Treatment and prevention of cytokine release syndrome using chimeric antigen receptors in combination with kinase inhibitors

Abstract

The present disclosure provides compositions and methods for treating a disease associated with antigen expression or for treating or preventing cytokine release syndrome, e.g., by administering a CAR therapy with a kinase inhibitor, such as a JAK-STAT inhibitor and / or a BTK inhibitor.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201780054186.0 (International Application No. PCT / US2017 / 042129), with an application date of July 14, 2017 and an invention title of "Treatment and Prevention of Cytokine Release Syndrome Using Chimeric Antigen Receptors in Combination with Kinase Inhibitors".

[0002] This application claims priority to U.S. Serial No. 62 / 362,659, filed July 15, 2016, U.S. Serial No. 62 / 366,997, filed July 26, 2016, and U.S. Serial No. 62 / 381,230, filed August 30, 2016, the entire contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention generally relates to the use of immune effector cells (e.g., T cells or NK cells) engineered to express chimeric antigen receptors (CARs) in combination with kinase inhibitors (e.g., JAK-STAT or BTK inhibitors) to treat diseases and / or prevent cytokine release syndrome (CRS). Background Art

[0004] Many patients with hematological malignancies (e.g., B cell malignancies) cannot be cured with standard therapies. In addition, traditional treatment regimens often produce severe side effects. Recent developments in the use of autologous T cells modified with chimeric antigen receptors (CARs) (a therapy that relies on redirecting T cells to appropriate cell surface molecules on cancer cells (e.g., B cell malignancies)) have shown promising results in harnessing the power of the immune system to treat B cell malignancies and other cancers (see, e.g., Sadelain et al., Cancer Discovery 3:388-398 (2013)). Clinical results with murine-derived CART19 (i.e., “CTL019”) have shown promise for achieving complete remission in patients with CLL as well as pediatric ALL (see, e.g., Kalos et al., Sci Transl Med 3:95ra73 (2011), Porter et al., NEJM 365:725-733 (2011), Grupp et al., NEJM 368:1509-1518 (2013)). In addition to the ability of the chimeric antigen receptor on the genetically modified T cell to recognize and destroy target cells, successful therapeutic T cell therapy requires the ability to proliferate and persist over time, as well as the ability to further monitor for escape of leukemic cells. The variable quality of T cells (whether as a result of dysfunction, inhibition, or exhaustion) can impact the performance of CAR-converted T cells, but there is limited control over this performance at this time for skilled practitioners. For it to be effective, CAR-converted patient T cells need to continue and maintain the ability to proliferate in response to the target antigen. It has been shown that ALL patient T cells can do this with CART19 containing murine scFv (see, e.g., Grupp et al., NEJM 368:1509-1518 (2013)).

[0005] Cytokine release syndrome (CRS) is a serious and common adverse side effect of immune cell-based therapies, such as CAR T cell therapy. Severe CRS is a life-threatening toxicity. Deaths in severe CRS cases have been reported. Diagnosis and management of CRS in response to immune cell-based therapies are routinely based on clinical parameters and symptoms, e.g., see CRS grading as described by Lee, D. et al. (2014) Blood 124(2):188-195. Although the interleukin-6 receptor blocker tocilizumab and steroids can reverse CRS, there remains concern that these approaches may compromise the anti-tumor effect. Moreover, there is a lack of preclinical models of CRS following human CART. A preclinical model of CRS is needed following human CART administration. Moreover, there is also a need for CRS prevention modalities - such modalities can enhance the clinical feasibility of CART therapy. Summary of the Invention

[0006] This disclosure is at least partially based on the finding that JAK-STAT kinase inhibitors (such as ruxolitinib) can improve the severity of cytokine release syndrome (CRS) or prevent CRS following CART cell therapy for hematological cancers (such as acute myeloid leukemia (AML)) without significantly compromising the anti-tumor effect of CART therapy. This disclosure is also at least partially based on the finding that BTK inhibitors (such as ibrutinib) can improve or prevent CRS following CD19 CAR therapy for B cell tumors. Additionally, this disclosure is at least partially based on the finding that IL-6 inhibitors (such as those useful for CRS prevention / treatment) can be administered in combination with CAR therapy (e.g., before, concurrently, or after) without reducing the anti-cancer efficacy of CAR therapy.

[0007] Without wishing to be bound by theory, it is believed that treating a subject having a disease (such as a cancer described herein) with a combination therapy comprising a cell expressing a CAR and a JAK-STAT or BTK inhibitor, as compared to treating the subject with the cell expressing a CAR or the JAK-STAT or BTK inhibitor alone, results in improved inhibition or reduction of tumor progression in the subject and / or reduced adverse reactions (such as reduced CRS).

[0008] Accordingly, the present disclosure relates at least in part to compositions and methods for treating disorders (such as cancer (such as hematological cancer or other B cell malignancies)) using immune effector cells (such as T cells or NK cells) expressing chimeric antigen receptor (CAR) molecules (such as CARs that bind B cell antigens, such as CD123 or cluster of differentiation 19 protein (CD19) (such as OMIM accession number 107265, Swiss Prot. accession number P15391)). The compositions include, and the methods include administering in combination immune effector cells (such as T cells or NK cells) expressing a CAR (such as a B cell-targeted CAR) with a kinase inhibitor (such as one or more of a JAK-STAT inhibitor and / or a BTK inhibitor). In some embodiments, the combination maintains, has better clinical efficacy, and / or has lower toxicity (such as due to prevention of CRS) compared to either therapy alone. In some embodiments, the subject is at risk of CRS or has CRS; or the subject has been identified as having CRS or being at risk of developing CRS.

[0009] The present disclosure further relates to the use of engineered cells (such as immune effector cells (such as T cells or NK cells)) to express antigen-binding CAR molecules (such as the tumor antigens described herein, such as B cell antigens, such as CD123 or CD19), in combination with a kinase inhibitor (such as at least one JAK-STAT inhibitor) to treat disorders (such as cancer, such as hematological cancer) associated with the expression of B cell antigens (such as CD123 or CD19).

[0010] Compositions and methods are also provided for preventing CRS in a subject by using a combination of a JAK-STAT inhibitor and a CAR-expressing cell (such as a B cell-targeted CAR-expressing cell, such as a CD123 CAR-expressing cell).

[0011] Compositions and methods are also provided for preventing CRS in a subject by using a combination of a BTK inhibitor and a CAR-expressing cell (such as a B cell-targeted CAR-expressing cell, such as a CD19 CAR-expressing cell), such as where the subject is at risk of CRS or has CRS; or the subject has been identified as having CRS or being at risk of developing CRS.

[0012] In one aspect, provided herein are methods of treating a subject (e.g., a mammal) having a disease associated with the expression of an antigen (e.g., a tumor antigen, such as a tumor antigen described herein). The method comprises administering to the subject an effective amount of a combination of a cell (e.g., an immune effector cell (e.g., a T cell or an NK cell) expressing a CAR molecule that binds to an antigen (e.g., an antigen described herein, such as a tumor antigen, such as a B cell antigen)) and a JAK-STAT inhibitor (e.g., a JAK-STAT inhibitor described herein, such as ruxolitinib).

[0013] In another aspect, provided herein are methods of providing anti-tumor immunity to a subject (e.g., a mammal) having a disease associated with the expression of an antigen (e.g., a tumor antigen, such as a tumor antigen described herein). The method comprises administering to the subject an effective amount of a combination of a cell (e.g., an immune effector cell (e.g., a T cell or an NK cell) expressing a CAR molecule that binds to an antigen (e.g., an antigen described herein, such as a tumor antigen, such as a B cell antigen)) and a JAK-STAT inhibitor (e.g., a JAK-STAT inhibitor described herein, such as ruxolitinib).

[0014] In one embodiment, the CAR molecule binds CD123, such as a CAR molecule that binds to CD123 as described herein.

[0015] In another aspect, provided herein are methods of treating and / or preventing cytokine release syndrome (CRS) (e.g., CRS associated with CAR therapy (e.g., CAR-expressing cells described herein)) in a subject in need thereof, the method comprising administering a JAK-STAT inhibitor (e.g., ruxolitinib) alone or in combination with CAR therapy to the subject, thereby treating and / or preventing CRS in the subject.

[0016] In embodiments, the subject is at risk of developing CRS, has CRS, or is diagnosed with CRS. In embodiments, the subject has been, is being, or will be administered CAR therapy, such as CAR-expressing cells described herein.

[0017] In embodiments, the method further comprises administering to the subject an IL-6 inhibitor (e.g., an anti-IL6 receptor inhibitor, such as tocilizumab). In embodiments, the method comprises administering to the subject (i) a JAK-STAT inhibitor (e.g., ruxolitinib), (ii) CAR therapy (e.g., CAR-expressing cells described herein), and (iii) an IL-6 inhibitor (e.g., an anti-IL6 receptor inhibitor, such as tocilizumab).

[0018] In another aspect, provided herein are methods for preventing cytokine release syndrome (CRS) (e.g., CRS associated with CAR therapy (e.g., B cell antigen CAR therapy, such as CD19 CAR therapy)) in a subject in need thereof, the method comprising administering a BTK inhibitor (e.g., ibrutinib) alone or in combination with CAR therapy to the subject, thereby preventing CRS in the subject.

[0019] In embodiments, the subject is at risk of developing CRS, has CRS, or is diagnosed with CRS. In embodiments, the subject has been, is being, or will be administered CAR therapy, such as CAR therapy described herein. In embodiments, the subject is identified or was previously identified as being at risk of CRS.

[0020] In embodiments, the method comprises selecting a subject for administration of a BTK inhibitor. In embodiments, the subject is selected based on: (i) his or her risk of developing CRS, (ii) his or her CRS diagnosis, and / or (iii) whether he or she has been, is being, or will be administered CAR therapy (e.g., CAR therapy described herein, such as CAR19 therapy, such as CTL019). In embodiments, if the subject is diagnosed with CRS (e.g., severe or non-severe CRS), then the subject is selected for administration of a BTK inhibitor. In embodiments, if the subject is at risk of developing CRS (e.g., identified as being at risk of developing CRS), then the subject is selected for administration of a BTK inhibitor. In embodiments, if the subject has been, is being, or will be administered CAR therapy (e.g., CAR therapy described herein, such as CAR19 therapy, such as CTL019), then the subject is selected for administration of a BTK inhibitor.

[0021] In embodiments, the method further comprises administering an IL-6 inhibitor (e.g., an anti-IL6 receptor inhibitor, such as tocilizumab) to the subject. In embodiments, the method comprises administering to the subject (i) a BTK inhibitor (e.g., ibrutinib), (ii) CAR therapy (e.g., CAR-expressing cells described herein), and (iii) an IL-6 inhibitor (e.g., an anti-IL6 receptor inhibitor, such as tocilizumab).

[0022] In yet another aspect, provided herein are methods for treating or preventing CRS associated with the administration of CAR-expressing cells (e.g., cell populations) in a subject.

[0023] In yet another aspect, provided herein are methods of treating or preventing CRS associated with the administration of T cell inhibitor therapies (e.g., CD19 inhibition or depletion therapies, such as therapies comprising a CD19 inhibitor). In embodiments, CD19 inhibition or depletion therapies are associated with CRS.

[0024] Methods of treating or preventing CRS include administering an IL-6 inhibitor (e.g., an anti-IL6 receptor inhibitor, such as tocilizumab) to a subject before, concurrently with, or within 1 day (e.g., within 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, or less) of administering a dose (or a first dose) of the CAR-expressing cells (e.g., the cell population) or the therapy.

[0025] In embodiments, an IL-6 inhibitor (e.g., tocilizumab) is administered to a subject within (e.g., within 1 hour, 30 minutes, 20 minutes, 15 minutes, or less) after the first sign of CRS symptoms (e.g., fever, such as characterized by: e.g., two consecutive measurements (e.g., taken at least 4, 5, 6, 7, 8 hours, or more apart) within 24 hours, temperature of at least 38°C (e.g., at least 38.5°C)).

[0026] The following examples relate to any of the methods and compositions described herein.

[0027] CAR molecule

[0028] In embodiments, the CAR molecule comprises an antigen-binding domain (e.g., a B cell antigen-binding domain, a CD123-binding domain, or a CD19-binding domain), a transmembrane domain, and an intracellular signaling domain (e.g., an intracellular signaling domain comprising a co-stimulatory domain and / or a primary signaling domain).

[0029] In an embodiment, the CAR comprises an antigen-binding domain that binds to one or more of the following: CD19; CD123; CD22; CD30; CD171; CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin 11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21 (prostasin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine protein kinase ERBB2 (Her2 / neu); mucin 1, cell surface-associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostate enzyme; prostate acid phosphatase (PAP); mutant elongation factor 2 (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (Prosome, Macropain) subunit, beta type, 9 (LMP2); glycoprotein 100 (gp100); oncogenic fusion protein composed of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin A type receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5);High-molecular-weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide moiety of globoH glycosphingolipid (GloboH); breast differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor beta-3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternative reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1); ETS translocation variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer-testis antigen-1 (MAD-CT-1); melanoma cancer-testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostate-specific protein (prostein); survivin; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or galectin-8), melanoma antigen recognized by T cells 1 (MelanA or MART1); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma apoptosis inhibitor (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 1B1 (CYP1B1);CCCTC-binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of Imprinted Sites), squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosomal protein-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; mutant heat shock protein 70-2 (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); mucin-like hormone receptor-like 2 containing EGF-like modules (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); or immunoglobulin lambda-like polypeptide 1 (IGLL1).;

[0030] In other embodiments, the CAR molecule is capable of binding an antigen described herein, such as an antigen described in the Antigen section below.

[0031] In one embodiment, the antigen comprises a B cell antigen, such as CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, and / or CD79a.

[0032] In an embodiment, the antigen is CD123. In an embodiment, the antigen is CD19.

[0033] In other embodiments, the antigen is BCMA. In an embodiment, the antigen is CLL.

[0034] Exemplary CAR molecules

[0035] In one embodiment, the CAR molecule comprises the CD123 CAR described herein, such as the CD123 CAR described in US 2014 / 0322212A1 or US2016 / 0068601 A1 (both incorporated herein by reference). In an embodiment, the CD123 CAR comprises amino acids or has the nucleotide sequence shown in US 2014 / 0322212A1 or US2016 / 0068601 A1 (both incorporated herein by reference).

[0036] In an embodiment, the CAR molecule comprises the CD19 CAR molecule described herein, such as the CD19 CAR molecule described in US-2015-0283178-A1, such as CTL019. In an embodiment, the CD19 CAR comprises amino acids or has the nucleotide sequence shown in US-2015-0283178-A1 (incorporated herein by reference).

[0037] In one embodiment, the CAR molecule comprises the BCMACAR molecule described herein, such as the BCMACAR described in US-2016-0046724-A1. In an embodiment, the BCMACAR comprises amino acids or has the nucleotide sequence shown in US-2016-0046724-A1 (incorporated herein by reference).

[0038] In one embodiment, the CAR molecule comprises the CLL1 CAR described herein, such as the CLL1 CAR described in US 2016 / 0051651A1 (incorporated herein by reference). In an embodiment, the CLL1 CAR comprises amino acids or has the nucleotide sequence shown in US2016 / 0051651 A1 (incorporated herein by reference).

[0039] In one embodiment, the CAR molecule comprises the CD33 CAR described herein, such as the CD33 CAR described in US 2016 / 0096892A1 (incorporated herein by reference). In an embodiment, the CD33 CAR comprises amino acids or has the nucleotide sequence shown in US2016 / 0096892 A1 (incorporated herein by reference).

[0040] In one embodiment, the CAR molecule comprises the EGFRvIIICAR molecule described herein, such as the EGFRvIIICAR described in US2014 / 0322275 A1 (incorporated herein by reference). In an embodiment, the EGFRvIIICAR comprises amino acids or has the nucleotide sequence shown in US2014 / 0322275 A1 (incorporated herein by reference).

[0041] In an embodiment, the CAR molecule comprises the mesothelin CAR described herein, such as the mesothelin CAR described in WO 2015 / 090230 (incorporated herein by reference). In an embodiment, the mesothelin CAR comprises amino acids or has the nucleotide sequence shown in WO 2015 / 090230 (incorporated herein by reference).

[0042] CD123 CAR antigen-binding domain

[0043] In an embodiment, the CAR molecule is capable of binding to CD123 (e.g., wild-type or mutant CD123). In an embodiment, the CAR molecule comprises an anti-CD123 binding domain that comprises one or more (e.g., all three) of the light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of the anti-CD123 binding domain described herein (e.g., as described in US2014 / 0322212 A1 or US2016 / 0068601 A1), and / or one or more (e.g., all three) of the heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of the anti-CD123 binding domain described herein (e.g., as described in US2014 / 0322212A1 or US2016 / 0068601 A1), such as an anti-CD123 binding domain comprising one or more (e.g., all three) LC CDRs and one or more (e.g., all three) HC CDRs.

