Chimeric antigen receptors and uses thereof

Abstract

The present disclosure provides chimeric antigen receptors comprising novel intracellular domain pairs. It further provides cells expressing such chimeric antigen receptors, compositions of cells expressing such chimeric antigen receptors, as well as methods of making and using the same.

Description

[0001] CHIMERIC ANTIGEN RECEPTORS AND USES THEREOF

[0002] RELATED APPLICATION

[0003] [1] This application claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 63 / 635,184, filed April 17, 2024, the contents of which are hereby incorporated by reference.

[0004] BACKGROUND

[0005] [2] Chimeric antigen receptor (CAR)-T cell therapies have shown remarkable progress in the treatment of certain hematologic cancers. Translation of this therapeutic modality more broadly within oncology has been limited by several factors, including T cell susceptibility to various immunosuppressive factors in the tumor microenvironment (TME).

[0006] [3] Chimeric antigen receptors (CARs) are comprised of several domains, including a targeting domain that determines its antigen specificity (e.g., an antigen-binding domain) and an intracellular signaling region, which comprises intracellular domains (ICDs) that activate cell signaling. Signaling cascades triggered by ICDs of CARs in immune cells drive cell behaviors that correspond to different therapeutic outcomes.

[0007] Currently approved cell therapies contain one of two combinations of ICDs: a 4- IBB costimulatory domain combined with a CD3-zeta activation domain (BBz), or a CD28 costimulatory domain combined with a CD3-zeta activation domain (28z). While these canonical CAR-T ICD combinations have achieved clinical success in a limited number of hematologic cancer indications, CAR-based cell therapies have not yet been successful in other indications, such as the treatment of solid tumors. Thus, there is a need for the CARs with different ICDs that drive more favorable T cell phenotypes, such as improved T cell persistence and resistance to immunosuppressive TME factors.

[0008] SUMMARY

[0009] [4] In certain aspects, provided herein are chimeric antigen receptors (CARs) comprising novel combinations of intracellular domains (ICDs). In certain embodiments, immune cells expressing the CARs provided herein have favorable therapeutic properties, such as reduced cellular exhaustion and / or improved effector function against solid tumors. CARs are comprised of several domains, including a targeting domain that determines its antigen specificity (e.g., an antigen-binding domain) and an intracellular signaling region, which comprises ICDs that activate cell signaling. Signaling cascades triggered by ICDs of CARs in immune cells drive cell behaviors that correspond to different therapeutic outcomes. Currently approved cell therapies contain one of two combinations of ICDs: a 4- 1BB costimulatory domain combined with a CD3-zeta activation domain (BBz), or a CD28 costimulatory domain combined with a CD3-zeta activation domain (28z). While these canonical CAR-T ICD combinations have achieved clinical success in a limited number of hematologic cancer indications, CAR-based cell therapies have not yet been successful in other indications, such as the treatment of solid tumors. Thus, there is a need for CARs with different ICDs that drive more favorable T cell phenotypes, such as more favorable exhaustion profiles.

[0010] [5] In certain aspects, the disclosure provides a chimeric antigen receptor (CAR) polypeptide comprising: (1) an antigen -binding domain; (2) a hinge domain; (3) a transmembrane domain; and (4) an intracellular signaling region, wherein the intracellular signaling region comprises an intracellular domain (ICD) pair, the intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD pair is selected from the ICD pairs listed in Table 1.

[0011] [6] In some embodiments, the ICD1 and ICD2 are each, respectively: (i) CD3d_ITAM and CD22, (ii) CD3z_D12mut and FCGR3A, (iii) CD3z_D23mut and CD3d, (iv) CD3z_D23mut and DR3, (v) CD3z_D23mut and MyD88_CD40, (vi) CD3z_D23mut and NKp46, (vii) CD3z_D23mut and 0X40, (viii) CD3z_ITAMl and CD8a, (ix) CD79a and CD3g_ITAM, (x) CD79a and NKp30, (xi) CD79b_ITAM and MyD88_CD40, (xii) CTLA4 and CD3g_ITAM, (xiii) DAP12JTAM and 0X40, (xiv) FCER1G and FCER1GJTAM, (xv) FCER1G and MyD88_CD40, or (xvi) NKp46 and DAP12JTAM.

[0012] [7] In some embodiments, the ICD1 and ICD2 are each, respectively: (i) CD3d_ITAM and CD22, (ii) CD3z_D12mut and FCGR3A, (iii) CD3z_D23mut and CD3d, (iv) CD3z_D23mut and DR3, (v) CD3z_D23mut and MyD88_CD40, (vi) CD3z_D23mut and NKp46, (vii) CD3z_D23mut and 0X40, (viii) CD3z_ITAMl and CD8a, (ix) CD79a and CD3g_ITAM, (x) CD79a and NKp30, (xi) CD79b_ITAM and MyD88_CD40, (xii) CTLA4 and CD3g_ITAM, (xiii) DAP12JTAM and 0X40, (xiv) FCER1G and FCER1GJTAM, (xv) FCER1G and MyD88_CD40, or (xvi) NKp46 and DAP12JTAM, wherein the ICD1 is positioned between the transmembrane domain and the ICD2.

[0013] [8] In some embodiments, the sequences of the ICD1 and ICD2, respectively, are or comprise: (i) SEQ ID NO: 44 and SEQ ID NO: 33, (ii) SEQ ID NO: 25 and SEQ ID NO: 59, (iii) SEQ ID NO: 26 and SEQ ID NO: 43, (iv) SEQ ID NO: 26 and SEQ ID NO: 56, (v) SEQ ID NO: 26 and SEQ ID NO: 10, (vi) SEQ ID NO: 26 and SEQ ID NO: 82, (vii) SEQ ID NO: 26 and SEQ ID NO: 13, (viii) SEQ ID NO: 47 and SEQ ID NO: 51, (ix) SEQ ID NO: 34 and SEQ ID NO: 46, (x) SEQ ID NO: 34 and SEQ ID NO: 80, (xi) SEQ ID NO: 49 and SEQ ID NO: 10, (xii) SEQ ID NO: 7 and SEQ ID NO: 46, (xiii) SEQ ID NO: 55 and SEQ ID NO: 13, (xiv) SEQ ID NO: 17 and SEQ ID NO: 12, (xv) SEQ ID NO: 17 and SEQ ID NO: 10, or (xvi) SEQ ID NO: 82 and SEQ ID NO: 55.

[0014] [9] In some embodiments, the sequences of the ICD1 and ICD2 are each, respectively, at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to: (i) SEQ ID NO: 44 and SEQ ID NO: 33, (ii) SEQ ID NO: 25 and SEQ ID NO: 59, (iii) SEQ ID NO: 26 and SEQ ID NO: 43, (iv) SEQ ID NO: 26 and SEQ ID NO: 56, (v) SEQ ID NO: 26 and SEQ ID NO: 10, (vi) SEQ ID NO: 26 and SEQ ID NO: 82, (vii) SEQ ID NO: 26 and SEQ ID NO: 13, (viii) SEQ ID NO: 47 and SEQ ID NO: 51, (ix) SEQ ID NO: 34 and SEQ ID NO: 46, (x) SEQ ID NO: 34 and SEQ ID NO: 80, (xi) SEQ ID NO: 49 and SEQ ID NO: 10, (xii) SEQ ID NO: 7 and SEQ ID NO: 46, (xiii) SEQ ID NO: 55 and SEQ ID NO: 13, (xiv) SEQ ID NO: 17 and SEQ ID NO: 12, (xv) SEQ ID NO: 17 and SEQ ID NO: 10, or (xvi) SEQ ID NO: 82 and SEQ ID NO: 55, wherein the ICD1 is positioned between the transmembrane domain and the ICD2.

[0015]

[0010] In some embodiments, the intracellular signaling region comprises an amino acid sequence that is at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to any one of the sequences listed in Table 3.

[0016]

[0011] In some embodiments, the intracellular signaling region comprises an amino acid sequence listed in Table 3.

[0017]

[0012] In some embodiments, the intracellular signaling region comprises an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to any of: (i) SEQ ID NO: 451, (ii) SEQ ID NO: 452, (iii) SEQ ID NO: 453, (iv) SEQ ID NO: 454, (v) SEQ ID NO: 455, (vi) SEQ ID NO: 456, (vii) SEQ ID NO: 457, (viii) SEQ ID NO: 458, (ix) SEQ ID NO: 459, (x) SEQ ID NO: 460, (xi) SEQ ID NO: 461, (xii) SEQ ID NO: 462, (xiii) SEQ ID NO: 463, (xiv) SEQ ID NO: 464, (xv) SEQ ID NO: 465, or (xvi) SEQ ID NO: 466.

[0013] In some embodiments, the antigen-binding domain is a single chain fragment variable (scFv) domain.

[0018]

[0014] In some embodiments, the scFv domain comprises any one of the scFv domains set forth in Table 4.

[0019]

[0015] In some embodiments, the scFv domain comprises 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to any one of the scFv domains set forth in Table 4.

[0020]

[0016] In some embodiments, the sequence of intracellular signaling region is or comprises any of: (i) SEQ ID NO: 451, (ii) SEQ ID NO: 452, (iii) SEQ ID NO: 453, (iv) SEQ ID NO: 454, (v) SEQ ID NO: 455, (vi) SEQ ID NO: 456, (vii) SEQ ID NO: 457, (viii) SEQ ID NO: 458, (ix) SEQ ID NO: 459, (x) SEQ ID NO: 460, (xi) SEQ ID NO: 461, (xii) SEQ ID NO: 462, (xiii) SEQ ID NO: 463, (xiv) SEQ ID NO: 464, (xv) SEQ ID NO: 465, or (xvi) SEQ ID NO: 466.

[0021]

[0017] In some embodiments, the antigen-binding domain binds a cancer-associated antigen.

[0022]

[0018] In some embodiments, the cancer-associated antigen is selected from any one of the cancer-associated antigens listed in Table 5.

[0023]

[0019] In some embodiments, the cancer-associated antigen is GD2.

[0024]

[0020] In some embodiments, the antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 485.

[0025]

[0021] In some embodiments, the transmembrane domain is or comprises a sequence listed in Table 7.

[0026]

[0022] In some embodiments, the transmembrane domain is or comprises a sequence at least 90% identical to a sequence listed in Table 7.

[0027]

[0023] In some embodiments, the hinge domain is selected from any one of the hinge domains of Table 6.

[0028]

[0024] In some embodiments, the sequence of the hinge domain is at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to a sequence listed in Table 6.

[0029]

[0025] In some embodiments, the hinge domain is an IgG4 hinge domain.

[0026] In certain aspects, the disclosure provides a chimeric antigen receptor (CAR) polypeptide comprising: (1) an antigen -binding domain; (2) a hinge domain; (3) a transmembrane domain; and (4) an intracellular signaling region, wherein the antigenbinding domain binds to antigen GD2, and wherein the intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD1 and ICD2 are each, respectively, any of: (i) SEQ ID NO: 44 and SEQ ID NO: 33, (ii) SEQ ID NO: 25 and SEQ ID NO: 59, (iii) SEQ ID NO: 26 and SEQ ID NO: 43, (iv) SEQ ID NO: 26 and SEQ ID NO: 56, (v) SEQ ID NO: 26 and SEQ ID NO: 10, (vi) SEQ ID NO: 26 and SEQ ID NO: 82, (vii) SEQ ID NO: 26 and SEQ ID NO: 13, (viii) SEQ ID NO: 47 and SEQ ID NO: 51, (ix) SEQ ID NO: 34 and SEQ ID NO: 46, (x) SEQ ID NO: 34 and SEQ ID NO: 80, (xi) SEQ ID NO: 49 and SEQ ID NO: 10, (xii) SEQ ID NO: 7 and SEQ ID NO: 46, (xiii) SEQ ID NO: 55 and SEQ ID NO: 13, (xiv) SEQ ID NO: 17 and SEQ ID NO: 12, (xv) SEQ ID NO: 17 and SEQ ID NO: 10, or (xvi) SEQ ID NO: 82 and SEQ ID NO: 55, wherein the ICD1 is positioned between the transmembrane domain and the ICD2.

[0030]

[0027] In some embodiments, the sequence of the CAR polypeptide is or comprises any sequence in Table 8.

[0031]

[0028] In some embodiments, the sequence of the CAR polypeptide is or comprises any of SEQ ID NO: 486 to 501.

[0032]

[0029] In some embodiments, the sequence of the CAR polypeptide is at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to any of SEQ ID NO: 486 to 501.

[0033]

[0030] In certain aspects, the disclosure provides a nucleic acid encoding a CAR polypeptide described herein.

[0034]

[0031] In certain aspects, the disclosure provides a vector comprising a nucleic acid described herein.

[0035]

[0032] In some embodiments, the vector is an expression vector.

[0036]

[0033] In some embodiments, the vector is a viral vector.

[0037]

[0034] In some embodiments, the viral vector is a retroviral vector, lentiviral vector or an adeno-associated viral (AAV) vector.

[0038]

[0035] In certain aspects, the disclosure provides a cell comprising a nucleic acid described herein.

[0036] In certain aspects, the disclosure provides a cell expressing a CAR polypeptide described herein.

[0039]

[0037] In some embodiments, the cell is a T cell.

[0040]

[0038] In some embodiments, the T cell is a cytotoxic T lymphocyte (CTL).

[0041]

[0039] In some embodiments, the T cell is a primary CD8+ T cell.

[0042]

[0040] In some embodiments, the cell proliferates in the presence of antigenexpressing target cells.

[0043]

[0041] In some embodiments, the cell is resistant to exhaustion.

[0044]

[0042] In certain aspects, the disclosure provides a method of generating a CAR- expressing cell comprising contacting the cell with a nucleic acid described herein, or a vector described herein.

[0045]

[0043] In certain aspects, the disclosure provides a composition comprising any cells described herein.

[0046]

[0044] In certain aspects, the disclosure provides a method of treating a tumor in a subject, the method comprising administering a composition described herein.

[0047]

[0045] In some embodiments, the tumor is a blood cancer.

[0048]

[0046] In some embodiments, the blood cancer is a B cell chronic lymphocytic leukemia, non-Hodgkin’s lymphoma, a mantle cell lymphoma, multiple myeloma, or an acute lymphoblastic leukemia.

[0049]

[0047] In some embodiments, the tumor is a solid tumor.

[0050]

[0048] In some embodiments, the solid tumor is a neuroblastoma, osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, leiomyosarcoma, medulloblastoma, glioblastoma or diffuse midline glioma.

[0051]

[0049] In some embodiments, the solid tumor is an epithelial cancer.

[0052]

[0050] In some embodiments, the epithelial cancer is a non-small cell lung cancer, a triple negative breast cancer, melanoma, or an ovarian cancer.

[0053]

[0051] In some embodiments, the tumor expresses the antigen recognized by the antigen-binding domain of the CAR polypeptide.

[0054]

[0052] In some embodiments, the tumor expresses the antigen recognized by the antigen-binding domain of the CAR polypeptide, wherein the tumor expresses the antigen GD2.

[0055]

[0053] In certain aspects, the disclosure provides a cell bank comprising cells for adoptive immunotherapy, wherein the cells express a CAR polypeptide described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0056]

[0054] The drawings included herein, which are composed of the following Figures, are for illustration purposes only and not for limitation.

[0057]

[0055] FIG. 1 is a schematic diagram showing the experimental design using the 14g2a-2ICD library: CAR T donor libraries derived from healthy human donors 2745 (d2745), 6903 (d6903) and 6904 (d6904). These were screened against human MG-63 osteosarcoma and SK-N-AS neuroblastoma cells, as described below.

[0058]

[0056] FIG. 2A is a graph showing a pooled in vitro screen, showing that the CAR library (“14g2alib”) exhibits reduced T cell exhaustion marker PD1 expression, as screened in both human MG-63 osteosarcoma and SK-N-AS neuroblastoma cells, which both express GD2. In FIGs. 2A to 2D controls are: UTD: Untransduced T cells (negative control); and 28O40Z: Previously-described CAR-T (positive control). Each dot represents the results from the pooled data from one of the three donors.

[0059]

[0057] FIG. 2B is a graph showing a pooled in vitro screen, showing that the CAR library (“14g2alib”) exhibits reduced T cell exhaustion marker TIM3 expression, as screened in MG-63 and SK-N-AS cells. Each dot represents the results from the pooled data from one of the three donors.

[0060]

[0058] FIG. 2C is a graph showing a pooled in vitro screen, showing that the CAR library (“14g2alib”) exhibits reduced T cell exhaustion marker LAG3 expression, as screened in both MG-63 and SK-N-AS cells, compared to the controls.

[0061]

[0059] FIG. 2D is a graph showing a pooled in vitro screen, showing that the CAR library (“14g2alib”) exhibits reduced expression of T cell exhaustion markers PD1, TIM3 and LAG3, as screened in both MG-63 and SK-N-AS cells, compared to the controls.

[0062]

[0060] FIG. 3A is a schematic showing the methodology for hit picking (in vitro). ICDs that appear most often in top 100 enriched designs were selected, be: barcode. DO and D23: Day 0 and Day 23.

[0063]

[0061] FIG. 3B is a schematic showing the methodology for hit picking (in vivo).

[0064]

[0062] FIG. 4 is a plot showing the number of tracked unique CAR polypeptide designs at each step of in vivo pooled screen process. Details related to FIGs. 4 to 6 are provided in Example 3.

[0065]

[0063] FIG. 5 is a graph showing the number of CAR polypeptide designs post-next generation sequencing (NGS) filtering for each in vivo pooled screen group.

[0064] FIGs. 6A, B, C, and D represent fold-enrichment (X-axis) and the extent of variation across replicate mice (Y-axis) of each CAR polypeptide design post NGS (next generation sequencing) filtering.

[0066]

[0065] FIGs. 7A and 7B show results of in vitro hit validation in MG-63 osteosarcoma cells, with various individual CAR polypeptides highlighted. CAR polypeptides were evaluated for tumor control (e.g., control of tumor growth) and expansion. For FIGs. 7A, 7B, 8, 9A, 9B, 10A and 10B, the identity of various CAR polypeptides and controls are described in Example 4.

