in the primary sequence but in close proximity in the three-dimensional folded structure of an antigen. Accordingly, different epitopes on an antigen may contain overlapping primary amino acid sequences. It is also possible to combine peptide epitopes containing substantially overlapping sequences to create longer peptide sequences carrying multiple epitopes in one single continuous peptide chain. In certain circumstance, an epitope may include moieties of saccharides, phosphoryf groups, or sulfonyl groups on the antigen.

[0056] The term“cancer” refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often observed aggregated into a tumor, but such cells can exist alone within an animal subject, or can be a non-tumorigenic cancer cell, such as a leukemia cell. Thus, the terms“cancer” or can encompass reference to a solid tumor, a soft tissue tumor, or a metastatic lesion. As used herein, the term“cancer” includes premalignant, as well as malignant cancers. In some embodiments, the cancer is a solid tumor, a soft tissue tumor, or a metastatic lesion.

[0057] The terms“host cell” and“recombinant cell” are used interchangeably herein. It is understood that such terms, as well as“cell culture” and“cell line”, refer not only to the particular subject cell or cell line but also to the progeny or potential progeny of such a cell 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 or cell line.

[0058] The term“operably linked”, as used herein, denotes a physical or functional linkage between two or more elements, e.g., polypeptide sequences or polynucleotide sequences, which permits them to operate in their intended fashion.

[0059] 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 et al, 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.

[0060] As used herein, and unless otherwise specified, a“therapeutically effective amount” or a“therapeutically effective number” of an agent is an amount sufficient to provide a therapeutic benefit in the treatment or management of a disease, e.g., cancer, or to delay or minimize one or more symptoms associated with the disease. A therapeutically effective amount of a compound means an amount 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 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 a symptom 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); and Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0061] As used herein, an“individual” or a“subject” includes animals, such as human (e.g., human individuals) and non-human animals. In some embodiments, an“individual” or “subject” 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, non-human primates, and other mammals, such as e.g., sheep, dogs, cows, chickens, and non-mammals, such as amphibians, reptiles, etc.

[0062] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also

encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0063] 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. 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 sub-combination. 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 was individually and explicitly disclosed herein.

B COMPOSITIONS OF THE DISCLOSURE

[0064] As described in greater detail below, one aspect of the present disclosure relates to a new class of CARs comprising single or dual antigen-binding moieties capable of binding different epitopes on a same antigen. Without being bound to any particular theory, this represent a new approach to increase the number of engaged CAR molecules per target cell by simultaneously targeting two or more different epitopes on a same antigen associated with or a same antigen specific for a target cell, e.g., cancer cell. Also provided, in other related aspects of the disclosure, are nucleic acids encoding the CARs as disclosed herein, recombinant cells expressing the CARs as disclosed herein, pharmaceutical compositions containing the nucleic acids and/or recombinant cells as disclosed herein.

Chimeric polypeptides

[0065] In one aspect, some embodiments of the disclosure relate to a chimeric polypeptide including, from N-terminus to C-terminus: (a) a first polypeptide segment including an extracellular domain (ECD) including a first antigen-binding moiety capable of binding to a first epitope of an antigen, and a second antigen-binding moiety capable of binding to a second epitope of the antigen; (b) a first polypeptide segment including a transmembrane domain (TMD); and (c) a third polypeptide segment an intracellular signaling domain (ICD), wherein the first and the second antigen-binding moieties are in reverse orientation relative to the spatial orientation of their respective epitopes on the antigen. In some embodiments, the first and the second antigen-binding moieties are operably linked to each other in tandem. In some embodiments, the first and the second antigen-binding moieties are operably linked to each other in tandem and in reverse orientation relative to the spatial orientation of their respective epitopes on the antigen.

[0066] The binding of the antigen-binding moieties to their respective target epitopes can be either in a competitive or non-competitive fashion with a natural ligand of the target antigen. Accordingly, in some embodiments of the disclosure, the binding of the antigen binding moieties to their respective target epitopes can be ligand-blocking. In some other embodiments, the binding of the antigen-binding moieties to their respective target epitopes does not block binding of the natural ligand. In some embodiments, the chimeric polypeptide includes at least one polypeptide segment operably linked to a second polypeptide segment to which it is not naturally linked in nature. The chimeric polypeptide segments may normally exist in separate proteins that are brought together in the chimeric polypeptide disclosed herein or they may normally exist in the same protein but are placed in a new arrangement in the chimeric polypeptide disclosed herein. A chimeric polypeptide 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.

[0067] Designation of the polypeptide segments of the disclosed chimeric polypeptide as the "first",“second”,“third”, or“fourth” polypeptide segments is not intended to imply any particular structural arrangement of the "first",“second”,“third”, or“fourth” polypeptide segments within the chimeric polypeptide.

[0068] In some embodiments, at least two of polypeptide segments of the disclosed chimeric polypeptide are directly linked to one another. In some embodiments, at least two of polypeptide segments of the disclosed chimeric polypeptide are linked to one another via at least one covalent bond. In some embodiments, at least two of polypeptide segments of the disclosed chimeric polypeptide are linked to one another via at least one peptide bond. In some embodiments, at least two of polypeptide segments of the disclosed chimeric polypeptide 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. Non-limiting 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).

[0069] The linker can also be a linker peptide sequence. Accordingly, in some embodiments, at least two of the polypeptide domains 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. In some embodiments, the peptide linker includes an amino acid sequence having at least about 80%, about 90%, about 95%, about 96%, about 97, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 10 in the Sequence Listing.

Chimeric anti sen receptors (CARs)

[0070] As described above, the chimeric polypeptides of the instant disclosure include, from N-terminus to C-terminus: (a) an extracellular domain (ECD) including a first antigen binding moiety capable of binding to a first epitope of an antigen, and a second antigen binding moiety capable of binding to a second epitope of the antigen; (b) a transmembrane domain (TMD); and (c) an intracellular signaling domain (ICD), wherein the first and the second antigen-binding moieties are in reverse orientation relative to the spatial orientation of their respective epitopes on the antigen. In some embodiments, chimeric polypeptides disclosed herein are chimeric antigen receptors (CARs). In some embodiments, the first and the second antigen-binding moieties are operably linked to each other in tandem. In some embodiments, the first and the second antigen-binding moieties are operably linked to each other in tandem and in reverse orientation relative to the spatial orientation of their respective epitopes on the antigen.

Extracellular domain (ECD)

[0071] As outlined above, the chimeric polypeptides and CARs of the disclosure contain an ECD including (i) a first antigen-binding moiety capable of binding to a first epitope of an antigen, and (ii) a second antigen-binding moiety capable of binding to a second epitope of the antigen. In some embodiments, the first and the second antigen-binding moieties are independently selected from the group consisting of an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), a nanobody, a VH domain, a VL domain, a single domain antibody (dAb), a VNAR domain, and a VHH domain, or a functional fragment thereof. In some embodiments, at least one of the first antigen-binding moiety and the second antigen-binding moiety is a scFv or a functional fragment thereof. In some embodiments, both the first and the second antigen-binding moiety are scFv or a functional fragment thereof, which bind to different epitopes on a same target antigen. In some embodiments, the first and the second antigen-binding moieties can independently include a heavy chain variable region and a light chain variable region.

[0072] The first and the second antigen-binding moieties of 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, binding affinity can be used as a measure of the strength of a non-covalent interaction between two molecules, e.g., an antibody or portion thereof and an antigen (e.g., CD22 antigen or HER2 antigen). In some cases, binding affinity can be used to describe monovalent interactions (intrinsic activity). Binding affinity between two molecules may be quantified by

determination of the dissociation constant (KD). In turn, KD can be determined by

measurement of the kinetics of complex formation and dissociation using, e.g., the surface plasmon resonance (SPR) method (Biacore). The rate constants corresponding to the association and the dissociation of a monovalent complex are referred to as the association rate constants k_a (or k_on) and dissociation rate constant k_d (or k_0ff), respectively. KD is related to k_a and k_d through the equation KD = k_d / k_a. The value of the dissociation constant can be determined directly by well-known methods, and can be computed even for complex mixtures by methods such as those set forth in Caceci et al. (1984, Byte 9: 340-362). For example, the KD may be established using a double-filter nitrocellulose filter binding assay such as that disclosed by Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428- 5432). Other standard assays to evaluate the binding ability of engineered antibodies of the present disclosure towards target antigens are known in the art, including for example, ELISAs, Western blots, RIAs, and flow cytometry analysis, and other assays exemplified elsewhere herein. The binding kinetics and binding affinity of the antibody also can be assessed by standard assays known in the art, such as Surface Plasmon Resonance (SPR), e.g. by using a Biacore™ system, or KinExA. In some embodiments, the binding affinity of an antibody or an antigen-binding moiety for a target antigen (e.g., CD22 antigen or HER2 antigen) can be calculated by the Scatchard method described by Frankel et al, Mol. Immunol, 16: 101-106, 1979. It will be understood that an antigen-binding moiety that “specifically binds” an antigen (such as CD22 antigen or HER2) is an antigen-binding moiety that does not significantly bind other antigens but binds the target 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.

[0073] In some embodiments, the first and the second antigen-binding moieties bind different epitopes with overlapping primary amino acid sequences. In some embodiments, the first and the second antigen-binding moieties bind different epitopes having less than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 60% overlapping primary amino acid sequences. In some embodiments, the first and the second antigen binding moieties bind non-overlapping epitopes on the antigen. In some embodiments, the epitope binding of the first antigen-binding moiety does not significantly interfere (e.g., block, inhibit, compete, and/or reduce) with epitope binding of the second antigen-binding moiety. In some embodiments, epitope binding of the first antigen-binding moiety does not significantly compete with epitope binding of the second antigen-binding moiety.