[0044] In one embodiment, the encoded CD123 binding domain comprises one or more (e.g., all three) of the light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of the CD123 binding domain described herein, and / or one or more (e.g., all three) of the heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of the CD123 binding domain described herein, e.g., a CD123 binding domain comprising one or more (e.g., all three) LC CDRs and one or more (e.g., all three) HC CDRs. In one embodiment, the encoded CD123 binding domain (e.g., a human or humanized CD123 binding domain) comprises the light chain variable region (e.g., in Table 11A, 12A, or 12B) described herein and / or the heavy chain variable region (e.g., in Table 11A, 12A, or 12B) described herein. In one embodiment, the encoded CD123 binding domain is a scFv that comprises the light and heavy chains of the amino acid sequences of Table 11A, 12A, or 12B. In one embodiment, the CD123 binding domain (e.g., scFv) comprises: a light chain variable region that comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequence of the light chain variable region provided in Table 11A, 12A, or 12B, but no more than 30, 20, or 10 modifications (e.g., substitutions, e.g., conservative substitutions), or a sequence having at least 95%, e.g., 95%-99% identity to the amino acid sequence of Table 11A, 12A, or 12B; and / or a heavy chain variable region that comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequence of the heavy chain variable region provided in Table 11A, 12A, or 12B, but no more than 30, 20, or 10 modifications (e.g., substitutions, e.g., conservative substitutions), or a sequence having at least 95% (e.g., 95%-99%) identity to the amino acid sequence of Table 11A, 12A, or 12B.

[0045] In other embodiments, the encoded CD123 binding domain comprises HC CDR1, HC CDR2, and HC CDR3 of any of the CD123 heavy chain binding domain amino acid sequences listed in Table 11A, 12A, or 12B. In an embodiment, the CD33 binding domain further comprises LC CDR1, LC CDR2, and LC CDR3. In an embodiment, the CD123 binding domain comprises LC CDR1, LC CDR2, and LC CDR3 of any of the CD123 light chain binding domain amino acid sequences listed in Table 11A, 12A, or 12B.

[0046] In some embodiments, the encoded CD123 binding domain comprises one, two, or all of the LC CDR1, LC CDR2, and LC CDR3 of any of the CD123 light chain binding domain amino acid sequences listed in Table 11A or 12B, and one, two, or all of the HC CDR1, HC CDR2, and HCCDR3 of any of the CD123 heavy chain binding domain amino acid sequences listed in Table 11A, 12A, or 12B.

[0047] In one embodiment, the encoded CD123 binding domain comprises an amino acid sequence selected from SEQ ID NOs: 157 - 160, 184 - 215, 478, 480, 483, and 485. In one embodiment, the encoded CD123 binding domain (e.g., scFv) comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, such as conservative substitutions) of the amino acid sequences of 157 - 160, 184 - 215, 478, 480, 483, and 485, but not more than 30, 20, or 10 modifications (e.g., substitutions, such as conservative substitutions), or a sequence having at least 95% identity (e.g., having 95% - 99% identity) to the amino acid sequences of SEQ ID NOs: 157 - 160, 184 - 215, 478, 480, 483, and 485.

[0048] In another embodiment, the encoded CD123 binding domain comprises a heavy chain variable region that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 216 - 219 or 243 - 274, or an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, such as conservative substitutions) of SEQ ID NOs: 216 - 219 or 243 - 274, but not more than 30, 20, or 10 modifications (e.g., substitutions, such as conservative substitutions), or an amino acid sequence having at least 95% identity (e.g., having 95% - 99% identity) to SEQ ID NOs: 216 - 219 or 243 - 274. In another embodiment, the encoded CD123 binding domain comprises a heavy chain variable region that comprises an amino acid sequence corresponding to the heavy chain variable region of SEQ ID NO: 478, 480, 483, or 485, or comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, such as conservative substitutions) of the corresponding portion of SEQ ID NOs: 478, 480, 483, or 485, but not more than 30, 20, or 10 modifications (e.g., substitutions, such as conservative substitutions), or comprises an amino acid sequence having at least 95% identity (e.g., having 95% - 99% identity) to the corresponding portion of SEQ ID NOs: 478, 480, 483, or 485.

[0049] In another embodiment, the encoded CD123 binding domain comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 275-278 or 302-333, or an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, such as conservative substitutions) of SEQ ID NOs: 275-278 or 302-333 but no more than 30, 20, or 10 modifications (e.g., substitutions, such as conservative substitutions), or an amino acid sequence having at least 95% identity (e.g., having 95%-99% identity) to SEQ ID NOs: 275-278 or 302-333. In another embodiment, the encoded CD123 binding domain comprises a light chain variable region comprising an amino acid sequence corresponding to the light chain variable region of SEQ ID NO: 478, 480, 483, or 485, or comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, such as conservative substitutions) of the corresponding portion of SEQ ID NO: 478, 480, 483, or 485 but no more than 30, 20, or 10 modifications (e.g., substitutions, such as conservative substitutions), or comprising an amino acid sequence having at least 95% identity (e.g., having 95%-99% identity) to the corresponding portion of SEQ ID NO: 478, 480, 483, or 485.

[0050] In one embodiment, the nucleic acid molecule encoding the scFv comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO:479, 481, 482, or 484, or a sequence having at least 95% identity, such as 95%-99% identity, thereto. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a heavy chain variable region and / or a light chain variable region, wherein the nucleotide sequence comprises a nucleotide sequence portion selected from the group consisting of: SEQ ID NO:479, 481, 482, or 484 corresponding to the heavy chain variable region and / or the light chain variable region, or a sequence having at least 95% identity, such as 95%-99% identity, thereto. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a heavy chain variable region and / or a light chain variable region, wherein the encoded amino acid sequence is selected from the group consisting of: SEQ ID NO:157-160, or a sequence having at least 95% identity, such as having 95%-99% identity, thereto. In one embodiment, the nucleic acid molecule encodes an scFv, which scFv comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:184-215, or a sequence having at least 95% identity, such as 95%-99% identity, thereto. In one embodiment, the nucleic acid molecule comprises a sequence encoding a heavy chain variable region and / or a light chain variable region, wherein the encoded amino acid sequence is selected from the group consisting of: SEQ ID NO:184-215, or a sequence having at least 95% identity, such as 95%-99% identity, thereto.

[0051] In one embodiment, the encoded CD123 binding domain comprises a (Gly4-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO:26). The light chain variable region and the heavy chain variable region of the scFv can be in any of the following orientations, for example: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0052] CD19 CAR antigen-binding domain

[0053] In embodiments, the CAR molecule is capable of binding CD19 (e.g., wild-type or mutant CD19). In embodiments, the CAR molecule comprises an anti-CD19 binding domain that comprises one or more (e.g., all three) of the light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of the anti-CD123 binding domain described herein, and / or one or more (e.g., all three) of the heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of the anti-CD19 binding domain described herein, such as an anti-CD19 binding domain that comprises one or more (e.g., all three) LC CDRs and one or more (e.g., all three) HCCDRs.

[0054] In one embodiment, the anti-CD19 binding domain comprises one or more (e.g., all three) of the heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of the anti-CD19 binding domain described herein. For example, the anti-CD19 binding domain has two variable heavy chain regions, each variable heavy chain region comprising HC CDR1, HC CDR2, and HC CDR3 as described herein. In one embodiment, the anti-CD19 binding domain comprises the murine light chain variable region (e.g., in Table 14A) and / or the murine heavy chain variable region (e.g., in Table 14A) described herein. In one embodiment, the anti-CD19 binding domain is a scFv that comprises the murine light chain and murine heavy chain of the amino acid sequence of Table 14A. In one embodiment, the anti-CD19 binding domain (e.g., scFv) comprises: a light chain variable region that comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence of the light chain variable region provided in Table 14A but no more than 30, 20, or 10 modifications (e.g., substitutions), or a sequence having at least 95% identity, e.g., 95%-99% identity, to the amino acid sequence of Table 14A; and / or a heavy chain variable region that comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence of the heavy chain variable region provided in Table 14A but no more than 30, 20, or 10 modifications (e.g., substitutions), or a sequence having at least 95% identity, e.g., 95%-99% identity, to the amino acid sequence of Table 14A. In one embodiment, the anti-CD19 binding domain comprises the sequence of SEQ ID NO:774, or a sequence having at least 95% identity, e.g., 95%-99% identity, thereto. In one embodiment, the anti-CD19 binding domain is a scFv and comprises a light chain variable region of the amino acid sequence described herein (e.g., in Table 14A) attached via a linker (e.g., the linker described herein) to a heavy chain variable region of the amino acid sequence described herein (e.g., in Table 14A). In one embodiment, the anti-CD19 binding domain comprises a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO:26). The light chain variable region and heavy chain variable region of the scFv can be in any orientation, for example: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0055] In one embodiment, the CAR molecule comprises a humanized anti-CD19 binding domain that includes one or more (e.g., all three) of the light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of the humanized anti-CD19 binding domain described herein, and one or more (e.g., all three) of the heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of the humanized anti-CD19 binding domain described herein, e.g., a humanized anti-CD19 binding domain that includes one or more (e.g., all three) LC CDRs and one or more (e.g., all three) HC CDRs. In one embodiment, the humanized anti-CD19 binding domain comprises at least HC CDR2. In one embodiment, the humanized anti-CD19 binding domain includes one or more (e.g., all three) of the heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of the humanized anti-CD19 binding domain described herein; e.g., the humanized anti-CD19 binding domain has two variable heavy chain regions, each variable heavy chain region comprising HC CDR1, HC CDR2, and HC CDR3 described herein. In one embodiment, the humanized anti-CD19 binding domain comprises at least HC CDR2. In one embodiment, the light chain variable region comprises one, two, three, or all four framework regions of the VK3_L25 germline sequence. In one embodiment, the light chain variable region has modifications (e.g., substitutions, such as substitutions of one or more amino acids found at corresponding positions in the murine light chain variable region of SEQ ID NO:773, e.g., substitutions at one or more of positions 71 and 87). In one embodiment, the heavy chain variable region comprises one, two, three, or all four framework regions of the VH4_4-59 germline sequence. In one embodiment, the heavy chain variable region has modifications (e.g., substitutions, such as substitutions of one or more amino acids found at corresponding positions in the murine heavy chain variable region of SEQ ID NO:773, e.g., substitutions at one or more of positions 71, 73, and 78). In one embodiment, the humanized anti-CD19 binding domain comprises the light chain variable region described herein (e.g., in Table 13A) and / or the heavy chain variable region described herein (e.g., in Table 13A). In one embodiment, the humanized anti-CD19 binding domain is a scFv that comprises the light chain and heavy chain of the amino acid sequence of Table 13A.In one embodiment, a humanized anti-CD19 binding domain (e.g., scFv) comprises: a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence of the light chain variable region provided in Table 13A but no more than 30, 20, or 10 modifications (e.g., substitutions), or a sequence having at least 95% identity, such as 95%-99% identity, to the amino acid sequence of Table 13A; and / or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence of the heavy chain variable region provided in Table 13A but no more than 30, 20, or 10 modifications (e.g., substitutions), or a sequence having at least 95% identity, such as 95%-99% identity, to the amino acid sequence of Table 13A. In one embodiment, the humanized anti-CD19 binding domain comprises a sequence selected from the group consisting of SEQ ID NOs: 710-721, or a sequence having at least 95% identity, such as 95%-99% identity, thereto. In one embodiment, the humanized anti-CD19 binding domain is an scFv and comprises a light chain variable region of the amino acid sequence described herein (e.g., in Table 13A) attached via a linker (e.g., a linker described herein) to a heavy chain variable region of the amino acid sequence described herein (e.g., in Table 13A).

[0056] In an embodiment, the antigen recognition domain binds CD19. In an embodiment, the CAR comprises the amino acid sequence of the CD19 CAR described herein. In an embodiment, the CAR comprises the amino acid sequence of SEQ ID NO: 773.

[0057] In one embodiment, the humanized anti-CD19 binding domain includes a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO: 26). The light chain variable region and the heavy chain variable region of the scFv can be in any orientation, such as: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0058] Other CAR domains

[0059] In one embodiment, the CAR molecule comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In one embodiment, the transmembrane domain comprises the sequence of SEQ ID NO:6. In one embodiment, the transmembrane domain comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence of SEQ ID NO:6 but not more than 20, 10, or 5 modifications (e.g., substitutions), or a sequence having at least 95% identity, such as 95%-99% identity, to the amino acid sequence of SEQ ID NO:6.

[0060] In one embodiment, the antigen-binding domain (e.g., the CD123 or CD19-binding domain) is linked to the transmembrane domain by a hinge region (e.g., the hinge region described herein). In one embodiment, the encoded hinge region comprises SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:3, or a sequence having at least 95% identity, such as 95%-99% identity, thereto.

[0061] In one embodiment, the CAR molecule further comprises a sequence encoding a co-stimulatory domain, such as the co-stimulatory domain described herein. In one embodiment, the co-stimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS, and 4-1BB (CD137). In one embodiment, the co-stimulatory domain comprises the sequence of SEQ ID NO:7. In one embodiment, the co-stimulatory domain comprises the sequence of SEQ ID NO:8. In one embodiment, the co-stimulatory domain comprises the sequence of SEQ ID NO:43. In one embodiment, the co-stimulatory domain comprises the sequence of SEQ ID NO:45. In one embodiment, the co-stimulatory domain comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence of SEQ ID NO:7, 8, 43, or 45 but not more than 20, 10, or 5 modifications (e.g., substitutions), or a sequence having at least 95% identity, such as 95%-99% identity, to the amino acid sequence of SEQ ID NO:7, 8, 43, or 45.

[0062] In one embodiment, the CAR molecule further comprises a sequence encoding an intracellular signaling domain (such as the intracellular signaling domains described herein). In one embodiment, the intracellular signaling domain comprises the functional signaling domain of 4-1BB and / or the functional signaling domain of CD3ζ. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO:7 and / or the sequence of SEQ ID NO:9 or 10. In one embodiment, the intracellular signaling domain comprises the functional signaling domain of CD27 and / or the functional signaling domain of CD3ζ. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO:8 and / or the sequence of SEQ ID NO:9 or 10. In one embodiment, the intracellular signaling domain comprises an amino acid sequence having at least one, two or three modifications (such as substitutions) but not more than 20, 10 or 5 modifications (such as substitutions) of the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8 and / or the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10, or a sequence having at least 95% identity, such as 95%-99% identity, with the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8 and / or the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO:7 or SEQ ID NO:8, and the sequence of SEQ ID NO:9 or SEQ ID NO:10, wherein the sequence comprising the intracellular signaling domain is expressed in the same frame and expressed as a single polypeptide chain.

[0063] In one embodiment, the CAR molecule further comprises a leader sequence, such as the leader sequence described herein. In one embodiment, the leader sequence comprises the amino acid sequence of SEQ ID NO:1, or a sequence having at least 95% identity, such as 95%-99% identity, with the amino acid sequence of SEQ ID NO:1.

[0064] CD123 CAR construct

[0065] In an embodiment, the CAR molecule comprises a leader sequence (such as the leader sequences described herein, such as a leader sequence having SEQ ID NO: 1 (or having at least 95% identity thereto, such as 95%-99% identity)), a CD123 binding domain as described herein (such as a CD123 binding domain comprising LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2, and HC CDR3 as described herein, such as the CD123 binding domain described in Table 11A or 12A, or a sequence having at least 95% identity thereto, such as 95%-99% identity), a hinge region (such as the hinge region described herein, such as a hinge region having SEQ ID NO: 2 (or having at least 95% identity thereto, such as 95%-99% identity)), a transmembrane domain (such as the transmembrane domain described herein, such as a transmembrane domain having the sequence of SEQ ID NO: 6 or a sequence having at least 95% identity thereto, such as 95%-99% identity), and an intracellular signaling domain (such as the intracellular signaling domain described herein (e.g., an intracellular signaling domain comprising a co-stimulatory domain and / or a primary signaling domain)). In one embodiment, the intracellular signaling domain comprises a co-stimulatory domain (such as the co-stimulatory domain described herein, such as a 4-1BB co-stimulatory domain having the sequence of SEQ ID NO: 7 (or having at least 95% identity thereto, such as 95%-99% identity)) and / or a primary signaling domain (such as the primary signaling domain described herein, such as a CD3ζ stimulatory domain having the sequence of SEQ ID NO: 9 or SEQ ID NO: 10 (or having at least 95% identity thereto, such as 95%-99% identity)). In one embodiment, the intracellular signaling domain comprises a co-stimulatory domain (such as the co-stimulatory domain described herein, such as a 4-1BB co-stimulatory domain having the sequence of SEQ ID NO: 7) and / or a primary signaling domain (such as the primary signaling domain described herein, such as a CD3ζ stimulatory domain having SEQ ID NO: 9 or SEQ ID NO: 10).