[0067]

[0066] FIG. 8 shows CAR-T cell fold-expansion across 3 serial MG-63 tumor challenges over the course of 2 weeks.

[0068]

[0067] FIGs. 9A and 9B are plots showing the tumor-killing potential of novel CAR polypeptides. CAR-T cells in either donor 6903 (d6903, FIG. 9A) or donor 8089 (d8089, FIG. 9B) were challenged with MG-63 osteosarcoma cells 3 times over the course of 2 weeks.

[0069]

[0068] FIGs. 10A and 10B are plots showing fold-expansion of CAR-T cells in 2 different T cell donors during 2 serial MG-63 tumor challenges over the course of 8 days.

[0070] DETAILED DESCRIPTION

[0071] General

[0072]

[0069] Chimeric antigen receptors (CAR) have shown great clinical success in reprogramming cytotoxic T cells to target tumor antigens (Sterner and Sterner (2021) Blood Cancer Journal 11:69). Typically, CARs include an extracellular domain specific for the tumor antigen, a hinge and transmembrane domain, and an intracellular domain (ICD) that transmits downstream signals. First generation CAR-T designs had a single ICD derived from the CD3 zeta chain. Second generation CAR-T designs included a co- stimulatory domain in addition to the CD3 zeta chain. Newer designs include multiple co- stimulatory or signaling domains in addition to CD3z, as well as other accessory sequences like cytokine expression. All of the six currently-approved CAR-T products are of the second generation design and all contain either CD28 or 4- IBB as the costimulatory domain.

[0073]

[0070] The intracellular signaling domain can dramatically impact cell function and clinical outcomes (Kawalekar et al. (2016) Immunity 44(2):380-390). As one example, the CD28 signaling domain leads to rapid, early cell proliferation in patients, while cells engineered with the 4- IBB domain tend to have more moderate but extended proliferation profiles. The different signaling domains also lead to differences in T cell exhaustion, which is a state of dysfunction arising from excessive antigen stimulation and a barrier to increased therapeutic efficacy for T cell therapies. T cell exhaustion is correlated to a fingerprint of expression of transmembrane proteins PD-1 (PD1), LAG-3 (LAG3) and Tim- 3 (TIM-3 or TIM3). See: Wherry 2011 Nature Immunol. 12: 492; and Terris et al. 2014 J. Immunol. 193: 1525. As CAR-T cells engage antigen-expressing tumor cells, T cell signaling is initiated through the CAR and can lead to T cell exhaustion, especially in the case of bulky solid tumors. CAR-T cells engineered with CD28 and 4- IBB have different exhaustion profiles, with 4- IBB CAR-T cells generally exhibiting less exhaustion as compared to CD28 CAR-T cells (Majzner and Mackall (2019) Nature Medicine 25:1341- 1355).

[0074]

[0071] While canonical CAR-T ICD combinations 4-lBB-CD3z (BBz) and CD28- CD3z (28z) have achieved clinical success in a limited number of hematologic indications, CAR-based cell therapies have not yet been successful in more challenging indications, such as bulky solid tumors, where T cell exhaustion is one of the limiting factors. BBz is described in, for example, Johnson et al. 2015 Sci. Trans. Med. 7:275; and Ying et al. 2019 Nature Med. 25: 947, and also referenced as BB.z. In certain embodiments, the present disclosure provides CAR polypeptides with new ICD combinations that, for example, can confer more favorable T cell phenotypes compared to the canonical CAR-T ICD combinations. As disclosed herein, cytotoxic T cells expressing the CARs disclosed herein (e.g., CARs with the various ICD combinations disclosed herein) can, in some embodiments, exhibit superior T cell proliferation and survival, and less exhaustive phenotypes (e.g., lower populations of PD-1+TIM3+or PDl+LAG-3+), compared to FDA- approved BBz control CARs.

[0075]

[0072] The present disclosure demonstrates that provided CAR-based cell therapies are effective in treating solid tumors. As shown in the Figures and elsewhere herein, Applicant has identified and developed signaling modules driving favorable phenotypes of T cells against solid tumors.

[0076]

[0073] In some aspects, provided herein are CAR polypeptides and polynucleotides encoding said CAR polypeptides, wherein the CAR polypeptides comprise an intracellular signaling region, wherein the intracellular signaling region comprises an ICD pair, wherein the intracellular domain pair comprises a first intracellular domain (e.g., ICD1) and a second intracellular domain (e.g., ICD2), and wherein the first ICD and the second ICD are individually any ICD disclosed herein, and / or the intracellular domain pair is any pair of ICD1 and ICD2 disclosed herein.

[0077]

[0074] In some aspects, provided herein are CAR polypeptides and polynucleotides encoding said CAR polypeptides comprising an intracellular signaling region, wherein the intracellular signaling region comprises an ICD pair, wherein the intracellular domain pair comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), and wherein the ICD pair is selected from the ICD pairs listed in Table 1. In some aspects, the ICD1 and / or ICD2 may comprise an ICD amino acid sequence set forth in Table 2. In some aspects, the intracellular signaling region may comprise an amino acid sequence set forth in Table 3. In some embodiments, the CAR polypeptide further comprises an antigenbinding domain, e.g., a scFv domain set forth in Table 4. In certain embodiments, the antigen-binding domain binds to a cancer antigen, such as disialoganglioside GD2. The CAR polypeptide may also comprise a hinge domain (e.g., a hinge domain selected from Table 6) and a transmembrane domain (e.g., a transmembrane domain selected from Table 7). Also provided herein are immune cells expressing a CAR polypeptide disclosed herein, as well as methods of treating a tumor in a subject by administering a composition comprising cells (e.g., immune cells) expressing a CAR polypeptide disclosed herein.

[0078] Certain Definitions

[0079]

[0075] For convenience, certain terms employed in the specification, examples, and appended claims are collected here. Additional definitions for the following terms and other terms are set forth throughout the specification.

[0080]

[0076] As used herein, the term “administering” means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administering by a medical professional and self-administering. Such an agent can contain, for example, a cell expressing a CAR provided herein.

[0081]

[0077] The term “amino acid” is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally-occurring amino acids. Example amino acids include naturally-occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of the foregoing.

[0082]

[0078] The term “binding” or “interacting” refers to an association, which may be a stable association, between two molecules, e.g., between a peptide and a binding partner or agent, e.g., small molecule, due to, for example, electrostatic, hydrophobic, ionic and / or hydrogen-bond interactions under physiological conditions.

[0083]

[0079] As used herein, the term “cancer” includes, but is not limited to, solid tumors and blood borne tumors. The term cancer includes, but is not limited to, diseases of the skin, tissues, organs, bone, cartilage, nerves, blood cells, vessels, and organs, including the cervix, anus, vagina, vulva, penis, tongue base, larynx, and tonsil. The term “cancer” further encompasses primary and metastatic cancers.

[0084]

[0080] The term “chimeric antigen receptor” (CAR) refers to molecules that combine a binding domain against a component present on the target cell, for example an antibodybased specificity for a desired antigen (e.g., a tumor antigen) with an immune cellactivating intracellular domain to generate a chimeric protein. Generally, CARs comprise an extracellular single chain antigen-binding domain (e.g., an scFv) fused to the intracellular signaling domain by a hinge domain (which is optional) and a transmembrane domain.

[0085]

[0081] The term “epitope” means a protein determinant capable of specific binding to an antibody or immune cell (e.g., T cell). Epitopes usually include chemically active surface groupings of molecules such as lipids, amino acids or sugar side chains. Certain epitopes can be defined by a particular sequence of amino acids to which a CAR or antibody is capable of binding.

[0086]

[0082] ‘Gene construct” refers to a nucleic acid, such as a vector, plasmid, viral genome or the like which includes a “coding sequence” for a polypeptide or which can otherwise transcribe to a biologically active RNA (e.g., antisense, decoy, ribozyme, etc.), may be transfected into cells, e.g., mammalian cells, and may cause expression of the coding sequence in cells transfected with the construct. The gene construct may include one or more regulatory elements operably linked to the coding sequence, as well as intronic sequences, poly adenylation sites, origins of replication, marker genes, etc.

[0087]

[0083] The terms “ligand-binding domain” and “antigen-binding domain” are used interchangeably herein, and refer to that portion of a CAR that binds specifically to a predetermined antigen.

[0088]

[0084] The term “linker” refers to a molecule or group of molecules connecting two compounds, such as two polypeptides. The linker may be comprised of a single linking molecule or may comprise a linking molecule and a spacer molecule, intended to separate the linking molecule and a compound by a specific distance.

[0085] The term “operably linked to” refers to the functional relationship of a nucleic acid with another nucleic acid sequence. Promoters, enhancers, transcriptional and translational stop sites, and other signal sequences are examples of nucleic acid sequences operably linked to other sequences. For example, operable linkage of DNA to a transcriptional control element refers to the physical and functional relationship between the DNA and promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.

[0089]

[0086] As used herein, the phrase “pharmaceutically acceptable” refers to those agents, compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0090]

[0087] As used herein, the phrase “pharmaceutically-acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting an agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or poly anhydrides; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

[0091]

[0088] The terms “polynucleotide”, and “nucleic acid” are used interchangeably. They refer to a natural or synthetic molecule, or some combination thereof, comprising a single nucleotide or two or more nucleotides linked by a phosphate group at the 3’ position of one nucleotide to the 5’ end of another nucleotide. The polymeric form of nucleotides is not limited by length and can comprise either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. A polynucleotide may be further modified, such as by conjugation with a labeling component. In all nucleic acid sequences provided herein, U nucleotides are interchangeable with T nucleotides. The polynucleotide is not necessarily associated with the cell in which the nucleic acid is found in nature, and / or operably linked to a polynucleotide to which it is linked in nature.

[0092]

[0089] The term “precancerous lesions” or “precancerous condition” refers to atypical cells and / or tissues that are associated with an increased risk of cancer. The term “precancerous lesions” may refer, for example, to dysplasia, benign neoplasia, or carcinoma in situ.

[0093]

[0090] As used herein, a therapeutic that “prevents” a condition refers to a compound that, when administered to a statistical sample prior to the onset of the disorder or condition, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.

[0094]

[0091] An “intracellular signaling region” of a CAR, as used herein, is the part of the chimeric antigen receptor protein that is located within the cell and that is responsible for intracellular signaling following the binding of an extracellular antigen-binding domain to the target. In some embodiments, the intracellular signal region can include multiple intracellular domains (ICDs) that may each convey a separate intracellular signal. In some cases, the intracellular signaling region comprises a pair of an intracellular domain 1 (ICD1) and an intracellular domain 2 (ICD2) listed in Tables 1-3, and / or the ICD1 and ICD2 are independently selected from any ICD described herein.

[0092] The term “specifically binds” or “specific binding”, as used herein, when referring to a polypeptide (including CAR polypeptides) refers to a binding reaction which is determinative of the presence of the protein or polypeptide or receptor in a heterogeneous population of proteins and other biologies. Thus, under designated conditions (e.g., immunoassay conditions in the case of an antibody), a specified ligand or antibody “specifically binds” to its particular “target” (e.g., an antibody specifically binds to an endothelial antigen) when it does not bind in a significant amount to other proteins present in the sample or to other proteins to which the ligand or antibody may come in contact in an organism. Generally, a first molecule that “specifically binds” a second molecule has an affinity constant (Ka) greater than about 105M1(e.g., 106M ( 107M ( 108M ( 109M ( IO10M ( 1011M ( and 1012M1or more) with that second molecule. For example, in the case of the ability of a PIG-specific CAR to bind to a peptide presented on an MHC (e.g., class I MHC or class II MHC); typically, a CAR specifically binds to its peptide / MHC with an affinity of at least a KD of about 10'4M or less, and binds to the predetermined antigen / binding partner with an affinity (as expressed by KD) that is at least 10 fold less, at least 100 fold less or at least 1000 fold less than its affinity for binding to a non-specific and unrelated peptide / MHC complex (e.g., one comprising a BSA peptide or a casein peptide).

[0095]

[0093] As used herein, the term “subject” means a human or non-human animal selected for treatment or therapy.

[0096]

[0094] The terms “transformation”, “transfection”, or “transduction” mean the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell (e.g., a mammalian cell) including introduction of a nucleic acid to the chromosomal DNA of said cell.

[0097]

[0095] As used herein, the term “treatment” refers to clinical intervention designed to alter the natural course of the individual being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of progression, ameliorating or palliating the pathological state, and remission or improved prognosis of a particular disease, disorder, or condition. An individual is successfully “treated,” for example, if one or more symptoms associated with a particular disease, disorder, or condition are mitigated or eliminated.

[0098]

[0096] The term “vector” refers to the means by which a nucleic acid can be propagated and / or transferred between organisms, cells, or cellular components. Vectors include plasmids, viruses, bacteriophage, pro-viruses, phagemids, transposons, and artificial chromosomes, and the like, to which the nucleic acid has been linked, and may or may not be able to replicate autonomously or integrate into a chromosome of a host cell. Such vectors may include any vector, (e.g., a plasmid, cosmid or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to a transcriptional control element).

[0099]

[0097] In certain embodiments, agents may be used alone or conjointly administered with another type of therapeutic agent. As used herein, the phrase “conjoint administration” or “administered conjointly” refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the subject, which may include synergistic effects of the two agents). For example, the different therapeutic agents can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially. In certain embodiments, the different therapeutic agents can be administered within about one hour, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about a week of one another. Thus, a subject who receives such treatment can benefit from a combined effect of different therapeutic agents.

[0100] Chimeric Antigen Receptors (CARs)

[0101]

[0098] Chimeric antigen receptors (CARs) are receptors comprising a targeting moiety that is associated with an intracellular signaling region comprising one or more intracellular domains (ICDs) in a single fusion molecule. In certain embodiments, the binding moiety of a CAR comprises an antigen -binding domain, e.g., a single-chain fragment variable (scFv) comprising the light and heavy chain variable fragments of a monoclonal antibody joined by a flexible linker. In certain embodiments, the binding moiety further comprises transmembrane and hinge domains.

[0102]

[0099] In some embodiments, a CAR comprises: an antigen-binding domain; a transmembrane domain; and an intracellular signaling region, wherein the intracellular signaling region comprises an ICD1 and an ICD2. In some embodiments, a CAR further comprises an optional hinge domain positioned between the antigen-binding domain and the transmembrane domain. In some embodiments, a CAR comprises, in N- to C-terminal order: an antigen-binding domain; a transmembrane domain; and an intracellular signaling region, wherein the intracellular signaling region comprises an ICD1 and an ICD2.

[0100] In various embodiments, a CAR (or a library thereof) can be evaluated using any of a number of different assays. In some embodiments, an assay can determine if a T cell expressing a CAR is capable of reducing the number or growth of a population of target cells (e.g., cancer cells) which express an antigen recognized by the CAR. In some embodiments, an assay can determine if a T cell expressing a CAR expresses a marker indicative or suggestive of T cell exhaustion (e.g., PD1, LAG3 and / or TIM3). In some embodiments, an assay can determine if a T cell expressing a CAR is capable of expansion (e.g., continued growth of the T cell). In some embodiments, a T cell expressing a CAR is capable of: (1) mediating a reduction in the number or growth of a population of target cells which express an antigen recognized by the CAR; (2) demonstrating low (or decreased, relative to a control cell) levels of expression of a marker indicative or suggestive of T cell exhaustion (e.g., PD1, LAG3 and / or TIM3); and / or demonstrating expansion (e.g., continued growth of the T cell).

[0103]

[0101] In some embodiments, an assay can be performed in vitro or in vivo. In some embodiments, an in vitro assay can be performed with a cell line. In some embodiments, an in vitro assay can be performed with a cell line which is a tumor cell line. In some embodiments, an in vitro assay can be performed with a cell line which is a tumor cell line expressing (or over-expressing) a particular antigen of interest. In some embodiments, an in vitro assay can be performed with a cell line which is a tumor cell line expressing (or overexpressing) a particular antigen of interest which is bound by the CAR. In some embodiments, an in vivo assay involves the use of an animal model. In some embodiments, an in vivo assay involves the use of an immunocompromised animal model which is also tumor-bearing. In some embodiments, an in vivo assay involves the use of an immunocompromised animal model which is also tumor-bearing, wherein the tumor cells express or over-express an antigen of interest. In some embodiments, an in vivo assay involves the use of an immunocompromised animal model which is also tumor-bearing, wherein the tumor cells express or over-express an antigen of interest which is bound by a CAR. In some embodiments, an assay can be a pooled or an arrayed screen. In some embodiments, in a pooled screen, two or more CAR polypeptides are tested simultaneously. In some embodiments, in a pooled screen, a library or other plurality of CAR polypeptides is tested simultaneously. In some embodiments, in a pooled screen, a library or other plurality of CAR polypeptides is tested simultaneously, and then the results de-convoluted. As a non-limiting example, a library of T cells, each expressing a different CAR, can be tested simultaneously for expansion. The T cells demonstrating the greatest expansion can be identified (and the results thus deconvoluted) by bar-coded sequencing. In some embodiments, an assay can be an array or individual assay. In some embodiments, in an array or individual assay, one or more individual CAR polypeptides is tested individually and not in a group. In some embodiments, a pooled assay can be performed to select individual CAR polypeptides which can then be further tested or validated in an array or individual assay.

[0104]

[0102] In certain embodiments, the CARs provided herein comprise an intracellular signaling region. In certain aspects, the intracellular signaling region comprising an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the intracellular domain pair is selected from the intracellular domain pairs listed in Table 1 below.

[0105] Table 1: Example intracellular domain pairs.

[0106]

[0103] In some embodiments, in Table 1, an ICD listed in the one column and another

[0107] ICD listed in the other column on the same line constitute an intracellular domain pair. For example, CD3d_ITAM and MyD88_CD40 (on line 1) constitute an intracellular domain pair.

[0108]

[0104] In some embodiments, the ICD1 and / or the ICD2 comprises a full-length of the ICD listed in Table 1 above. In some embodiments, the ICD1 and / or the ICD2 comprises a functionally active fragment of the ICD listed in Tables 1 above. The ICD1 and / or the ICD2 may be an activating domain, a co- stimulatory domain, an inhibitory domain, or a domain from other immune cell types, such as B cell, NK cell, or macrophage.