[0074] Methods and assays suitable to determine whether different epitopes on an antigen are non-overlapping and/or non-competing epitopes, , are known in the art and include FACS-based competition assays and ELISA-based competition assays In general, competitive binding between antigen-binding molecules can be determined by an assay in which the antigen-binding proteins to be assayed (e.g., antigen-binding moieties, antibodies or immunologically functional fragment thereol) under test prevents or inhibits specific binding of a reference antigen-binding molecule to a common antigen. An antigen-binding molecule to be assayed that binds to the same epitope as the reference antigen-binding molecule will be able to effectively compete for binding and thus will be expected to significantly interfere (e.g., block, inhibit, compete, and/or reduce) binding of the reference binding molecule. Antigen-binding molecules that bind different, non-competing epitopes on an antigen generally show no interference in their binding affinity to the antigen, as well as in their therapeutic, diagnostic, prophylactic, or research uses. In some embodiments, epitope binding of the first antigen-binding moiety reduces epitope binding of the second antigen binding moiety by less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1%. In some embodiments, epitope binding of the first antigen-binding moiety does not significantly reduce epitope binding of the second antigen-binding moiety, e.g., epitope binding of the first antigen-binding moiety reduces epitope binding of the second antigen-binding moiety by less than about 5%, less than about 4%, less than about 3%, less than about 1%, or less than about 1%. In some embodiments, the first and the second antigen-binding moieties bind non competing epitopes on the antigen, in which case epitope binding of the first antigen-binding moiety does not reduce epitope binding of the second antigen-binding moiety.

[0075] In some embodiments, epitope binding of the first antigen-binding moiety inhibits epitope binding of the second antigen-binding moiety by less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1%. In some embodiments, epitope binding of the first antigen binding moiety does not significantly inhibits epitope binding of the second antigen-binding moiety, e.g., epitope binding of the first antigen-binding moiety inhibits epitope binding of the second antigen-binding moiety by less than about 5%, less than about 4%, less than about 3%, less than about 1%, or less than about 1%. In some embodiments, the first and the second antigen-binding moieties bind non-competing epitopes on the antigen, in which case epitope binding of the first antigen-binding moiety does not reduce epitope binding of the second antigen-binding moiety.

[0076] In some embodiments, epitope binding of the first antigen-binding moiety does not compete with epitope binding of the second antigen-binding moiety (e.g., the first epitope and the second epitope are non-competing epitopes). One of ordinary skill in the art will understand that“non-overlapping epitope(s)” or“non-competing epitope(s)” of an antigen generally refer to epitope(s) that are recognized by one member of a pair of antigen-binding moieties but not the other member.

[0077] In some embodiments, the first and the second antigen-binding moieties are capable of binding the same antigen expressed by a target cell. In principle, there are no particular limitations to the type of target cell. In some embodiments, the target cell is a cancer cell. Non-limiting examples of cancer cells suitable for the compositions and methods disclosed herein include leukemia cells, acute myeloma leukemia cells, lymphoma cells, anaplastic lymphoma cells, astrocytoma cells, B-cell cancer cells, T-cell cancers, epithelial type cancer cells, mesenchymal type cancer cells, breast cancer cells, colon cancer cells, ependymoma cells, esophageal cancer cells, glioblastoma cells, glioma cells, leiomyosarcoma cells, and liposarcoma cells. Additional cancer cells for the compositions and methods disclosed herein include, but are not limited to, liver cancer cells, lung cancer cells, mantle cells, lymphoma cells, melanoma cells, neuroblastoma cells, non-small cell lung cancer cells. Also suitable for the compositions and methods disclosed herein include, but are not limited to, oligodendroglioma cells, ovarian cancer cells, pancreatic cancer cells, peripheral T-cell lymphoma cells, renal cancer cells, stomach cancer cells, carcinoma cells, mesothelioma cells, and sarcoma cells.

[0078] In some embodiments, the antigen is expressed at low density by the target cell, 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.

Antisens

[0079] In principle, there are no particular limitations with regard to target antigens suitable for the compositions and methods disclosed herein. Non-limiting examples of suitable target antigens include tumor associated-antigen (TAA) or a tumor-specific antigen (TSA). The term“tumor associated antigen” or“TAA” generally refers to a molecule, such as e.g., protein, present on tumor cells and on normal cells, or on many normal cells, but at much lower concentration than on tumor cells. In contrast, the term“tumor specific antigen” or“TSA” generally refers to a molecule, such as e.g., protein which is present on tumor cells but absent from normal cells. In some embodiments, the antigen is associated with or specific for a hematologic malignancy. In some embodiments, the antigen is associated with or specific for a solid tumor.

[0080] Non-limiting examples of suitable target antigens include CD22, human epidermal growth factor receptor 2 (HER2/neu/ErbB-2), CD276 (B7-H3), MUC1, PSMA, Glypican 2 (GPC2), IL- 13 -receptor alpha 1, IL- 13 -receptor alpha 2, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, and melanoma-associated antigen (MAGE). Other suitable target antigens include, but are not limited to, CD34, CD45, CD123, CD93, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), ALK, DLK1, FAP, NY-ESO, WT1, HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-Dl, muscle-specific actin (MSA), neurofil ament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor- 1, and the dimeric form of the pyruvate kinase isoenzyme type M2 (tumor M2-PK). Also suitable for the compositions and methods disclosed herein include, but are not limited to, CD19, CD20, CD5, CD7, CD3, TRBC1, TRBC2, BCMA, CD38, CD123, CD93, CD34, CDla, SLAMF7/CS1, FLT3, CD33, CD123, TALLA-1, CSPG4, DLL3, IgG Kappa light chain, IgA Lamba light chain, CD16/ FcyRIII, CD64, FITC, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), EGFR and isovariants thereof, TEM-8, sperm protein 17 (Spl7), mesothelin, PAP (prostatic acid phosphatase), prostate stem cell antigen (PSCA), prostein, NKG2D, TARP (T cell receptor gamma alternate reading frame protein), Trp-p8, STEAP1 (six-transmembrane epithelial antigen of the prostate 1), an abnormal ras protein, an abnormal p53 protein, integrin b3 (CD61), galactin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), and Ral-B.

[0081] In some embodiments, the antigen is selected from the group consisting of CD22, HER2/neu/ErbB-2, and CD276 (B7-H3). In some embodiments, the antigen is CD22. In some embodiments, the antigen is HER2/neu/ErbB-2. In some embodiments, the antigen is CD276 (B7-H3). Transmembrane domains ( TMD )

[0082] As outlined above, the chimeric polypeptides and CARs of the disclosure include a transmembrane domain (TMD). Generally, the transmembrane domain (also referred to as transmembrane region) suitable for the chimeric polypeptides and CARs disclosed herein can be any one of the transmembrane domains known in the art. Without being bound to theory, it is believed that the transmembrane domain traverses the cell membrane, anchors the CAR to the cell surface, and connects the extracellular domain to the intracellular signaling domain, thus impacting expression of the CAR on the cell surface. Examples of suitable transmembrane domains include, but are not limited to, a CD28 transmembrane domain, a CD8a transmembrane domain, a CD3 transmembrane domain, a CD4 transmembrane domain, a CTLA4 transmembrane domain, and a PD-1 transmembrane domain. Accordingly, in some embodiments, the transmembrane domain is derived from a CD28 transmembrane domain, a CD8a transmembrane domain, a CD3 transmembrane domain, a CD4 transmembrane domain, a CTLA4 transmembrane domain, and a PD-1 transmembrane domain. In some embodiments, the chimeric polypeptides and CARs disclosed herein include a transmembrane domain derived from a CD8a. In some embodiments, the chimeric polypeptides and CARs disclosed herein include a

transmembrane domain derived from a CD28. In some embodiments, the chimeric polypeptides and CARs disclosed herein include a transmembrane domain an amino acid sequence exhibiting at least about 80%, about 90%, about 95%, about 96%, about 97, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 15 in the Sequence Listing.

Intracellular domains (ICD)

[0083] In some embodiments, the intracellular signaling domain includes one or more costimulatory domains. 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 4-1BB (CD137), 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- IBB (CD 137) 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 4-1BB (CD137) 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 domain having an amino acid sequence exhibiting at least about 80%, about 90%, about 95%, about 96%, about 97, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 16 in the Sequence Listing.

[0084] In some embodiments, the intracellular signaling domain includes a CD3z intracellular signaling domain which, without being bound to any particular theory, is believed to mediate downstream signaling during T cell activation. In some embodiments, the CD3z intracellular signaling domain includes an amino acid sequence having at least about 80%, about 90%, about 95%, about 96%, about 97, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 17 in the Sequence Listing.

[0085] In some embodiments, the chimeric polypeptide of the disclosure includes, in N- terminus to C-terminus direction: (a) an extracellular domain including a first and a second antigen-binding moieties capable of binding to different epitopes of an antigen selected from the group consisting of CD22, HER2, and CD276; (b) a transmembrane domain derived from CD8a; (c) an intracellular domain including a costimulatory domain from 4-1BB; and d) a 6Ό3z intracellular signaling domain, wherein the first and the second antigen-binding moieties are in reverse orientation relative to the spatial orientation of their respective epitopes on the antigen. In some embodiments, the first and the second antigen-binding moieties are operably linked to each other in tandem. In some embodiments, the first and the second antigen-binding moieties are operably linked to each other in tandem and in reverse orientation relative to the spatial orientation of their respective epitopes on the antigen.

[0086] In some embodiments, the chimeric polypeptide of the disclosure includes, from N-terminus to C-terminus: (a) an extracellular domain including a first antigen-binding moiety having an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 6 and a second antigen-binding moiety having an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 11 ; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first and the second antigen-binding moieties are in reverse orientation relative to the spatial orientation of their respective epitopes on HER2. In some embodiments, the first and the second antigen-binding moieties are operably linked to each other in tandem. In some embodiments, the first and the second antigen-binding moieties are operably linked to each other in tandem and in reverse orientation relative to the spatial orientation of their respective epitopes on HER2.

[0087] In some embodiments, the chimeric polypeptide of the disclosure includes, from N-terminus to C-terminus: (a) an extracellular domain including a first antigen-binding moiety having an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 7 and a second antigen-binding moiety having an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 12; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first and the second antigen-binding moieties are in reverse orientation relative to the spatial orientation of their respective epitopes on HER2. In some embodiments, the first and the second antigen-binding moieties are operably linked to each other in tandem. In some embodiments, the first and the second antigen-binding moieties are operably linked to each other in tandem and in reverse orientation relative to the spatial orientation of their respective epitopes on HER2.