[0066] CD19 CAR construct

[0067] In one embodiment, the CAR molecule comprises a leader sequence, such as the leader sequences described herein, such as SEQ ID NO:1 or a leader sequence having at least 95% identity thereto, such as 95%-99% identity; an anti-CD19 binding domain as described herein, e.g., an anti-CD19 binding domain comprising the LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2, and HC CDR3 described herein, e.g., the murine anti-CD19 binding domain described in Table 14A, the humanized anti-CD19 binding domain described in Table 13A, or a sequence having 95%-99% identity thereto; a hinge region, such as the hinge regions described herein, such as a hinge region having SEQ ID NO:2, 3, or 4 or having at least 95% identity thereto, such as 95%-99% identity; a transmembrane domain, such as the transmembrane domains described herein, e.g., a transmembrane domain having the sequence of SEQ ID NO:6 or having at least 95% identity thereto, such as 95%-99% identity; and an intracellular signaling domain, such as the intracellular signaling domains described herein (e.g., an intracellular signaling domain comprising a co-stimulatory domain and / or a primary signaling domain). In one embodiment, the intracellular signaling domain comprises a co-stimulatory domain (e.g., the co-stimulatory domains described herein, such as the 4-1BB co-stimulatory domain having the sequence of SEQ ID NO:7, the CD28 co-stimulatory domain having the sequence of SEQ ID NO:43, the CD27 co-stimulatory domain having the sequence of SEQ ID NO:8, or the ICOS co-stimulatory domain having the sequence of SEQ ID NO:45, or having at least 95% identity thereto, such as 95%-99% identity), and / or a primary signaling domain (e.g., the primary signaling domains described herein, such as the CD3ζ stimulatory domain having the sequence of SEQ ID NO:9 or SEQ ID NO:10 or having at least 95% identity thereto, such as 95%-99% identity).

[0068] Other exemplary CAR constructs

[0069] In one embodiment, the CAR molecule comprises the following (e.g., consists of): the amino acid sequence described in US-2015-0283178-A1, US-2016-0046724-A1, US2014 / 0322212 A1, US 2016 / 0068601A1, US2016 / 0051651 A1, US2016 / 0096892 A1, US 2014 / 0322275A1, or WO 2015 / 090230; or an amino acid sequence having at least one, two, three, four, five, 10, 15, 20, or 30 modifications (e.g., substitutions) of the amino acid sequence described in US-2015-0283178-A1, US-2016-0046724-A1, US2014 / 0322212 A1, US 2016 / 0068601A1, US2016 / 0051651 A1, US2016 / 0096892 A1, US 2014 / 0322275A1, or WO 2015 / 090230 but not more than 60, 50, or 40 amino acid modifications (e.g., substitutions); or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence described in US-2015-0283178-A1, US-2016-0046724-A1, US2014 / 0322212 A1, US2016 / 0068601A1, US2016 / 0051651A1, US2016 / 0096892A1, US 2014 / 0322275A1, or WO 2015 / 090230.

[0070] vector

[0071] In one embodiment, the cell expressing the CAR molecule comprises a vector that includes a nucleic acid sequence encoding the CAR molecule. In one embodiment, the vector is selected from the group consisting of: DNA, RNA, plasmid, lentiviral vector, adenoviral vector, or retroviral vector. In one embodiment, the vector is a lentiviral vector. In one embodiment, the vector further comprises a promoter. In one embodiment, the promoter is the EF-1 promoter. In one embodiment, the EF-1 promoter comprises the sequence of SEQ ID NO:11. In one embodiment, the vector is an in vitro transcribed vector, such as a vector that transcribes the RNA of the nucleic acid molecule described herein. In one embodiment, the nucleic acid sequence in the in vitro vector further comprises a poly(A) tail, such as the poly(A) tail described herein (e.g., comprising approximately 150 adenosine bases (SEQ ID NO:30)). In one embodiment, the nucleic acid sequence in the in vitro vector further comprises a 3'UTR, such as the 3'UTR described herein, for example, comprising at least one repeat of the 3'UTR derived from human β-globin. In one embodiment, the nucleic acid sequence in the in vitro vector further comprises a promoter, such as the T2A promoter.

[0072] Cells expressing CAR

[0073] In certain embodiments of the compositions and methods disclosed herein, the cell expressing the CAR molecule (also referred to herein as "cells expressing CAR") is a cell or cell population as described herein, such as a human immune effector cell or cell population (e.g., human T cells or human NK cells, such as the human T cells described herein or the human NK cells described herein). In one embodiment, the human T cells are CD8+ T cells. In one embodiment, the cell is an autologous T cell. In one embodiment, the cell is an allogeneic T cell. In one embodiment, the cell is a T cell and the T cell is defective in diacylglycerol kinase (DGK). In one embodiment, the cell is a T cell and the T cell is defective in Ikaros. In one embodiment, the cell is a T cell and the T cell is defective in both DGK and Ikaros. It should be understood that the compositions and methods described herein using the narrative term "cell" encompass compositions and methods comprising one or more cells (e.g., cell populations).

[0074] In some embodiments, the administered CAR-expressing cells comprise an inducible CAR (iCAR), such as the iCAR described herein. The iCAR can comprise, for example, an intracellular signaling member comprising an intracellular signaling domain and a first switch domain, an antigen-binding member comprising an antigen-binding domain that binds to an antigen (e.g., an antigen described herein, such as a B cell antigen, such as CD123 or CD19) and a second switch domain; and a transmembrane domain. The method can further comprise administering a dimerizing molecule, for example, in an amount sufficient to cause dimerization of the first switch domain and the second switch domain.

[0075] inhibitor

[0076] In embodiments, the JAK-STAT inhibitor comprises / is an antibody molecule, small molecule, polypeptide (e.g., a fusion protein), or inhibitory nucleic acid (e.g., siRNA or shRNA). In embodiments, the JAK-STAT inhibitor is a small molecule, such as ruxolitinib, AG490, AZD1480, tofacitinib (tasocitinib or CP-690550), CYT387, fedratinib, baricitinib (INCB039110), lestaurtinib (CEP701), pacritinib (SB1518), XL019, gandotinib (LY2784544), BMS911543, fedratinib (SAR302503), decemotinib (V-509), INCB39110, GEN1, GEN2, GLPG0634, NS018, and N-(cyanomethyl)-4-[2-(4-morpholinylanilino)pyrimidin-4-yl]benzamide or a pharmaceutically acceptable salt thereof. In embodiments, the JAK-STAT inhibitor is ruxolitinib or a pharmaceutically acceptable salt thereof.

[0077] In embodiments, the BTK inhibitor comprises / is an antibody molecule, small molecule, polypeptide (e.g., a fusion protein), or inhibitory nucleic acid (e.g., siRNA or shRNA). In embodiments, the BTK inhibitor is a small molecule, such as ibrutinib, GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774, or LFM-A13, or a pharmaceutically acceptable salt thereof, or a combination thereof. In embodiments, the BTK inhibitor is ibrutinib or a pharmaceutically acceptable salt thereof.

[0078] In embodiments, an IL-6 inhibitor (e.g., used according to any of the compositions or methods described herein) comprises an inhibitor of IL-6 signaling (e.g., comprising an IL-6 inhibitor or an inhibitor of the IL-6 receptor (IL-6R)). Exemplary IL-6 inhibitors include tocilizumab, siltuximab, bazedoxifene, and soluble glycoprotein 130 (sgp130) blockers. Exemplary IL-6 inhibitors are described in International Application WO 2014011984, which is hereby incorporated by reference. Tocilizumab is described in more detail herein, e.g., in the "CRS Therapy" section herein. In one embodiment, the IL-6 inhibitor is an anti-IL-6 antibody, e.g., an anti-IL-6 chimeric monoclonal antibody, e.g., siltuximab. In other embodiments, the inhibitor comprises a soluble gp130 or a fragment thereof capable of blocking IL-6 signaling. In some embodiments, the sgp130 or its fragment is fused to a heterologous domain (e.g., an Fc domain, e.g., is a gp130-Fc fusion protein, such as FE301). In embodiments, the IL-6 inhibitor comprises an antibody, e.g., an antibody to the IL-6 receptor, such as sarilumab, olokizumab (CDP6038), elsilimomab, sirukumab (CNTO136), ALD518 / BMS-945429, ARGX-109, or FM101. In some embodiments, the IL-6 inhibitor comprises a small molecule, such as CPSI-2364.

[0079] Disease

[0080] In embodiments, a disease associated with antigen expression is a hyperproliferative disorder, such as cancer. In embodiments, the cancer is a solid cancer. In other embodiments, the cancer is a hematological cancer.

[0081] In embodiments, the hematological cancer is leukemia. In embodiments, the hematological cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), or chronic lymphocytic leukemia (CLL). In embodiments, the hematological cancer is lymphoma, such as mantle cell lymphoma (MCL).

[0082] In embodiments, the hematological cancer is a B cell malignancy, such as B cell leukemia or B cell lymphoma.

[0083] In embodiments, the hematological cancer is selected from: chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphoblastic leukemia (ALL), Hodgkin lymphoma, B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), small lymphocytic leukemia (SLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma (extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue), marginal zone lymphoma, myelodysplasia and myelodysplastic syndromes, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell tumors, Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, splenic lymphoma / leukemia, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemia variant, lymphoplasmacytic lymphoma, heavy chain disease, plasmacytic myeloma, solitary plasmacytoma of bone, extramedullary plasmacytoma, nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, primary cutaneous follicle center lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, primary effusion lymphoma, or lymphoma, unclassifiable.

[0084] In embodiments, the hematological cancer is selected from: acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), acute lymphoblastic B-cell leukemia (B-cell acute lymphoblastic leukemia, BALL), acute lymphoblastic T-cell leukemia (T-cell acute lymphoblastic leukemia (TALL)), B-cell prolymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia (CML), hairy cell leukemia, Hodgkin lymphoma, histiocytic disorders, mast cell disorders, myelodysplasia, myelodysplastic syndromes, myeloproliferative neoplasms, plasmacytic myeloma, plasmacytoid dendritic cell tumors, or a combination thereof.

[0085] In an embodiment, the disease is a disease associated with B cell antigen expression (e.g., expression of one or more of CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, and / or CD79a). In an embodiment, the disease associated with B cell antigen expression is selected from proliferative diseases such as cancer, malignancy, or pre-cancerous conditions such as myelodysplasia, myelodysplastic syndrome, or pre-leukemia, or the disease is a non-cancer-related indication associated with B cell antigen (e.g., one or more of CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, and / or CD79a) expression. In certain embodiments, the disease associated with B cell antigen expression is "pre-leukemia", which is a diverse collection of hematological disorders associated with ineffective production (or dysplasia) of myeloid blood cells. In some embodiments, the disease associated with B cell antigen expression includes but is not limited to atypical and / or non-classical cancers, malignancies, pre-cancerous conditions, or proliferative diseases expressing B cell antigens (e.g., one or more of CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, and / or CD79a). In an embodiment, the disease associated with B cell antigen expression is a hematological cancer, leukemia, lymphoma, MCL, CLL, ALL, Hodgkin lymphoma, or multiple myeloma. Any combination of diseases associated with B cell antigen expression as described herein can be treated with the methods and compositions described herein.

[0086] CRS

[0087] In an embodiment, the CRS is severe CRS, such as grade 4 or 5 CRS. In an embodiment, the CRS is CRS of lower severity, such as grade 1, 2, or 3 CRS. Additional descriptions of CRS are provided in the section entitled "Cytokine Release Syndrome".

[0088] In an embodiment of any of the methods described herein, the CRS is CRS that is distinguishable from sepsis, for example, by the methods described herein, such as by the method of distinguishing CRS and sepsis in a subject as described herein. In an embodiment, the method of distinguishing CRS and sepsis includes obtaining a measure of one or more of the following:

[0089] (i) The level or activity of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all) of GM-CSF, HGF, IFN-γ, IFN-α, IL-10, IL-15, IL-5, IL-6, IL-8, IP-10, MCP1, MIG, MIP-1β, sIL-2Rα, sTNFRI, and sTNFRII, wherein a level or activity higher than a reference indicates CRS; or

[0090] (ii) The level or activity of one or more (e.g., 2, 3, 4, 5, 6, or all) of CD163, IL-1β, sCD30, sIL-4R, sRAGE, sVEGFR-1, and sVEGFR-2, wherein a level or activity higher than a reference indicates sepsis. Additional embodiments of methods for differentiating CRS and sepsis in a subject are described herein.

[0091] Dosing regimen

[0092] In some embodiments, the CAR-expressing cells and an inhibitor (e.g., a JAK-STAT or BTK inhibitor) are administered sequentially, in parallel, or with a therapeutic interval, such as as described herein.

[0093] In one embodiment, the CAR-expressing cells and an inhibitor (e.g., a JAK-STAT or BTK inhibitor) are administered sequentially. In one embodiment, the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered before the CAR-expressing cells. In one embodiment, the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered after the CAR-expressing cells.

[0094] In one embodiment, the inhibitor (e.g., a JAK-STAT or BTK inhibitor) and the CAR-expressing cells are administered simultaneously or in parallel.

[0095] In an embodiment, the CAR-expressing cells and an inhibitor (e.g., a JAK-STAT or BTK inhibitor) are administered with a therapeutic interval. In one embodiment, the therapeutic interval comprises a single dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) and a single dose of the CAR-expressing cells (e.g., in any order). In another embodiment, the therapeutic interval comprises multiple doses of the inhibitor (e.g., a first and a second dose) and a dose of the CAR-expressing cells (e.g., in any order).

[0096] When the treatment interval comprises a single dose of an inhibitor (e.g., a JAK-STAT or BTK inhibitor) and a single dose of the CAR-expressing cells, in certain embodiments, the dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) and the dose of the CAR-expressing cells are administered simultaneously or in parallel. For example, the dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) and the dose of the CAR-expressing cells are administered within 2 days of each other (e.g., within 2 days, 1 day, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, 1 hour, or less). In an embodiment, the treatment interval begins after the first administered dose and is completed after the subsequently administered dose.

[0097] When the treatment interval comprises a single dose of an inhibitor (e.g., a JAK-STAT or BTK inhibitor) and a single dose of the CAR-expressing cells, in certain embodiments, the dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) and the dose of the CAR-expressing cells are administered sequentially. In an embodiment, the dose of the CAR-expressing cells is administered before the dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor), and the treatment interval begins after the dose of the CAR-expressing cells is administered and is completed after the dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered. In other embodiments, the dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered before the dose of the CAR-expressing cells, and the treatment interval begins after the dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered and is completed after the dose of the CAR-expressing cells is administered. In one embodiment, the treatment interval further comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) subsequent doses of the inhibitor (e.g., a JAK-STAT or BTK inhibitor). In such embodiments, the treatment interval comprises two, three, four, five, six, seven, eight, nine, ten or more doses of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) and one dose of the CAR-expressing cells. In one embodiment, the dose of the CAR-expressing cells is administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or 2 weeks before or after the dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered. In an embodiment, in the case of administering more than one dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor), the dose of the CAR-expressing cells is administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or 2 weeks before or after the first dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered or after the treatment interval begins. In an embodiment, in the case of administering more than one dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor), the second inhibitor (e.g., a JAK-STAT or BTK inhibitor) dose is administered about 10h, 12h, 14h, 16h, 18h, 20h, 24h, 1 day, 1.5 days, 2 days, 3 days or 4 days after the first dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered.

[0098] In cases where multiple doses of an inhibitor (e.g., a JAK-STAT or BTK inhibitor) (e.g., a first dose and a second dose, and optionally subsequent doses) and a dose of CAR-expressing cells are included in a treatment interval, in certain embodiments, the dose of CAR-expressing cells and the first dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) are administered simultaneously or concurrently with each other, e.g., within 2 days (e.g., within 2 days, 1 day, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, or less). In an embodiment, the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered after (i) the dose of CAR-expressing cells or (ii) the first dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor), whichever is administered later. In an embodiment, the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered at least 8 h after (i) or (ii) (e.g., at least 8 h, 9 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 24 h, 1 day, 1.5 days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or longer). In an embodiment, subsequent doses of the inhibitor (e.g., a third, fourth, or fifth dose, etc.) of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) are administered after the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor). In an embodiment, subsequent doses of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) are administered at least 8 h after the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) (e.g., at least 8 h, 9 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 24 h, 1 day, 1.5 days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or longer). In such embodiments, the treatment interval begins after the first administered dose and is completed after the second dose (or subsequent dose) of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered. In an embodiment, the dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered once daily (QD) or twice daily (BID), and the treatment interval is at least 7 days, 8 days, 9 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, or longer. Any treatment interval described herein may include one or more doses of CAR-expressing cells.