[0109]

[0105] In some embodiments, the ICD1 is positioned between the transmembrane domain and the ICD2. In some embodiments, the ICD2 is positioned between the transmembrane domain and the ICD1.

[0110]

[0106] In some embodiments, the CAR disclosed herein may comprise an ICD1 and ICD2 pair listed in Table 1 above, and the ICD1 and / or the ICD2 consists of or comprises an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an ICD amino acid sequence set forth in Table 2 below. In certain embodiments, the CAR disclosed herein comprises an ICD1 and ICD2 pair listed in Tables 1 above, and the ICD1 and / or the ICD2 consists of or comprises an amino acid sequence set forth in Table 2 below.

[0111]

[0107] In some embodiments, the CAR disclosed herein comprises an ICD1 listed in Table 2 below. In some embodiments, the CAR disclosed herein comprises an ICD2 listed in Table 2 below. In some embodiments, the CAR disclosed herein comprises any combination of two ICDs listed in Table 2.

[0112]

[0108] In some embodiments, the CAR disclosed herein comprises an ICD1 comprising an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an ICD amino acid sequence set forth in Table 2 below.

[0113]

[0109] In some embodiments, the CAR disclosed herein comprises an ICD2 comprising an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an ICD amino acid sequence set forth in Table 2 below.

[0110] In some embodiments, the CAR disclosed herein comprises any combination of two ICDs, wherein the two ICDs each comprise an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any two ICD amino acid sequences set forth in Table 2 below.

[0114] Table 2: Example ICD amino acid sequences

[0115]

[0111] Note that CTLA-4_ICM is also known as CTLA4. Example polynucleotide sequences corresponding to these amino acid sequences are provided in Table 9. Note also that 0X40 is represented by two sequences: a longer sequence, SEQ ID NO: 13; and an overlapping shorter sequence, SEQ ID NO: 21. The polynucleotide sequence corresponding to the amino acid sequence of SEQ ID NO: 13 is provided as SEQ ID NO: 391. The polynucleotide sequence corresponding to the amino acid sequence of SEQ ID NO: 21 is provided as SEQ ID NO: 468.

[0116]

[0112] In some embodiments, a CAR comprises: an antigen-binding domain, a hinge domain, a transmembrane domain, and an intracellular signaling region, wherein the intracellular signaling region comprises an ICD1 and an ICD2, wherein the CAR was identified in an vitro screen and then validated in one or more subsequent in vitro assay(s). These embodiments include CAR polypeptides comprising the following ICD1 and ICD2, respectively: FCER1G and MyD88_CD40, CD3z_D23mut and MyD88_CD40, CD79b_ITAM and MyD88_CD40, DAP12JTAM and 0X40, CD3z_D23mut and 0X40, and CD3z_D23mut and DR3, wherein the ICD1 is positioned between the transmembrane domain and the ICD2. In some embodiments, a CAR comprises: an antigen-binding domain, a transmembrane domain, and an intracellular signaling region, wherein the intracellular signaling region comprises an ICD1 and an ICD2, wherein the CAR was identified in an in vivo screen and then validated in one or more subsequent in vitro assay(s). These embodiments include CAR polypeptides comprising the following ICD1 and ICD2, respectively: CD3z_D23mut and CD3d, CD3d_ITAM and CD22, NKp46 and DAP12_ITAM, CD3z_D23mut and NKp46, CTLA-4_ICM and CD3g_ITAM, CD3z_ITAMl and CD8a, CD3z_D12mut and FCGR3A, CD79a and CD3g_ITAM, CD79a and NKp30, and FCER1G and FCER1G_ITAM, wherein the ICD1 is positioned between the transmembrane domain and the ICD2.

[0117]

[0113] In some embodiments, an intracellular signaling region comprises an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the intracellular domain pair is any of: CD3d_ITAM-CD22 (also known as: D3 or CD3d_ITAM.CD22); CD3z_D12mut-FCGR3A (also known as: D8 or CD3z_D12mut.FCGR3A); CD3z_D23mut-CD3d (also known as: B3 or CD3z_D23mut.CD3d); CD3z_D23mut-DR3 (also known as: CIO, CAR_C10 or CD3z_D23mut.DR3); CD3z_D23mut-MyD88_CD40 (also known as: Cl, CAR_C1 or CD3z_D23mut.MyD88_CD40); CD3z_D23mut-NKp46 (also known as: E10 or CD3z_D23mut.NKp46); CD3z_D23mut-OX40 (also known as: G8, CAR_G8, or CD3z_D23mut.OX40); CD3z_ITAMl-CD8a (also known as: B4 or CD3z_ITAMl.CD8a); CD79a-CD3g_ITAM (also known as: CD79a.CD3g_ITAM); CD79a-NKp30 (also known as: C6 or CD79a.NKp30); CD79b_ITAM-MyD88_CD40 (also known as: DI, CAR_D1 or CD79b_ITAM.MyD88_CD40); CTLA4-CD3g_ITAM (also known as: G3, CTLA_4.CD3g_ITAM or CTLA4.CD3g_ITAM); DAP12_ITAM-OX40 (also known as: G5, CAR_G5, or DAP12_ITAM.OX40); FCER1G-FCER1G_ITAM (also known as: D5 or FCER IG.FCER IGJTAM); FCERlG-MyD88_CD40 (also known as: Al, CAR_A1, or FCERlG.MyD88_CD40); or NKp46-DAP12_ITAM (also known as: G4 or

[0118] NKp46.DAP12_ITAM), wherein a hyphen (“-”) separates an ICD1 and ICD2, respectively.

[0119]

[0114] In some embodiments, a CAR (chimeric antigen receptor) comprises an intracellular signaling region.

[0120]

[0115] In some embodiments, an intracellular signaling region comprises two intracellular domains, wherein the two intracellular domains are: CD3d_ITAM and CD22.

[0121]

[0116] In some embodiments, an intracellular signaling region comprises two intracellular domains, wherein the two intracellular domains are: CD3z_D12mut and FCGR3A.

[0122]

[0117] In some embodiments, an intracellular signaling region comprises two intracellular domains, wherein the two intracellular domains are: CD3z_D23mut and CD3d.

[0123]

[0118] In some embodiments, an intracellular signaling region comprises two intracellular domains, wherein the two intracellular domains are: CD3z_D23mut and DR3.

[0124]

[0119] In some embodiments, an intracellular signaling region comprises two intracellular domains, wherein the two intracellular domains are: CD3z_D23mut and MyD88_CD40.

[0125]

[0120] In some embodiments, an intracellular signaling region comprises two intracellular domains, wherein the two intracellular domains are: CD3z_D23mut and NKp46. 1

[0121] In some embodiments, an intracellular signaling region comprises two intracellular domains, wherein the two intracellular domains are: CD3z_D23mut and 0X40.

[0126]

[0122] In some embodiments, an intracellular signaling region comprises two intracellular domains, wherein the two intracellular domains are: CD3z_ITAMl and CD8a.

[0127]

[0123] In some embodiments, an intracellular signaling region comprises two intracellular domains, wherein the two intracellular domains are: CD79a and CD3g_ITAM.

[0128]

[0124] In some embodiments, an intracellular signaling region comprises two intracellular domains, wherein the two intracellular domains are: CD79a and NKp30.

[0129]

[0125] In some embodiments, an intracellular signaling region comprises two intracellular domains, wherein the two intracellular domains are: CD79b_ITAM and MyD88_CD40.

[0130]

[0126] In some embodiments, an intracellular signaling region comprises two intracellular domains, wherein the two intracellular domains are: CTLA4 and CD3g_ITAM.

[0131]

[0127] In some embodiments, an intracellular signaling region comprises two intracellular domains, wherein the two intracellular domains are: DAP12_ITAM and 0X40.

[0132]

[0128] In some embodiments, an intracellular signaling region comprises two intracellular domains, wherein the two intracellular domains are: FCER1G and FCER I GJTAM.

[0133]

[0129] In some embodiments, an intracellular signaling region comprises two intracellular domains, wherein the two intracellular domains are: FCER1G and MyD88_CD40.

[0134]

[0130] In some embodiments, an intracellular signaling region comprises two intracellular domains, wherein the two intracellular domains are: NKp46 and DAP12_ITAM.

[0135]

[0131] In some embodiments, an intracellular signaling region comprises an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD1 and ICD2 are, in N- to C-terminal order: CD3d_ITAM and CD22, respectively.

[0136]

[0132] In some embodiments, an intracellular signaling region comprises an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD1 and ICD2 are, in N- to C-terminal order: CD3z_D12mut and FCGR3A, respectively.

[0137]

[0133] In some embodiments, an intracellular signaling region comprises an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD1 and ICD2 are, in N- to C-terminal order: CD3z_D23mut and CD3d, respectively.

[0138]

[0134] In some embodiments, an intracellular signaling region comprises an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD1 and ICD2 are, in N- to C-terminal order: CD3z_D23mut and DR3, respectively.

[0139]

[0135] In some embodiments, an intracellular signaling region comprises an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD1 and ICD2 are, in N- to C-terminal order: CD3z_D23mut and MyD88_CD40, respectively.

[0140]

[0136] In some embodiments, an intracellular signaling region comprises an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD1 and ICD2 are, in N- to C-terminal order: CD3z_D23mut and NKp46, respectively.

[0141]

[0137] In some embodiments, an intracellular signaling region comprises an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD1 and ICD2 are, in N- to C-terminal order: CD3z_D23mut and 0X40, respectively.

[0142]

[0138] In some embodiments, an intracellular signaling region comprises an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD1 and ICD2 are, in N- to C-terminal order: CD3z_ITAMl and CD8a, respectively.

[0143]

[0139] In some embodiments, an intracellular signaling region comprises an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD1 and ICD2 are, in N- to C-terminal order: CD79a and CD3g_ITAM, respectively.

[0144]

[0140] In some embodiments, an intracellular signaling region comprises an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD1 and ICD2 are, in N- to C-terminal order: CD79a and NKp30, respectively.

[0145]

[0141] In some embodiments, an intracellular signaling region comprises an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD1 and ICD2 are, in N- to C-terminal order: CD79b_ITAM and MyD88_CD40, respectively.

[0146]

[0142] In some embodiments, an intracellular signaling region comprises an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD1 and ICD2 are, in N- to C-terminal order: CTLA4 and CD3g_ITAM, respectively.

[0147]

[0143] In some embodiments, an intracellular signaling region comprises an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD1 and ICD2 are, in N- to C-terminal order: DAP12_ITAM and 0X40, respectively.

[0148]

[0144] In some embodiments, an intracellular signaling region comprises an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD1 and ICD2 are, in N- to C-terminal order: FCER1G and FCER1G_ITAM, respectively.

[0149]

[0145] In some embodiments, an intracellular signaling region comprises an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD1 and ICD2 are, in N- to C-terminal order: FCER1G and MyD88_CD40, respectively.

[0150]

[0146] In some embodiments, an intracellular signaling region comprises an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD1 and ICD2 are, in N- to C-terminal order: NKp46 and DAP12_ITAM, respectively.

[0151]

[0147] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are, in N- to C-terminal order: SEQ ID NO: 44 and SEQ ID NO: 33, respectively.

[0152]

[0148] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are, in N- to C-terminal order: SEQ ID NO: 25 and SEQ ID NO: 59, respectively.

[0153]

[0149] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are, in N- to C-terminal order: SEQ ID NO: 26 and SEQ ID NO: 43, respectively.

[0154]

[0150] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are, in N- to C-terminal order: SEQ ID NO: 26 and SEQ ID NO: 56, respectively.

[0155]

[0151] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are, in N- to C-terminal order: SEQ ID NO: 26 and SEQ ID NO: 10, respectively.

[0156]

[0152] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are, in N- to C-terminal order: SEQ ID NO: 26 and SEQ ID NO: 82, respectively.

[0157]

[0153] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are, in N- to C-terminal order: SEQ ID NO: 26 and SEQ ID NO: 13, respectively.

[0158]

[0154] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are, in N- to C-terminal order: SEQ ID NO: 47 and SEQ ID NO: 51, respectively.

[0159]

[0155] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are, in N- to C-terminal order: SEQ ID NO: 34 and SEQ ID NO: 46, respectively.

[0160]

[0156] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are, in N- to C-terminal order: SEQ ID NO: 34 and SEQ ID NO: 80, respectively.

[0161]

[0157] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are, in N- to C-terminal order: SEQ ID NO: 49 and SEQ ID NO: 10, respectively.

[0162]

[0158] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are, in N- to C-terminal order: SEQ ID NO: 7 and SEQ ID NO: 46, respectively.

[0163]

[0159] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are, in N- to C-terminal order: SEQ ID NO: 55 and SEQ ID NO: 13, respectively.

[0164]

[0160] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are, in N- to C-terminal order: SEQ ID NO: 17 and SEQ ID NO: 12, respectively.

[0165]

[0161] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are, in N- to C-terminal order: SEQ ID NO: 17 and SEQ ID NO: 10, respectively.

[0166]

[0162] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are, in N- to C-terminal order: SEQ ID NO: 82 and SEQ ID NO: 55, respectively.

[0167]

[0163] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are independently at least 90% identical to, in N- to C- terminal order: SEQ ID NO: 44 and SEQ ID NO: 33, respectively.

[0168]

[0164] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are independently at least 90% identical to, in N- to C- terminal order: SEQ ID NO: 25 and SEQ ID NO: 59, respectively.

[0169]

[0165] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are independently at least 90% identical to, in N- to C- terminal order: SEQ ID NO: 26 and SEQ ID NO: 43, respectively.

[0170]

[0166] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are independently at least 90% identical to, in N- to C- terminal order: SEQ ID NO: 26 and SEQ ID NO: 56, respectively.

[0171]

[0167] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are independently at least 90% identical to, in N- to C- terminal order: SEQ ID NO: 26 and SEQ ID NO: 10, respectively.

[0172]

[0168] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are independently at least 90% identical to, in N- to C- terminal order: SEQ ID NO: 26 and SEQ ID NO: 82, respectively.

[0173]

[0169] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are independently at least 90% identical to, in N- to C- terminal order: SEQ ID NO: 26 and SEQ ID NO: 13, respectively.

[0174]

[0170] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are independently at least 90% identical to, in N- to C- terminal order: SEQ ID NO: 47 and SEQ ID NO: 51, respectively.

[0175]

[0171] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are independently at least 90% identical to, in N- to C- terminal order: SEQ ID NO: 34 and SEQ ID NO: 46, respectively.

[0176]

[0172] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are independently at least 90% identical to, in N- to C- terminal order: SEQ ID NO: 34 and SEQ ID NO: 80, respectively.

[0177]

[0173] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are independently at least 90% identical to, in N- to C- terminal order: SEQ ID NO: 49 and SEQ ID NO: 10, respectively.

[0178]

[0174] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are independently at least 90% identical to, in N- to C- terminal order: SEQ ID NO: 7 and SEQ ID NO: 46, respectively.

[0179]

[0175] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are independently at least 90% identical to, in N- to C- terminal order: SEQ ID NO: 55 and SEQ ID NO: 13, respectively.

[0180]

[0176] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are independently at least 90% identical to, in N- to C- terminal order: SEQ ID NO: 17 and SEQ ID NO: 12, respectively.

[0181]

[0177] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are independently at least 90% identical to, in N- to C- terminal order: SEQ ID NO: 17 and SEQ ID NO: 10, respectively.

[0182]

[0178] In some embodiments, an intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the sequences of the ICD1 and ICD2 are independently at least 90% identical to, in N- to C- terminal order: SEQ ID NO: 82 and SEQ ID NO: 55, respectively.

[0183]

[0179] As used herein, a person of skill in the art would understand that “at least 90%” also includes at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity.

[0184]

[0180] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is or comprises: SEQ ID NO: 451.

[0181] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is or comprises: SEQ ID NO: 452.

[0185]

[0182] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is or comprises: SEQ ID NO: 453.

[0186]

[0183] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is or comprises: SEQ ID NO: 454.

[0187]

[0184] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is or comprises: SEQ ID NO: 455.

[0188]

[0185] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is or comprises: SEQ ID NO: 456.

[0189]

[0186] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is or comprises: SEQ ID NO: 457.

[0190]

[0187] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is or comprises: SEQ ID NO: 458.

[0191]

[0188] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is or comprises: SEQ ID NO: 459.

[0192]

[0189] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is or comprises: SEQ ID NO: 460.

[0193]

[0190] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is or comprises: SEQ ID NO: 461.

[0194]

[0191] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is or comprises: SEQ ID NO: 462.

[0192] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is or comprises: SEQ ID NO: 463.

[0195]

[0193] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is or comprises: SEQ ID NO: 464.

[0196]

[0194] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is or comprises: SEQ ID NO: 465.

[0197]

[0195] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is or comprises: SEQ ID NO: 466.

[0198]

[0196] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is at least 90% identical to: SEQ ID NO: 451.

[0199]

[0197] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is at least 90% identical to: SEQ ID NO: 452.

[0200]

[0198] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is at least 90% identical to: SEQ ID NO: 453.

[0201]

[0199] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is at least 90% identical to: SEQ ID NO: 454.

[0202]

[0200] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is at least 90% identical to: SEQ ID NO: 455.

[0203]

[0201] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is at least 90% identical to: SEQ ID NO: 456.

[0204]

[0202] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is at least 90% identical to: SEQ ID NO: 457.

[0203] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is at least 90% identical to: SEQ ID NO: 458.

[0205]

[0204] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is at least 90% identical to: SEQ ID NO: 459.

[0206]

[0205] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is at least 90% identical to: SEQ ID NO: 460.

[0207]

[0206] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is at least 90% identical to: SEQ ID NO: 461.

[0208]

[0207] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is at least 90% identical to: SEQ ID NO: 462.

[0209]

[0208] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is at least 90% identical to: SEQ ID NO: 463.

[0210]

[0209] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is at least 90% identical to: SEQ ID NO: 464.

[0211]

[0210] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is at least 90% identical to: SEQ ID NO: 465.