[0088] In some embodiments, the chimeric polypeptide of the disclosure includes, from N-terminus to C-terminus: (a) an extracellular domain including a first antigen-binding moiety having an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 8 and a second antigen-binding moiety having an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 13; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first and the second antigen-binding moieties are in reverse orientation relative to the spatial orientation of their respective epitopes on CD22. In some embodiments, the first and the second antigen-binding moieties are operably linked to each other in tandem. In some embodiments, the first and the second antigen-binding moieties are operably linked to each other in tandem and in reverse orientation relative to the spatial orientation of their respective epitopes on CD22.

[0089] In some embodiments, the chimeric polypeptide of the disclosure includes, from N-terminus to C-terminus: (a) an extracellular domain including a first antigen-binding moiety having an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 9 and a second antigen-binding moiety having an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 14; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first and the second antigen-binding moieties are in reverse orientation relative to the spatial orientation of their respective epitopes on CD22. In some embodiments, the first and the second antigen-binding moieties are operably linked to each other in tandem. In some embodiments, the first and the second antigen-binding moieties are operably linked to each other in tandem and in reverse orientation relative to the spatial orientation of their respective epitopes on CD22.

[0090] In some embodiments, the chimeric polypeptides and CARs of the disclosure includes an amino acid sequence having at least about 80%, about 90%, about 95%, about 96%, about 97, about 98%, about 99%, or about 100% sequence identity to a chimeric receptor disclosed herein. In some embodiments, provided herein are chimeric polypeptides and CARs including an amino acid sequence having at least about 80%, about 90%, about 95%, about 96%, about 97, about 98%, about 99%, or about 100% sequence identity to any one of SEQ ID NOS: 1-4 identified in the Sequence Listing. In some embodiments, the chimeric polypeptide or CAR includes an amino acid sequence having at least about 80%, about 90%, about 95%, about 96%, about 97, about 98%, about 99%, or about 100% sequence identity to any one of SEQ ID NO: 1. In some embodiments, the chimeric polypeptide or CAR includes an amino acid sequence having at least about 80%, about 90%, about 95%, about 96%, about 97, about 98%, about 99%, or about 100% sequence identity to any one of SEQ ID NO: 2. In some embodiments, the chimeric polypeptide or CAR includes an amino acid sequence having at least about 80%, about 90%, about 95%, about 96%, about 97, about 98%, about 99%, or about 100% sequence identity to any one of SEQ ID NO: 3. In some embodiments, the chimeric polypeptide or CAR includes an amino acid sequence having at least about 80%, about 90%, about 95%, about 96%, about 97, about 98%, about 99%, or about 100% sequence identity to any one of SEQ ID NO: 4.

[0091] In some embodiments, provided herein are chimeric polypeptides and CARs including an amino acid sequence having at least about 90% sequence identity to any one of SEQ ID NOS: 1-4 identified in the Sequence Listing. In some embodiments, the chimeric polypeptide or CAR includes an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 1. In some embodiments, the chimeric polypeptide or CAR includes an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 2. In some embodiments, the chimeric polypeptide or CAR includes an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 3. In some embodiments, the chimeric polypeptide or CAR includes an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 4. [0092] In some embodiments, provided herein are chimeric polypeptides and CARs including an amino acid sequence having at least about 95% sequence identity to any one of SEQ ID NOS: 1-4 identified in the Sequence Listing. In some embodiments, the chimeric polypeptide or CAR includes an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 1. In some embodiments, the chimeric polypeptide or CAR includes an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 2. In some embodiments, the chimeric polypeptide or CAR includes an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 3. In some embodiments, the chimeric polypeptide or CAR includes an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 4.

[0093] In some embodiments, provided herein are chimeric polypeptides and CARs including an amino acid sequence having at least about 96%, 97, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOS: 1-4 identified in the Sequence Listing. In some embodiments, the chimeric polypeptide or CAR includes an amino acid sequence having at least about 96%, 97, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the chimeric polypeptide or CAR includes an amino acid sequence having at least about 96%, 97, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the chimeric polypeptide or CAR includes an amino acid sequence having at least about 96%, 97, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3. In some embodiments, the chimeric polypeptide or CAR includes an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4.

Nucleic Acid Molecules

[0094] In another aspect, provided herein are various nucleic acid molecules including nucleotide sequences encoding the chimeric polypeptides and CARs 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, regulatory sequences which allow in vivo expression of the receptor in a host cell.

[0095] Nucleic acid molecules of the present disclosure can be of any length, including for example, 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.

[0096] In some embodiments, provided herein is a nucleic acid molecule including a nucleotide sequence encoding a chimeric polypeptide or CAR as disclosed herein. In some embodiments, the nucleic acid molecule includes a nucleotide sequence encoding a chimeric polypeptide including, in N-terminus to C-terminus: (a) an extracellular domain including a first antigen-binding moiety capable of binding to a first epitope of an antigen, and a second antigen-binding moiety capable of binding to a second epitope of the antigen; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first and the second antigen-binding moieties are in reverse orientation relative to the spatial orientation of their respective epitopes on the antigen. In some embodiments, the first and the second antigen-binding moieties are operably linked to each other in tandem. In some embodiments, the first and the second antigen-binding moieties are operably linked to each other in tandem and in reverse orientation relative to the spatial orientation of their respective epitopes on the antigen.

[0097] In some embodiments, the nucleotide sequence is incorporated into an expression cassette or an expression vector. It will be understood that an expression cassette generally includes a construct of genetic material that contains coding sequences and enough 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 or CAR as disclosed herein, which is operably linked to expression control elements, such as a promoter, and optionally, any or a combination of other nucleic acid sequences that affect the transcription or translation of the coding sequence.

[0098] 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. [0099] 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 typically 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 typically 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. 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“lenti viral vector” refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, including LTRs that are primarily derived from a lentivirus, which is a genus of retrovirus.

[0100] In some embodiments, provided herein are nucleic acid molecules encoding a chimeric polypeptide with an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97, 98%, 99%, or 100% sequence identity to a CAR disclosed herein. In some embodiments, provided herein are nucleic acid molecules encoding a polypeptide with an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOS: 1-4 in the Sequence Listing. In some embodiments, the nucleic acid molecules encode a chimeric 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 chimeric 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 chimeric 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 chimeric 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.

[0101] In addition or alternatively, two or more separate CARs can be expressed on a single T cell using a single vector by taking advantage of ribosomal skip sequences or internal ribosomal entry sites (“bicistronic CAR”). Accordingly, in some embodiments, nucleic acids of the disclosure can encode two or more chimeric polypeptides or CARs as disclosed herein. For example, a nucleic acid that encodes two or more chimeric polypeptides or CARs can be a multi-cistronic nucleic acid, wherein the two or more coding sequences are separated by a sequence encoding an IRES (internal ribosome entry site) or a 2A sequence, which provide for expression of each chimeric polypeptide or CAR separately, or for the immediate cleavage into two separate chimeric polypeptides upon expression. Examples of 2A sequences include T2A, P2A, E2A, and F2A. In some embodiments, the nucleic acid is a bicistronic nucleic acid that encodes (a) a first chimeric polypeptide including, from N- terminus to C-terminus: (i) an extracellular domain including a first antigen-binding moiety capable of binding to a first epitope of an antigen; (ii) a transmembrane domain; and (iii) an intracellular signaling domain; and (b) a second chimeric polypeptide including, from N- terminus to C-terminus: (i) an extracellular domain including a second antigen-binding moiety capable of binding to a second epitope of the antigen; (ii) a transmembrane domain; and (iii) an intracellular signaling domain, wherein the first and the second antigen-binding moieties are capable of binding to different epitopes of the antigen, and wherein the first and the second chimeric polypeptides are separated by a 2A sequence. In an embodiment, the 2A sequence is a P2A sequence.

[0102] The nucleic acid sequences encoding the chimeric polypeptides and CARs as disclosed herein 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.

[0103] Some embodiments disclosed herein relate to vectors or expression cassettes including a recombinant nucleic acid molecule encoding the chimeric receptors disclosed herein. The expression cassette generally 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. The expression cassette may be inserted into a vector for targeting to a desired host cell and/or into an individual. An expression cassette can be inserted into a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, as a linear or circular, single-stranded or double-stranded, DNA or RNA

polynucleotide molecule, derived from any source, capable of genomic integration or autonomous replication, including a nucleic acid molecule where one or more nucleic acid sequences has been linked in a functionally operative manner, i.e., operably linked.

[0104] Also provided herein are vectors, plasmids, or viruses containing one or more of the nucleic acid molecules encoding any chimeric polypeptide or CAR disclosed herein. The nucleic acid molecules can be contained within a vector that is capable of directing their expression in, for example, a cell that has been transformed/transduced with the vector. Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available, or readily prepared by a skilled artisan. See for example, 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. et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (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. et al. (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).

[0105] DNA vectors can be introduced into eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host 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.

[0106] Viral vectors that can be used in the disclosure include, for example, retrovirus vectors, adenovirus vectors, and adeno-associated virus vectors, lentivirus vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, for example,

Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.).

[0107] The precise components of the expression system are not critical. For example, a chimeric receptor as disclosed herein can be produced in a eukaryotic host, such as a mammalian cells (e.g., COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, VA). In selecting an expression system, it matters only that the components are compatible with one another. Artisans or ordinary skill are able to make such a determination. Furthermore, if guidance is required in selecting an expression system, skilled artisans may consult P. Jones, “Vectors: Cloning Applications”, John Wiley and Sons, New York, N.Y., 2009).

[0108] 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).

[0109] 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

[0110] The nucleic acid of the present disclosure can be introduced into a host cell, such as, for example, a human T lymphocyte, to produce a recombinant cell containing the nucleic acid molecule. 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.