[0099] In other embodiments, in the case of a treatment interval comprising multiple doses of an inhibitor (e.g., a JAK-STAT or BTK inhibitor) (e.g., a first dose and a second dose, and optionally subsequent doses) and a dose of CAR-expressing cells, the dose of CAR-expressing cells and the first dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) are administered sequentially. In an embodiment, the dose of CAR-expressing cells is administered after the first dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) but before the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor). In an embodiment, subsequent doses of the inhibitor (e.g., a third, fourth, or fifth dose, etc.) are administered after the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor). In such embodiments, the treatment interval begins after the first dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered and is completed after the second, third, fourth, fifth, or sixth dose (or subsequent doses) of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered. In one embodiment, the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered at least 8 h (e.g., at least 8 h, 9 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 24 h, 1 day, 1.5 days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or longer) after the first dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered. In one embodiment, subsequent doses of the inhibitor (e.g., a third, fourth, or fifth dose, etc.) are administered at least 8 h (e.g., at least 8 h, 9 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 24 h, 1 day, 1.5 days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or longer) after the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered. In one embodiment, the dose of CAR-expressing cells is administered at least 1 day (e.g., at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or longer) after the first dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered. In one embodiment, the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered within 1 day (e.g., within 24 h, 20 h, 18 h, 16 h, 14 h, 12 h, 10 h, 8 h, 6 h, or less) after the dose of CAR-expressing cells is administered.In an embodiment, a second dose of an inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered in parallel with a dose of the CAR-expressing cells. In one embodiment, the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered at least 1 day (e.g., at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or longer) after the dose of the CAR-expressing cells is administered. In an embodiment, the treatment interval comprises consecutive administrations of the inhibitor (e.g., a JAK-STAT or BTK inhibitor), e.g., once a day, twice a day, three times a day, once every 2 days, once every 3 days or once every 4 days. In an embodiment, in the case of consecutive administration of the inhibitor, the dose of the CAR-expressing cells (e.g., the first dose) is administered, for example, at least 1 day (e.g., at least 1, 2, 3, 4, 5, 6, 7 days, e.g., at least 1, 2, 3, 4, 5, 6 weeks, 1, 2, 3, 4, 5, 6 months or longer) after the first dose of the inhibitor. In an embodiment, in the case of consecutive administration of the inhibitor, the dose of the CAR-expressing cells (e.g., the first dose) is administered in parallel with the first dose of the inhibitor (e.g., within 1 day (e.g., within 24h, 20h, 18h, 16h, 14h, 12h, 10h, 8h, 6h, or less time)). In an embodiment, in the case of consecutive administration of the inhibitor, the inhibitor is administered, for example, at least 1 day (e.g., at least 1, 2, 3, 4, 5, 6, 7 days, e.g., at least 1, 2, 3, 4, 5, 6 weeks, 1, 2, 3, 4, 5, 6 months or longer) after the first dose of the CAR-expressing cells. In other embodiments, the dose of the CAR-expressing cells is administered before the first dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor). In such embodiments, the treatment interval begins after the CAR-expressing cells are administered and is completed after the second dose (or subsequent dose) of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered. In an embodiment, the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered at least 8h (e.g., at least 8h, 9h, 10h, 12h, 14h, 16h, 18h, 20h, 24h, 1 day, 1.5 days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or longer) after the first dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered.In an embodiment, a subsequent dose of an inhibitor (e.g., a JAK-STAT or BTK inhibitor), such as a third, fourth, or fifth dose, etc., is administered at least 8 h (e.g., at least 8 h, 9 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 24 h, 1 day, 1.5 days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or longer) after a second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor). In an embodiment, a first dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered at least 1 day (e.g., at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or longer) after administration of the CAR-expressing cells. In an embodiment, the dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered once daily (QD) or twice daily (BID), and the treatment interval is at least 7 days, 8 days, 9 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, or longer.

[0100] In one embodiment, any treatment interval described herein can be repeated one or more times, such as 1, 2, 3, 4, 5 times, or more. In one embodiment, the treatment interval is repeated once, resulting in a treatment regimen comprising two treatment intervals. In one embodiment, the repeated treatment interval is administered at least 1 day, such as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 2 weeks or longer, after completion of the first or previous treatment interval. In one embodiment, the repeated treatment interval is administered at least 3 days after completion of the first or previous treatment interval.

[0101] In one embodiment, one or more (e.g., 1, 2, 3, 4, or 5) subsequent treatment intervals may follow any treatment interval described herein. The one or more subsequent treatment intervals are different from the first or previous treatment interval. By way of example, a first treatment interval consisting of a single dose of an inhibitor (e.g., a JAK-STAT or BTK inhibitor) and a single dose of CAR-expressing cells is followed by a second treatment interval consisting of multiple doses (e.g., two, three, four, or more doses) of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) and a single dose of CAR-expressing cells. In one embodiment, the one or more subsequent treatment intervals are administered at least 1 day, such as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 2 weeks, after completion of the first or previous treatment interval.

[0102] In any of the methods described herein, one or more subsequent doses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses) of an inhibitor (e.g., a JAK-STAT or BTK inhibitor) are administered after completion of one or more treatment intervals. In embodiments, in the case of repeated treatment intervals or administration of two or more treatment intervals, one or more subsequent doses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses) of an inhibitor (e.g., a JAK-STAT or BTK inhibitor) are administered after completion of one treatment interval and before the start of another treatment interval. In one embodiment, the dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered every 8 h, 10 h, 12 h, 14 h, 16 h, 20 h, 24 h, 1 day, 1.5 days, 2 days, 3 days, 4 days, 5 days, 7 days, 2 weeks, 3 weeks, or 4 weeks after completion of one or more or each treatment interval. In one embodiment, one, two, or three doses of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) are administered daily after completion of one or more or each treatment interval.

[0103] In any of the methods described herein, one or more (e.g., 1, 2, 3, 4, 5, or more) subsequent doses of cells expressing a CAR are administered after completion of one or more treatment intervals. In embodiments, in the case of repeated treatment intervals or administration of two or more treatment intervals, one or more subsequent doses (e.g., 1, 2, 3, 4, or 5, or more doses) of cells expressing a CAR are administered after completion of one treatment interval and before the start of another treatment interval. In one embodiment, the dose of cells expressing a CAR is administered every 2 days, 3 days, 4 days, 5 days, 7 days, 2 weeks, 3 weeks, or 4 weeks after completion of one or more or each treatment interval.

[0104] In one embodiment, a treatment interval comprises a single dose of cells expressing a CAR (e.g., cells expressing a CD123 CAR or cells expressing a CD19 CAR), which single dose is administered in parallel with a first dose of an inhibitor (e.g., a JAK-STAT inhibitor such as ruxolitinib; or a BTK inhibitor such as ibrutinib) within 2 days (e.g., within 2 days, 1 day, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, or less). In embodiments, during the treatment interval, the JAK-STAT inhibitor (e.g., ruxolitinib) or the BTK inhibitor (e.g., ibrutinib) is administered twice daily (BID). In embodiments, during the treatment interval, the JAK-STAT inhibitor (e.g., ruxolitinib) or the BTK inhibitor (e.g., ibrutinib) is administered once daily (QD).

[0105] In other embodiments, the treatment interval comprises a single dose of CAR-expressing cells (e.g., cells expressing a CD123 CAR or cells expressing a CD19 CAR), which single dose is administered after (e.g., within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks or longer) a first dose of an inhibitor (e.g., a JAK-STAT inhibitor such as ruxolitinib; or a BTK inhibitor such as ibrutinib). In an embodiment, a second dose of the inhibitor (e.g., a JAK-STAT inhibitor such as ruxolitinib; or a BTK inhibitor such as ibrutinib) is administered after the first dose of the inhibitor (e.g., a JAK-STAT inhibitor such as ruxolitinib; or a BTK inhibitor such as ibrutinib). In an embodiment, subsequent doses of the inhibitor (e.g., a JAK-STAT inhibitor such as ruxolitinib; or a BTK inhibitor such as ibrutinib) are administered. In an embodiment, the dose of the inhibitor (e.g., a JAK-STAT inhibitor such as ruxolitinib; or a BTK inhibitor such as ibrutinib) is administered twice a day (BID). In an embodiment, the dose of the inhibitor (e.g., a JAK-STAT inhibitor such as ruxolitinib; or a BTK inhibitor such as ibrutinib) is administered once a day (QD). In an embodiment, the treatment interval comprises at least 5 (e.g., at least 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20 or more) doses of the inhibitor (e.g., a JAK-STAT inhibitor such as ruxolitinib; or a BTK inhibitor such as ibrutinib). In an embodiment, the treatment interval comprises continuous administration (e.g., QD or BID) of the inhibitor. In an embodiment, the treatment interval lasts 1-7 days, 1-5 weeks or 1-12 months.

[0106] In any of the methods described herein, a single dose of CAR-expressing cells and a single dose of an inhibitor (e.g., a JAK-STAT inhibitor such as ruxolitinib; or a BTK inhibitor such as ibrutinib) are administered to a subject. In one embodiment, the single dose of CAR-expressing cells is administered at least 1 day (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 20, 25, 30, 35, 40 days, or 2 weeks, 3 weeks, 4 weeks or longer) after the single dose of the inhibitor (e.g., a JAK-STAT inhibitor such as ruxolitinib; or a BTK inhibitor such as ibrutinib).

[0107] In one embodiment, after an initial dose of cells expressing a CAR, one or more (e.g., 1, 2, 3, 4, or 5) subsequent doses of cells expressing a CAR are administered to a subject. In one embodiment, one or more subsequent doses of cells expressing a CAR are administered at least 2 days (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 20, 25, 30, 35, 40 days, or 2 weeks, 3 weeks, 4 weeks, or longer) after a previous dose of cells expressing a CAR. In one embodiment, one or more subsequent doses of cells expressing a CAR are administered at least 5 days after a previous dose of cells expressing a CAR. In one embodiment, three doses of cells expressing a CAR are administered to the subject weekly or one dose every 2 days.

[0108] In one embodiment, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) subsequent doses of an inhibitor (e.g., a JAK-STAT inhibitor such as ruxolitinib; or a BTK inhibitor such as ibrutinib) are administered after a single dose of the inhibitor (e.g., a JAK-STAT inhibitor such as ruxolitinib; or a BTK inhibitor such as ibrutinib). In one embodiment, one or more subsequent doses of an inhibitor (e.g., a JAK-STAT inhibitor such as ruxolitinib; or a BTK inhibitor such as ibrutinib) are administered at least 5 days, 7 days, 10 days, 14 days, 20 days, 25 days, 30 days, 2 weeks, 3 weeks, 4 weeks, or 5 weeks after a previous dose of the inhibitor (e.g., a JAK-STAT inhibitor such as ruxolitinib; or a BTK inhibitor such as ibrutinib). In other embodiments, one or more subsequent doses of an inhibitor (e.g., a JAK-STAT inhibitor such as ruxolitinib; or a BTK inhibitor such as ibrutinib) are administered every other day, once a day, or twice a day after a previous dose of the inhibitor (e.g., a JAK-STAT inhibitor such as ruxolitinib; or a BTK inhibitor such as ibrutinib).

[0109] In one embodiment, one or more subsequent doses of an inhibitor (e.g., a JAK-STAT inhibitor such as ruxolitinib; or a BTK inhibitor such as ibrutinib) are administered at least 1, 2, 3, 4, 5, 6, or 7 days after a dose of cells expressing a CAR (e.g., an initial dose of cells expressing a CAR).

[0110] In one embodiment, one or more (e.g., 1, 2, 3, 4, or 5) doses of an inhibitor (e.g., a JAK-STAT inhibitor such as ruxolitinib; or a BTK inhibitor such as ibrutinib) are administered before a first dose of cells expressing a CAR.

[0111] In one embodiment, one or more doses of cells expressing a CAR and one or more doses of an inhibitor (e.g., a JAK-STAT inhibitor such as ruxolitinib; or a BTK inhibitor such as ibrutinib) are administered repeatedly, e.g., 1, 2, 3, 4, 5 times, or more.

[0112] The doses and treatment regimens of the therapeutic agents disclosed herein can be determined by one of ordinary skill in the art.

[0113] In any dosing regimen or treatment interval described herein, in some embodiments, the dose of cells expressing a CAR (e.g., cells expressing a CD19 CAR or a CD123 CAR) comprises at least about 1x10 5 、5x10 6 、1x10 7 、1.5x10 7 、2x10 7 、2.5x10 7 、3x10 7 、3.5x10 7 、4x10 7 、5x10 7 、1x10 8 、1.5x10 8 、2x10 8 、2.5x10 8 、3x10 8 、3.5x10 8 、4x10 8 、5x10 8 、1x10 9 、2x10 9 、or 5x10 9 cells. In some embodiments, the dose of cells expressing a CAR comprises at least about 1 - 5x10 7 to 1 - 5x10 8 . In some embodiments, about 1 - 5x10 7 cells expressing a CAR are administered to a subject. In other embodiments, about 1 - 5x10 8 cells expressing a CAR are administered to a subject.

[0114] In an embodiment, cells expressing a CAR are administered at a dose (e.g., total dose) of 1.5x10 7 to 5x10 9 cells / kg (e.g., 0.3x10 6 to 1x10 8 cells / kg). In an embodiment, the total dose does not exceed 1.5x10 10cells / kg (e.g., administered over time in multiple doses), e.g., not more than 1.5x10 9 cells / kg, e.g., not more than 1.5x10 8 cells / kg.

[0115] In one embodiment, up to 10, 9, 8, 7, 6, 5, 4, 3, or 2 doses of cells are administered. In other embodiments, e.g., in treatment intervals of one, two, three, four, or more weeks, one, two, three, four, five, or 6 doses of cells are administered to a mammal. In one embodiment, up to 6 doses are administered within two weeks. The doses can be the same or different. In one embodiment, a lower dose is initially administered, followed by one or more higher doses. In an exemplary embodiment, the lower dose is about 1x10 5 to 1x10 9 cells / kg, or 1x10 6 to 1x10 8 cells / kg; and the higher dose is about 2x10 5 to 2x10 9 cells / kg or 2x10 6 to 2x10 8 cells / kg, followed by about 4x10 5 to 4x10 9 cells / kg, or 4x10 6 to 4x10 8 cells / kg for 3 - 6 doses.

[0116] In embodiments, CAR-expressing cells are administered to a subject according to a dosing regimen (comprising the total dose of cells administered to the subject by dose escalation (e.g., one, two, three, or more individual administrations of partial doses)). In embodiments, a first percentage of the total dose is administered on the first day of treatment, a second percentage of the total dose is administered on subsequent (e.g., second, third, fourth, fifth, sixth, seventh, or later) days of treatment, and optionally, a third percentage (e.g., the remaining percentage) of the total dose is administered on yet subsequent (e.g., third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or later) days of treatment. For example, 10% of the total cell dose is delivered on the first day, 30% of the total cell dose is delivered on the second day, and 60% of the remaining total cell dose is delivered on the third day of treatment. For example, the total cell dose comprises 1 to 5x10 7 or 1 to 5x10 8 CAR-expressing cells.

[0117] In embodiments, the total dose is administered in multiple doses (e.g., a first dose, a second dose, and optionally a third dose, etc.).

[0118] In an embodiment, the first dose comprises, for example, about 10% of the total dose administered on the first day (e.g., about 1x10 7 cells / kg). In an embodiment, the second dose comprises, for example, about 30% of the total dose administered on subsequent days (e.g., 1, 2, 3, 4, 5, 6, or 7 days after the first dose) (e.g., about 3x10 7 cells / kg). In an embodiment, the second dose is administered if the subject is clinically stable after the first dose. In an embodiment, a subsequent dose (e.g., a third dose, optionally a fourth dose, etc.) is administered to the subject, for example, when the sum of the first dose, the second dose, and the subsequent dose(s) totals the total dose. In an embodiment, when the total dose is administered in multiple doses, the time between each dose is at least 1 day (e.g., at least 1, 2, 3, 4, 5, 6, 7 days, 1, 2, or 3 weeks, or longer). In an embodiment, the time between the second dose and the third dose, and / or between the third dose and the fourth dose, and / or between the fourth dose and the fifth dose is at least 1 week (e.g., at least 1, 2, 3, 4 weeks, or longer).

[0119] In an embodiment, in any dosing regimen described herein, the dose of an inhibitor (e.g., a JAK-STAT inhibitor or a BTK inhibitor) is administered every 1, 2, 3, 4, 5, 6, or 7 days, or twice daily, or three times daily.

[0120] In an embodiment, the JAK-STAT inhibitor (e.g., ruxolitinib) is administered orally at a dose of 2.5 mg to 50 mg (e.g., 2.5 - 5 mg, 5 - 10 mg, 10 - 15 mg, 15 - 20 mg, 20 - 25 mg, 25 - 30 mg, 30 - 35 mg, 35 - 40 mg, 40 - 45 mg, or 45 - 50 mg) twice daily (e.g., a total of 5 mg to 100 mg per day).

[0121] In an embodiment, the BTK inhibitor (e.g., ibrutinib (PCI-32765)) is administered orally at a dose of about 250 mg, 300 mg, 350 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600 mg (e.g., 250 mg, 420 mg, or 560 mg) daily for a period of time, e.g., daily for a 21-day cycle, or daily for a 28-day cycle. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more cycles of the BTK inhibitor (e.g., ibrutinib) are administered.