[0212]

[0211] In some embodiments, a CAR comprises an antigen-binding region, a transmembrane domain, and an intracellular signaling region, wherein the sequence of the intracellular signaling region is at least 90% identical to: SEQ ID NO: 466.

[0213]

[0212] In some embodiments, the CAR described herein comprises an intracellular signaling region consisting of or comprising an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence set forth in Table 3 below. In certain embodiments, the CAR comprises an intracellular signaling region that is or comprises an amino acid sequence that is identical to a sequence set forth in Table 3, but for 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 sequence differences (amino acid additions, deletions, and / or substitutions). In some embodiments, the sequence differences are amino acid substitutions (e.g., conservative amino acid substitutions). In certain embodiments, the CAR disclosed herein comprises an intracellular signaling region consisting of or comprising an amino acid sequence as set forth in Table 3 below.

[0214] Table 3: Example intracellular signaling regions.

[0215]

[0213] In some embodiments, the ICD1 and the ICD2 are connected with a peptide linker. The peptide linker may be flexible and may not affect ICD functions. In certain embodiments, the peptide linker is glycine-glycine (GG). In some embodiments, longer and flexible linker sequences, such as GGGGS (SEQ ID NO: 201), can also be utilized. In some embodiments, the linker is GGAGGC (SEQ ID NO: 202). In some embodiments, linkers at the front of ICD1 and the back of ICD2 that bridge ICD combinations to the CAR backbone are single glycine residues to minimize any potential effect from linker sequences.

[0216]

[0214] In certain embodiments, the binding domain and / or extracellular domain of a CAR provided herein provides the CAR with the ability to bind to the target antigen of interest. A binding domain (e.g., a ligand-binding domain or antigen-binding domain) can be any protein, polypeptide, oligopeptide, or peptide that possesses the ability to specifically recognize and bind to a biological molecule (e.g., a cell surface receptor or tumor protein, or a component thereof). A binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule of interest. For example, and as further described herein, a binding domain may be antibody light chain and heavy chain variable regions, or the light and heavy chain variable regions can be joined together in a single chain and in either orientation (e.g., VL- Vnor VH-VL). A variety of assays are known for identifying binding domains of the present disclosure that specifically bind with a particular target, including Western blot, ELISA, flow cytometry, or surface plasmon resonance analysis (e.g., using BIACORE analysis). The target may be an antigen of clinical interest against which it would be desirable to trigger an effector immune response that results in tumor killing.

[0217]

[0215] In one embodiment, the binding domain of the CAR is a single chain fragment variable (scFv) specific for a cancer antigen, and may be a murine, human or humanized scFv. Single chain antibodies may be cloned from the V region genes of a hybridoma specific for a desired target. A technique which can be used for cloning the variable region heavy chain (VH) and variable region light chain (VL) has been described, for example, in Orlandi et al., PNAS, 1989; 86: 3833-3837. Thus, in certain embodiments, a binding domain comprises an antibody-derived binding domain but can be a non-antibody derived binding domain. An antibody-derived binding domain can be a fragment of an antibody or a genetically engineered product of one or more fragments of the antibody, which fragment is involved in binding with the antigen.

[0218]

[0216] In some embodiments, the CAR further comprises a single chain fragment variable (scFv) domain. The scFv domain may be a scFv domain set forth in Table 4 below, or a scFv that includes complementarity determining region (CDR) and / or variable region sequences set forth in Table 4. In some embodiments, the CAR comprises an antigenbinding domain (e.g., a scFv domain) comprising heavy chain and light chain CDR sequences of an antibody or scFv provided in Table 4. In some embodiments, the CAR comprises an antigen-binding domain (e.g., a scFv domain) comprising a heavy chain variable region sequence and a light chain variable region sequence of an antibody or scFv provided in Table 4. In some embodiments, the CAR further comprises a scFv domain consisting of or comprising an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence set forth in Table 4.

[0219]

[0217] In certain embodiments, the CAR polypeptide comprises a means for binding a target antigen. Example structures corresponding to means for binding antigens are provided in Table 4.

[0220] Table 4: Example binding domain sequences

[0221]

[0218] In some embodiments, the CAR described herein further comprises an antigen-binding domain. The term “antigen-binding domain” or “antigen binding fragment' of an antibody as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., ROR1). It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments may also be bispecific, dual specific, or multi- specific formats; specifically binding to two or more different antigens. Examples of binding fragments encompassed within the term “antigen-binding domain” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546, Winter et al., PCT publication WO 90 / 05144 Al herein incorporated by reference), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding domain” of an antibody. In certain embodiments, scFv molecules may be incorporated into a fusion protein. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R.J., et al. (1994) Structure 2:1121-1123). Such antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5). In some embodiments, the antigen-binding domain is a scFv domain described herein.

[0222]

[0219] In some embodiments, the antigen-binding domain binds to a cancer antigen.

[0223] The cancer antigen may be selected from the cancer antigens listed in Table 5 below. In some embodiments, as is known to one of ordinary skill in the art, an antigen such as a cancer antigen can comprise one or more antigenic domains, and an individual antigenic domain may be sufficient for recognition by an antigen-binding domain.

[0224] Table 5: Example cancer antigens

[0220] In certain embodiments, the CARs of the present disclosure may comprise a linker between the various domains, added for appropriate spacing and conformation of the molecule. For example, in one embodiment, there may be a linker between the binding domain VH or VL which may be between 1-10 amino acids long. In other embodiments, the linker between any of the domains of the chimeric antigen receptor may be between 1-20 or more than 20 amino acids long. In this regard, the linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids long. In further embodiments, the linker may be 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids long. Ranges including the numbers described herein are also included herein, e.g., a linker 10-30 amino acids long.

[0225]

[0221] In certain embodiments, linkers suitable for use in the CAR described herein are flexible linkers. Suitable linkers can be readily selected and can be of any of a suitable of different lengths, such as from 1 amino acid e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0226]

[0222] Example flexible linkers include glycine polymers (G)n, glycine- serine polymers, where n is an integer of at least one, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine- serine polymers are relatively unstructured, and therefore may be able to serve as a neutral tether between domains of fusion proteins such as the CARs described herein. Glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains. The ordinarily skilled artisan will recognize that design of a CAR can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure to provide for a desired CAR structure. The linker may be placed between any two components of the CAR. In some embodiments, the CAR may comprise a plurality of linkers.

[0227]

[0223] In some embodiments, the binding domain of the CAR may be followed by a “spacer,” or, “hinge,” which refers to the region that moves the antigen-binding domain away from the effector cell surface to enable proper cell / cell contact, antigen-binding and activation (Patel et al., Gene Therapy, 1999; 6: 412-419). The hinge region in a CAR is generally between the transmembrane (TM) and the binding domain. In certain embodiments, a hinge region is an immunoglobulin hinge region and may be a wild type immunoglobulin hinge region or an altered wild type immunoglobulin hinge region. Other example hinge regions used in the CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8a, CD4, CD28 and CD7, which may be wild-type hinge regions from these molecules or may be altered. In some embodiments, a hinge domain is referenced as a hinge region. In some embodiments, any domain described herein can also be referenced as a region.

[0228]

[0224] In some embodiments, the CAR described herein further comprises a hinge domain. The hinge domain may be selected from the hinge domains of Table 6 below. In certain embodiments, the hinge domain is an IgG4 hinge domain. In some embodiments, the CAR further comprises a hinge domain comprising an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence set forth in Table 6.

[0229] Table 6: Example hinge domains

[0230]

[0225] In some embodiments, the CAR described herein further comprises a transmembrane domain. The “transmembrane” region or domain is the portion of the CAR that anchors the extracellular binding portion to the plasma membrane of the immune effector cell, and facilitates binding of the binding domain to the target antigen. In some embodiments, the transmembrane domain may be a CD3(^ transmembrane domain. Other transmembrane domains that may be employed in some embodiments include those obtained from CD8, CD8a, CD4, CD28, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD134, CD137, and CD154. In certain embodiments, the transmembrane domain is synthetic in which case it would comprise predominantly hydrophobic residues such as leucine and valine.

[0231]

[0226] In some embodiments, the transmembrane domain is selected from a transmembrane domain of CD28, CD8a, ICOS, 4- IBB, CD4, Tim4, 0X40, CD27, CD2, LFA-1, CD30, CD40, PD-1, CD7, LIGHT, NKG2C, B7-H3, NKG2D, NKp44, NKp46, DAP12, CD16, NKp30, FcRy, DAP10, 2B4, or DNAM-1. In some embodiments, the transmembrane domain may be selected from the transmembrane domains of Table 7 below. In certain embodiments, the transmembrane domain is a CD28 transmembrane domain. In some embodiments, the CAR further comprises a transmembrane domain comprising an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence set forth in Table 7.

[0232] Table 7: Example transmembrane domains

[0233]

[0234]

[0227] The CAR polypeptides encompassed by the present disclosure may comprise an amino acid sequence set forth in Table 8 below.

[0235]

[0228] In various non-limiting example CAR polypeptides provided in Table 8, the CAR polypeptide comprises, in N- to C-terminal order: an antigen-binding domain, a transmembrane domain, and an intercellular region comprising, in N- to C-terminal order, an ICD1 and an ICD2.

[0229] In various non-limiting example CAR polypeptides provided in Table 8, the CAR polypeptide comprises an antigen-binding domain which binds to GD2. In various non-limiting example CAR polypeptides provided in Table 8, the CAR polypeptide comprises an antigen-binding domain which binds to GD2, which antigen-binding domain comprises the sequence:

[0236] EFGLSWLFLVAILKGVQCSRDYKDDDDKDILLTQTPLSLPVSLGDQASISCRSSQS LVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRV EAEDLGVYFCSQSTHVPPLTFGAGTKLELKRADAAPTVSIFPGSGGGGSGGEVKL QQSGPSLVEPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTS YNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVS SAKTTPPSVYGRVTVSSA (SEQ ID NO: 485).

[0237]

[0230] In various non-limiting example CAR polypeptides provided in Table 8, the CAR polypeptide comprises a hinge domain represented by SEQ ID NO: 211; and a transmembrane domain which is a human CD28-TMD1 (SEQ ID NO: 227).

[0238] Table 8. Example CAR polypeptides

[0239]

[0231] In some embodiments, a CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling region, wherein the antigen-binding domain binds the antigen GD2, and wherein the intracellular signaling region comprises an ICD1 and an ICD2 , wherein the ICD1 is positioned between the transmembrane domain and the ICD2; and wherein the sequence of the CAR is, comprises, or is at least 90% identical to, any sequence in Table 8. In some embodiments, an optional hinge domain is positioned between the antigen-binding domain and the transmembrane domain.

[0240]

[0232] In some embodiments, a CAR comprises an antigen-binding domain, a hinge domain, a transmembrane domain, and an intracellular signaling region, wherein the antigen-binding domain binds the antigen GD2, and wherein the intracellular signaling region comprises an ICD1 and an ICD2, wherein the ICD1 is positioned between the transmembrane domain and the ICD2; and wherein the sequence of the CAR is, comprises, or is at least 90% identical to, any of SEQ ID NO: 269-371 or 486 to 501. In some embodiments, a CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling region, wherein the antigen-binding domain binds the antigen GD2, and wherein the intracellular signaling region comprises an ICD1 and an ICD2, wherein the ICD1 is positioned between the transmembrane domain and the ICD2; and wherein the sequence of the CAR is, comprises, or is at least 90% identical to, any of SEQ ID NO: 269-371 or 486 to 501.

[0241]

[0233] Polypeptides having substantial sequence similarities can have the same or similar catalytic and / or functional activity. Accordingly, in some embodiments, a derivative, equivalent, variant, fragment, or mutant of a CAR described herein or fragment thereof may also be suitable for the methods and compositions provided herein. In some embodiments, the CAR further comprises an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence set forth in Table 8. In certain embodiments, the CAR is or comprises an amino acid sequence that is identical to a sequence set forth in Table 8, but for 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 sequence differences (amino acid additions, deletions, and / or substitutions). In some embodiments, the sequence differences are amino acid substitutions (e.g., conservative amino acid substitutions).

[0242]

[0234] In some embodiments, variations or derivatives of the CARs are provided herein. The altered CAR polypeptide may have an altered amino acid sequence, for example by conservative substitution, yet still induces antigen- specific proliferation, cytotoxicity, cytokine secretion, and / or other immune cell phenotypes, and are considered functional equivalents. As used herein, the term “conservative substitution” denotes the replacement of an amino acid residue by another, biologically similar residue. It is well known in the art that the amino acids within the same conservative group may typically substitute for one another without substantially affecting the function of a protein. According to certain embodiments, the derivatives, equivalents, variants, or mutants of a CAR are polypeptides that are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to an amino acid sequence of a CAR described herein (e.g., the amino acid sequences listed in Table 8) or fragment thereof. In some embodiments, the identity is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more.

[0243]

[0235] In some embodiments, the CAR polypeptides encompassed by the present disclosure may consist of or comprise an amino acid sequence derived from a CAR polypeptide set forth in Table 8. In some embodiments, the CAR polypeptides may include conservative or non-conservative mutations. A CAR polypeptide may comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more mutations. In some embodiments, a CAR polypeptide may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more mutations.

[0244] Nucleic Acids and Vectors

[0245]

[0236] In certain aspects, also disclosed are nucleic acids and polynucleotide vectors encoding the CAR polypeptides disclosed herein.

[0246]

[0237] In some embodiments, the nucleic acids and / or polynucleotide vectors encoding the CAR polypeptides disclosed herein comprise an ICD1 -encoding nucleic acid sequence set forth in Table 9 below, and / or an ICD2-encoding nucleic acid sequence set forth in Table 9 below. As is known to one of ordinary skill in the art, an amino acid sequence may be encoded by any number of different polynucleotide sequences; the polynucleotides can be, for example, codon-optimized to optimize expression in a particular cell or cell line.

[0247] Table 9: Example ICD-encoding nucleic acid sequences

[0248]

[0238] Non-limiting examples of some polynucleotide sequences encoding selected intracellular signaling regions are provided below, in Table 10. Table 10: Example intracellular signaling region-encoding nucleic acid sequences

[0249]

[0239] Nucleic acid sequences encoding the disclosed CARs, and regions thereof, can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than cloned.

[0250]

[0240] Expression of nucleic acids encoding CARs is typically achieved by operably linking a nucleic acid encoding the CAR polypeptide to a promoter, and incorporating the construct into an expression vector. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

[0251]

[0241] In certain embodiments, the polynucleotide encoding the CAR described herein is inserted into a vector. The vector is a vehicle into which a polynucleotide encoding a protein may be covalently inserted so as to bring about the expression of that protein and / or the cloning of the polynucleotide. Such vectors may also be referred to as “expression vectors”. The isolated polynucleotide may be inserted into a vector using any suitable methods known in the art, for example, without limitation, the vector may be digested using appropriate restriction enzymes and then may be ligated with the isolated polynucleotide having matching restriction ends. Expression vectors have the ability to incorporate and express heterologous or modified nucleic acid sequences coding for at least part of a gene product capable of being transcribed in a cell. In most cases, RNA molecules are then translated into a protein. Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operatively linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are discussed infra. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.

[0252]

[0242] The expression vector may have the necessary 5' upstream and 3' downstream regulatory elements such as promoter sequences such as CMV, PGK and EFla promoters, ribosome recognition and binding TATA box, and 3' UTR AAUAAA transcription termination sequence for the efficient gene transcription and translation in its respective host cell. Other suitable promoters include the constitutive promoter of simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), HIV LTR promoter, MoMuLV promoter, avian leukemia virus promoter, EBV immediate early promoter, and Rous Sarcoma Virus promoter. Human gene promoters may also be used, including, but not limited to the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. In certain embodiments, inducible promoters are also contemplated as part of a vector expressing a chimeric antigen receptor. This provides a molecular switch capable of turning on expression of the polynucleotide sequence of interest or turning off expression. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, or a tetracycline promoter.

[0253]

[0243] The expression vector may have additional sequences such as 6x-histidine, c- Myc, and FLAG tags which are incorporated into the expressed CARs. Thus, the expression vector may be engineered to contain 5' and 3' untranslated regulatory sequences that sometimes can function as enhancer sequences, promoter regions and / or terminator sequences that can facilitate or enhance efficient transcription of the nucleic acid(s) of interest carried on the expression vector. An expression vector may also be engineered for replication and / or expression functionality (e.g., transcription and translation) in a particular cell type, cell location, or tissue type. Expression vectors may include a selectable marker for maintenance of the vector in the host or recipient cell.

[0254]

[0244] In various embodiments, the vectors are plasmids, autonomously replicating sequences, and transposable elements. Additional example vectors include, without limitation, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or Pl-derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses. Examples of categories of animal viruses useful as vectors include, without limitation, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40). Examples of expression vectors are Lenti-X™ Bicistronic Expression System (Neo) vectors (Clontech), pClneo vectors (Promega) for expression in mammalian cells; pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2N5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. The coding sequences of the CARs disclosed herein can be ligated into such expression vectors for the expression of the chimeric protein in mammalian cells.

[0255]

[0245] In certain embodiments, the nucleic acids encoding the CAR are provided in a viral vector. A viral vector can be derived from, for example, a retrovirus (e.g., a foamy virus) or lentivirus. As used herein, the term, “viral vector,” refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain the coding sequence for the various chimeric proteins described herein in place of nonessential viral genes. The vector and / or particle can be utilized for the purpose of transferring DNA, RNA or other nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0256]

[0246] In certain embodiments, the viral vector containing the coding sequence for a CAR described herein is a retroviral vector or a lentiviral vector. The term “retroviral vector” refers to a vector containing structural and functional genetic elements that are primarily derived from a retrovirus.

[0257]

[0247] Retroviral vectors for use herein can be derived from any known retrovirus (e.g., type c retroviruses, such as Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)). Retroviruses” also include human T cell leukemia viruses, HTLV-1 and HTLV-2, and the lentiviral family of retroviruses, such as Human Immunodeficiency Viruses, HIV-1, HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine immunodeficiency virus (EIV), and other classes of retroviruses.