[0111] Accordingly, in some embodiments, the nucleic acid molecules can be delivered by viral or non-viral delivery vehicles known in the art. 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 mini-circle expression vector for transient expression. Accordingly, in some embodiments, 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 achieved using classical random genomic recombination techniques or with more precise techniques such as guide RNA-directed CRISPR/Cas9 genome editing, or DNA- guided endonuclease genome editing with NgAgo (Natronobacterium gregoryi Argonaute), or TALENs genome editing (transcription activator-like effector nucleases). In some embodiments, the nucleic acid molecule is present in the recombinant host cell as a mini circle expression vector for transient expression.

[0112] 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, adeno-associated virus (AAV) is 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.

[0113] Lenti viral-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.

[0114] 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 polypeptides of interest. Host cells can be either untransformed cells or cells that have already been transfected with at least one nucleic acid molecule.

[0115] 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 a eukaryotic cell. 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 cell is a human cell. In some embodiments, the cell is a non-human primate cell. In some

embodiments, the mammalian cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell. In some embodiments, the cell is a stem cell. In some embodiments, the cell is a hematopoietic stem cell.

[0116] In some embodiments, the recombinant cell is an immune system cell, e.g., a lymphocyte (e.g., a T cell or NK cell), or a dendritic cell. In some embodiments, the immune cell is a 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 regulatory T cell, a helper T cell (TH), a cytotoxic T cell (TCTL), or other T cell. In some embodiments, the immune system cell is a T lymphocyte. In some embodiments, the cell is a precursor T cell or a T regulatory (Treg) cell. In some embodiments, the cell is a CD34+, CD8+, or a CD4+ cell. In some embodiments, the cell is a CD8+ T cytotoxic lymphocyte cell selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and bulk CD8+ T cells. In some embodiments of the cell, the cell is a CD4+ T helper lymphocyte cell selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some embodiments, the cell can be obtained by leukapheresis performed on a sample obtained from an individual. In some embodiments, the subject is a human patient.

[0117] In some embodiments, the recombinant cell includes: (a) a chimeric polypeptide as described herein; and/or (b) a recombinant nucleic acid molecule as described herein. In some embodiments, the recombinant cell includes a nucleic acid molecule including a nucleic acid sequence encoding a chimeric polypeptide which has at least about 80%, about 90%, about 95%, about 96%, about 97, about 98%, about 99%, or about 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-4. In some embodiments, the recombinant cell includes a nucleic acid molecule including a nucleic acid sequence encoding a chimeric polypeptide which has at least about 80%, about 90%, about 95%, about 96%, about 97, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 1. In some embodiments, the recombinant cell includes a nucleic acid molecule including a nucleic acid sequence encoding a chimeric polypeptide which has at least about 80%, about 90%, about 95%, about 96%, about 97, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 2. In some embodiments, the recombinant cell includes a nucleic acid molecule including a nucleic acid sequence encoding a chimeric polypeptide which has at least about 80%, about 90%, about 95%, about 96%, about 97, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 3. In some embodiments, the recombinant cell includes a nucleic acid molecule including a nucleic acid sequence encoding a chimeric polypeptide which has at least about 80%, about 90%, about 95%, about 96%, about 97, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 4.

[0118] In some embodiments, the recombinant cell of the disclosure has been engineered to express the following: (a) a first chimeric polypeptide comprising, from N- terminus to C-terminus: (i) an extracellular domain comprising a first antigen-binding moiety capable of binding to a first epitope of an antigen; (ii) a transmembrane domain; and (iii) an intracellular signaling domain; and (b) a second chimeric polypeptide comprising, from N- terminus to C-terminus: (i) an extracellular domain comprising a second antigen-binding moiety capable of binding to a second epitope of the antigen; (ii) a transmembrane domain; and (iii) an intracellular signaling domain, wherein the first and the second antigen-binding moieties are capable of binding to different epitopes of the antigen. [0119] In another aspect, some embodiments of the disclosure relate to methods for making a recombinant cell, including (a) providing a cell capable of protein expression and (b) contacting the provided cell with a recombinant nucleic acid of the disclosure.

[0120] In another aspect, provided herein are cell cultures including at least one recombinant cell as disclosed herein, and a culture medium. Generally, the culture medium can be any suitable culture medium for culturing the cells described herein. 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. 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

[0121] 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 described herein and a pharmaceutically acceptable excipient, e.g., carrier. Accordingly, one aspect of the present disclosure relates to pharmaceutical compositions that include a pharmaceutical acceptable carrier and one or more of the following: (a) a recombinant nucleic acid as disclosed herein, and (b) a recombinant cell as disclosed herein. In some

embodiments, the pharmaceutical compositions of the disclosure are formulated for the treating, preventing, ameliorating a disease such as cancer, or for reducing or delaying the onset of the disease.

[0122] In some embodiments, the disclosed pharmaceutical 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.

[0123] 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 pharmacopeiae 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 or an animal (some non-limiting examples include a human, or a mammal). In some embodiments, the individual is a human.

[0124] 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. One of ordinary skilled in the art will appreciate that the formulation should suit the mode of administration.

[0125] For example, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions 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 syringability exists. It can be stabilized under the conditions of manufacture and storage, and can 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.

[0126] 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.

C. METHODS OF THE DISCLOSURE

[0127] 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 treatment of relevant diseases, such as 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 therapeutic agents for use in methods of treating an individual who has, who is suspected of having, or who may be at high risk for developing one or more diseases. In some embodiments, the individual is a patient under the care of a physician.

[0128] Accordingly, in one aspect, some embodiments of the disclosure relate to methods for modulating (e.g., inhibiting) an activity of a target cell in an individual, the methods include administering to the individual a first therapy including one or more of nucleic acids, recombinant cells, and pharmaceutical compositions as disclosed herein, wherein the first therapy modulates (e.g., inhibits) an activity of the target cell.

[0129] In another aspect, some embodiments of the disclosure relate to methods for modulating (e.g. , inhibiting) an activity of a target cell in an individual, the methods include

(a) providing a composition including at least one recombinant cell as disclosed herein; and

(b) contacting a target cell with the provided composition. In a related aspect, provided herein are methods for modulating an activity of a target cell, the method including providing composition including at least one recombinant cell engineered to express the following: (a) a first chimeric polypeptide including, from N-terminus to C-terminus: (i) an extracellular domain including a first antigen-binding moiety capable of binding to a first epitope of an antigen; (ii) a transmembrane domain; and (iii) an intracellular signaling domain; and (b) a second chimeric polypeptide including, from N-terminus to C-terminus: (i) an extracellular domain including a second antigen-binding moiety capable of binding to a second epitope of the antigen; (ii) a transmembrane domain; and (iii) an intracellular signaling domain, wherein the first and the second antigen-binding moieties are capable of binding to different epitopes of the antigen; and contacting a target cell with the provided composition. In some embodiments, the provided composition modulates (e.g., inhibits) an activity of the target cell. In some embodiments, the provided composition includes a therapeutically effective number of the recombinant cells disclosed herein, wherein the recombinant cells modulates (e.g., inhibits) an activity of the target cells in the individual.

[0130] Non-limiting exemplary cellular activities that can be modulated using the methods provide herein include, but are not limited to, cell growth, proliferation, apoptosis, non-apoptotic death, differentiation, dedifferentiation, migration, secretion of a gene product, cellular adhesion, and cytolytic activity. 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 a therapeutically effective number of the recombinant cells disclosed herein, wherein the recombinant cells inhibit an activity of the target cells in the individual. Generally, the target cells of the disclosed methods can be any cell type in an individual and can be, for example a leukemia cell, an acute myeloma leukemia cell, a lymphoma cell, an anaplastic lymphoma cell, an astrocytoma cell, a B-cell cancer cell, a T- cell cancer, an epithelial type cancer cell, a mesenchymal type cancer cell, a breast cancer cell, a colon cancer cell, an ependymoma cell, an esophageal cancer cell, a glioblastoma cell, a glioma cell, a leiomyosarcoma cell, a liposarcoma cell, a liver cancer cell, a lung cancer cell, a mantle cell lymphoma cell, a melanoma cell, a neuroblastoma cell, a non-small cell lung cancer cell, an oligodendroglioma cell, an ovarian cancer cell, a pancreatic cancer cell, a peripheral T-cell lymphoma cell, a renal cancer cell, a stomach cancer cell, a carcinoma cell, a mesothelioma cell, and a sarcoma cell. In some embodiments, the target cell is a pathogenic cell. In some embodiments, the target cell is a cancer cell. In some embodiments, the modulation of the activity the target cell results in the death of the target cell. [0131] In another aspect, provided herein are methods for the treatment of a disease in an individual in need thereof, which includes administering to the individual a first therapy including a composition that includes at least one recombinant cell as disclosed herein. In some embodiments, the provided composition includes a therapeutically effective number of the recombinant cells disclosed herein, wherein the recombinant cells treat the disease in the individual.

[0132] Another aspect relates to methods for the treatment of a disease in an individual in need thereof, the method including administering to the individual a first therapy including a composition that includes at least one recombinant cell engineered to express the following: (a) a first chimeric polypeptide including, from N-terminus to C-terminus: (i) an extracellular domain including a first antigen-binding moiety capable of binding to a first epitope of an antigen; (ii) a transmembrane domain; and (iii) an intracellular signaling domain; and (b) a second chimeric polypeptide including, from N-terminus to C-terminus: (i) an extracellular domain including a second antigen-binding moiety capable of binding to a second epitope of the antigen; (ii) a transmembrane domain; and (iii) an intracellular signaling domain, wherein the first and the second antigen-binding moieties are capable of binding to different epitopes of the antigen. In some embodiments, the provided composition includes a therapeutically effective number of the recombinant cells disclosed herein, wherein the recombinant cells treat the disease in the individual.

[0133] In some embodiments, the antigen is expressed at low density by the target cell, 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, 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.

[0134] In some embodiments, the disease in the individual is a cancer. In some embodiments, the cancer is a pediatric cancer. In some embodiments, the cancer is a pancreatic cancer, a colon cancer, an ovarian cancer, a prostate cancer, a lung cancer, mesothelioma, a breast cancer, a urothelial cancer, a liver cancer, a head and neck cancer, a sarcoma, a cervical cancer, a stomach cancer, a gastric cancer, a melanoma, a uveal melanoma, a cholangiocarcinoma, multiple myeloma, leukemia, lymphoma, and

glioblastoma. Administration of recombinant cells to an individual

[0135] In some embodiments, the methods of the disclosure involve administering an effective amount or number of the recombinants cells to an individual in need of such treatment. 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 individual’s bloodstream or otherwise administered to the individual.