[0122] In some embodiments of any of the methods disclosed herein, the method comprises administering an inhibitor (e.g., a BTK inhibitor such as ibrutinib; or a JAK-STAT inhibitor such as ruxolitinib) to a subject, reducing the amount of the inhibitor (e.g., stopping administration), and subsequently administering cells expressing a CAR (e.g., cells expressing CAR19 or CAR123) to the subject.

[0123] In some embodiments, the method comprises administering an inhibitor (e.g., a BTK inhibitor such as ibrutinib; or a JAK-STAT inhibitor such as ruxolitinib) to a subject, and subsequently administering a combination of the inhibitor and cells expressing a CAR (e.g., cells expressing CAR19 or CAR123) to the subject.

[0124] In some embodiments, the method comprises administering an inhibitor (e.g., a BTK inhibitor such as ibrutinib or a JAK-STAT inhibitor such as ruxolitinib) to a subject, reducing the amount of the inhibitor (e.g., stopping or discontinuing administration), and subsequently administering a combination of cells expressing a CAR (e.g., cells expressing CAR19 or CAR123) and a second inhibitor (e.g., a second inhibitor other than the first inhibitor) to the subject. In some embodiments, the first inhibitor is a BTK inhibitor and the second inhibitor is a BTK inhibitor other than the first BTK inhibitor (e.g., other than ibrutinib). In some embodiments, the first inhibitor is a JAK-STAT inhibitor and the second inhibitor is a JAK-STAT inhibitor other than the first JAK-STAT inhibitor (e.g., other than ruxolitinib). In some embodiments, the first inhibitor is a JAK-STAT inhibitor and the second inhibitor is a BTK inhibitor. In some embodiments, the first inhibitor is a BTK inhibitor and the second inhibitor is a JAK-STAT inhibitor. In some embodiments, the second BTK inhibitor is selected from one or more of GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774, or LFM-A13, or a combination thereof. In embodiments, the second JAK-STAT inhibitor is selected from one or more of AG490, AZD1480, tofacitinib (tasocitinib or CP-690550), or CYT387.

[0125] In one embodiment, cells expressing a CAR molecule (e.g., a CAR molecule as described herein) are administered at a dose and / or dosing schedule as described herein.

[0126] In one embodiment, any method described herein further comprises administering a therapy for preventing or treating CRS. In an embodiment, the therapy comprises an IL-6 inhibitor (e.g., an anti-IL6 receptor inhibitor, such as an anti-IL6 receptor inhibitor, such as tocilizumab). In other embodiments, the therapy comprises an IL-6 inhibitor in combination with one or more (or all) of a vasoactive drug, an immunosuppressant, a corticosteroid, or mechanical ventilation. In an embodiment, the method comprises administering an IL-6 inhibitor (e.g., tocilizumab) before a dose (e.g., a first dose) of the CAR-expressing cell (e.g., the CAR-expressing cell described herein) (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or 1, 2, 3, or 4 weeks before). In an embodiment, the method comprises administering an IL-6 inhibitor (e.g., tocilizumab) in parallel with a dose (e.g., a first dose) of the CAR-expressing cell (e.g., the CAR-expressing cell described herein). In an embodiment, the method comprises administering an IL-6 inhibitor (e.g., tocilizumab) after a dose (e.g., a first dose) of the CAR-expressing cell (e.g., the CAR-expressing cell described herein), but before or within 1 week of the first sign of fever in the subject (e.g., within 1 week, 7, 6, 5, 4, 3, 2, 1 days, or less). In an embodiment, the method comprises administering an IL-6 inhibitor (e.g., tocilizumab) after a dose (e.g., a first dose) of the CAR-expressing cell (e.g., the CAR-expressing cell described herein), and within 1 week of a temperature of at least 38°C (e.g., at least 38.5°C) in the subject (e.g., measured twice consecutively within 24 hours (e.g., at least 4 hours apart)). In an embodiment, prior to treatment with the CAR-expressing cell, the subject has (e.g., is diagnosed or identified as having) a high tumor burden. In an embodiment, prior to administering the CAR-expressing cell (e.g., about 1-5 days before administering the CAR-expressing cell), the high tumor burden comprises at least 40% blasts in the subject's bone marrow (e.g., at least 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or more blasts).

[0127] In an embodiment, the method comprises administering a dose of tocilizumab of about 5-15 mg / kg, such as 8-12 mg / kg (e.g., about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, 11 mg / kg, or about 12 mg / kg).

[0128] In one embodiment, a CAR molecule is introduced into T cells (e.g., using in vitro transcription), and a subject (e.g., a human) receives an initial administration of cells comprising the CAR molecule and one or more subsequent administrations of cells comprising the CAR molecule, wherein the one or more subsequent administrations are given less than 15 days (e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days) after the previous administration. In one embodiment, more than one administration of cells comprising the CAR molecule is given to the subject (e.g., a human) per week, such as 2, 3, or 4 administrations of cells comprising the CAR molecule per week. In one embodiment, a subject (e.g., a human subject) receives more than one administration of cells comprising the CAR molecule per week (e.g., 2, 3, or 4 administrations per week) (also referred to herein as a cycle), then no cells comprising the CAR molecule are given for one week, and then one or more additional administrations of cells comprising the CAR molecule are given to the subject (e.g., more than one administration of cells comprising the CAR molecule per week). In another embodiment, a subject (e.g., a human subject) receives more than one cycle of cells comprising the CAR molecule, and the time between each cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, cells comprising the CAR molecule are given every other day, 3 times per week. In one embodiment, administration of cells comprising the CAR molecule continues for at least two, three, four, five, six, seven, eight, or more weeks.

[0129] In one embodiment, a combination of a kinase inhibitor and cells expressing a CAR molecule (e.g., a CAR molecule as described herein) is given as first-line treatment for a disease (e.g., cancer, such as cancer as described herein). In another embodiment, a combination of a kinase inhibitor and cells expressing a CAR molecule (e.g., a CAR molecule as described herein) is given as first-line, second-line, third-line, fourth-line treatment for a disease (e.g., cancer, such as cancer as described herein).

[0130] In an embodiment, any method described herein further comprises lymphodepletion of the subject, e.g., before administration of one or more cells expressing a CAR molecule as described herein (e.g., a CAR molecule that binds CD19 or CD123). Lymphodepletion can include, for example, administration of one or more of melphalan, cyclophosphamide, cyclophosphamide and fludarabine.

[0131] Subject

[0132] In an embodiment, the subject is at risk of developing CRS (e.g., is identified as being at risk of developing CRS), has CRS, or is diagnosed with CRS.

[0133] In an embodiment, a subject has been, is being, or will be administered a CAR therapy, such as the CAR therapy described herein. In an embodiment, a subject has been, is being, or will be administered cells expressing CAR123 or cells expressing CAR19.

[0134] In an embodiment, the method includes identifying (and optionally selecting) a subject who: i) is at risk of developing CRS; or ii) has CRS.

[0135] In an embodiment, the method includes selecting a subject for administration of an inhibitor (e.g., a JAK-STAT inhibitor or a BTK inhibitor). In an embodiment, the subject is selected based on: (i) his or her risk of developing CRS, (ii) his or her CRS diagnosis, and / or (iii) whether he or she has been, is being, or will be administered a CAR therapy (e.g., the CAR therapy described herein, such as CAR19 therapy, such as CTL019 or CD123 CAR therapy). In an embodiment, if a subject is diagnosed with CRS (e.g., severe or non-severe CRS), then the subject is selected for administration of a JAK-STAT or BTK inhibitor. In an embodiment, if a subject is at risk of developing CRS (e.g., is identified as being at risk of developing CRS), then the subject is selected for administration of a JAK-STAT or BTK inhibitor. In an embodiment, if a subject has been, is being, or will be administered a CAR therapy (e.g., the CAR therapy described herein, such as CAR19 therapy, such as CTL019; or CAR123 therapy), then the subject is selected for administration of a JAK-STAT or BTK inhibitor.

[0136] Subjects at risk of CRS

[0137] In an embodiment, if a subject has a high tumor burden (e.g., prior to administration of a CAR therapy (e.g., the CAR therapy described herein)), then the subject is identified as being at risk of having CRS.

[0138] In an embodiment, a subject is identified as being at risk of CRS by obtaining the CRS risk status of the subject, wherein the CRS risk status comprises a measure of one, two, three, four, five, six, seven, eight, nine, ten, or more (all) of the following:

[0139] (i) The level or activity of sgp130 or IFN-γ or a combination thereof in the subject (e.g., in a sample (e.g., a blood sample), such as where the subject is an adult or pediatric subject);

[0140] (ii) The level or activity of sgp130, IFN-γ, or IL1Ra, or a combination thereof (e.g., a combination of any two or all three of sgp130, IFN-γ, and IL1Ra) in a subject (e.g., a sample (e.g., a blood sample), e.g., where the subject is an adult or pediatric subject);

[0141] (iii) The level or activity of sgp130 or IFN-γ, or a combination thereof, in a subject (e.g., in a sample (e.g., a blood sample)), and the level of a bone marrow disorder in the subject (e.g., where the subject is a pediatric subject);

[0142] (iv) The level or activity of sgp130, IFN-γ, or MIP1-α, or a combination thereof (e.g., a combination of any two or all three of sgp130, IFN-γ, and MIP1-α) in a subject (e.g., a sample (e.g., a blood sample), e.g., where the subject is a pediatric subject);

[0143] (v) The level or activity of sgp130, MCP1, or eosinophil chemotactic factor, or a combination thereof (e.g., a combination of any two or all three of sgp130, MCP1, or eosinophil chemotactic factor) in a subject (e.g., in a sample (e.g., a blood sample), e.g., where the subject is an adult or pediatric subject);

[0144] (vi) The level or activity of IL-2, eosinophil chemotactic factor, or sgp130, or a combination thereof (e.g., a combination of any two or all three of IL-2, eosinophil chemotactic factor, or sgp130) in a subject (e.g., in a sample (e.g., a blood sample), e.g., where the subject is an adult or pediatric subject);

[0145] (vii) The level or activity of IFN-γ, IL-2, or eosinophil chemotactic factor, or a combination thereof (e.g., a combination of any two or all three of IFN-γ, IL-2, or eosinophil chemotactic factor) in a subject (e.g., in a sample (e.g., a blood sample), e.g., where the subject is a pediatric subject);

[0146] (viii) The level or activity of IL-10 in a subject (e.g., in a sample (e.g., a blood sample), e.g., where the subject is a pediatric subject) and the level of the subject's disease burden, or a combination thereof;

[0147] (ix) The level or activity of IFN-γ or IL-13, or a combination thereof, in a subject (e.g., where the subject is a pediatric subject); or

[0148] (x) The level or activity of IFN-γ, IL-13, or MIP1-α, or a combination thereof (e.g., a combination of any two or all three of IFN-γ, IL-13, and MIP1-α) in a sample (e.g., a blood sample, such as where the subject is a pediatric subject); or

[0149] (xi) The level or activity of IFN-γ or MIP1-α, or a combination thereof, in a sample (e.g., a blood sample, such as where the subject is a pediatric subject);

[0150] Wherein the CRS risk status indicates the risk that the subject will develop CRS (e.g., severe CRS).

[0151] Any of the above methods may further comprise: in response to determining the CRS risk status, performing one, two, or more (all) of the following:

[0152] Identifying the subject as being at high risk or low risk of developing severe CRS;

[0153] Administering a BTK inhibitor (e.g., ibrutinib) or a JAK-STAT inhibitor (e.g., ruxolitinib);

[0154] Administering an altered dose of the CAR-expressing cell therapy;

[0155] Altering the schedule or course of the CAR-expressing cell therapy;

[0156] Administering a therapy for treating CRS, e.g., a therapy selected from one or more of the following: an IL-6 inhibitor (e.g., an anti-IL6 receptor inhibitor, such as tocilizumab), a vasoactive drug, an immunosuppressant, a corticosteroid, or mechanical ventilation; and / or

[0157] Administering an alternative therapy, e.g., for a subject at high risk of developing severe CRS, such as the standard of care for a particular cancer type.

[0158] In some embodiments of these methods, the CRS risk status comprises a measure of the level or activity of sgp130, IFN-γ, or IL-13, or a combination thereof (e.g., a combination of any two or all three of sgp130, IFN-γ, and IL-13) in the subject (e.g., in a sample (e.g., a blood sample), such as where the subject is an adult or pediatric subject).

[0159] In some embodiments of these methods, the CRS risk status indicates whether the subject is at high risk or low risk of developing severe CRS. For example, the CRS can be clinical grade 1-3, or can be severe CRS of clinical grade 4-5.

[0160] In some embodiments, these methods are performed on a subject who does not have symptoms of CRS (e.g., clinical symptoms), such as one or more of hypotension or fever; or severe CRS, such as one or more of grade 4 organ toxicity or the need for mechanical ventilation.

[0161] In some embodiments of these methods, high levels or activities of IFN-γ, sgp130, MCP1, IL-10, or disease burden, or any combination thereof, indicate a high risk of severe CRS. In some embodiments, low levels or activities of IL13, IL1Ra, MIP1a, or eosinophil chemotactic factor, or any combination thereof, indicate a high risk of severe CRS.

[0162] In some embodiments of these methods, for example, relative to a reference, a subject at high risk of severe CRS has or is identified as having a higher level or activity of sgp130 or IFN-γ, or a combination thereof (e.g., in a sample (such as a blood sample)).

[0163] In other embodiments of the method, for example, relative to a reference, a subject at high risk of severe CRS has or is identified as having a higher level or activity of sgp130, a higher level or activity of IFN-γ, a lower level or activity of IL1Ra, or a combination thereof (e.g., in a sample (such as a blood sample)). In one embodiment, a subject at high risk of severe CRS is identified as having a higher level or activity of sgp130 and a higher level or activity of IFN-γ; a higher level or activity of sgp130, and a lower level or activity of IL1Ra; a higher level or activity of IFN-γ, and a lower level or activity of IL1Ra; or a higher level or activity of sgp130, and a lower level or activity of IFN-γ, and a lower level or activity of IL1Ra, for example, compared to a reference. In some embodiments, the reference is a subject at low risk of severe CRS or a control level or activity. The subject can be a human, such as an adult or pediatric subject.

[0164] In some embodiments of these methods, in a subject (e.g., in a sample (e.g., a blood sample)), a subject at high risk of severe CRS, for example relative to a reference, such as compared to a subject at low risk of severe CRS, or compared to a control level or activity, has or is identified as having a higher level or activity of sgp130 or IFN-γ or a combination thereof, and a higher level of myeloid disease. In one embodiment, a subject at high risk of severe CRS is identified as having a higher level of sgp130 and IFN-γ; sgp130 and myeloid disease; IFN-γ and myeloid disease; or sgp130, IFN-γ, and myeloid disease, for example compared to a reference (e.g., a subject at low risk of severe CRS, or a control level or activity). The subject can be a human, such as a pediatric subject.

[0165] In some embodiments of these methods, a subject at high risk of severe CRS (e.g., a pediatric subject) is identified as having a higher level or activity of sgp130, a higher level or activity of IFN-γ, or a lower level or activity of MIP1-α or a combination thereof (e.g., in a sample (e.g., a blood sample)), compared to a reference (e.g., a subject at low risk of severe CRS) or compared to a control level or activity. In one embodiment, a subject at high risk of severe CRS is identified as having a higher level or activity of sgp130 and a higher level or activity of IFN-γ; a higher level or activity of sgp130, and a lower level or activity of MIP1-α; a higher level or activity of IFN-γ, and a lower level or activity of MIP1-α; a higher level or activity of sgp130, a higher level or activity of IFN-γ, and a lower level or activity of MIP1-α, for example compared to a reference (e.g., a subject at low risk of severe CRS) or compared to a control level or activity.

[0166] In some embodiments of these methods, subjects at high risk of severe CRS are identified as having a higher level or activity of sgp130, a higher level or activity of MCP1, or a lower level or activity of eosinophil chemokine, or a combination thereof (e.g., in a sample such as a blood sample), compared to a reference (e.g., a subject at low risk of severe CRS) or compared to a control level or activity. In some embodiments, subjects at high risk of severe CRS are identified as having: a higher level or activity of sgp130 and a higher level or activity of MCP1, a higher level or activity of sgp130 and a lower level or activity of eosinophil chemokine, a higher level or activity of MCP1 and a lower level or activity of eosinophil chemokine, a higher level or activity of sgp130, a higher level or activity of MCP1, and a lower level or activity of eosinophil chemokine, compared to a reference (e.g., a subject at low risk of severe CRS) or compared to a control level or activity.

[0167] In some embodiments of these methods, subjects at high risk of severe CRS are identified as having an altered (e.g., higher) level or activity of IL-2, a lower level or activity of eosinophil chemokine, or a higher level or activity of sgp130, or a combination thereof (e.g., in a sample such as a blood sample), compared to a reference (e.g., a subject at low risk of severe CRS) or compared to a control level or activity. In some embodiments, subjects at high risk of severe CRS are identified as having: an altered (e.g., higher) level or activity of IL-2 and a lower level or activity of eosinophil chemokine, an altered (e.g., higher) level or activity of IL-2 and a higher level or activity of sgp130, a lower level or activity of eosinophil chemokine and a higher level or activity of sgp130, an altered (e.g., higher) level or activity of IL-2, a lower level or activity of eosinophil chemokine, and a higher level or activity of sgp130, compared to a reference (e.g., a subject at low risk of severe CRS) or compared to a control level or activity.