[0258]

[0248] A lentiviral vector refers to a vector derived from a lentivirus, a group (or genus) of retroviruses that give rise to slowly developing disease. Viruses included within this group include HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2); visna-maedi; a caprine arthritis-encephalitis virus; equine infectious anemia virus; feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV). Preparation of the recombinant lentivirus can be achieved using the methods according to Dull et al. and Zufferey et al. (Dull et al., J. Virol., 1998; 72: 8463-8471 and Zufferey et al., J. Virol. 1998; 72:9873-9880).

[0259]

[0249] Retroviral vectors (i.e., both lentiviral and non-lentiviral) can be formed using standard cloning techniques by combining the desired DNA sequences in the order and orientation described herein (Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals; Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141- 6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science 254:1802- 1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol 150:4104-4115; U.S. Pat. Nos. 4,868,116; 4,980,286; PCT Application WO 89 / 07136; PCT Application WO 89 / 02468; PCT Application WO 89 / 05345; and PCT Application WO 92 / 07573).

[0260]

[0250] Suitable sources for obtaining retroviral (i.e., both lentiviral and non-lentiviral) sequences for use in forming the vectors include, for example, genomic RNA and cDNAs available from commercially available sources, including the Type Culture Collection (ATCC), Rockville, Md. The sequences also can be synthesized chemically.

[0261]

[0251] For expression of a CAR, the vector may be introduced into a host cell to allow expression of the polypeptide within the host cell. The expression vectors may contain a variety of elements for controlling expression, including without limitation, promoter sequences, transcription initiation sequences, enhancer sequences, selectable markers, and signal sequences. These elements may be selected as appropriate by a person of ordinary skill in the art, as described above. For example, the promoter sequences may be selected to promote the transcription of the polynucleotide in the vector. Suitable promoter sequences include, without limitation, T7 promoter, T3 promoter, SP6 promoter, beta- actin promoter, EFla promoter, CMV promoter, and SV40 promoter. Enhancer sequences may be selected to enhance the transcription of the polynucleotide. Selectable markers may be selected to allow selection of the host cells inserted with the vector from those not, for example, the selectable markers may be genes that confer antibiotic resistance. Signal sequences may be selected to allow the expressed polypeptide to be transported outside of the host cell.

[0262]

[0252] For cloning of the polynucleotide, the vector may be introduced into a host cell (an isolated host cell) to allow replication of the vector itself and thereby amplify the copies of the polynucleotide contained therein. The cloning vectors may contain sequence components generally include, without limitation, an origin of replication, promoter sequences, transcription initiation sequences, enhancer sequences, and selectable markers. These elements may be selected as appropriate by a person of ordinary skill in the art. For example, the origin of replication may be selected to promote autonomous replication of the vector in the host cell.

[0263]

[0253] In certain embodiments, the present disclosure provides isolated host cells containing the vectors provided herein. The host cells containing the vector may be useful in expression or cloning of the polynucleotide contained in the vector. Suitable host cells can include, without limitation, prokaryotic cells and eukaryotic cells such as mammalian cells, such as human cells.

[0264]

[0254] The CARs are introduced into a host cell using transfection and / or transduction techniques known in the art. As used herein, the terms, “transfection,” and, “transduction,” refer to the processes by which an exogenous nucleic acid sequence is introduced into a host cell. The nucleic acid may be integrated into the host cell DNA or may be maintained extrachromosomally. The nucleic acid may be maintained transiently or may be a stable introduction. Transfection may be accomplished by a variety of means known in the art including but not limited to calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics. Transduction refers to the delivery of a gene(s) using a viral or retroviral vector by means of viral infection rather than by transfection. In certain embodiments, retroviral vectors are transduced by packaging the vectors into virions prior to contact with a cell. For example, a nucleic acid encoding a CAR carried by a retroviral vector can be transduced into a cell through infection and pro virus integration.

[0265]

[0255] In order to assess the expression of a CAR polypeptide or portions thereof, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes.

[0266]

[0256] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene. Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter- driven transcription.

[0267]

[0257] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0258] In the case where a non-viral delivery system is utilized, an example delivery vehicle is a liposome. In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc, (Birmingham, Ala.).

[0268] Immune effector cells

[0269]

[0259] In certain aspects, also disclosed herein are cells that are engineered to express the disclosed CAR polypeptides. In some embodiments, the cells engineered to express the disclosed CAR polypeptides are immune effector cells. In some embodiments, other types of cells, such as induced pluripotent stem cells (iPSC) or hematopoietic stem cells (HSC), are engineered and differentiated into immune effector cells that express the disclosed CAR polypeptides.

[0270]

[0260] In some embodiments, the iPSC, HSC, or immune effector cells are obtained from the subject to be treated (i.e., are autologous). However, in certain embodiments, immune effector cell lines or donor effector cells (allogeneic) are used.

[0271]

[0261] Immune effector cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. Immune effector cells can be obtained from blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. For example, cells from the circulating blood of an individual may be obtained by apheresis. In some embodiments, immune effector cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. A specific subpopulation of immune effector cells can be further isolated by positive or negative selection techniques. For example, immune effector cells can be isolated using a combination of antibodies directed to surface markers unique to the positively selected cells, e.g., by incubation with antibody-conjugated beads for a time period sufficient for positive selection of the desired immune effector cells. Alternatively, enrichment of immune effector cells population can be accomplished by negative selection using a combination of antibodies directed to surface markers unique to the negatively selected cells.

[0272]

[0262] In certain embodiments, the present disclosure provides methods for making the immune effector cells which express the CARs described herein. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from a subject, such as a subject having a cancer antigen e.g., GD2) expressing tumor cell, such that the immune effector cells express one or more CAR as described herein. In certain embodiments, the immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro. Such cells can then be directly readministered into the individual. In further embodiments, the immune effector cells are first activated and stimulated to proliferate in vitro prior to being genetically modified to express a CAR. In this regard, the immune effector cells may be cultured before or after being genetically modified (i.e., transduced or transfected to express a CAR as described herein).

[0273]

[0263] Prior to in vitro manipulation or genetic modification of the immune effector cells described herein, the source of cells may be obtained from a subject. In particular, the immune effector cells for use with the CARs as described herein comprise T cells. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cell can be obtained from a unit of blood collected from the subject using any number of techniques known to the skilled person, such as FICOLL separation. In one embodiment, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing. In one embodiment, the cells are washed with PBS. In an alternative embodiment, the washed solution lacks calcium, and may lack magnesium or may lack many, if not all, divalent cations. As would be appreciated by those of ordinary skill in the art, a washing step may be accomplished by methods known to those in the art, such as by using a semiautomated flowthrough centrifuge. After washing, the cells may be resuspended in a variety of biocompatible buffers or other saline solution with or without buffer. In certain embodiments, the undesirable components of the apheresis sample may be removed in the cell directly resuspended culture media.

[0274]

[0264] In certain embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient. A specific subpopulation of T cells, such as CD28+, CD4+, CD8+, CD45RA+, and CD45RO+T cells, can be further isolated by positive or negative selection techniques. For example, enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method for use herein is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD 14, CD20, CD1 b, CD 16, HLA-DR, and CD8. Flow cytometry and cell sorting may also be used to isolate cell populations of interest.

[0275]

[0265] PBMCs may be used directly for genetic modification with the CARs using methods as described herein. In certain embodiments, after isolation of PBMC, T lymphocytes are further isolated and in certain embodiments, both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after genetic modification and / or expansion. CD8+cells can be obtained by using standard methods. In some embodiments, CD8+cells are further sorted into naive, central memory, and effector cells by identifying cell surface antigens that are associated with each of those types of CD8+cells. In embodiments, memory T cells are present in both CD62L+and CD62L" subsets of CD8+peripheral blood lymphocytes. PBMC are sorted into CD62L" CD8+and CD62L+CD8+fractions after staining with anti-CD8 and anti-CD62L antibodies. In some embodiments, the expression of phenotypic markers of central memory TCM include CD45RO, CD62L, CCR7, CD28, CD3, and CD 127 and are negative for granzyme B. In some embodiments, central memory T cells are CD45RO+, CD62L+, CD8+T cells. In some embodiments, effector T cells are negative for CD62L, CCR7, CD28, and CD 127, and positive for granzyme B and perforin. In some embodiments, naive CD8+T lymphocytes are characterized by the expression of phenotypic markers of naive T cells including CD62L, CCR7, CD28, CD3, CD 127, and CD45RA.

[0276]

[0266] In certain embodiments, CD4+T cells are further sorted into subpopulations. For example, CD4+T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+T lymphocytes are CD45RO-, CD45RA+, CD62L+CD4+T cell. In some embodiments, central memory CD4+cells are CD62L positive and CD45RO positive. In some embodiments, effector CD4+cells are CD62L and CD45RO negative.

[0277]

[0267] The immune effector cells, such as T cells, can be genetically modified following isolation using known methods, or the immune effector cells can be activated and expanded (or differentiated in the case of progenitors) in vitro prior to being genetically modified. In another embodiment, the immune effector cells, such as T cells, are genetically modified with the chimeric antigen receptors described herein (e.g., transduced with a viral vector comprising a nucleic acid encoding a CAR) and then are activated and expanded in vitro. Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. Pat. Nos. 6,905,874; 6,867,041; 6,797,514; W02012079000.

[0278] Generally, such methods include contacting PBMC or isolated T cells with a stimulatory agent and costimulatory agent, such as anti-CD3 and anti-CD28 antibodies, generally attached to a bead or other surface, in a culture medium with appropriate cytokines, such as IL-2 (e.g., recombinant human IL-2). Anti-CD3 and anti-CD28 antibodies attached to the same bead serve as a “surrogate” antigen presenting cell (APC). In other embodiments, the T cells may be activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines using methods such as those described in U.S. Pat. Nos.

[0279] 6,040,177; 5,827,642; and WO2012129514.

[0280]

[0268] In some embodiments, the immune effector cells comprise any leukocyte involved in defending the body against infectious disease and foreign materials. For example, the immune effector cells can comprise lymphocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, or any combinations thereof. For example, the immune effector cells can comprise T lymphocytes, preferably cytotoxic T lymphocytes (CTLs). In some embodiments, the immune effector cell is an NK cell, a regulatory T cell (Treg), a gamma delta T cell, a macrophage, a B cell, an NKT cell, or a plasmacytoid dendritic cell.

[0281]

[0269] T helper cells (TH cells) assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. These cells are also known as CD4+T cells because they express the CD4 glycoprotein on their surface. Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response. These cells can differentiate into one of several subtypes, including Thl, Th2, Th3, Thl7, Th9, or Tfh, which secrete different cytokines to facilitate a different type of immune response.

[0282]

[0270] Cytotoxic T cells (Tc cells, or CTLs) destroy virally infected cells and tumor cells, and are also implicated in transplant rejection. These cells are also known as CD8+T cells since they express the CD8 glycoprotein at their surface. These cells recognize their targets by binding to antigen associated with MHC class I molecules, which are present on the surface of all nucleated cells. Through IL- 10, adenosine and other molecules secreted by regulatory T cells, the CD8+cells can be inactivated to an anergic state, which prevents autoimmune diseases.

[0283]

[0271] Memory T cells are a subset of antigen- specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen, thus providing the immune system with “memory” against past infections. Memory cells may be either CD4+or CD8+. Memory T cells typically express the cell surface protein CD45RO.

[0272] Regulatory T cells (Tregcells), formerly known as suppressor T cells, are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell-mediated immunity toward the end of an immune reaction and to suppress auto- reactive T cells that have escaped the process of negative selection in the thymus. Two major classes of CD4+Tregcells have been described: naturally occurring Tregcells and adaptive Tregcells.

[0284]

[0273] Natural killer T (NKT) cells (not to be confused with natural killer (NK) cells) bridge the adaptive immune system with the innate immune system. Unlike conventional T cells that recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize a glycolipid antigen presented by a molecule called CD Id.

[0285]

[0274] In some embodiments, the T cells comprise a mixture of CD4+cells. In other embodiments, the T cells are enriched for one or more subsets based on cell surface expression. For example, in some cases, the T cells are cytotoxic CD8+T lymphocytes.

[0286]

[0275] Natural-killer (NK) cells are CD56+CD3 large granular lymphocytes that can kill virally infected and transformed cells, and constitute a critical cellular subset of the innate immune system (Godfrey J, et al. Leuk Lymphoma 2012 53:1666-1676). Unlike cytotoxic CD8+T lymphocytes, NK cells launch cytotoxicity against tumor cells without the requirement for prior sensitization, and can eradicate MHC-I-negative cells (Nami- Mancinelli E, et al. Int Immunol 2011 23:427-431). NK cells are safer effector cells, as they may avoid the potentially lethal complications of cytokine storms (Morgan RA, et al. Mol Ther 2010 18:843-851), tumor lysis syndrome (Porter DL, et al. N Engl J Med 2011 365:725-733), and on-target, off-tumor effects.

[0287] Binding Properties of Chimeric Antigen Receptors

[0288]

[0276] As used herein, the term “binding” in the context of the binding of a chimeric antigen receptor to, e.g., a predetermined antigen, such as a cell surface protein or fragment thereof (or to an antigen bound to a cell surface protein such as an HLA molecule). Binding typically refers to an interaction or association between a minimum of two entities or molecular structures, such as an antigen-binding domain: antigen interaction. For instance, binding affinity typically corresponds to a KD value of about 10'7M or less, such as about 10'8M or less, such as about 10'9M or less when determined by, for instance, surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the antibody or chimeric antigen receptor as the analyte (or antiligand). The term “KD” (M) refers to the dissociation equilibrium constant of a particular antigen-binding domain: antigen interaction. There is an inverse relationship between KD and binding affinity, therefore the smaller the KD value, the higher, i.e., stronger, the affinity. Thus, the terms “higher affinity” or “stronger affinity” relate to a higher ability to form an interaction and therefore a smaller KD value, and conversely the terms “lower affinity” or “weaker affinity” relate to a lower ability to form an interaction and therefore a larger KD value. In some circumstances, a higher binding affinity (or KD) of a particular molecule (e.g., a chimeric antigen receptor) to its interactive partner molecule (e.g., antigen X) compared to the binding affinity of the molecule (e.g., chimeric antigen receptor) to another interactive partner molecule (e.g., antigen Y) may be expressed as a binding ratio determined by dividing the larger KD value (lower, or weaker, affinity) by the smaller KD (higher, or stronger, affinity), for example, expressed as 5-fold or 10-fold greater binding affinity, as the case may be. Cell-based binding strategies, such as fluorescent-activated cell sorting (FACS) binding assays, are also routinely used, and FACS data correlates well with other methods such as radioligand competition binding and SPR (Benedict, CA, J Immunol Methods. 1997, 201(2):223-31; Geuijen, CA, et al. J Immunol Methods. 2005, 302(l-2):68- 77).

[0289]

[0277] Accordingly, in some embodiments, a chimeric antigen receptor of the present disclosure binds to the predetermined antigen or cell surface molecule (receptor) having an affinity corresponding to a KD value that is at least ten-fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein). As described herein, a chimeric antigen receptor of the present disclosure can bind to an HLA-presented antigen described herein. According to the present disclosure, in some embodiments, the affinity of a chimeric antigen receptor with a KD value that is equal to or less than ten-fold lower than a non-specific antigen may be considered non-detectable binding.

[0290]

[0278] The term “kd” (sec-1or 1 / s) refers to the dissociation rate constant of a particular antigen-binding domain: antigen interaction, or the dissociation rate constant of a chimeric antigen receptor. Said value is also referred to as the koff value.

[0291]

[0279] The term “ka” (M'1x sec'1or 1 / M x 1 / s) refers to the association rate constant of a particular antigen-binding domain: antigen interaction, or the association rate constant of a chimeric antigen receptor.

[0292]

[0280] The term “KA” (M'1or 1 / M) refers to the association equilibrium constant of a particular antigen-binding domain: antigen interaction, or the association equilibrium constant of a chimeric antigen receptor. The association equilibrium constant is obtained by dividing the kaby the kd.

[0293]

[0281] The term “EC50” or “EC50” refers to the half-maximal effective concentration, which includes the concentration of a chimeric antigen receptor that induces a response halfway between the baseline and maximum after a specified exposure time. The EC50 essentially represents the concentration of a chimeric antigen receptor where 50% of its maximal effect is observed. In certain embodiments, the EC50 value equals the concentration of a chimeric antigen receptor of the present disclosure that gives half- maximal binding to cells expressing an antigen (e.g., a tumor-associated antigen), as determined by e.g., a FACS binding assay. Thus, reduced or weaker binding is observed with an increased EC50, or half-maximal effective concentration value.

[0294]

[0282] In one embodiment, decreased binding can be defined as an increased EC50 chimeric antigen receptor concentration that enables binding to the half-maximal amount of target cells.

[0295]

[0283] In certain embodiments, the present disclosure provides chimeric antigen receptors with antigen-binding domains derived from antibodies that bind an antigen (e.g., a human antigen, a cancer antigen) with high affinity (e.g., nanomolar or sub-nanomolar KD values).

[0296]

[0284] According to certain embodiments, the present disclosure provides chimeric antigen receptors with antigen-binding domains derived from corresponding antibodies that bind human antigen (e.g., at 25°C) with a KD of less than about 5 nM as measured by surface plasmon resonance. In certain embodiments, the corresponding antibodies bind an antigenic protein with a KD of less than about 20 nM, less than about 10 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 800 pM, less than about 700 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 50 pM, or less than about 25 pM as measured by surface plasmon resonance.

[0297]

[0285] In certain embodiments, the present disclosure also provides chimeric antigen receptors with antigen-binding domains derived from corresponding antibodies that bind the antigenic protein with a dissociative half-life (d / i) of greater than about 10 minutes or greater than about 125 minutes as measured by surface plasmon resonance at 25°C. In certain embodiments, the corresponding antibodies bind the antigenic protein with a tkz of greater than about 3 minutes, greater than about 4 minutes, greater than about 10 minutes, greater than about 20 minutes, greater than about 30 minutes, greater than about 40 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, greater than about 110 minutes, or greater than about 120 minutes, as measured by surface plasmon resonance at 25°C.