[0136] 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 an individual 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.

[0137] When provided prophylactically, the recombinant cells described herein can be administered to an individual 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.

[0138] 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.

[0139] For use in the various embodiments described herein, an effective amount of recombinant cells as disclosed herein, can be at least 10² cells, at least 5 ^c 10² cells, at least 10³ cells, at least 5 ^c 10³ cells, at least 10⁴ cells, at least 5 ^c 10⁴ cells, at least 10⁵ cells, at least 2 ^c 10⁵ cells, at least 3 ^c 10⁵ cells, at least 4 ^c 10⁵ cells, at least 5 ^c 10⁵ cells, at least 6 ^c 10⁵ cells, at least 7 ^c 10⁵ cells, at least 8 ^c 10⁵ cells, at least 9 ^c 10⁵ cells, at least 1 ^c 10⁶ cells, at least 2 ^c 10⁶ cells, at least 3 ^c 10⁶ cells, at least 4 ^c 10⁶ cells, at least 5 ^c 10⁶ cells, at least 6 x 10⁶ cells, at least 7 ^c 10⁶ cells, at least 8 ^c 10⁶ cells, at least 9 ^c 10⁶ cells, 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 an individual in need thereof. [0140] 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 an individual 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 individual, e.g., administration results in delivery to a desired location in the individual where at least a portion of the composition delivered, e.g., at least 1 ^c 10³ cells, 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 intrastemal 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.

[0141] In some embodiments, the recombinant cells are administered systemically, e.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 individual’s circulatory system and, thus, is subject to metabolism and other similar biological processes.

[0142] The efficacy of a treatment including any of the compositions provided herein for the treatment of a disease or condition can be determined by a skilled clinician. However, one skilled in the art will appreciate that a 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 an individual 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 an individual 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.

[0143] As discussed above, a therapeutically effective number of recombinant cells refers to a number of recombinant cells that is sufficient to promote a provide a therapeutic benefit in the treatment or management of a disease, e.g., cancer, or to delay or minimize one or more symptoms associated with the disease when administered to an individual, such as one who has, is suspected of having, or is at risk for the disease. In some embodiments, an effective number includes a number 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. In some embodiments, an effective number includes a number sufficient to inhibit tumor growth or metastasis of a cancer in the individual. In some embodiments, an effective number includes a number sufficient to increase cytokine production and enhance killing of a cancer cell.

[0144] In some embodiments of the disclosed methods, the individual is a mammal. In some embodiments, the mammal is a human. In some embodiments, the individual has or is suspected of having a disease associated with perturbation (e.g., inhibition or hyper activation) of cell signaling mediated by a cell surface ligand or antigen. The diseases suitable for being treated by the compositions and methods of the disclosure include, but are not limited to, cancers, autoimmune diseases, inflammatory diseases, and infectious diseases. In some embodiments, the disease is a cancer or a chronic infection. Non-limiting examples of cancers that can suitably treated by the compositions and methods of the disclosure include pancreatic cancers, colon cancers, ovarian cancers, prostate cancers, lung cancers, mesothelioma, breast cancers, urothelial cancers, liver cancers, head and neck cancers.

Additional cancers that can suitably treated by the compositions and methods of the disclosure include, but are not limited to, sarcoma, cervical cancers, stomach cancers, gastric cancers, melanoma, uveal melanoma, cholangiocarcinoma, multiple myeloma, leukemia, lymphoma, B-cell cancers, T-cell cancers, epithelial type cancers, mesenchymal type cancers, and glioblastoma.

Additional therapies

[0145] As discussed above, the recombinant cells, and pharmaceutical compositions described herein can be administered in combination with one or more additional therapies such as, for example, chemotherapeutics or anti-cancer agents or anti-cancer therapies.

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 therapeutic agents, chemotherapeutics, anti-cancer agents, or anti-cancer therapies is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, and surgery.“Chemotherapy” and“anti cancer agent” are used interchangeably herein. Various classes of anti-cancer agents can be used. Non-limiting examples 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.

[0146] In some embodiments, the disclosed treatment methods further include administering to the individual 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 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.

Methods for modulating an activity of a target cell

[0147] In another aspect, provided herein are various methods for modulating an activity of a cell. The methods include the steps of: (a) providing a composition including at least one recombinant cell as disclosed herein; and (b) contacting a target cell with the provided composition. In a related aspect, provided herein are methods for modulating an activity of a target cell, the method including (A) providing composition including at least one recombinant cell engineered to express the following: (a) a first chimeric polypeptide including, from N-terminus to C-terminus: (i) an extracellular domain including a first antigen-binding moiety capable of binding to a first epitope of an antigen; (ii) a

transmembrane domain; and (iii) an intracellular signaling domain; and (b) a second chimeric polypeptide including, from N-terminus to C-terminus: (i) an extracellular domain including a second antigen-binding moiety capable of binding to a second epitope of the antigen; (ii) a transmembrane domain; and (iii) an intracellular signaling domain, wherein the first and the second antigen-binding moieties are capable of binding to different epitopes of the antigen; and (B) contacting a target cell with the provided composition. In some embodiments, the provided composition modulates an activity of the target cell. One skilled in the art upon reading the present disclosure will appreciate that the disclosed methods can be carried out in vivo, ex vivo, or in vitro. In some embodiments, the provided composition modulates an activity of the target cell.

[0148] Non-limiting exemplary cellular activities that can be modulated using the methods provide herein include, but are not limited to, cell growth, proliferation, apoptosis, non-apoptotic death, differentiation, dedifferentiation, migration, secretion of a molecule, cellular adhesion, and cytolytic activity. In some embodiments, the target cell is a cancer cell. In some embodiments, the cancer cell is selected from the group consisting of a leukemia cell, an acute myeloma leukemia cell, a lymphoma cell, an anaplastic lymphoma cell, an astrocytoma cell, a B-cell cancer cell, a T-cell cancer, an epithelial type cancer cell, a mesenchymal type cancer cell, a breast cancer cell, a colon cancer cell, an ependymoma cell, an esophageal cancer cell, a glioblastoma cell, a glioma cell, a leiomyosarcoma cell, a liposarcoma cell, a liver cancer cell, a lung cancer cell, a mantle cell lymphoma cell, a melanoma cell, a neuroblastoma cell, a non-small cell lung cancer cell, an oligodendroglioma cell, an ovarian cancer cell, a pancreatic cancer cell, a peripheral T-cell lymphoma cell, a renal cancer cell, a stomach cancer cell, a carcinoma cell, a mesothelioma cell, and a sarcoma cell.

[0149] In some embodiments, the antigen is expressed at low density by the target cell, 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.

SYSTEMS AND KITS

[0150] Also provided herein are systems and kits including the chimeric polypeptides, CARs, recombinant nucleic acids, recombinant cells, or pharmaceutical compositions provided and described herein as well as written instructions for making and using the same. For example, provided herein, in some embodiments, are systems and/or kits that include one or more of the following: (i) an chimeric polypeptide as described herein, (ii) CAR as described herein, (iii) a recombinant nucleic acids as described herein, (iv) a recombinant cell as described herein, and (v) a pharmaceutical composition as described herein. In some embodiments, the systems and/or kits of the disclosure further include one or more syringes (including pre-filled syringes) and/or catheters (including pre-filled syringes) used to administer one any of the provided recombinant nucleic acids, recombinant cells, or pharmaceutical compositions to a subject. In some embodiments, a kit can have one or more additional therapeutic agents that can be administered simultaneously or sequentially with the other kit components for a desired purpose, e.g., for modulating an activity of a cell, inhibiting a target cancer cell, or treating a disease in a subject in need thereof.

[0151] Any of the above-described systems and 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 for in vitro production of the chimeric receptor polypeptides.

[0152] In some embodiments, a system or kit can further include instructions for using the components of the kit to practice the methods. 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 kits as a package insert, in the labeling of the container of the kit or components thereof ( /. e.. 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), are 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.

[0153] 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 to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0154] 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.

[0155] 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.

EXAMPLES

[0156] 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 1

Assessing functionality of T cells co-transduced with two anti-CD22 single-targeting CAR constructs

[0157] This Example describes experiments performed to evaluate functionality of human T cells co-transduced with two anti-CD22 single-targeting CAR constructs each containing a single chain antibody fragments (scFv) targeting different epitopes on the antigen CD22.

[0158] It is hypothesized that two different epitopes of the same target antigen can increase the number of CAR receptors at the immune synapse, increasing CAR T cell reactivity at low antigen density (see, e.g. FIG. 2). Two single-targeting CAR molecules were constructed with scFv’s m971 and HA22 which bind two different epitopes on the antigen CD22. Each of the CAR constructs were each generated with the incorporation of a cyan fluorescent protein (CFB or mCherry) used for convenient detection and cell sorting (see, e.g., FIG. 3, which is adopted from Haso W. et al, Blood. 2013 Feb 14; 121(7): 1165-74. In this scenario, HA22 scFv targets a distal epitope of CD22 while m971 scFv targets a proximal epitope. In these experiments, retroviral vectors encoding the above CARs with the indicated structures were synthesized and assembled by using standard methods.

[0159] CAR T cells were then transduced with the single-targeting CARs described above. FIGS. 4A-4C schematically summarize the results from experiments performed to isolate CAR-T cells containing one or both CAR constructs described in FIG. 3. In these experiments, retroviral vectors encoding these CARs with the indicated structures were synthesized and assembled by using standard methods. Viral supernatant was produced in 293GP cells after transient transfection of the retroviral plasmids. CARs were transduced into human T cells. Primary human T cells were transduced with viral supernatant after physiological activation with Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher) and were cultured in the presence of interleukin-2 (IL-2). After a period of seven days, the CAR-T cells were FACS sorted based on fluorescence to achieve a 100% pure single-positive CAR T cells (i.e., expressing one CAR construct) or double-positive CAR T cell populations (i.e., expressing both CAR constructs).