[0168] In some embodiments of these methods, subjects at high risk of severe CRS are identified as having a higher level or activity of IFN-γ, an altered (e.g., higher) level or activity of IL-2, or a lower level or activity of eosinophil chemokine or a combination thereof (e.g., in a sample such as a blood sample), compared to a reference (e.g., a subject at low risk of severe CRS) or compared to a control level or activity. In some embodiments, the subject is a pediatric subject. In some embodiments, subjects at high risk of severe CRS are identified as having: a higher level or activity of IFN-γ and an altered (e.g., higher) level or activity of IL-2, a higher level or activity of IFN-γ and a lower level or activity of eosinophil chemokine, an altered (e.g., higher) level or activity of IL-2 and a lower level or activity of eosinophil chemokine, a higher level or activity of IFN-γ, an altered (e.g., higher) level or activity of IL-2 and a lower level or activity of eosinophil chemokine, which is compared to a reference (e.g., a subject at low risk of severe CRS) or compared to a control level or activity.

[0169] In some embodiments of these methods, subjects at high risk of severe CRS are identified as having a higher level or activity of IL-10, or a higher level or activity of disease burden or a combination thereof (e.g., in a sample such as a blood sample), compared to a reference (e.g., a subject at low risk of severe CRS) or compared to a control level or activity. In some embodiments, the subject is a pediatric subject.

[0170] In some embodiments of these methods, subjects at high risk of severe CRS are identified as having a higher level or activity of IFN-γ, or a lower level of IL-13 or a combination thereof (e.g., in a sample such as a blood sample), compared to a reference (e.g., a subject at low risk of severe CRS) or compared to a control level or activity. In some embodiments, the subject is a pediatric subject.

[0171] In some embodiments of these methods, a subject at high risk of severe CRS is identified as having a higher level or activity of IFN-γ, a lower level or activity of IL-13, or a lower level or activity of MIP1-α or a combination thereof (e.g., in a sample such as a blood sample), compared to a reference (e.g., a subject at low risk of severe CRS) or compared to a control level or activity. In some embodiments, the subject is a pediatric subject. In some embodiments, a subject at high risk of severe CRS is identified as having: a higher level or activity of IFN-γ or a lower level or activity of IL-13, a higher level or activity of IFN-γ or a lower level or activity of MIP1-α, a lower level or activity of IL-13 or a lower level or activity of MIP1-α, a higher level or activity of IFN-γ, a lower level or activity of IL-13, and a lower level or activity of MIP1-α, compared to a reference (e.g., a subject at low risk of severe CRS) or compared to a control level or activity.

[0172] In some embodiments of these methods, a subject at high risk of severe CRS is identified as having a higher level or activity of IFN-γ, or a lower level or activity of MIP1-α or a combination thereof (e.g., in a sample such as a blood sample), compared to a reference (e.g., a subject at low risk of severe CRS) or compared to a control level or activity. In some embodiments, the subject is a pediatric subject.

[0173] In some embodiments, e.g., in a 3-biomarker panel (e.g., containing IL2, eotaxin, and sgp130), or in a 3-biomarker panel containing IFN-γ, IL2, and eotaxin (e.g., in pediatric patients), a higher level or activity of IL2 indicates that the subject is at high risk of severe CRS. In other embodiments, e.g., in a 2-biomarker panel, e.g., for pediatric patients, a higher level or activity of IL2 indicates that the subject is at low risk of severe CRS.

[0174] In some embodiments of these methods, a higher level of the markers described herein is a level greater than or equal to 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, 100,000, 200,000, or 500,000 pg / ml. In some embodiments, a higher level of sgp130 is greater than or equal to 150,000, 200,000, 210,000, 215,000, 218,000, 218,179, 220,000, 225,000, 230,000, or 250,000 pg / ml. In some embodiments, a higher level of IFN-γ is greater than or equal to 6, 7, 8, 9, 10, 10.4272, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 27.6732, 28, 29, 30, 31, 32, 33, 34, 35, 40, 50, 60, 70, 75, 80, 85, 90, 91, 92, 93, 94, 94.8873, 95, 96, 97, 98, 99, 100, 105, 110, 115, or 120 pg / ml. In some embodiments, a higher level of IL-10 is greater than or equal to 5, 6, 7, 8, 9, 10, 11, 11.7457, 12, 13, 14, 15, 16, 17, 18, 19, or 20 pg / ml. In some embodiments, a greater tumor burden is greater than or equal to 25%, 30%, 35%, 40%, 45%, 50%, 51.9%, 55%, 60%, 65%, 70%, or 75%. In some embodiments, a lower level of sgp130, IFN-γ, IL-10, or tumor burden is less than or equal to any value in this paragraph.

[0175] In some embodiments of these methods, the lower levels of the markers described herein are levels greater than or equal to 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, 100,000, 200,000, or 500,000 pg / ml. In some embodiments, the lower levels of IL1Ra are less than or equal to 550, 575, 600, 625, 650, 657.987, 675, 700, 720, or 750 pg / ml. In some embodiments, the lower levels of MCP1 are less than or equal to 3500, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4636.52, 4700, 4800, 4900, 5000, or 5500 pg / ml. In some embodiments, the lower levels of eosinophil chemotactic factor are less than or equal to 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 29.0902, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 pg / ml. In some embodiments, the lower levels of MIP1a are less than or equal to 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30.1591, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 pg / ml. In some embodiments, the higher levels of IL1Ra, MCP1, eosinophil chemotactic factor, or MIP1a are greater than or equal to any value in this paragraph.

[0176] In some embodiments of these methods, the sensitivity is at least 0.75, 0.79, 0.80, 0.82, 0.85, 0.86, 0.90, 0.91, 0.93, 0.95, 0.96, 0.97, 0.98, 0.99, or 1.0. In some embodiments, the specificity is at least 0.75, 0.77, 0.80, 0.85, 0.86, 0.89, 0.90, 0.92, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or 1.0. In some embodiments, the PPV is at least 0.62, 0.65, 0.70, 0.71, 0.75, 0.80, 0.82, 0.83, 0.85, 0.90, 0.91, 0.92, 0.95, 0.96, 0.97, 0.98, 0.99, or 1.0. In some embodiments, the NPV is at least 0.80, 0.85, 0.90, 0.92, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or 1.0.

[0177] In some embodiments of these methods, the measurement of eosinophil-activating chemokines includes the measurement of one or more (e.g., two or all) of eosinophil chemokine-1, eosinophil chemokine-2, and eosinophil chemokine-3. In some embodiments, the measurement of eosinophil chemokines includes the measurement of eosinophil chemokine-1 and eosinophil chemokine-2, eosinophil chemokine-1 and eosinophil chemokine-3, or eosinophil chemokine-2 and eosinophil chemokine-3.

[0178] Any method disclosed herein may further comprise the step of obtaining measurements of the levels and activities of one, two, three, four, five, ten, twenty, or more of the following cytokines in a subject (e.g., in a sample from the subject (e.g., a blood sample)), the cytokines being selected from sTNFR2, IP10, sIL1R2, sTNFR1, M1G, VEGF, sILR1, TNFα, IFNα, GCSF, sRAGE, IL4, IL10, IL1R1, IFN-γ, IL6, IL8, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, or GM-CSF, or combinations thereof. In some embodiments, a subject with severe CRS or at high risk of developing severe CRS has or is identified as having a higher level or activity of one or more (e.g., two, three, four, five, ten, fifteen, twenty, or all) of the following cytokines, compared to a reference (e.g., a subject at low risk of severe CRS) or compared to a control level or activity, the cytokines being selected from sTNFR2, IP10, sIL1R2, sTNFR1, M1G, VEGF, sILR1, TNFα, IFNα, GCSF, sRAGE, IL4, IL10, IL1R1, IFN-γ, IL6, IL8, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, or GM-CSF, or combinations thereof.

[0179] Any method disclosed herein may further comprise the step of obtaining a material for the level and activity of one, two, three, four, five, six, seven, eight, or all of the following cytokines in a subject (e.g., in a sample from the subject, such as a blood sample), the cytokines being selected from IFN-γ, IL10, IL6, IL8, IP10, MCP1, M1G, sIL2Rα, GM-CSF, or TNFα, or a combination thereof. In some embodiments, a subject with severe CRS or at high risk of developing severe CRS has or is identified as having a higher level or activity of one or more (e.g., two, three, four, five, six, seven, eight, or all) of the following cytokines, selected from IFN-γ, IL10, IL6, IL8, IP10, MCP1, M1G, sIL2Rα, GM-CSF, or TNFα, or a combination thereof, compared to a reference (e.g., a subject at low risk of severe CRS) or compared to a control level or activity.

[0180] Any method disclosed herein may further comprise the step of obtaining a material for the level and activity of one, two, three, four, five, six, or all of the following cytokines in a subject (e.g., in a sample from the subject, such as a blood sample), the cytokines being selected from IFN-γ, IL10, IL6, IL8, IP10, MCP1, M1G, or sIL2Rα, or a combination thereof. In some embodiments, a subject with severe CRS or at high risk of developing severe CRS has or is identified as having a higher level or activity of one or more (e.g., two, three, four, five, six, or all) of the following cytokines, selected from IFN-γ, IL10, IL6, IL8, IP10, MCP1, M1G, or sIL2Rα, or a combination thereof, compared to a reference (e.g., a subject at low risk of severe CRS) or compared to a control level or activity.

[0181] In some embodiments, any method disclosed herein may further include the step of determining the level of C-reactive protein (CRP) in a sample (e.g., a blood sample) from a subject. In one embodiment, a subject at low risk of severe CRS has or is identified as having a CRP level of less than 7 mg / dL (e.g., 7, 6.8, 6, 5, 4, 3, 2, 1 mg / dL or less). In one embodiment, a subject at high risk of severe CRS has or is identified as having a higher level of CRP in a sample (e.g., a blood sample) compared to a subject at low risk of severe CRS or compared to a control level or activity. In one embodiment, the higher level or activity is at least 2-fold higher (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100, 500, 1000-fold or more) compared to a subject at low risk of severe CRS or compared to a control level or activity.

[0182] In other embodiments, the methods disclosed herein further include the step of selecting or altering a therapy (e.g., a CAR-expressing cell therapy) for a subject based on the obtained CRS risk status. In an embodiment, in the case where the obtained CRS risk status is that the subject is at high risk of severe CRS, the therapy is altered to discontinue it, or a subsequent (e.g., second, third, or fourth) dose of the therapy (e.g., a CAR-expressing cell) is at a lower dose that is lower than the previous dose. In other embodiments, a subsequent (e.g., second, third, or fourth) dose of the CAR-expressing cells comprises a different CAR or a different cell type than the previous CAR-expressing cell therapy administered to the subject.

[0183] In other embodiments of these methods, the measurement of one or more of the biomarkers (e.g., one or more of the biomarkers in (i)-(xi)) is obtained from a sample (e.g., a blood sample) obtained from a subject. In some embodiments, the subject is evaluated while receiving CAR-expressing cell therapy, e.g., a sample from the subject. In other embodiments, the subject is evaluated after receiving CAR-expressing cell therapy, e.g., a sample from the subject. For example, the subject is evaluated, e.g., a sample from the subject, within 10 days or less (e.g., 1-10 days, 1-9 days, 1-8 days, 1-7 days, 1-6 days, 1-5 days, 1-4 days, 1-3 days, or 1-2 days, 5 days or less, 4 days or less, 3 days or less, 2 days or less, 1 day or less (e.g., 1, 3, 5, 10, 12, 15, 20 hours)) after infusion with CAR-expressing cell therapy. In some embodiments, the subject is evaluated 5 days or less, 4 days or less, 3 days or less, 2 days or less, 1 day or less (e.g., but not earlier than 1, 3, 5, 10, 12, 15, 20 hours) after infusion with CAR-expressing therapy. In other embodiments, the measurement of one or more of the biomarkers includes detecting one or more of nucleic acid (e.g., mRNA) levels or protein levels.

[0184] In embodiments, the methods include determining whether a subject has severe CRS. The method includes obtaining a CRS risk status, e.g., in response to an immune cell-based therapy, e.g., CAR-expressing cell therapy for a subject (e.g., CAR19-expressing cell therapy or CAR123-expressing cell therapy), wherein the CRS risk status includes measuring one, two, or more (all) of the following:

[0185] (i) the level or activity of one or more (e.g., 3, 4, 5, 10, 15, 20, or more) or a combination of the following cytokines or analytes in a sample (e.g., a blood sample), the cytokine selected from: sTNFR2, IP10, sIL1R2, sTNFR1, M1G, VEGF, sILR1, TNFα, IFNα, GCSF, sRAGE, IL4, IL10, IL1R1, IFN-γ, IL6, IL8, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, or GM-CSF, the analyte selected from: C-reactive protein (CRP), ferritin, lactate dehydrogenase (LDH), aspartate aminotransferase (AST) or blood urea nitrogen (BUN), alanine aminotransferase (ALT), creatinine (Cr) or fibrinogen;

[0186] (ii) The level or activity of IL6, IL6R, or sgp130, or a combination thereof (e.g., a combination of any two or all three of IL6, IL6R, and sgp130) in a sample (e.g., a blood sample); or

[0187] (iii) The level or activity of IL6, IFN-γ, or IL2R, or a combination thereof (e.g., a combination of any two or all three of IL6, IFN-γ, and IL2R) in a sample (e.g., a blood sample);

[0188] wherein the value indicates the severe CRS status of the subject.

[0189] In embodiments, elevated levels of cytokines (i)-(iii) or all analytes other than fibrinogen indicate severe CRS. In embodiments, low fibrinogen indicates severe CRS.

[0190] Compositions and compositions for use

[0191] In another aspect, the present disclosure features a composition (e.g., one or more dosage formulations, combinations, or one or more pharmaceutical compositions) comprising cells expressing a CAR (e.g., CD123 CAR) as described herein and an inhibitor as described herein (e.g., a JAK-STAT inhibitor, such as ruxolitinib). The cells expressing the CAR and the inhibitor (e.g., a JAK-STAT inhibitor) can be the same or different formulations or pharmaceutical compositions. The cells expressing the CAR and one or more kinase inhibitors can be present in a single dosage form or in two or more dosage forms.

[0192] In embodiments, the compositions disclosed herein are used as a medicament.

[0193] In embodiments, the compositions disclosed herein are used to treat a disease associated with the expression of an antigen as described herein, such as a B cell antigen (e.g., CD123 or CD19).

[0194] In another aspect, the present disclosure features a composition (e.g., one or more dosage formulations, combinations, or one or more pharmaceutical compositions) comprising cells expressing a CAR (e.g., CD123 CAR) as described herein and an inhibitor as described herein (e.g., a JAK-STAT inhibitor) for use in a method of treating (or preparing a medicament for treating) a disease (e.g., a cancer as described herein) associated with the expression of an antigen (e.g., a B cell antigen, such as CD123 or CD19).

[0195] In another aspect, the present disclosure features a composition (e.g., one or more dosage formulations, combinations, or one or more pharmaceutical compositions) comprising a cell expressing a CAR (e.g., a CD123 CAR or a CD19 CAR) as described herein and an inhibitor as described herein (e.g., a JAK-STAT inhibitor or a BTK inhibitor) for use in a method of preventing CRS in a subject.

[0196] In another aspect, the invention relates to a cell expressing a CAR molecule as described herein, which is used as a medicament in combination with a kinase inhibitor (e.g., a kinase inhibitor as described herein (e.g., a BTK inhibitor such as ibrutinib, or a JAK-STAT inhibitor such as ruxolitinib)), for example, to prevent CRS in a subject. In another aspect, the invention relates to a kinase inhibitor as described herein (e.g., a BTK inhibitor such as ibrutinib, or a JAK-STAT inhibitor such as ruxolitinib), which is used as a medicament in combination with a cell expressing a CAR molecule as described herein, for example, to prevent CRS in a subject.

[0197] In another aspect, the invention relates to a cell expressing a CAR molecule as described herein, which is used in combination with a kinase inhibitor (e.g., a kinase inhibitor as described herein (e.g., a BTK inhibitor such as ibrutinib, or a JAK-STAT inhibitor such as ruxolitinib)) to treat a disease expressing a B cell antigen (e.g., CD19 or CD123).

[0198] In another aspect, the invention relates to a kinase inhibitor as described herein (e.g., a BTK inhibitor such as ibrutinib, or a JAK-STAT inhibitor such as ruxolitinib), which is used in combination with a cell expressing a CAR molecule as described herein to treat a disease expressing a B cell antigen (e.g., CD19 or CD123).