[0298]

[0286] In certain embodiments, the present disclosure also provides chimeric antigen receptors with antigen-binding domains derived from corresponding antibodies that bind specifically to human cell lines which express endogenous GD2, as determined by a FACS binding assay.

[0299] Therapeutic Methods

[0300]

[0287] Immune effector cells expressing the CARs disclosed herein may in some embodiments elicit a therapeutically beneficial immune response against antigenexpressing (e.g., GD2-expressing) cancer cells. For example, an anti-tumor immune response elicited by the disclosed CAR-modified immune effector cells may be an active or a passive immune response. In addition, the CAR-mediated immune response may be part of an adoptive immunotherapy approach in which CAR-modified immune effector cells induce an immune response specific to a cancer antigen (e.g., GD2).

[0301]

[0288] CAR-expressing immune effector cells prepared as described in some embodiments herein can be utilized in methods and compositions for adoptive immunotherapy in accordance with known techniques, or variations thereof that will be apparent to those skilled in the art based on the instant disclosure. See, e.g., US Patent Application Publication No. 2003 / 0170238 to Gruenberg et al; see also U.S. Pat. No. 4,690,915 to Rosenberg.

[0302]

[0289] In some aspects, provided herein are methods of treating cancer e.g., a solid tumor) in a subject by administering to the subject a composition comprising cells expressing a CAR polypeptide disclosed herein. The present disclosure demonstrates that provided CAR-based cell therapies are effective in treating both blood cancer and solid tumors. In some embodiments, provided are methods for treating a solid tumor in a subject by administering to the subject a composition comprising cells expressing a CAR polypeptide as disclosed herein.

[0303] I l l

[0290] Provided herein are methods of treating a tumor in a subject, the method comprising administering a composition comprising cells expressing a CAR polypeptide comprising an intracellular signaling region, wherein the intracellular signaling region comprises an intracellular domain pair, wherein the intracellular domain pair comprises any one of the intracellular domain pairs in Table 1.

[0304]

[0291] Provided herein are methods of treating a tumor in a subject, the method comprising administering a composition comprising cells expressing a CAR polypeptide comprising an intracellular signaling region comprising an intracellular domain pair disclosed herein.

[0305]

[0292] In some embodiments, the cells are formulated by first harvesting them from their culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a “pharmaceutically acceptable” carrier) in a treatment-effective amount. Suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but also 5% dextrose in water or Ringer's lactate can be utilized. The infusion medium can be supplemented with human serum albumin.

[0306]

[0293] A treatment-effective amount of cells in the composition is at least 2 cells (for example, at least 1 CD8+central memory T cell and at least 1 CD4+helper T cell subset), or is more typically greater than 102cells and up to 106or up to and including 108or 109cells, and can be more than IO10cells. The number of cells will depend upon the ultimate use for which the composition is intended as will the type of cells included therein.

[0307]

[0294] The cells may be autologous or heterologous to the patient undergoing therapy. The cells may be allogeneic. If desired, the treatment may also include administration of mitogens (e.g., PHA) or lymphokines, cytokines, and / or chemokines (e.g., IFN-y, IL-2, IL-12, TNF-a, IL-18, and TNF-p, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIPla, etc.) as described herein to enhance induction of the immune response.

[0308]

[0295] The CAR expressing immune effector cell populations may be administered either alone, or as a pharmaceutical composition in combination with diluents and / or with other components such as IL-2 or other cytokines or cell populations. Pharmaceutical compositions disclosed herein may comprise a CAR-expressing immune effector cell population, such as T cells, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions disclosed herein may be formulated for intravenous administration.

[0309]

[0296] The anti-tumor immune response induced in a subject by administering CAR expressing T cells described herein using the methods described herein, or other methods known in the art, may include cellular immune responses mediated by cytotoxic T cells capable of killing infected cells, by regulatory T cells, and / or by helper T cells. Humoral immune responses, mediated primarily by helper T cells capable of activating B cells thus leading to antibody production, may also be induced. A variety of techniques may be used for analyzing the type of immune responses induced by the compositions disclosed herein, which are well described in the art; e.g., Current Protocols in Immunology, Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, N.Y., N.Y.

[0310]

[0297] Thus, in some aspects, provided herein are methods of treating an individual diagnosed with or suspected of having, or at risk of developing a malignancy, comprising administering to the individual a therapeutically effective amount of the CAR-expressing immune effector cells as described herein.

[0311]

[0298] The administration of the disclosed compositions may be carried out in any convenient manner, including by injection, transfusion, or implantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumor ally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, intracerebroventricularly, or intraperitoneally. In some embodiments, the disclosed compositions are administered to a patient by intradermal or subcutaneous injection. In some embodiments, the disclosed compositions are administered by i.v. injection. The compositions may also be injected directly into a tumor, lymph node, or site of infection.

[0312]

[0299] In certain embodiments, the disclosed CAR-modified immune effector cells are administered to a patient in conjunction with e.g., before, simultaneously or following) any number of relevant treatment modalities, including but not limited to additional cancer treatments. In some embodiments, the CAR- modified immune effector cells may be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. In some embodiments, the CAR-modified immune effector cells are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as 0KT3 or CAMPATH. In other embodiments, the cell compositions are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan. For example, in some embodiments, subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following the transplant, subjects receive an infusion of the expanded immune cells. In additional embodiments, expanded cells are administered before or following surgery to treat cancer or pre-cancerous lesions in the subject.

[0313] Administration Regimens

[0314]

[0300] According to certain embodiments of the present disclosure, multiple doses of the engineered cells may be administered to a subject over a defined time course. The methods according to this aspect comprise sequentially administering to a subject multiple doses of the cells. As used herein, “sequentially administering” means that each dose is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval e.g., hours, days, weeks or months). In some embodiments, the present disclosure provides methods which comprise sequentially administering to the patient a single initial dose, followed by one or more secondary doses, and optionally followed by one or more tertiary doses.

[0315]

[0301] The terms “initial dose,” “secondary doses,” and “tertiary doses,” refer to the temporal sequence of administration of the engineered cells of the present disclosure. Thus, the “initial dose” is the dose which is administered at the beginning of the treatment regimen (also referred to as the “baseline dose”); the “secondary doses” are the doses which are administered after the initial dose; and the “tertiary doses” are the doses which are administered after the secondary doses. The initial, secondary, and tertiary doses may all contain the same amount of engineered cells, but generally may differ from one another in terms of frequency of administration. In certain embodiments, however, the amount of engineered cells contained in the initial, secondary and / or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as “loading doses” followed by subsequent doses that are administered on a less frequent basis (e.g., “maintenance doses”).

[0316]

[0302] In one embodiment of the present disclosure, each secondary and / or tertiary dose is administered 1 to 26 (e.g., 1, 156, 2, 2*6, 3, 3!6, 4, 4* / z, 5, 5* / z, 6, 6V2, 7, 7*6, 8, 8 / 2, 9, 9* , 10, 10* / 2, 11, 11*6, 12, 12 / 2, 13, 13 / 2, 14, 14 / 2, 15, 15*6, 16, 16*6, 17, 17*6, 18, 18*6, 19, 19 / 2, 20, 20 / 2, 21, 21 / 2, 22, 22 / 2, 23, 23 / 2, 24, 24*6, 25, 25*6, 26, 26*6, or more) weeks after the immediately preceding dose. The phrase “the immediately preceding dose,” as used herein, means, in a sequence of multiple administrations, the dose which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.

[0317]

[0303] The methods according to this aspect of the present disclosure may comprise administering to a patient any number of secondary and / or tertiary doses. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Likewise, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.

[0318]

[0304] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 4 weeks after the immediately preceding dose. Alternatively, the frequency at which the secondary and / or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.

[0319] Indications

[0320]

[0305] In certain aspects, provided herein are methods of treating cancer using a CAR-expressing cell provided herein. In certain embodiments, the cancer to be treated expresses a cancer antigen (e.g., GD2) to which the CAR expressed by the immune effector cells (e.g., T cells) specifically binds.

[0321]

[0306] In some embodiments, the cells are expressing GD2 targeting CARs described herein, and cancers that may be treated by methods and compositions comprising such cells include, but are not limited to epithelial cancers (e.g., non-small cell lung cancer, triple negative breast cancer, and ovarian cancer). In some embodiments, the methods and compositions provided herein relate to the treatment of solid tumors.

[0322]

[0307] In some embodiments, the methods and compositions provided herein relate to the treatment of a carcinoma. The term “carcinoma” refers to a malignant growth made up of epithelial cells tending to infiltrate the surrounding tissues, and / or resist physiological and non-physiological cell death signals, and that gives rise to metastases. Non-limiting examples of carcinomas include: acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, carcinoma villosum, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypernephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, Schneiderian carcinoma, scirrhous carcinoma, and carcinoma scroti.

[0323]

[0308] In some embodiments, the methods and compositions provided herein relate to the treatment of a sarcoma. The term “sarcoma” generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar, heterogeneous, or homogeneous substance. Sarcomas include, but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, rhabdomyosarcoma, leiomyosarcoma, endometrial sarcoma, stromal sarcoma, Ewing' s sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, Abernethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymal sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous Sarcoma, serocystic sarcoma, synovial sarcoma, and telangiectatic sarcoma.

[0324]

[0309] Additional example neoplasias that can be treated using the methods and compositions described herein include Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, Wilms tumor, breast cancer, ovarian cancer, lung cancer, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain and spinal cord tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, and adrenal cortical cancer.

[0325]

[0310] In some embodiments, the cancer treated is a melanoma. The term “melanoma” is taken to mean a tumor arising from the melanocytic system of the skin and other organs. Non-limiting examples of melanomas are Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, nodular melanoma subungual melanoma, and superficial spreading melanoma.

[0326]

[0311] In some embodiments, the methods and compositions provided herein relate to the treatment of a leukemia. The term “leukemia” is meant broadly progressive, malignant diseases of the hematopoietic organs / systems and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Non-limiting examples of leukemia diseases include, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, undifferentiated cell leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, plasmacytic leukemia, and promyelocytic leukemia.

[0327]

[0312] Particular categories of tumors that can be treated using methods and compositions described herein include lymphoproliferative disorders, breast cancer, ovarian cancer, prostate cancer, cervical cancer, endometrial cancer, bone cancer, liver cancer, stomach cancer, colon cancer, colorectal cancer, pancreatic cancer, cancer of the thyroid, head and neck cancer, cancer of the central nervous system, cancer of the peripheral nervous system, skin cancer, kidney cancer, as well as metastases of all the above. Particular types of tumors include hepatocellular carcinoma, hepatoma, hepatoblastoma, rhabdomyosarcoma, esophageal carcinoma, thyroid carcinoma, ganglioblastoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, Ewing's tumor, leimyosarcoma, rhabdotheliosarcoma, invasive ductal carcinoma, papillary adenocarcinoma, melanoma, pulmonary squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (well differentiated, moderately differentiated, poorly differentiated or undifferentiated), bronchioloalveolar carcinoma, renal cell carcinoma, hypernephroma, hypemephroid adenocarcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, lung carcinoma including small cell, non-small and large cell lung carcinoma, bladder carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, retinoblastoma, neuroblastoma, colon carcinoma, rectal carcinoma, hematopoietic malignancies including all types of leukemia and lymphoma including: acute myelogenous leukemia, acute myelocytic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, mast cell leukemia, multiple myeloma, myeloid lymphoma, Hodgkin' s lymphoma, nonHodgkin' s lymphoma.

[0328] Non-Limiting Embodiments

[0329]

[0313] Embodiment 1. A chimeric antigen receptor (CAR) polypeptide comprising: (1) an antigen -binding domain; (2) a hinge domain; (3) a transmembrane domain; and (4) an intracellular signaling region, wherein the intracellular signaling region comprises an intracellular domain (ICD) pair, the ICD pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD 2), wherein the ICD pair is selected from the ICD pairs listed in Table 1.

[0330]

[0314] Embodiment 2. The CAR polypeptide of embodiment 1, wherein the ICD1 and ICD2 are each, respectively:

[0331] (i) CD3d_ITAM and CD22,

[0332] (ii) CD3z_D12mut and FCGR3A,

[0333] (iii) CD3z_D23mut and CD3d,

[0334] (iv) CD3z_D23mut and DR3,

[0335] (v) CD3z_D23mut and MyD88_CD40,

[0336] (vi) CD3z_D23mut and NKp46,

[0337] (vii) CD3z_D23mut and 0X40,

[0338] (viii) CD3z_ITAMl and CD8a,

[0339] (ix) CD79a and CD3g_ITAM,

[0340] (x) CD79a and NKp30,

[0341] (xi) CD79b_ITAM and MyD88_CD40,

[0342] (xii) CTLA4 and CD3g_ITAM, (xiii) DAP12_ITAM and 0X40,

[0343] (xiv) FCER1G and FCERIGJTAM,

[0344] (xv) FCER1G and MyD88_CD40, or

[0345] (xvi) NKp46 and DAP12JTAM.

[0346]

[0315] Embodiment 3. The CAR polypeptide of embodiment 2, wherein the ICD1 is positioned between the transmembrane domain and the ICD2.

[0347]

[0316] Embodiment 4. The CAR polypeptide of embodiment 1, wherein sequences of the ICD1 and ICD2, respectively, are or comprise:

[0348] (i) SEQ ID NO: 44 and SEQ ID NO: 33,

[0349] (ii) SEQ ID NO: 25 and SEQ ID NO: 59,

[0350] (iii) SEQ ID NO: 26 and SEQ ID NO: 43,

[0351] (iv) SEQ ID NO: 26 and SEQ ID NO: 56,

[0352] (v) SEQ ID NO: 26 and SEQ ID NO: 10,

[0353] (vi) SEQ ID NO: 26 and SEQ ID NO: 82,

[0354] (vii) SEQ ID NO: 26 and SEQ ID NO: 13,

[0355] (viii) SEQ ID NO: 47 and SEQ ID NO: 51,

[0356] (ix) SEQ ID NO: 34 and SEQ ID NO: 46,

[0357] (x) SEQ ID NO: 34 and SEQ ID NO: 80,

[0358] (xi) SEQ ID NO: 49 and SEQ ID NO: 10,

[0359] (xii) SEQ ID NO: 7 and SEQ ID NO: 46,

[0360] (xiii) SEQ ID NO: 55 and SEQ ID NO: 13,

[0361] (xiv) SEQ ID NO: 17 and SEQ ID NO: 12,

[0362] (xv) SEQ ID NO: 17 and SEQ ID NO: 10, or

[0363] (xvi) SEQ ID NO: 82 and SEQ ID NO: 55.

[0364]

[0317] Embodiment 5. The CAR polypeptide of embodiment 1, wherein the sequences of the ICD1 and ICD2 are each, respectively, at least 90% identical to:

[0365] (i) SEQ ID NO: 44 and SEQ ID NO: 33,

[0366] (ii) SEQ ID NO: 25 and SEQ ID NO: 59,

[0367] (iii) SEQ ID NO: 26 and SEQ ID NO: 43,

[0368] (iv) SEQ ID NO: 26 and SEQ ID NO: 56,

[0369] (v) SEQ ID NO: 26 and SEQ ID NO: 10,

[0370] (vi) SEQ ID NO: 26 and SEQ ID NO: 82,

[0371] (vii) SEQ ID NO: 26 and SEQ ID NO: 13, (viii) SEQ ID NO: 47 and SEQ ID NO: 51,

[0372] (ix) SEQ ID NO: 34 and SEQ ID NO: 46,

[0373] (x) SEQ ID NO: 34 and SEQ ID NO: 80,

[0374] (xi) SEQ ID NO: 49 and SEQ ID NO: 10,

[0375] (xii) SEQ ID NO: 7 and SEQ ID NO: 46,

[0376] (xiii) SEQ ID NO: 55 and SEQ ID NO: 13,

[0377] (xiv) SEQ ID NO: 17 and SEQ ID NO: 12,

[0378] (xv) SEQ ID NO: 17 and SEQ ID NO: 10, or

[0379] (xvi) SEQ ID NO: 82 and SEQ ID NO: 55, wherein the ICD1 is positioned between the transmembrane domain and the ICD2.

[0380]

[0318] Embodiment 6. The CAR polypeptide of any one of embodiments 1-5, wherein the intracellular signaling region comprises an amino acid sequence that is at least 90% identical to any one of the sequences listed in Table 3.

[0381]

[0319] Embodiment 7. The CAR polypeptide of any one of embodiments 1-6, wherein the intracellular signaling region comprises an amino acid sequence listed in Table 3.

[0382]

[0320] Embodiment 8. The CAR polypeptide of any one of embodiments 1-7, wherein the intracellular signaling region comprises an amino acid sequence at least 90% identical to any of: (i) SEQ ID NO: 451, (ii) SEQ ID NO: 452, (iii) SEQ ID NO: 453, (iv) SEQ ID NO: 454, (v) SEQ ID NO: 455, (vi) SEQ ID NO: 456, (vii) SEQ ID NO: 457, (viii) SEQ ID NO: 458, (ix) SEQ ID NO: 459, (x) SEQ ID NO: 460, (xi) SEQ ID NO: 461, (xii) SEQ ID NO: 462, (xiii) SEQ ID NO: 463, (xiv) SEQ ID NO: 464, (xv) SEQ ID NO: 465, or (xvi) SEQ ID NO: 466.

[0383]

[0321] Embodiment 9. The CAR polypeptide of any one of embodiments 1-8, wherein the antigen-binding domain is a single chain fragment variable (scFv) domain.

[0384]

[0322] Embodiment 10. The CAR polypeptide of embodiment 9, wherein the scFv domain comprises any one of the scFv domains set forth in Table 4.

[0385]

[0323] Embodiment 11. The CAR polypeptide of embodiment 9, wherein the scFv domain comprises 90% identity to any one of the scFv domains set forth in Table 4.