[0160] FIGS. 5A-5B schematically summarize the results of experiments performed to illustrate that engineered human T cells expressing both CAR constructs described in FIG. 3 demonstrate enhanced killing of tumor cells. In these experiments, on Day 10 post activation, the CAR T cells targeting individual epitope (i.e. , expressing one CAR construct) or dual epitopes (i.e. , expressing both CAR constructs) on the antigen CD22 with the indicated structures were co-cultured with NALM6 cells expressing native level of CD22 (NALM6 WT), which expressed approximately 4000 molecules CD22 per cell) or expressing low level of CD22 (LOW CD22 NALM6) which expressed approximately 1,500 molecules per cell. Tumor NALM6 cells were engineered to express a green fluorescent protein (GFP) reporter. Tumor cells remaining (survival) were measured over time in an Incucyte by measuring expression of the GFP reporter. The experimental results described in FIGS. 5A-5B indicate that CAR T cells expressing both CAR constructs and targeting dual epitopes on CD22 demonstrated enhanced killing of tumor cells.

[0161] Additional experiments were performed to investigate whether engineered human T cells expressing both CAR constructs described in FIG. 3 generate more cytokine secretion against CD22^low leukemia than control T cells expressing either individual CAR.

[0162] In these experiments, on Day 10 post activation, the CAR T cells targeting individual epitope (i.e., expressing one CAR construct) or dual epitopes (i.e.. expressing both CAR constructs) on the antigen CD22 with the indicated structures were co-cultured for 24 hours with NALM6 cells expressing low level of CD22 (22KO+22#2 NALM6), which expressed, approximately 1,500 molecules CD22 per cell. Interleukin-2 (IL-2) and IFN-g content was measured in the supernatant. The experimental results described in FIGS. 6A-6B indicate that CAR T cells expressing both CAR constructs and targeting dual epitopes on CD22 demonstrated an increase in cytokine production in response to low CD22 tumor cells.

EXAMPLE 2

Assessing functionality of T cells co-transduced with two anti-HER2 single-targeting CAR constructs

[0163] This Example describes experiments performed to evaluate functionality of human T cells co-transduced with two anti-HER2 single-targeting CAR constructs each containing a scFv targeting different epitopes on the antigen HER2.

[0164] In these experiments, two CARs were constructed with two scFv’s 4D5 and FRP5 which bind different epitopes on HER2. FRP5 targets a distal epitope while 4D5 targets a proximal epitope. Retroviral vectors encoding these CARs with the indicated structures were synthesized commercially and cloned by standard methods. Viral supernatant was produced in 293 GP cells after transient transfection of the retroviral plasmid. CARs were then individually introduced into human T cells, where primary human T cells were transduced with viral supernatant after activation with Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher) and were cultured in the presence of interleukin-2 (IL-2). After a period of seven days, the CAR-T cells were FACS sorted based on fluorescence to achieve a 100% pure single-positive CAR T cells (i.e., expressing one CAR construct) or double- positive CAR T cell populations (i.e., expressing both CAR constructs). On Day 10 post activation, the CAR T cells targeting individual epitope or dual epitopes on the antigen CD22 with the indicated structures were co-cultured for 24 hours with NALM6 engineered to express variable levels of HER2 as well as a tumor cell line that expresses low levels of HER2 (143b). Interleukin-2 (IL-2) and IFN-g content was measured in the supernatant. As shown in FIGS. 7A-7B, CAR T cells transduced with both anti-HER2 single-targeting CARs generated more cytokine than control T cells expressing either individual CARs, especially against low antigen density tumors.

EXAMPLE 3

Assessing functionality of T cells co-transduced with two anti-CD276 single-targeting CAR constructs

[0165] This Example describes experiments performed to evaluate functionality of human T cells co-transduced with two anti-CD276 single-targeting CAR constructs each containing a scFv targeting different epitopes on the antigen CD276 (B7-H3).

[0166] In these experiments, two CARs were constructed with two scFv’s MGA271 and N3 which bind different epitopes on CD276. Retroviral vectors encoding these CARs with the indicated structures were synthesized commercially and cloned by standard methods. Viral supernatant was produced in 293 GP cells after transient transfection of the retroviral plasmid. CARs were then individually introduced into human T cells, where primary human T cells were transduced with viral supernatant after activation with Dynabeads Human T- Activator CD3/CD28 (Thermo Fisher) and were cultured in the presence of interleukin-2 (IL- 2). After a period of seven days, the CAR-T cells were FACS sorted based on fluorescence to achieve a 100% pure single-positive CAR T cells (i.e., expressing one CAR construct) or double-positive CAR T cell populations (i.e., expressing both CAR constructs). On Day 10 post activation, the CAR T cells targeting individual epitope or dual epitopes on the antigen CD276 with the indicated structures were co-cultured for 24 hours with cell lines expressing CD276. Nalm6-B7H3 was a leukemia cell line on which CD276 was artificially expressed. MG63.3 and 143b were osteosarcoma cell lines that expressed CD276. U383 and U87 were glioblastoma multiforme cell lines that expressed CD276. Interleukin-2 (IL-2) was measured in the supernatant. As shown in FIG. 14, CAR T cells transduced with both anti-CD276 single-targeting CARs generated more cytokine than control T cells expressing either individual CARs. EXAMPLE 4

Functionality of T cells expressing a dual -targeting CAR construct with two svFv’s each recognizing different epitopes on a same antigen

[0167] This Example describes experiments performed to evaluate functionality of human T cells expressing a dual-targeting CAR molecule that includes two svFvs each recognizing different epitopes on a same antigen.

[0168] As discussed in Example 1 above, CAR T cells expressing two anti-CD22 CAR constructs each containing a scFv recognizing different epitopes on the antigen CD22 demonstrated an increase in cytokine production in response to low CD22 tumor cells and an enhancement in killing of tumor cells. It was hypothesized that using a tandem dual -targeting CAR with m971 and HA22 scFv’s linked to each other in an orientation consistent with the epitope spatial orientation on CD22 would yield the same results. The schematic of FIG. 8 illustrates how a tandem dual -targeting CAR that two scFv’s recognizing different epitopes on a antigen would target the antigen. In this scenario, both scFv’s are connected in a single CAR molecule in the correct spatial orientation based on the location of their target epitopes.

[0169] As shown in FIGS. 9A-9B, it was observed that a tandem dual-targeting CAR design with m971 and HA22 scFv’s linked to each other in“normal orientation” does not enhance activity against CD22^low tumor cells. In these experiments, retroviral vectors encoding two single targeting CARs (m971-BBz and HA22-BBz) and a tandem dual targeting CAR (m971-HA22-BBz) with the indicated structures were synthesized commercially and cloned by standard methods. Viral supernatant was produced in 293GP cells after transient transfection of the retroviral plasmid. CARs were then individually introduced into human T cells, where primary human T cells were transduced with viral supernatant after activation with Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher) and were cultured in the presence of IL-2. On Day 10 post activation, the CD22 targeting individual or tandem dual-targeting CAR T cells with the indicated structures (“normal orientation”, i.e. with the scFv’s linked to each other in tandem and in a spatial orientation consistent with the spatial orientation of their respective epitopes on CD22) were co-cultured with NALM6 cells expressing low level of CD22 (LOW CD22 NALM6) with approximately 1,500 molecules per cell. Tumor cells remaining (survival) were measured over time in an Incucyte by measuring GFP (the NALM6 cells express GFP). As shown in FIGS. 9A-9B, tandem dual-targeting CAR T cells with the correct spatial orientation demonstrated reduced killing of tumor cells as compared to T cells expressing single-targeting CAR constructs (FIG. 9A). In addition, the CD22 single-targeting CAR T cells or tandem-dual-targeting (correct spatial orientation) CAR T cells with the indicated structures were co-cultured for 24 hours with NALM6 cells expressing native level of CD22 (NALM6 WT) with approximately 4000 molecules CD22 per cell or low CD22 (LOW CD22 NALM6, approximately 1,500 molecules per cell). IL-2 content was measured in the supernatant. As shown in FIG. 9B, tandem-dual -targeting CAR T cells with the correct spatial orientation demonstrated reduced cytokine production as compared to T cells expressing single-targeting CAR constructs.

[0170] Surprisingly, it was observed that a tandem dual-targeting CAR design with m971 and HA22 scFv’s linked to each other in“reverse orientation” relative to the spatial orientation of their respective epitopes on the antigen significantly enhanced T cell activity against CD22^low tumor cells.

[0171] The schematic of FIG. 10 illustrates how a reverse oriented tandem dual targeting CAR would recognize and/or bind an antigen. In this scenario, a tandem dual targeting CAR molecule was constructed with m971 and HA22 scFv’s linked to each other in the orientation opposite to epitope spatial location. Without being bound to any particular theory, it is believed that the orientation m971 and HA22 scFv’s was reversed to force engagement of more CAR molecules per T cell. Accordingly, this would result in the recruitment of more CAR molecules to the synapse (as each CAR molecule can bind only one epitope due to reverse orientation) and provide greater activation.

[0172] FIGS. 11A-11B schematically summarize the results of experiments performed to illustrate that a tandem dual -targeting CAR design with m971 and HA22 scFv’s linked to each other in“reverse orientation” demonstrate enhance activity against CD22^low tumor cells. In these experiments, retroviral vectors encoding these CARs with the indicated structures were synthesized commercially and cloned by standard methods. Viral supernatant was produced in 293GP cells after transient transfection of the retroviral plasmid. CARs were then individually introduced into human T cells, where primary human T cells were transduced with viral supernatant after activation with Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher) and were cultured in IL-2. On Day 10 post activation, the CD22 targeting individual or tandem dual-targeting CAR T cells with the indicated structures (“reverse orientation”, i.e. with the scFv’s linked to each other in tandem and in a reverse orientation relative to the spatial orientation of their respective epitopes on CD22) were co cultured with NALM6 cells expressing low level of CD22 (LOW CD22 NALM6) with approximately 1,500 molecules per cell. Tumor cells remaining (survival) were measured over time in an Incucyte by measuring GFP (the NALM6 cells express GFP). As shown in FIGS. 11A-11B, tandem dual-targeting CAR T cells with the reverse spatial orientation demonstrated equivalent killing of tumor cells as compared to T cells expressing single targeting CAR constructs (FIG. 11 A).