[0199] In another aspect, the invention relates to a kinase inhibitor as described herein (e.g., a BTK inhibitor such as ibrutinib, or a JAK-STAT inhibitor such as ruxolitinib), which is used in combination with a cell expressing a CAR molecule as described herein to reduce one or more side effects of the CAR therapy as described herein.

[0200] In another aspect, the invention relates to a CAR molecule as described herein, which is used in combination with a cell and a cytokine (e.g., IL-7, IL-15, and / or IL-21 as described herein) (e.g., as a medicament). In another aspect, the invention relates to a cytokine as described herein, which is used in combination with a cell expressing a CAR molecule as described herein (e.g., as a medicament).

[0201] In another aspect, the invention relates to cells expressing a CAR molecule as described herein for use in combination with a cytokine (e.g., IL-7, IL-15, and / or IL-21 as described herein) for treating (e.g., as a medicament) a disease expressing a B cell antigen (e.g., CD123 or CD19). In another aspect, the invention relates to a cytokine as described herein for use in combination with cells expressing a CAR molecule as described herein for treating (e.g., as a medicament) a disease expressing a B cell antigen (e.g., CD123 or CD19).

[0202] In some aspects, the present disclosure provides a method for differentiating CRS from sepsis in a subject, the method comprising obtaining a measurement of one or more of the following:

[0203] (i) the level or activity of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all) of GM-CSF, HGF, IFN-γ, IFN-α, IL-10, IL-15, IL-5, IL-6, IL-8, IP-10, MCP1, MIG, MIP-1β, sIL-2Rα, sTNFRI, and sTNFRII, wherein a level or activity above a reference indicates CRS; or

[0204] (ii) the level or activity of one or more (e.g., 2, 3, 4, 5, 6, or all) of CD163, IL-1β, sCD30, sIL-4R, sRAGE, sVEGFR-1, and sVEGFR-2, wherein a level or activity above a reference indicates sepsis.

[0205] In an embodiment, if the measurement indicates sepsis, the method comprises administering a therapy for treating CRS (e.g., a therapy as described herein). In an embodiment, if the measurement indicates sepsis, the method comprises administering a therapy for treating sepsis.

[0206] In some aspects, the present disclosure further provides a kit for differentiating CRS from sepsis in a patient, the kit comprising a set of reagents for specifically detecting the level or activity of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2, 22, or all) genes or proteins selected from the following:

[0207] GM-CSF, HGF, IFN-γ, IFN-α, IL-10, IL-15, IL-5, IL-6, IL-8, IP-10, MCP1, MIG, MIP-1β, sIL-2Rα, sTNFRI, sTNFRII, CD163, IL-1β, sCD30, sIL-4R, sRAGE, sVEGFR-1, and sVEGFR-2; and

[0208] Instructions for using the kit;

[0209] wherein the instructions provide that if the detected level or activity of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all) of GM-CSF, HGF, IFN-γ, IFN-α, IL-10, IL-15, IL-5, IL-6, IL-8, IP-10, MCP1, MIG, MIP-1β, sIL-2Rα, sTNFRI, or sTNFRII is greater than a reference value, the subject may have CRS,

[0210] and / or if the detected level or activity of one or more (e.g., 2, 3, 4, 5, 6, or all) of CD163, IL-1β, sCD30, sIL-4R, sRAGE, sVEGFR-1, or sVEGFR-2 is greater than a reference value, the subject may have sepsis.

[0211] In some aspects, the present disclosure also provides a reaction mixture that comprises:

[0212] A set of reagents that specifically detect the level or activity of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or all) genes or proteins selected from: GM-CSF, HGF, IFN-γ, IFN-α, IL-10, IL-15, IL-5, IL-6, IL-8, IP-10, MCP1, MIG, MIP-1β, sIL-2Rα, sTNFRI, sTNFRII, CD163, IL-1β, sCD30, sIL-4R, sRAGE, sVEGFR-1, and sVEGFR-2, and

[0213] A biological sample (e.g., a blood sample).

[0214] In an embodiment, the biological sample is from a subject treated with CAR-expressing cell therapy and / or having symptoms of CRS and / or sepsis.

[0215] In some aspects, the present disclosure also provides a method of identifying sepsis in a subject, the method comprising obtaining a measurement of one or more of the following:

[0216] (i) the level or activity of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or all) of ANG2, GCSF, IFNα, IL1RA, IL4, IL6, MIG, MIP1α, PTX3, TNFα, sCD163, sCD30, sIL-1RI, sIL-1RII, sIL-2Rα, sIL-4R, sRAGE, sTNFRI, sTNFRII, sVEGFR1, sVEGFR2, sVEGFR3 and VEGF, wherein a higher level or activity relative to a reference indicates sepsis;

[0217] (ii) the level or activity of one or more (e.g., both) of IL13 and RANTES, wherein a lower level or activity relative to a reference indicates sepsis.

[0218] In some aspects, the present disclosure provides a method of treating one or more of neurotoxicity, CRS or posterior reversible encephalopathy syndrome (PRES), the method comprising administering to a subject in need thereof a therapeutically effective amount of cyclophosphamide. In related aspects, the present disclosure provides cyclophosphamide for use in treating neurotoxicity, CRS or posterior reversible encephalopathy syndrome (PRES). In embodiments, cyclophosphamide is administered after a cell-based therapy (e.g., a cell-based therapy for cancer, a CD19 inhibitory therapy or a CD19 depletion therapy), or the subject has been previously treated with a cell-based therapy (e.g., a cell-based therapy for cancer, a CD19 inhibitory therapy or a CD19 depletion therapy). In embodiments, cyclophosphamide is administered before, concurrently with or after the cell-based therapy.

[0219] In an embodiment, the patient has or is identified as having CRS, PRES, or both. In some embodiments, the subject has been treated with a CD19 inhibition or depletion therapy. In some embodiments, the CD19 inhibitor is a CD19 antibody, such as a CD19 bispecific antibody (e.g., a bispecific T cell engager targeting CD19, e.g., blinatumomab). In some embodiments, the therapy comprises cells expressing a CAR, such as an anti-BCMACAR or an anti-CD19CAR. In an embodiment, the subject has neurotoxicity, such as focal deficits (e.g., cranial nerve palsy or hemiplegia) or global abnormalities (e.g., generalized seizures, confusion) or status epilepticus. In an embodiment, the subject does not have any clinical symptoms of CRS. In an embodiment, the subject has one or more clinical symptoms of CRS. In an embodiment, the subject has or is identified as having elevated IL-6 relative to a reference (e.g., the subject's IL-6 level prior to therapy with cells expressing a CAR). In an embodiment, the subject has or is identified as having an elevated serum level of cytokines associated with CRS (e.g., IL-6 and / or IL-8) relative to a reference. In an embodiment, the subject has or is identified as having an elevated level of cytokines associated with CRS (e.g., CSF IL-6 and / or IL-8) relative to a reference. In an embodiment, the subject is treated or has been treated with a therapy for CRS, such as tocilizumab or corticosteroids (e.g., (methylprednisolone, hydrocortisone, or both). In an embodiment, the subject has or is identified as having an increase in circulating, activated CR-expressing cells. In an embodiment, the subject has or is identified as having cells expressing a CAR in the CSF.

[0220] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the following describes suitable methods and materials. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Additionally, the materials, methods, and examples are illustrative only and not intended to be limiting. Headings, subheadings, or numbered or lettered elements, such as (a), (b), (i), etc., are presented only for ease of reading. The use of headings or numbered or lettered elements in this document does not require that steps or elements be in alphabetical order, or that steps or elements must be discontinuous from each other. Other features, objects, and advantages of the present invention will be apparent from the specification and drawings and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0221] The following detailed description of the preferred embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present invention, the presently preferred embodiments are shown in the drawings. However, it should be understood that the present invention is not limited to the exact arrangements and instrumentalities shown in the embodiments of the drawings.

[0222] Figure 1A is a schematic diagram illustrating an experiment conducted as described in Example 1 (e.g., in a mouse model that develops CRS after CART). Figure 1B is a graph showing the expansion of CART cells after AML injection. Figure 1C is a survival curve showing the survival of mice after high-dose CART123. Figure 1D is a set of graphs showing the levels of various cytokines in mice treated with high-dose CART123.

[0223] Figure 2A is a schematic diagram illustrating an experiment conducted as described in Example 1 (e.g., to determine the effect of ruxolitinib on CRS after CART therapy). Figure 2B is a graph showing the change in mouse body weight measured as % change relative to baseline (plotted on the y-axis against time on the x-axis). Figure 2C is a graph showing the disease burden from serial retro-orbital bleeds measured as leukemia cells / μl (huCD45 dim cells) (plotted on the y-axis against time on the x-axis). Figure 2D is a graph showing the change in mouse body weight when treated with ruxolitinib. The body weight measured as % change relative to baseline is plotted on the y-axis against time on the x-axis. Figure 2E is a graph showing the absolute CD3+ cell count from serial retro-orbital bleeds from mice. Serial retro-orbital bleeds were performed at the indicated time points on the x-axis. The absolute CD3+ cell count is plotted on the Y-axis. Figure 2F is a set of graphs showing the levels of inflammatory cytokines from mouse serum obtained by retro-orbital bleed of mice one week after CAR123 injection. Figure 2G is a survival graph showing the survival of mice treated with a combination of 60 mg / kg ruxolitinib and CART123. Figure 2H is a flow cytometry plot showing the peripheral blood analysis (gated on live human CD45 positive cells) of surviving mice treated with ruxolitinib 70 days after AML injection.

[0224] Figure 3A is a schematic diagram of the experiment described in Example 2, particularly a model of CRS development after CART19 treatment in B cell tumors. Figure 3B is an image of the spleen from a representative mouse sacrificed before T cell treatment, showing high tumor burden. Figure 3CIt is a flow cytometry plot showing a high level of circulating tumor B cells present in peripheral blood (PB) at randomization (gating strategy: time gate, lymphocytes, single cells, live gate, huCD45+muCD45-). Figure 3D It is a survival curve showing a significant decrease in overall survival of mice treated with CART19. Figure 3E It is a set of plots showing Luminex analysis of serum human cytokines, which shows a significant increase in cytokines in the PB of mice receiving CART19 compared to untreated. For Figures 3C - 3E , all graphs represent two independent experiments (5 mice per group). The Student t-test was used to compare two groups. The log-rank test was used to compare survival curves. Asterisks represent p-values (* = <0.05, ** = <0.01, *** = <0.001, **** = <0.0001), and "ns" means "not significant" (p > 0.05).

[0225] Figure 4A It is a schematic diagram showing the experiment in Example 2. For example, a combination of CART19 with ibrutinib or vehicle was administered in the mouse model generated in Example 2. Figure 4B It is a survival curve showing a significant increase in overall survival of mice treated with CART19 plus ibrutinib. Figure 4C It is a plot showing the number of CD19+ cells in peripheral blood after treatment with vehicle or ibrutinib. Figure 4D It is a plot showing that ibrutinib treatment does not have a negative impact on T cell expansion (on the contrary, T cell expansion is enhanced by ibrutinib treatment). Figure 4E It is a plot showing serum cytokine levels from mice treated with CART19 or CART19 + ibrutinib by Luminex analysis; a significant decrease in all cytokines involved in CRS was observed. Figure 4F It is a set of plots showing significant cytokine production in a dose-dependent manner in primary MCL cells incubated with ibrutinib for 24 hours. Figures 4B - 4F All graphs in represent two independent experiments (5 mice per group). The Student t-test was used to compare two groups; in the analysis comparing multiple groups, one-way ANOVA with Holm-Sidak correction for multiple comparisons was used. The log-rank test was used to compare survival curves. Asterisks represent p-values (* = <0.05, ** = <0.01, *** = <0.001, **** = <0.0001), and "ns" means "not significant" (p > 0.05).

[0226] Figure 5It is a graph showing serum cytokine concentrations in xenograft mice bearing primary pediatric ALL treated with CD19 CAR T cells. Seven days later, 10 6 primary ALLs and 5x10 6 autologous CD19 CAR T cells were administered to NSG mice. Serum was collected 3 days after T cell delivery, and tocilizumab was administered to subgroups of animals on days 1 and 3 after T cells. Cytokine concentrations were measured in pg / mL.

[0227] Figure 6 It is a graph showing serum cytokine concentrations in xenograft mice bearing ALL cell lines treated with CD19 CAR T cells. NSG mice were transplanted with 10 6 Nalm-6 ALL cells, and 5x10 6 CD19 CAR T cells derived from normal donors were administered 7 days later. Serum was collected 3 days after T cell delivery, and tocilizumab was administered to subgroups of animals on days 1 and 3 after T cells. Cytokine concentrations were measured in pg / mL.

[0228] Figure 7 A-7J is a graph showing cytokine expression after cell co-culture. T cells, targets, and APCs were combined at a ratio of 10:50:1, respectively. Supernatants were collected after 18 hours of co-culture. Cytokine levels were measured in pg / mL. Significant differences were indicated by * or ** and represent p values < 0.05.

[0229] Figure 8 A-8E is a graph showing cytokine secretion in a co-culture experiment combining monocyte lineage cells with T cells and targets. Monocyte lineage cells were differentiated in vitro, and T cells, targets, and APCs were combined at a ratio of 10:50:1, respectively. Supernatants were collected at 18 and 48 hours and analyzed for cytokine concentrations, measured in pg / mL.

[0230] Figure 9 A-9C is a graph showing transcriptional analysis of isolated cell populations. T cells and targets were separated from APCs using trans-well inserts and co-cultured for 18 hours. 697 RNA transcripts were quantified from each cell population, and the log counts per cell were shown. (A) Transcriptional profiles when CD19 CAR T cells were combined with targets and when combined with targets and pooled monocytes, (B) transcriptional profiles when APCs were combined with targets and when combined with targets and non-targeted T cells, and (C) transcriptional profiles when APCs were combined with targets and non-targeted T cells and when combined with targets and targeted T cells.

[0231] Figure 10It is a figure showing the transcript profiles of activated CD19 CAR T cells and monocyte lineage APCs. After 18 hours, cells were harvested from a transwell co-culture of CD19 CAR T cells, Nalm-6 leukemia, and pooled monocytes. Transcript counts from T cells are shown in blue, and counts from APCs are shown in red.

[0232] Figure 11 A-11C is a figure showing T cell degranulation in the presence of APCs. T cells expressing (A) no CAR molecule, (B) a CAR targeting GD2, or (C) a CD19-targeting CAR were combined with the CD19+ target ALL cell line Nalm-6. Degranulation was measured by quantifying CD107a surface expression.

[0233] Figure 12 It is a figure showing NanoString analysis of PBMCs collected from ALL patients treated with CD19 CAR T cells. Peripheral blood was collected on the first day of fever after engineered T cell infusion. T cells from the first seven patients were detectable in peripheral blood and there was no detectable ALL, while the last three patients had only ALL cells and no detectable T cells.

[0234] Figure 13 It is a set of images showing microscopic analysis of peripheral blood T cells collected at the first fever after infusion of CD19 CAR T cells in patients with acute lymphoblastic leukemia. Images were captured at 1000x magnification. Detailed Description

[0235] Definitions

[0236] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0237] The terms "a" and "an" refer to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0238] When referring to measurable values such as amounts, time intervals, etc., the term "about" is intended to cover variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the specified value, as such variations are appropriate for carrying out the disclosed methods.

[0239] The term "chimeric antigen receptor" or alternatively "CAR" refers to a recombinant polypeptide construct that comprises at least an extracellular antigen-combining domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as the "intracellular signaling domain") (the signaling domain comprising a functional signaling domain derived from a stimulatory molecule as defined below). In some embodiments, the domains in the CAR polypeptide construct are in the same polypeptide chain, such as comprising a chimeric fusion protein. In some embodiments, the domains in the CAR polypeptide construct are not contiguous with each other, such as being in different polypeptide chains (e.g., as provided in RCAR as described herein).

[0240] In one aspect, the stimulatory molecule of the CAR is the ζ chain associated with the T cell receptor complex. In one aspect, the cytoplasmic signaling domain comprises a primary signaling domain (e.g., the primary signaling domain of CD3-ζ). In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one co-stimulatory molecule as defined below. In one aspect, the co-stimulatory molecule is selected from 4-1BB (i.e., CD137), CD27, ICOS, and / or CD28. In one aspect, the CAR comprises a chimeric fusion protein (which comprises an extracellular antigen recognition domain), a transmembrane domain, and an intracellular signaling domain (which comprises a functional signaling domain derived from a stimulatory molecule). In one aspect, the CAR comprises a chimeric fusion protein (which comprises an extracellular antigen recognition domain), a transmembrane domain, and an intracellular signaling domain (which comprises a functional signaling domain derived from a co-stimulatory molecule and a functional signaling domain derived from a stimulatory molecule). In one aspect, the CAR comprises a chimeric fusion protein (which comprises an extracellular antigen recognition domain) a transmembrane domain, and an intracellular signaling domain (which comprises two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulatory molecule). In one aspect, the CAR comprises a chimeric fusion protein (which comprises an extracellular antigen recognition domain) a transmembrane domain, and an intracellular signaling domain (which comprises at least two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulatory molecule). In one aspect, the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (e.g., aa scFv) during cellular processing and CAR localization to the cell membrane.