[0386]

[0324] Embodiment 12. The CAR polypeptide of any one of embodiments 1-11, wherein the sequence of the intracellular signaling region is or comprises any of:

[0387] (i) SEQ ID NO: 451,

[0388] (ii) SEQ ID NO: 452, (iii) SEQ ID NO: 453,

[0389] (iv) SEQ ID NO: 454,

[0390] (v) SEQ ID NO: 455,

[0391] (vi) SEQ ID NO: 456,

[0392] (vii) SEQ ID NO: 457,

[0393] (viii) SEQ ID NO: 458,

[0394] (ix) SEQ ID NO: 459,

[0395] (x) SEQ ID NO: 460,

[0396] (xi) SEQ ID NO: 461,

[0397] (xii) SEQ ID NO: 462,

[0398] (xiii) SEQ ID NO: 463,

[0399] (xiv) SEQ ID NO: 464,

[0400] (xv) SEQ ID NO: 465, or

[0401] (xvi) SEQ ID NO: 466.

[0402]

[0325] Embodiment 13. The CAR polypeptide of any one of embodiments 1-12, wherein the antigen-binding domain binds a cancer-associated antigen.

[0403]

[0326] Embodiment 14. The CAR polypeptide of embodiment 13, wherein the cancer-associated antigen is selected from any one of the cancer-associated antigens listed in Table 5.

[0404]

[0327] Embodiment 15. The CAR polypeptide of embodiment 13, wherein the cancer-associated antigen is GD2.

[0405]

[0328] Embodiment 16. The CAR polypeptide of embodiment 15, wherein the antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 485.

[0406]

[0329] Embodiment 17. The CAR polypeptide of any one of embodiments 1-16, wherein the transmembrane domain is or comprises a sequence listed in Table 7.

[0407]

[0330] Embodiment 18. The CAR polypeptide of any one of embodiments 1-16, wherein the transmembrane domain is or comprises a sequence at least 90% identical to a sequence listed in Table 7.

[0408]

[0331] Embodiment 19. The CAR polypeptide of any one of embodiments 1-18, wherein the hinge domain is selected from any one of the hinge domains of Table 6.

[0409]

[0332] Embodiment 20. The CAR polypeptide of any one of embodiments 1-18, wherein the sequence of the hinge domain is at least 90% identical to a sequence listed in Table 6.

[0333] Embodiment 21. The CAR polypeptide of embodiment 19 or 20, wherein the hinge domain is an IgG4 hinge domain.

[0410]

[0334] Embodiment 22. A chimeric antigen receptor (CAR) polypeptide comprising: (1) an antigen-binding domain; (2) a transmembrane domain; and (3) an intracellular signaling region, wherein the antigen-binding domain binds to antigen GD2, and wherein the intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD1 and ICD2 are each, respectively, any of:

[0411] (i) SEQ ID NO: 44 and SEQ ID NO: 33,

[0412] (ii) SEQ ID NO: 25 and SEQ ID NO: 59,

[0413] (iii) SEQ ID NO: 26 and SEQ ID NO: 43,

[0414] (iv) SEQ ID NO: 26 and SEQ ID NO: 56,

[0415] (v) SEQ ID NO: 26 and SEQ ID NO: 10,

[0416] (vi) SEQ ID NO: 26 and SEQ ID NO: 82,

[0417] (vii) SEQ ID NO: 26 and SEQ ID NO: 13,

[0418] (viii) SEQ ID NO: 47 and SEQ ID NO: 51,

[0419] (ix) SEQ ID NO: 34 and SEQ ID NO: 46,

[0420] (x) SEQ ID NO: 34 and SEQ ID NO: 80,

[0421] (xi) SEQ ID NO: 49 and SEQ ID NO: 10,

[0422] (xii) SEQ ID NO: 7 and SEQ ID NO: 46,

[0423] (xiii) SEQ ID NO: 55 and SEQ ID NO: 13,

[0424] (xiv) SEQ ID NO: 17 and SEQ ID NO: 12,

[0425] (xv) SEQ ID NO: 17 and SEQ ID NO: 10, or

[0426] (xvi) SEQ ID NO: 82 and SEQ ID NO: 55, wherein the ICD1 is positioned between the transmembrane domain and the ICD2.

[0427]

[0335] Embodiment 23. The CAR polypeptide of embodiment 22, wherein the sequence of the CAR is or comprises any sequence in Table 8.

[0428]

[0336] Embodiment 24. The CAR polypeptide of embodiment 22, wherein the sequence of the CAR is or comprises any of SEQ ID NO: 486 to 501.

[0429]

[0337] Embodiment 25. The CAR polypeptide of embodiment 22, wherein the sequence of the CAR is at least 90% identical to any of SEQ ID NO: 486 to 501.

[0430]

[0338] Embodiment 26. A nucleic acid encoding the CAR polypeptide of any one of embodiments 1-25.

[0431]

[0339] Embodiment 27. A vector comprising the nucleic acid of embodiment 26.

[0340] Embodiment 28. The vector of embodiment 27, wherein the vector is an expression vector.

[0432]

[0341] Embodiment 29. The vector of embodiment 28, wherein the vector is a viral vector.

[0433]

[0342] Embodiment 30. The vector of embodiment 29, wherein the viral vector is a retroviral vector, a lentiviral vector or an adeno-associated viral (AAV) vector.

[0434]

[0343] Embodiment 31. A cell comprising the nucleic acid of embodiment 26.

[0435]

[0344] Embodiment 32. A cell expressing the CAR polypeptide of any one of embodiments 1-25.

[0436]

[0345] Embodiment 33. The cell of embodiment 31 or 32, wherein the cell is a T cell.

[0437]

[0346] Embodiment 34. The T cell of embodiment 33, wherein the T cell is a cytotoxic T lymphocyte (CTL).

[0438]

[0347] Embodiment 35. The T cell of embodiment 33, wherein the T cell is a primary CD8+ T cell.

[0439]

[0348] Embodiment 36. The cell of any one of embodiments 31-35, wherein the cell proliferates in the presence of antigen-expressing target cells.

[0440]

[0349] Embodiment 37. The cell of any one of embodiments 31-36, wherein the cell is resistant to exhaustion.

[0441]

[0350] Embodiment 38. A method of generating a CAR-expressing cell comprising contacting a cell with a nucleic acid of embodiment 28, or a vector of any one of embodiments 27-30.

[0442]

[0351] Embodiment 39. A composition comprising cells of any one of embodiments 31-37.

[0443]

[0352] Embodiment 40. A method of treating a tumor in a subject, the method comprising administering the composition of embodiment 39.

[0444]

[0353] Embodiment 41. The method of embodiment 40, wherein the tumor is a blood cancer.

[0445]

[0354] Embodiment 42. The method of embodiment 41, wherein the blood cancer is a B cell chronic lymphocytic leukemia, non-Hodgkin’s lymphoma, a mantle cell lymphoma, multiple myeloma, or an acute lymphoblastic leukemia.

[0446]

[0355] Embodiment 43. The method of embodiment 40, wherein the tumor is a solid tumor.

[0356] Embodiment 44. The method of embodiment 43, wherein the solid tumor is a neuroblastoma, osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, glioblastoma, diffuse midline glioma or leiomyosarcoma.

[0447]

[0357] Embodiment 45. The method of embodiment 43, wherein the solid tumor is an epithelial cancer.

[0448]

[0358] Embodiment 46. The method of embodiment 45, wherein the epithelial cancer is a non-small cell lung cancer, a triple negative breast cancer, melanoma, or an ovarian cancer.

[0449]

[0359] Embodiment 47. The method of any one of embodiments 40-46, wherein the tumor expresses the antigen recognized by the antigen-binding domain of the CAR polypeptide.

[0450]

[0360] Embodiment 48. The method of any one of embodiments 40-46, wherein the tumor expresses the antigen recognized by the antigen-binding domain of the CAR polypeptide, wherein the tumor expresses the antigen GD2.

[0451]

[0361] Embodiment 49. A cell bank comprising cells for adoptive immunotherapy, wherein the cells express the CAR polypeptide of any one of embodiments 1 to 25.

[0452] Exemplification

[0453] Example 1: Identification of Novel CAR Intracellular Domains

[0454]

[0362] Novel CAR polypeptide designs were discovered via both in vitro and in vivo pooled screens of a CAR library. Example combinations identified in the screens are provided in this disclosure in Table 1.

[0455]

[0363] While Chimeric Antigen Receptor-expressing T cells (CAR-T cells) have shown high response rates against hematological cancers in the clinic, they have shown limited efficacy against solid tumors due to lack of persistence and susceptibility to various immunosuppressive factors in the tumor microenvironment (TME). To address the suppressive TME, recent efforts to systemically discover novel intracellular signaling domains (ICDs) at scale have demonstrated success in engineering CAR polypeptide designs with enhanced solid tumor efficacy. However, in vitro screens are inherently limited in mimicking the TME and other physiologically relevant aspects of adoptive cell therapy (ACT).

[0456]

[0364] An anti-GD2 CAR library was generated using peripheral blood mononuclear cell (PBMC)-derived T cells obtained from three different donors and a combinatorial 2nd- generation GD2-targeting CAR library containing a theoretical diversity of 10,000 different CAR polypeptide designs. The donors were designated 2745, 6903 and 6904 and were healthy human donors, and cells obtained from Stemcell Technologies, Cambridge, MA. These libraries were screened in parallel in the context of both in vitro serial tumor rechallenge and an in vivo tumor xenograft mouse model. Novel CAR polypeptide designs that endow long-term persistence to primary T cells were discovered. The final candidates identified from the novel CAR polypeptide designs could be utilized for pre-clinical research and clinical applications.

[0457]

[0365] The in vitro co-culture assay was performed as illustrated in FIG. 1. The CAR library was enriched by repeated co-culture with human MG-63 osteosarcoma or SK-N-AS neuroblastoma cells. MG-63 is a cell that has fibroblast morphology isolated from the bone of a White, 14-year-old male patient with osteosarcoma, and SK-N-AS is a neuroblast isolated from the brain of a female human neuroblastoma patient; both are available from: American Type Culture Collection (ATCC, Manassas, VA, USA). As shown in FIG. 2 (e.g., FIGs. 2A, 2B, 2C and 2D), the enriched anti-GD2 CAR library exhibited reduced exhaustion marker (PD1, LAG3 and / or TIM3) expression as compared to a conventional 3rd-generation anti-GD2 CAR polypeptide design (28OX40z). 28OX40z (also known as 28OX40Z, 28.OX.Z and 28OXz) is described in: Behandi et al. 2012 Mol. Immunol. 50: 35; Taheri et al. 2024 BMC Biotech. 24:1; and WO2022083668A1. In FIG. 2, only data related to CAR polypeptides that bind to GD2 are shown. GD2 is a tumor marker previously- described; see: M.S. Io Piccolo et al. 2001 Cancer 92: 924; Battula et al. 2012 J. Clin. Invest. 122: 2066; Mansoori et al. 2019 Exp. Mol. Pathol. 109: 25; and Galan et al. Front. Oncol. Vol. 13 https: / / doi.org / 10.3389 / fonc.2023.1134763.

[0458]

[0366] FIG. 3 of the priority document, U.S. Provisional Patent Application Ser. No. 63 / 635,184, filed 4 / 17 / 24, the contents of which are hereby incorporated by reference, shows an in vitro hit list. In vitro hit designs were generated based on frequency of ICD usage among enriched barcodes that were found in both screening replicates, and designs with 2 or 0 ITAM-containing domains were excluded. “Shared hit” means specific ICD2|ICD1 barcode passed QC (quality control) and was enriched in both target cell line screens. “Hits of interest” are hand-picked specific ICD2|ICD1 barcodes that passed QC, had >5 estimated cells screened based on surface CAR staining, had a final log2fc >3, and contained 1 IT AM domain. The hits identified in that screen were: PILRB-CD27_ICM, PILRB-CD3z, CD8a-NKp30, NKp44-CD3e_ITAM, CD3z_D23mut-DR3, DNAM1-ICOS, FCER1G-FCRL4, FCER1G-SLAMF6, CD3z_X2Xmut-SLAMF6, and CD3z_ITAM3-

[0459] HVEM.

[0460] Example 2: In vitro screens

[0461]

[0367] An in vitro screen was performed on mice with various CAR polypeptides.

[0462]

[0368] FIG. 3A provides a schematic for the process used, (be: barcode.)

[0463]

[0369] Table 11 shows the relative frequency that different ICDs appeared in either position (proximal or distal), in an in vitro assay. The in vitro assay used donor libraries from donors 6904 (d6904), 6903 (d6903) and 2745 (donor 2745). CARs bind to GD2 and were assayed against SK-N-AS tumor cells, which express GD2. The relative abundance of different ICDs at different positions were determined by bar code sequencing. The positions were: proximal (e.g., proximal to the membrane, or positioned between the transmembrane domain and the other ICD); or distal (e.g., distal to the membrane, or positioned further than the other ICD from the membrane). The assay was performed as described elsewhere herein.

[0464]

[0370] Numbers are provided as a frequency percentage X100. For example: in line 2, column 2, 0.0099 indicates that of all CARs sequenced, 0.99% featured 4- IBB in the proximal position (as ICD1, positioned between the transmembrane domain and ICD2).

[0465] Table 11: Results of in vitro screen in SK-N-AS tumor cells

[0466]

[0371] Some of the ICDs appearing more frequently at the proximal position, as determined from this assay are listed below in Table 11 A:

[0467] Table 11A: Average frequencies of appearance of selected ICDs at proximal position, in in vitro screen in SK-N-AS tumor cells

[0468]

[0372] Some of the ICDs appearing more frequently at the proximal position, as determined from this assay are listed below.

[0469] Table 11B: Average frequencies of appearance of selected ICDs at distal position, in in vitro screen in SK-N-AS tumor cells

[0470]

[0373] Another in vitro assay used the three donor libraries from donors (d6904, d6903 and d2745). CARs bind to GD2 and were assayed against human MG-63 osteosarcoma cells, which also expresses GD2. The relative abundance of different ICDs at different positions were determined by bar code sequencing. The positions were: proximal and distal, as defined above, and the assay was performed as described elsewhere herein. Numbers are provided as a frequency percentage X100.

[0471] Table 12: Results of in vitro screen in human MG-63 osteosarcoma cells

[0472] Table 12A: Average frequencies of appearance of selected ICDs at proximal position, in in vitro screen in human MG-63 osteosarcoma cells.

[0473] Table 12B: Average frequencies of appearance of selected ICDs at distal position, in in vitro screen in human MG-63 osteosarcoma cells

[0474]

[0374] Example 3: In vivo screen

[0475]

[0375] An in vivo screen was performed on mice with various CAR polypeptides.

[0476]

[0376] As diagrammed in FIG. 3B, an in vivo pooled screen approach was developed to identify potent CAR-T designs in a CHLA-20 neuroblastoma xenograft model. A DNA- barcoded 10,000-member CAR ICD library was used to identify novel CAR ICDs with enhanced anti-tumor function. This library was cloned into a GD2 CAR-T backbone. GD2 CAR library-expressing primary T cells from one donor manufactured for the in vitro pooled screen were injected into tumor-bearing mice at >100x coverage of the library size, and 3 weeks post- ACT, spleens and tumors were collected.

[0477]

[0377] Various exclusion criteria included excluding designs that meet any of the following conditions: Containing LILRB2 or TIM3 domains (which demonstrated ubiquitous poor T cell expression from in vitro hit arrayed screen); Containing no ITAM(s); Coeff. of variation for log2(fc) between replicate mice >0.8; and Designs already evaluated in in vitro hit arrayed screen.

[0478]

[0378] Sequencing genomic DNA quantified the abundance of barcodes linked to each CAR ICD combination. Based on previously-published literature, the hypothesis was that any remaining CAR-T cells at the tumor site and spleen would represent T cells with long-term persistence elicited by antigen-specific CAR ICD signaling. Novel ICD combinations were preferentially enriched across multiple replicate mice. The anti-tumor function of select in vivo hits that were shared with the in vitro screen hits were validated in an arrayed screen, assessing cytotoxicity, cytokine secretion profiles, proliferation, memory and exhaustion phenotypes in a serial tumor rechallenge assay. These functional datasets allowed for the dissection of the effects of particular signaling components in T cells that endow long-term persistence and potential resistance to immunosuppressive TME factors. In vivo hits are based on specific ICD2|ICD1 barcodes that pass QC and had log2fc>l in at least 2 replicate mice.

[0479] The parameters of the experiment were as follows:

[0379] 4e6 CHLA-20.Luc flank injection was performed. Then i.v. CAR-T injection was performed 48 hours later, with le7 total T cells per mouse.

[0480]

[0380] FIG. 4 is a plot showing the number of tracked unique CAR polypeptide designs at each step of the in vivo pooled screen process. Plasmid library indicates the number of CAR polypeptide designs prior to lentiviral library production. LVV library indicates the number of CAR polypeptide designs post-lentiviral production. CAR-T library (Day 0) and intratumoral CAR-T (Day 23) indicate the number of CAR polypeptide designs prior to and post in vivo pooled screen, respectively. Each data marker dot in intratumoral CAR-T (Day 23) represents barcodes from each group in the in vivo screen.

[0481]

[0381] FIG. 5 shows data filtering in in vivo pooled screen barcode QC (quality control) steps. This graph shows the number of CAR polypeptide designs post NGS filtering for each in vivo pooled screen group. Unique barcodes DO (Day 0) and D23 (Day 23) indicate the number of unique CAR polypeptide designs prior to and post in vivo pooled screen, respectively. >10 reads on DO and D23 indicate the number of CAR polypeptide designs that are present in both before and after the in vivo pooled screen that have NGS raw read counts over 10. Log2fc >1 indicates the number of CAR polypeptide designs that have enriched at least 2-fold during the in vivo pooled screen. Each data marker represents barcodes from each replicate mouse within each group in the in vivo screen.

[0482]

[0382] A few CAR polypeptide designs were excluded by minimum read count QC steps. Unique barcodes were recovered and the number of unique hits seemed to correlate positively with control CAR %spike-in.

[0483]

[0383] The relative amounts of CAR library and spike-in are represented by the % numbers in FIGs. 5, 6A to 6D and Table 13 and elsewhere herein, below. “0%” indicates that the tested material was 100% CAR library, and 0% of spike-in of a control CAR (28OX40Z). 0,1%, 1% and 10% indicate that the tested material was 99.9%, 99%, and 90% of CAR library, respectively, and 0.1%, 1% or 10% of spike-in of control CAR.