[0173] In addition, the CD22 single-targeting CAR T cells or tandem-dual-targeting (reverse spatial orientation) CAR T cells with the indicated structures were co-cultured for 24 hours with NALM6 cells expressing native level of CD22 (NALM6 WT) with approximately 4000 molecules CD22 per cell or low CD22 (LOW CD22 NALM6, approximately 1,500 molecules per cell). IL-2 content was measured in the supernatant. As shown in FIG. 11B, tandem-dual -targeting CAR T cells with the reverse spatial orientation demonstrated enhanced cytokine production, which is indicative of superior function, as compared to T cells expressing single-targeting CAR constructs. This unexpected and surprising result appears to indicate that only the reverse tandem configuration of dual-targeting CARs increases the number of engaged CAR molecules per target cell by simultaneously targeting multiple epitopes on a same antigen (while dual-targeting CARs with“normal orientation” configuration does not).

EXAMPLE 5

Assessing functionality of T cells expressing a dual-targeting CAR construct with two svFvs each recognizing different epitopes on HER2

[0174] This Example describes experiments performed to evaluate functionality of human T cells expressing a dual-targeting CAR molecule that includes two svFvs each recognizing different epitopes on HER2.

[0175] As discussed in Example 2 above, CAR T cells expressing two anti-HER2 CAR constructs each containing a scFv recognizing different epitopes on the antigen HER2 demonstrated an increase in cytokine production in response to low HER2 tumor cells and an enhancement in killing of tumor cells. It was hypothesized that using a tandem dual -targeting CAR with two scFv’s 4D5 and FRP5 linked to each other in an orientation consistent with the epitope spatial orientation on HER2 would yield the same results.

[0176] FIGS. 12A-12C schematically summarize the results of experiments performed to illustrate that a tandem dual -targeting CAR design with 4D5 and FRP5 scFv’s linked to each other in“normal orientation” does not enhance activity against HER2^low tumor cells. [0177] FIG. 12C depicts a tandem dual -targeting CAR in the correct spatial orientation targeting HER2. Both scFv’s were linked to each other in a single CAR molecule in the correct spatial orientation based on the location of their target epitopes. 4D5 and FRP5 scFv’s recognize different epitopes on HER2. FRP5 scFv targets a distal epitope while 4D5 scFv targets a proximal epitope. Retroviral vectors encoding these CARs with the indicated structures were synthesized commercially and cloned by standard methods. Viral supernatant was produced in 293 GP cells after transient transfection of the retroviral plasmid. CARs were then transduced into human T cells, where primary human T cells were transduced with viral supernatant after activation with Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher) and were cultured in the presence of IL-2. On Day 10 post activation, the HER2 targeting individual or tandem dual-targeting CAR T cells with the indicated structures (“normal orientation”, i.e. with the scFv’s linked to each other in tandem and in a spatial orientation consistent with the spatial orientation of their respective epitopes on HER2 were co-cultured for 24 hours with NALM6 engineered to express variable levels of HER2 as well as a tumor cell lines that expresses low levels of HER2 (143b). IL-2 and IFN-g content was measured in the supernatant. As shown in FIGS. 12A-12B, tandem dual-targeting CAR T cells with the correct spatial orientation did not demonstrate enhanced IL2 production against antigen low tumor cells (FIG. 12A). It was also observed that IFNy production was higher, but this was accounted for by a rise in baseline IFNy (in the absence of tumor cells), and not increased reactivity (FIG. 12A). Accordingly, the experimental results presented above indicate that tandem dual-targeting HER2 CAR T cells with the correct spatial orientation do not enhance activity against low antigen density.

[0178] Unexpectedly, it was observed that the“tandem dual-targeting” CARs of the present disclosure outperformed traditional CARs only when the scFv’s are linked to each other in tandem and in reverse orientation relative to the spatial orientation of their respective epitopes on the antigen. It was observed that a tandem dual-targeting CAR design with 4D5 and FRP5 scFv’s linked to each other in“reverse orientation” relative to the spatial orientation of their respective epitopes on the antigen significantly enhanced T cell activity against HER2^low tumor cells. Without being bound to any particular theory, this surprising result appears to indicate that only the reverse tandem configuration of dual-targeting CARs increases the number of engaged CAR molecules per target cell by simultaneously targeting multiple epitopes on a same antigen (while dual-targeting CARs with“normal orientation” configuration does not). [0179] FIGS. 13A-13C schematically summarize the results of experiments performed to illustrate that a tandem dual -targeting CAR design with 4D5 and FRP5 scFv’s linked to each other in“reverse orientation” (e.g. , in the orientation opposite to epitope spatial location) demonstrate enhance activity against HER2^low tumor cells.

[0180] FIG. 13C depicts a tandem dual -targeting CAR in the reverse spatial orientation targeting HER2. Both scFv’s are connected in a single CAR molecule in the reverse spatial orientation based on the location of their target epitopes. 4D5 and FRP5 scFv’s recognize different epitopes on HER2. FRP5 targets a distal epitope while 4D5 targets a proximal epitope. Retroviral vectors encoding these CARs with the indicated structures were synthesized commercially and cloned by standard methods. Viral supernatant was produced in 293GP cells after transient transfection of the retroviral plasmid. CARs were then transduced into human T cells, where primary human T cells were transduced with viral supernatant after activation with Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher) and were cultured in the presence of IL-2. On day 10 post activation, the HER2 targeting individual or tandem dual-targeting CAR T cells with the indicated structures (“reverse orientation”, i.e. with the scFv’s linked to each other in tandem and in a reverse orientation relative to the spatial orientation of their respective epitopes on HER2) were co-cultured for 24 hours with NALM6 engineered to express variable levels of HER2 as well as a tumor cell lines that expresses low levels of HER2 (143b). IL-2 and IFN-g content was measured in the supernatant. As shown in FIG. 13A, tandem-dual-targeting CAR T cells with the reverse spatial orientation demonstrated enhanced cytokine production against antigen low tumor cells. Therefore, tandem-dual-targeting CAR T cells with the reverse spatial orientation do enhance activity against low antigen density.

[0181] 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

CLAIMS WHAT IS CLAIMED IS:

1. A chimeric polypeptide comprising, from N-terminus to C-terminus:

a) an extracellular domain (ECD) comprising a first antigen-binding moiety capable of binding to a first epitope of an antigen, and a second antigen-binding moiety capable of binding to a second epitope of the antigen;

b) a transmembrane domain (TMD); and

c) an intracellular signaling domain (ICD),

wherein the first and the second antigen-binding moieties are in reverse orientation relative to the spatial orientation of their respective epitopes on the antigen.

2. The chimeric polypeptide of claim 1, wherein the first and the second antigen binding moieties are operably linked to each other in tandem.

3. The chimeric polypeptide of any one of claims 1 to 2, wherein the intracellular signaling domain further comprises a Oϋ3z intracellular signaling domain.

4. The chimeric polypeptide of claim 1 or 3, wherein the chimeric polypeptide is a chimeric antigen receptor (CAR).

5. The chimeric polypeptide of any one of claims 1 to 4, wherein the first and the second epitopes are non-overlapping epitopes.

6. The chimeric polypeptide of any one of claims 1 to 5, wherein epitope binding of the first antigen-binding moiety does not significantly interfere with epitope binding of the second antigen-binding moiety.

7. The chimeric polypeptide of claim 6, wherein epitope binding of the first antigen binding moiety does not significantly compete with epitope binding of the second antigen binding moiety.

8. The chimeric polypeptide of any one of claims 1 to 7, wherein the first and the second antigen-binding moieties are capable of binding the same antigen expressed by a target cell.

9. The chimeric polypeptide of claim 8, wherein the antigen is expressed at low density by the target cell.

10. The chimeric polypeptide of any one of claims 8 to 9, wherein the target cell is a cancer cell.

11. The chimeric polypeptide of claim 10, wherein the cancer cell is selected from the group consisting of a leukemia cell, an acute myeloma leukemia cell, a lymphoma cell, an anaplastic lymphoma cell, an astrocytoma cell, a B-cell cancer cell, a T-cell cancer, an epithelial type cancer cell, a mesenchymal type cancer cell, a breast cancer cell, a colon cancer cell, an ependymoma cell, an esophageal cancer cell, a glioblastoma cell, a glioma cell, a leiomyosarcoma cell, a liposarcoma cell, a liver cancer cell, a lung cancer cell, a mantle cell lymphoma cell, a melanoma cell, a neuroblastoma cell, a non-small cell lung cancer cell, an oligodendroglioma cell, an ovarian cancer cell, a pancreatic cancer cell, a peripheral T-cell lymphoma cell, a renal cancer cell, a stomach cancer cell, a carcinoma cell, a mesothelioma cell, and a sarcoma cell.

12. The chimeric polypeptide of any one of claims 1 to 11, wherein the antigen is a tumor associated-antigen (TAA) or a tumor-specific antigen (TSA).

13. The chimeric polypeptide of any one of claims 1 to 11, wherein the antigen is associated with or specific for a hematologic malignancy.

14. The chimeric polypeptide of any one of claims 1 to 11, wherein the antigen is associated with or specific for a solid tumor.

15. The chimeric polypeptide of any one of claims 1 to 14, wherein the antigen is selected from the group consisting of CD22, human epidermal growth factor receptor 2 (HER2/neu/ErbB-2), CD276 (B7-H3), MUC1, PSMA, Glypican 2 (GPC2), IL- 13 -receptor alpha 1, IL- 13 -receptor alpha 2, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen- 125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD123, CD93, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), ALK,

DLK1, FAP, NY-ESO, WT1, HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-Dl, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor- 1, the dimeric form of the pyruvate kinase isoenzyme type M2 (tumor M2-PK), CD 19, CD20, CD5, CD7, CD3, TRBC1, TRBC2, BCMA, CD38, CD123, CD93, CD34, CDla, SLAMF7/CS1, FLT3, CD33, CD123, TALLA- 1, CSPG4, DLL3, IgG Kappa light chain, IgA Lamba light chain, CD16/ FcyRIII, CD64, FITC, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), EGFR and isovariants thereof, TEM-8, sperm protein 17 (Spl7), mesothelin,

PAP (prostatic acid phosphatase), prostate stem cell antigen (PSCA), prostein, NKG2D, TARP (T cell receptor gamma alternate reading frame protein), Trp-p8, STEAP1 (six- transmembrane epithelial antigen of the prostate 1), an abnormal ras protein, an abnormal p53 protein, integrin b3 (CD61), galactin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), and Ral-B.

16. The chimeric polypeptide of claim 14, wherein the antigen is selected from the group consisting of CD22, HER2/neu/ErbB-2, and CD276 (B7-H3).

17. The chimeric polypeptide of claim 16, wherein the antigen is CD22 or

HER2/neu/ErbB-2.

18. The chimeric polypeptide of any one of claims 1 to 17, wherein the first antigen binding moiety and the second antigen-binding moiety are independently selected from the group consisting of an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), a full-length immunoglobulin, a nanobody, a single domain antibody (sdAb), a VNAR domain, and a VHH domain, and a diabody, or a functional fragment of any thereof.

19. The chimeric polypeptide of claim 18, wherein at least one of the first antigen binding moiety and the second antigen-binding moiety is a scFv or a functional fragment thereof.

20. The chimeric polypeptide of any one of claims 1 to 19, wherein the intracellular signaling domain comprises one or more costimulatory domains.

21. The chimeric polypeptide of claim 20, wherein the one or more costimulatory domains is selected from the group consisting of a costimulatory 4-1BB (CD137) polypeptide sequence, a costimulatory CD27 polypeptide sequence, a costimulatory 0X40 (CD 134) polypeptide sequence, a costimulatory inducible T-cell costimulatory (ICOS) polypeptide sequence, and a CD2 costimulatory polypeptide sequence.

22. The chimeric polypeptide of any one of claims 1 to 21, wherein the

transmembrane domain is derived from a CD28 transmembrane domain, a CD8a

transmembrane domain, a CD3 transmembrane domain, a CD4 transmembrane domain, a CTLA4 transmembrane domain, and a PD-1 transmembrane domain.

23. The chimeric polypeptide of claim 22, wherein the transmembrane domain is derived from a CD8a transmembrane domain.

24. The chimeric polypeptide of any one of claims 1 to 23, wherein the chimeric polypeptide comprises, in N-terminus to C-terminus direction:

a) an extracellular domain comprising a first and a second antigen-binding moieties capable of binding to different epitopes of an antigen selected from the group consisting of CD22, HER2, and CD276;

b) a transmembrane domain derived from CD8a;

c) an intracellular domain comprising a costimulatory domain from 4- IBB; and d) a Oϋ3z intracellular signaling domain,

25. The chimeric polypeptide of any one of claims 1 to 24, 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 NOS: 1-4.

26. A recombinant nucleic acid comprising a nucleotide sequence that encodes a chimeric polypeptide according to any one of claims 1 to 25.

27. The recombinant nucleic acid of claim 26, wherein the nucleotide sequence is in an expression cassette or an expression vector.

28. The recombinant nucleic acid of claim 27, wherein the expression vector is a viral vector.

29. The recombinant nucleic acid of claim 28, wherein the viral vector is a lentiviral vector, an adenovirus vector, an adeno-associated virus vector, or a retroviral vector.

30. A recombinant cell comprising:

a) a chimeric polypeptide according to any one of claims 1 to 25; and/or

b) a recombinant nucleic acid according to any one of claims 26 to 29.

31. A recombinant cell that has been engineered to express the following:

a) a first chimeric polypeptide comprising, from N-terminus to C-terminus: (i) an extracellular domain comprising a first antigen-binding moiety capable of binding to a first epitope of an antigen; (ii) a transmembrane domain; and (iii) an intracellular signaling domain; and

b) a second chimeric polypeptide comprising, from N-terminus to C-terminus: (i) an extracellular domain comprising a second antigen-binding moiety capable of binding to a second epitope of the antigen; (ii) a transmembrane domain; and (iii) an intracellular signaling domain,

wherein the first and the second antigen-binding moieties are capable of binding to different epitopes of the antigen.

32. The recombinant cell of any one of claims 30 to 31, wherein the recombinant cell is a eukaryotic cell.

33. The recombinant cell of claim 32, wherein the eukaryotic cell is a mammalian cell.

34. The recombinant cell of claim 33, wherein the mammalian cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell.

35. The recombinant cell of claim 34, wherein the immune cell is a B cell, a monocyte, a natural killer cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a T lymphocyte, a regulatory T cell, a helper T cell, a cytotoxic T cell, or other T cell.

36. The recombinant cell of claim 35, wherein the immune cell is a T lymphocyte.

37. A cell culture comprising a recombinant cell according to any one of claims 30 to 36, and a culture medium.

38. A method for making a recombinant cell, comprising: (a) providing a cell capable of protein expression; and (b) transducing the cell with a recombinant nucleic acid according to any one of claims 26 to 29.

39. A pharmaceutical composition comprising:

a) a recombinant nucleic acid according to any one of claims 26 to 29; and/or b) a recombinant cell according to any one of claims 30 to 36; and

c) a pharmaceutically acceptable carrier.

40. The pharmaceutical composition of claim 39, wherein the composition comprises a recombinant nucleic acid according to any one of claims 26 to 29, and a pharmaceutically acceptable carrier.

41. A method for the treatment of a disease in an individual in need thereof, the method comprising administering to the individual a first therapy comprising a composition that comprises at least one recombinant cell according to any one of claims 30 to 37.

42. A method for the treatment of a disease in an individual in need thereof, the method comprising administering to the individual a first therapy comprising a composition that comprises at least one recombinant cell engineered to express the following:

a) a first chimeric polypeptide comprising, from N-terminus to C-terminus: (i) an

extracellular domain comprising a first antigen-binding moiety capable of binding to a first epitope of an antigen; (ii) a transmembrane domain; and (iii) an intracellular signaling domain; and

43. The method of any one of claims 41 to 42, wherein the provided composition treats the disease in the individual.

44. The method of any one of claims 41 to 43, wherein the antigen is expressed at low density.

45. The method of any one of claims 41 to 43, wherein the disease is a cancer.

46. The method of claim 46, wherein the cancer is a pancreatic cancer, a colon cancer, an ovarian cancer, a prostate cancer, a lung cancer, mesothelioma, a breast cancer, a urothelial cancer, a liver cancer, a head and neck cancer, a sarcoma, a cervical cancer, a stomach cancer, a gastric cancer, a melanoma, a uveal melanoma, a cholangiocarcinoma, multiple myeloma, leukemia, lymphoma, or glioblastoma.

47. The method of any one of claims 41 to 46, wherein the administered first therapy inhibits tumor growth and/or metastasis of the cancer in the individual.

48. The method of any one of claims 41 to 47, wherein the administered first therapy increases cytokine production and/or enhances cancer cell killing.

49. The method of any one of claims 41 to 48, wherein the first therapy is

administered to the individual in conjunction with administration of a second therapy.

50. The method of claim 49, wherein the second therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, and surgery.

51. The method of any one of claims 49 to 50, wherein the first therapy and the second therapy are administered concomitantly.

52. The method of any one of claims 49 to 50, wherein the first therapy is

administered at the same time as the second therapy.

53. The method of any one of claims 49 to 50, wherein the first therapy and the second therapy are administered sequentially.

54. The method of claim 53, wherein the first therapy is administered before the second therapy.

55. The method of claim 53, wherein the first therapy is administered after the second therapy.

56. The method of any one of claims 49 to 50, wherein the first therapy is administered before and after the second therapy.

57. The method of any one of claims 49 to 50, wherein the first therapy and the second therapy are administered in rotation.

58. The method of any one of claims 49 to 50, wherein the first therapy and the second therapy are administered together in a single formulation.

59. A method for modulating an activity a target cell, the method comprising:

a) providing a composition comprising at least one recombinant cell according to any one of claims 30 to 36; and

b) contacting a target cell with the provided composition.

60. A method for modulating an activity of a target cell, the method comprising: providing a composition comprising at least one recombinant cell engineered to express the following:

b) a second chimeric polypeptide comprising, from N-terminus to C-terminus: (i) an extracellular domain comprising a second antigen-binding moiety capable of binding to a second epitope of the antigen; (ii) a transmembrane domain; and (iii) an intracellular signaling domain, wherein the first and the second antigen-binding moieties are capable of binding to different epitopes of the antigen; and

contacting a target cell with the provided composition.

61. The method of any one of claims 59 to 60, wherein the provided composition modulates an activity of the target cell.

62. The method of any one of claims 59 to 61, wherein the activity of the cell to be modulated is selected from the group consisting of: cell growth, proliferation, apoptosis, non- apoptotic death, differentiation, dedifferentiation, migration, secretion of a molecule, cellular adhesion, and cytolytic activity.

63. The method of any one of claims 59 to 62, wherein the target cell is a cancer cell.

64. The method of any one of claims 59 to 61, wherein the contacting is carried out in vivo, ex vivo, or in vitro.

65. The method of any one of claims 59 to 64, wherein the antigen is expressed at low density by the target cell.

66. A system for modulating an activity of a target cell, or for treating a disease in an individual in need thereof, the system comprising:

a) a chimeric polypeptide according to any one of claims 1 to 25;

b) a recombinant nucleic acid according to any one of claims 26 to 29;

c) a recombinant cell according to any one of claims 30 to 36; and/or

d) a pharmaceutical composition according to any one of claims 39 to 40.

67. Use of the following for the manufacture of a medicament for the treatment and/or prevention of a disease:

a) a chimeric polypeptide according to any one of claims 1 to 25;

b) a recombinant nucleic acid according to any one of claims 26 to 29;

c) a recombinant cell according to any one of claims 30 to 36; and/or

d) a pharmaceutical composition according to any one of claims 39 to 40.