[0484]

[0384] FIGs 6A, B, C, and D represent fold-enrichment (X-axis) and the extent of variation across replicate mice (Y-axis) of each CAR polypeptide design (represented by round, triangular and square data markers) post NGS (next generation sequencing) filtering. Log2fc and Coeff. of variation (cv) values are means across mouse replicates. Different shapes of markers indicate the number of replicate mice that each CAR polypeptide design is discovered from.

[0385] Table 13 shows the relative frequency that different ICDs appeared in either position, in the in vivo assay.

[0485]

[0386] CARs bind to GD2 and were assayed in NSG mice bearing CHLA-20 neuroblastoma tumor cells, which express GD2. The relative abundance of different ICDs at different positions were determined by bar code sequencing. The CAR library was mixed with a spike-in of a control CAR (28OX40Z).

[0486] Table 13: Results of in vivo screen

[0487] Table 13A: Average frequencies of appearance of selected ICDs at proximal position, in in vivo screen

[0488] Table 13B: Average frequencies of appearance of selected ICDs at distal position, in in vivo screen

[0489]

[0387] The information obtained in the in vitro and in vivo screens, and as described in Tables 11 to 13, were used to guide the design and selection of CARs for further evaluation.

[0490] Example 4: In vitro validation of various CAR polypeptides

[0491]

[0388] FIGs. 7A and 7B show results of in vitro hit validation in MG-63 tumor cells, with various individual CAR polypeptides highlighted. CAR polypeptides were evaluated for tumor control and expansion. The graphs show: CAR-T cell expansion (X-axis); and tumor control (Y-axis), which is represented by the area under the curve (AUC) of tumor growth across 3 serial tumor challenges over the course of 2 weeks.

[0492]

[0389] In Fig. 7A: Al: FCERlG-MyD88_CD40; Cl: CD3z_D23mut-MyD88_CD40; DI: CD79b_ITAM-MyD88_CD40; G5: DAP12_ITAM-OX40; G8: CD3z_D23mut-OX40; CIO: CD3z_D23mut-DR3; and CAR4: CD3z__D23mut-CD40.

[0493]

[0390] CAR polypeptide designs come from top hits of the in vitro pooled screen.

[0494]

[0391] Similar CAR hits that induce potent killing and proliferation were identified across two different GD2+ target cells, MG-63 and SK-N-AS. Al, Cl, and DI share a very similar design: a membrane-proximal IT AM domain, and a membrane-distal MyD88_CD40.

[0495]

[0392] Dotted lines were drawn on the data value of a positive control, 28.OX40.Z, to highlight CAR hits that perform better than the control. CAR polypeptides which appear on the graph to the right of the positive control (e.g., to the right of the vertical dotted line through the positive control) show increased expansion relative to control; and CAR polypeptides which appear on the graph below the positive control (e.g., below the horizontal dotted line passing through the positive control) show increased tumor control relative to control.

[0496]

[0393] Highlighted CAR polypeptides include:

[0497] FCERlG-MyD88_CD40 (designated Al or CAR_A1) CD3z_D23mut-MyD88_CD40 (Cl) CD79b_ITAM-MyD88_CD40 (DI)

[0498] DAP 12_ITAM- 0X40 (G5 or CAR_G5) CD3z_D23mut-OX40 (G8) CD3z_D23mut-DR3 (CIO or CAR_C10)

[0499]

[0394] FIG. 8 shows CAR-T cell fold-expansion across 3 serial MG-63 tumor challenges over the course of 2 weeks.

[0500]

[0395] Many novel CAR polypeptides outperformed 3rd generation CAR polypeptides (41BB-CD3z, CAR4, and CD28-OX40-CD3z).

[0501]

[0396] Numerous CAR polypeptide designs that encompass MyD88_CD40 ICD in the membrane-distal location showed potent expansion during the serial rechallenge.

[0502]

[0397] Some CAR polypeptides that have proliferated less than control CARs still robustly control tumor growth; CAR polypeptides with novel ICDs can repeatedly clear tumor challenges through different mechanisms.

[0503]

[0398] FIGs. 9 A and 9B are plots showing the tumor-killing potential of novel CAR polypeptides. CAR-T cells in either donor 6903 (d6903, FIG. 9A) or donor 8089 (d8089, FIG. 9B) were challenged with MG-63 cells 3 times over the course of 2 weeks. CAR polypeptide designs come from top hits of the in vivo pooled screen. Y-axis represents tumor counts normalized by the count on the first day of each tumor challenge. Overlapping data points for CAR polypeptides that completely clear tumors at each challenge are shown across two different donor data sets.

[0504]

[0399] In an experiment with d6903 (as shown in FIG. 9A), 5 novel CAR polypeptide designs repeatedly cleared tumor:

[0505] Complete clearance in round 3:

[0506] CD3z_D23mut-CD3d

[0507] CD3d_ITAM-CD22

[0508] NKp46-DAP12_ITAM

[0509] Partial clearance in round 3:

[0510] CD3z_D23mut-NKp46

[0511] CTLA4-CD3g_ITAM

[0512]

[0400] As shown in Fig. 9B, in an experiment with d8089 cells, 10 novel CAR polypeptides repeatedly cleared tumor.

[0513] CAR polypeptides which demonstrated complete tumor clearance in round 3 included: CD3z_D23mut-CD3d CD3d_ITAM-CD22 NKp46-DAP12_ITAM CD3z_D23mut-NKp46 CTLA4-CD3g_ITAM

[0514] CD3z_ITAMl-CD8a

[0515] CAR polypeptides which demonstrated complete tumor clearance in round 3 (only 1 replicate, whereas other experiments had 3 replicates) included:

[0516] CD79a-NKp30

[0517] CD79a-CD3g_ITAM

[0518] FCER 1 G-FCER 1 G_ITAM

[0519] CD3z_D12mut-FCGR3A

[0520]

[0401] FIGs. 10A and 10B are plots showing fold-expansion of CAR-T cells in 2 different T cell donors during 2 serial human MG-63 osteosarcoma challenges over the course of 8 days. CAR polypeptide designs come from top hits of the in vivo pooled screen. BBz and 28OXz are controls, as previously described.

[0521]

[0402] Several CAR polypeptides, such as CD3z_D23mut-CD3d, CD3z_ITAMl- CD8a, and NKp46-DAP12_ITAM, demonstrated particularly high expansion in at least one assay.

[0522] INCORPORATION BY REFERENCE

[0523]

[0403] All publications, patents, patent applications and sequence accession numbers mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0524] EQUIVALENTS

[0525]

[0404] A number of embodiments have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs.

[0526]

[0405] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

We claim:

1. A chimeric antigen receptor (CAR) polypeptide comprising: (1) an antigen-binding domain; (2) a hinge domain; (3) a transmembrane domain; and (4) an intracellular signaling region, wherein the intracellular signaling region comprises an intracellular domain (ICD) pair, the ICD pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD pair is selected from the ICD pairs listed in Table 1.

2. The CAR polypeptide of claim 1, wherein the ICD1 and ICD2 are each, respectively:(i) CD3d_ITAM and CD22,(ii) CD3z_D12mut and FCGR3A,(iii) CD3z_D23mut and CD3d,(iv) CD3z_D23mut and DR3,(v) CD3z_D23mut and MyD88_CD40,(vi) CD3z_D23mut and NKp46,(vii) CD3z_D23mut and 0X40,(viii) CD3z_ITAMl and CD8a,(ix) CD79a and CD3g_ITAM,(x) CD79a and NKp30,(xi) CD79b_ITAM and MyD88_CD40,(xii) CTLA4 and CD3g_ITAM,(xiii) DAP12JTAM and 0X40,(xiv) FCER1G and FCER1GJTAM,(xv) FCER1G and MyD88_CD40, or(xvi) NKp46 and DAP12JTAM.

3. The CAR polypeptide of claim 2, wherein the ICD1 is positioned between the transmembrane domain and the ICD2.

4. The CAR polypeptide of claim 1, wherein sequences of the ICD1 and ICD2, respectively, are or comprise:(i) SEQ ID NO: 44 and SEQ ID NO: 33,(ii) SEQ ID NO: 25 and SEQ ID NO: 59,(iii) SEQ ID NO: 26 and SEQ ID NO: 43,(iv) SEQ ID NO: 26 and SEQ ID NO: 56,(v) SEQ ID NO: 26 and SEQ ID NO: 10,(vi) SEQ ID NO: 26 and SEQ ID NO: 82,(vii) SEQ ID NO: 26 and SEQ ID NO: 13,(viii) SEQ ID NO: 47 and SEQ ID NO: 51,(ix) SEQ ID NO: 34 and SEQ ID NO: 46,(x) SEQ ID NO: 34 and SEQ ID NO: 80,(xi) SEQ ID NO: 49 and SEQ ID NO: 10,(xii) SEQ ID NO: 7 and SEQ ID NO: 46,(xiii) SEQ ID NO: 55 and SEQ ID NO: 13,(xiv) SEQ ID NO: 17 and SEQ ID NO: 12,(xv) SEQ ID NO: 17 and SEQ ID NO: 10, or(xvi) SEQ ID NO: 82 and SEQ ID NO: 55.

5. The CAR polypeptide of claim 1, wherein the sequences of the ICD1 and ICD2 are each, respectively, at least 90% identical to:(i) SEQ ID NO: 44 and SEQ ID NO: 33,(ii) SEQ ID NO: 25 and SEQ ID NO: 59,(iii) SEQ ID NO: 26 and SEQ ID NO: 43,(iv) SEQ ID NO: 26 and SEQ ID NO: 56,(v) SEQ ID NO: 26 and SEQ ID NO: 10,(vi) SEQ ID NO: 26 and SEQ ID NO: 82,(vii) SEQ ID NO: 26 and SEQ ID NO: 13,(viii) SEQ ID NO: 47 and SEQ ID NO: 51,(ix) SEQ ID NO: 34 and SEQ ID NO: 46,(x) SEQ ID NO: 34 and SEQ ID NO: 80,(xi) SEQ ID NO: 49 and SEQ ID NO: 10,(xii) SEQ ID NO: 7 and SEQ ID NO: 46,(xiii) SEQ ID NO: 55 and SEQ ID NO: 13,(xiv) SEQ ID NO: 17 and SEQ ID NO: 12,(xv) SEQ ID NO: 17 and SEQ ID NO: 10, or(xvi) SEQ ID NO: 82 and SEQ ID NO: 55, wherein the ICD1 is positioned between the transmembrane domain and the ICD2.

6. The CAR polypeptide of any one of claims 1-5, wherein the intracellular signaling region comprises an amino acid sequence that is at least 90% identical to any one of the sequences listed in Table 3.

7. The CAR polypeptide of any one of claims 1-6, wherein the intracellular signaling region comprises an amino acid sequence listed in Table 3.

8. The CAR polypeptide of any one of claims 1-7, wherein the intracellular signaling region comprises an amino acid sequence at least 90% identical to any of: (i) SEQ ID NO: 451, (ii) SEQ ID NO: 452, (iii) SEQ ID NO: 453, (iv) SEQ ID NO: 454, (v) SEQ ID NO: 455, (vi) SEQ ID NO: 456, (vii) SEQ ID NO: 457, (viii) SEQ ID NO: 458, (ix) SEQ ID NO: 459, (x) SEQ ID NO: 460, (xi) SEQ ID NO: 461, (xii) SEQ ID NO: 462, (xiii) SEQ ID NO: 463, (xiv) SEQ ID NO: 464, (xv) SEQ ID NO: 465, or (xvi) SEQ ID NO: 466.

9. The CAR polypeptide of any one of claims 1-8, wherein the antigen-binding domain is a single chain fragment variable (scFv) domain.

10. The CAR polypeptide of claim 9, wherein the scFv domain comprises any one of the scFv domains set forth in Table 4.

11. The CAR polypeptide of claim 9, wherein the scFv domain comprises 90% identity to any one of the scFv domains set forth in Table 4.

12. The CAR polypeptide of any one of claims 1-11, wherein the sequence of the intracellular signaling region is or comprises any of:(i) SEQ ID NO: 451,(ii) SEQ ID NO: 452,(iii) SEQ ID NO: 453,(iv) SEQ ID NO: 454,(v) SEQ ID NO: 455,(vi) SEQ ID NO: 456,(vii) SEQ ID NO: 457,(viii) SEQ ID NO: 458,(ix) SEQ ID NO: 459,(x) SEQ ID NO: 460,(xi) SEQ ID NO: 461,(xii) SEQ ID NO: 462,(xiii) SEQ ID NO: 463,(xiv) SEQ ID NO: 464,(xv) SEQ ID NO: 465, or(xvi) SEQ ID NO: 466.

13. The CAR polypeptide of any one of claims 1-12, wherein the antigen-binding domain binds a cancer-associated antigen.

14. The CAR polypeptide of claim 13, wherein the cancer-associated antigen is selected from any one of the cancer-associated antigens listed in Table 5.

15. The CAR polypeptide of claim 13, wherein the cancer-associated antigen is GD2.

16. The CAR polypeptide of claim 15, wherein the antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 485.

17. The CAR polypeptide of any one of claims 1-16, wherein the transmembrane domain is or comprises a sequence listed in Table 7.

18. The CAR polypeptide of any one of claims 1-16, wherein the transmembrane domain is or comprises a sequence at least 90% identical to a sequence listed in Table 7.

19. The CAR polypeptide of any one of claims 1-18, wherein the hinge domain is selected from any one of the hinge domains of Table 6.

20. The CAR polypeptide of any one of claims 1-18, wherein the sequence of the hinge domain is at least 90% identical to a sequence listed in Table 6.

21. The CAR polypeptide of claim 19 or 20, wherein the hinge domain is an IgG4 hinge domain.

22. A chimeric antigen receptor (CAR) polypeptide comprising: (1) an antigen-binding domain; (2) a transmembrane domain; and (3) an intracellular signaling region, wherein the antigen-binding domain binds to antigen GD2, and wherein the intracellular signaling region comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD1 and ICD2 are each, respectively, any of:(i) SEQ ID NO: 44 and SEQ ID NO: 33,(ii) SEQ ID NO: 25 and SEQ ID NO: 59,(iii) SEQ ID NO: 26 and SEQ ID NO: 43,(iv) SEQ ID NO: 26 and SEQ ID NO: 56,(v) SEQ ID NO: 26 and SEQ ID NO: 10,(vi) SEQ ID NO: 26 and SEQ ID NO: 82,(vii) SEQ ID NO: 26 and SEQ ID NO: 13,(viii) SEQ ID NO: 47 and SEQ ID NO: 51,(ix) SEQ ID NO: 34 and SEQ ID NO: 46,(x) SEQ ID NO: 34 and SEQ ID NO: 80,(xi) SEQ ID NO: 49 and SEQ ID NO: 10,(xii) SEQ ID NO: 7 and SEQ ID NO: 46,(xiii) SEQ ID NO: 55 and SEQ ID NO: 13,(xiv) SEQ ID NO: 17 and SEQ ID NO: 12,(xv) SEQ ID NO: 17 and SEQ ID NO: 10, or(xvi) SEQ ID NO: 82 and SEQ ID NO: 55, wherein the ICD1 is positioned between the transmembrane domain and the ICD2.

23. The CAR polypeptide of claim 22, wherein the sequence of the CAR is or comprises any sequence in Table 8.

24. The CAR polypeptide of claim 22, wherein the sequence of the CAR is or comprises any of SEQ ID NO: 486 to 501.

25. The CAR polypeptide of claim 22, wherein the sequence of the CAR is at least 90% identical to any of SEQ ID NO: 486 to 501.

26. A nucleic acid encoding the CAR polypeptide of any one of claims 1-25.

27. A vector comprising the nucleic acid of claim 26.

28. The vector of claim 27, wherein the vector is an expression vector.

29. The vector of claim 28, wherein the vector is a viral vector.

30. The vector of claim 29, wherein the viral vector is a retroviral vector, a lentiviral vector or an adeno-associated viral (AAV) vector.

31. A cell comprising the nucleic acid of claim 26.

32. A cell expressing the CAR polypeptide of any one of claims 1-25.

33. The cell of claim 31 or 32, wherein the cell is a T cell.

34. The T cell of claim 33, wherein the T cell is a cytotoxic T lymphocyte (CTL).

35. The T cell of claim 33, wherein the T cell is a primary CD8+ T cell.

36. The cell of any one of claims 31-35, wherein the cell proliferates in the presence of antigenexpressing target cells.

37. The cell of any one of claims 31-36, wherein the cell is resistant to exhaustion.

38. A method of generating a CAR-expressing cell comprising contacting a cell with a nucleic acid of claim 28, or a vector of any one of claims 27-30.

39. A composition comprising cells of any one of claims 31-37.

40. A method of treating a tumor in a subject, the method comprising administering the composition of claim 39.

41. The method of claim 40, wherein the tumor is a blood cancer.

42. The method of claim 41 , wherein the blood cancer is a B cell chronic lymphocytic leukemia, non-Hodgkin’s lymphoma, a mantle cell lymphoma, multiple myeloma, or an acute lymphoblastic leukemia.

43. The method of claim 40, wherein the tumor is a solid tumor.

44. The method of claim 43, wherein the solid tumor is a neuroblastoma, osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, glioblastoma, diffuse midline glioma or leiomyosarcoma.

45. The method of claim 43, wherein the solid tumor is an epithelial cancer.

46. The method of claim 45, wherein the epithelial cancer is a non-small cell lung cancer, a triple negative breast cancer, melanoma, or an ovarian cancer.

47. The method of any one of claims 40-46, wherein the tumor expresses the antigen recognized by the antigen-binding domain of the CAR polypeptide.

48. The method of any one of claims 40-46, wherein the tumor expresses the antigen recognized by the antigen-binding domain of the CAR polypeptide, wherein the tumor expresses the antigen GD2.

49. A cell bank comprising cells for adoptive immunotherapy, wherein the cells express the CAR polypeptide of any one of claims 1 to 25.

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