[0241] A CAR comprising an antigen-binding domain that specifically binds to a particular tumor marker X (e.g., a scFv (a single-domain antibody) or a TCR (e.g., a TCRα-binding domain or a TCRβ-binding domain)), where X can be a tumor marker as described herein, is also referred to as an XCAR. For example, a CAR comprising an antigen-binding domain that specifically binds to CD123 is referred to as a CD123CAR or CAR123. For example, a CAR comprising an antigen-binding domain that specifically binds to CD19 is referred to as a CD19 CAR or CAR19. In some embodiments, the CAR comprises a CTL019 CAR as described herein. The CAR can be expressed in any cell, e.g., an immune effector cell (e.g., a T cell or an NK cell) as described herein.

[0242] A therapy comprising a cell expressing a CAR is referred to herein as a CAR therapy. For example, a therapy comprising a cell expressing a CD123 CAR or a CD19 CAR is referred to herein as a CD123CAR therapy or a CD19 CAR therapy, respectively.

[0243] The term "signaling domain" refers to the functional portion of a protein that acts by regulating cell activity via a defined signaling pathway by transmitting information within the cell to act as an effector by generating a second messenger or by responding to such a messenger.

[0244] As used herein, the terms "alpha subunit of the IL-3 receptor", "IL3Rα", "CD123", "IL3Rα chain", and "IL3Rα subunit" are used interchangeably to refer to an antigenic determinant known to be detectable on pre-leukemic progenitor cells. Human and murine amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human IL3Rα can be found under accession number NP_002174, and the nucleotide sequence encoding human IL3Rα can be found under accession number NM_005191. In one aspect, the antigen-binding portion of the CAR recognizes and binds to an epitope within the extracellular domain of the CD123 protein. In one aspect, the CD123 protein is expressed on cancer cells. As used herein, "CD123" includes proteins containing mutations (e.g., point mutations), fragments, insertions, deletions, and splice variants of full-length wild-type CD123.

[0245] As used herein, the term "CD19" refers to cluster of differentiation 19 protein, which is an antigenic determinant detectable on pre-leukemic progenitor cells. The amino acid and nucleic acid sequences of human and murine can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD19 can be found as UniProt / Swiss-Prot accession number P15391, and the nucleotide sequence encoding human CD19 can be found with accession number NM_001178098. As used herein, "CD19" includes proteins containing mutations (e.g., point mutations), fragments, insertions, deletions, and splice variants of full-length wild-type CD19. CD19 is expressed on most B-lineage cancers, including, for example, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma. Other cells expressing CD19 are provided in the definition of "diseases associated with CD19 expression" below. It is also an early marker of B-cell progenitors. See, e.g., Nicholson et al. Mol. Immunol. [Molecular Immunology] 34(16-17):1157-1165 (1997). In one aspect, the antigen-binding portion of the CART recognizes and binds an antigen within the extracellular domain of the CD19 protein. In one aspect, the CD19 protein is expressed on cancer cells.

[0246] As used herein, the term "CD20" refers to an antigenic determinant known to be detectable on B cells. Human CD20 is also known as transmembrane 4-domains, subfamily A, member 1 (MS4A1). The amino acid and nucleic acid sequences of human and murine can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD20 can be found with accession numbers NP_690605.1 and NP_068769.2, and the nucleotide sequences encoding transcript variants 1 and 3 of human CD20 can be found with accession numbers NM_152866.2 and NM_021950.3, respectively. In one aspect, the antigen-binding portion of the CAR recognizes and binds an antigen within the extracellular domain of the CD20 protein. In one aspect, the CD20 protein is expressed on cancer cells.

[0247] As used herein, the term "CD22" refers to an antigenic determinant known to be detectable on pre-leukemic cells. Human and murine amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequences of isotypes 1-5 of human CD22 can be found under accession numbers NP 001762.2, NP 001172028.1, NP001172029.1, NP 001172030.1, and NP 001265346.1, respectively, and the nucleotide sequences encoding variants 1-5 of human CD22 can be found under accession numbers NM 001771.3, NM 001185099.1, NM 001185100.1, NM 001185101.1, and NM 001278417.1, respectively. In one aspect, the antigen-binding portion of the CAR recognizes and binds an antigen within the extracellular domain of the CD22 protein. In one aspect, the CD22 protein is expressed on cancer cells.

[0248] As used herein, the term "ROR1" refers to an antigenic determinant known to be detectable on pre-leukemic cells. Human and murine amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequences of isotypes 1 and 2 precursors of human ROR1 can be found under accession numbers NP_005003.2 and NP_001077061.1, respectively, and the mRNA sequences encoding them can be found under accession numbers NM_005012.3 and NM_001083592.1. In one aspect, the antigen-binding portion of the CAR recognizes and binds an antigen within the extracellular domain of the ROR1 protein. In one aspect, the ROR1 protein is expressed on cancer cells.

[0249] As used herein, the term "CD33" refers to the cluster of differentiation 33 protein, which is an antigenic determinant detectable on leukemic cells and on normal precursor cells of the myeloid lineage. Human and murine amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD33 can be found as UniProt / Swiss-Prot accession number P20138, and the nucleotide sequence encoding human CD33 can be found under accession number NM_001772.3. In one aspect, the antigen-binding portion of the CAR recognizes and binds an epitope within the extracellular domain of the CD33 protein or a fragment thereof. In one aspect, the CD33 protein is expressed on cancer cells. As used herein, "CD33" includes proteins containing mutations (e.g., point mutations), fragments, insertions, deletions, and splice variants of full-length wild-type CD33.

[0250] As used herein, the term "BCMA" refers to B cell maturation antigen. BCMA (also known as TNFRSF17, BCM, or CD269) is a member of the tumor necrosis factor receptor (TNFR) family and is expressed primarily on terminally differentiated B cells (such as memory B cells and plasma cells). Its ligands are called B cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL) of the TNF family. BCMA is involved in mediating the survival of plasma cells to maintain long-term humoral immunity. The gene for BCMA is encoded on chromosome 16 and gives rise to a primary mRNA transcript of 994 nucleotides in length (NCBI accession number NM_001192.2), which encodes a protein of 184 amino acids (NP_001183.2). A second antisense transcript derived from the BCMA locus has been described, which may play a role in regulating BCMA expression. (Laabi Y. et al., Nucleic Acids Res. [Nucleic Acid Research], 1994, 22:1147-1154). Additional transcript variants of unknown significance have been described (Smirnova AS et al. Mol Immunol. [Molecular Immunology], 2008, 45(4):1179-1183). A second isoform (also known as TV4) has been identified (Uniprot accession code Q02223-2). As used herein, "BCMA" includes proteins containing mutations (such as point mutations), fragments, insertions, deletions, and splice variants of full-length wild-type BCMA.

[0251] As used herein, the term "CLL-1" refers to C-type lectin-like molecule-1, which is an antigenic determinant detectable on leukemic precursor cells and normal immune cells. C-type lectin-like-1 (CLL-1) is also known as MICL, CLEC12A, CLEC-1, dendritic cell-associated lectin 1, and DCAL-2. Human and murine amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CLL-1 can be found as UniProt / Swiss-Prot accession number Q5QGZ9, and the nucleotide sequence encoding human CLL-1 can be found at accession numbers NM 001207010.1, NM138337.5, NM 201623.3, and NM 201625.1. In one embodiment, the antigen-binding portion of the CAR recognizes and binds to an epitope within the extracellular domain of the CLL-1 protein or a fragment thereof. In one embodiment, the CLL-1 protein is expressed on cancer cells.

[0252] The term "EGFR" refers to any mammalian mature full-length epidermal growth factor receptor, including human and non-human forms. The 1186 amino acid human EGFR is described in Ullrich et al., Nature 309:418-425 (1984)) and GenBank accession number AF125253 and SwissProt accession number P00533-2.

[0253] The term "EGFRvIII" refers to epidermal growth factor receptor variant III. EGFRvIII is the most common EGFR variant observed in human tumors, but is rarely observed in normal tissues. The protein results from an in-frame deletion of exons 2-7 and the generation of a new glycine residue at the junction of exons 1 and 8 within the extracellular domain of EGFR, thereby creating a tumor-specific epitope. EGFRvIII is expressed in 24% to 67% of GBMs, but not in normal tissues. EGFRvIII is also referred to as mutant type III, δ-EGFR, EGFRde2-7, and EGFR, and is described in U.S. Patent Nos. 6,455,498, 6,127,126, 5,981,725, 5,814,317, 5,710,010, 5,401,828, and 5,212,290. Expression of EGFRvIII may be caused by chromosomal deletions or by abnormal alternative splicing. See Sugawa et al., 1990, Proc. Natl. Acad. Sci. 87:8602-8606.

[0254] As used herein, the term "mesothelin" refers to the 40-kDa protein mesothelin, which is anchored to the cell membrane by a glycosylphosphatidylinositol (GPI) linkage and an amino-terminal 31-kDa shed fragment called megakaryocyte potentiating factor (MPF). Both fragments contain N-glycosylation sites. The term also refers to a soluble spliced variant of the 40-kDa carboxy-terminal fragment, also referred to as "soluble mesothelin / MPF-related". Preferably, the term refers to human mesothelin of GenBank accession number AAH03512.1, and its native cleavage products, e.g., as expressed on the cell membrane (e.g., cancer cell membrane).

[0255] As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. An antibody can be polyclonal or monoclonal, multi-chain or single-chain, or a complete immunoglobulin, and can be derived from a natural or recombinant source. An antibody can be a tetramer of immunoglobulin molecules.

[0256] The term "antibody fragment" refers to at least a portion of a full antibody or a recombinant variant thereof, and refers to antigen-combining domains, such as the antigen-determining variable regions of a full antibody (which are sufficient to confer upon the antibody fragment recognition and specific combination with a target, such as an antigen). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, scFv antibody fragments, linear antibodies, single-domain antibodies such as sdAb (VL or VH), camelid VHH domains, and multispecific antibodies formed from antibody fragments (e.g., bivalent fragments comprising two Fab fragments linked by a disulfide bond at the hinge region) and isolated CDRs or other epitope-binding fragments of an antibody. Antigen-binding fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetra-bodies, v-NAR, and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be grafted onto scaffolds based on polypeptides such as type III fibronectin (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide microantibodies).

[0257] The term "scFv" refers to a fusion protein that comprises at least one antibody fragment that contains a light-chain variable region and at least one antibody fragment that contains a heavy-chain variable region, wherein the light-chain and heavy-chain variable regions are joined in series via a short flexible polypeptide linker and are capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the full antibody from which it is derived. Unless otherwise specified, as used herein, the scFv can have the VL and VH variable regions in either order (e.g., with respect to the N-terminus and C-terminus of the polypeptide), and the scFv can comprise VL-linker-VH or can comprise VH-linker-VL.

[0258] As used herein, the term "complementary determining region" or "CDR" refers to the amino acid sequences within the variable regions of an antibody that confer antigen specificity and binding affinity. For example, typically, there are three CDRs in each heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3), and three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3). The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described in the following: Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme), or combinations thereof. Under the Kabat numbering scheme, in some embodiments, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under the Chothia numbering scheme, in some embodiments, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In the combined Kabat and Chothia numbering scheme, in some embodiments, the CDR corresponds to the amino acid residues that are part of the Kabat CDR, the Chothia CDR, or both. For example, in some embodiments, the CDR corresponds to the amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in VH (e.g., mammalian VH, e.g., human VH); and the amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in VL (e.g., mammalian VL, e.g., human VL).

[0259] The portion of the CAR compositions of the invention that comprises an antibody or an antibody fragment thereof can exist in a variety of forms, wherein the antigen-binding domain is expressed as part of a continuous polypeptide chain (including, for example, single-domain antibody fragments (sdAbs), single-chain antibodies (scFvs), and humanized or human antibodies) (Harlow et al., 1999, in: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, in: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one aspect, the antigen-binding domain of the CAR compositions of the invention comprises an antibody fragment. In another aspect, the CAR comprises an antibody fragment that contains an scFv).

[0260] As used herein, the term "binding domain" or "antibody molecule" (also referred to herein as "anti-target (e.g., CD123) binding domain") refers to a protein that comprises at least one immunoglobulin variable domain sequence, such as an immunoglobulin chain or a fragment thereof. The terms "binding domain" or "antibody molecule" encompass antibodies and antibody fragments. In embodiments, the antibody molecule is a multispecific antibody molecule, e.g., it comprises multiple immunoglobulin variable domain sequences, wherein the first immunoglobulin variable domain sequence of the multiple has binding specificity for a first epitope and the second immunoglobulin variable domain sequence of the multiple has binding specificity for a second epitope. In embodiments, the multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody is specific for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.

[0261] The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains that exist in their naturally occurring conformation in an antibody molecule and that generally determines the class to which the antibody belongs.

[0262] The term "antibody light chain" refers to the smaller of the two types of polypeptide chains that exist in their naturally occurring conformation in an antibody molecule. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

[0263] The term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, such as an antibody expressed by a phage or yeast expression system. The term should also be construed to mean an antibody produced by synthesizing a DNA molecule encoding the antibody and expressing the antibody protein or the amino acid sequence specifying the antibody, where the DNA or amino acid sequence is obtained using recombinant DNA or amino acid sequence techniques that are available and well known in the art.

[0264] The term "antigen" or "Ag" refers to a molecule that elicits an immune response. The immune response can involve antibody production or activation of specific immune reactive cells or both. One of ordinary skill in the art will appreciate that any macromolecule that actually includes all proteins or peptides can serve as an antigen. In addition, an antigen can be derived from recombinant or genomic DNA. One of ordinary skill in the art will appreciate that any DNA that contains a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response thus encodes an "antigen" (as the term is used herein). In addition, those of ordinary skill in the art will appreciate that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is obvious that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in various combinations to encode a polypeptide that elicits the desired immune response. Additionally, one of ordinary skill in the art will appreciate that an antigen need not be encoded by a "gene" at all. It is obvious that an antigen can be produced synthetically or can be derived from a biological sample, or can be a macromolecule other than a polypeptide. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or fluids with other biological components.

[0265] The term "antitumor effect" refers to a biological effect that can be manifested by various means, including but not limited to, for example, reducing tumor volume, reducing the number of tumor cells, reducing the number of tumor metastases, increasing life expectancy, reducing tumor cell proliferation, reducing tumor cell survival, or improving various physiological symptoms associated with a cancerous condition. The "antitumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the present invention to prevent tumorigenesis in the first place.

[0266] The term "anticancer effect" refers to a biological effect that can be manifested by various means, including but not limited to, for example, reducing cancer volume, reducing the number of cancer cells, reducing the number of tumor metastases, increasing life expectancy, reducing cancer cell proliferation, reducing tumor cell survival, or improving various physiological symptoms associated with a cancerous condition. The "anticancer effect" can also be manifested by the ability of peptides, polynucleotides, cells, and antibodies to prevent cancer development in the first place.

[0267] The term "anti - tumor effect" refers to a biological effect that can be manifested by various means, including but not limited to, for example, reducing tumor volume, decreasing the number of tumor cells, reducing tumor cell proliferation, or reducing tumor cell survival.

[0268] The term "autologous" refers to any material derived from the same individual who will later have it re - introduced into that individual.

[0269] The term "allogeneic" refers to any material derived from a different animal of the same species as the individual into whom the material is introduced. Two or more individuals are said to be allogeneic to each other when the genes at one or more loci are not identical. In some aspects, allogeneic materials from individuals of the same species can be genetically different enough to interact antigenically.

[0270] The term "xenogeneic" refers to a graft derived from an animal of a different species.

[0271] As used herein, the term "apheresis" refers to an in vitro process recognized in the art by which blood from a donor or patient is removed from the donor or patient and passed through a device that separates one or more selected specific components, and the remainder is returned to the circulation of the donor or patient (e.g., by re - infusion). Thus, in the context of an "apheresis sample" it refers to a sample obtained using apheresis.

[0272] The term "combination" refers to a fixed combination in the form of a dosage unit, or to combination administration (wherein the compound of the invention and a combination partner (e.g., another drug as explained below, also referred to as a "therapeutic agent" or "co-agent") can be administered independently at the same time or separately at time intervals, especially in cases where these time intervals allow the combination partner to exhibit cooperation, such as a synergistic effect). The individual components can be packaged in a kit or separately. One or both components (e.g., powder or liquid) can be reconstituted or diluted to the desired dosage prior to administration. As used herein, terms such as "co-administered" or "combination administered" are intended to cover the administration of the selected combination partner to a single subject (e.g., a patient) in need thereof, and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or simultaneously. As used herein, the term "pharmaceutical combination" means a product resulting from the mixing or combination of more than one active ingredient, and includes both fixed and non-fixed combinations of active ingredients. The term "fixed combination" means that the active ingredients (e.g., the compound of the invention and the combination partner) are administered to a patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that the active ingredients (e.g., the compound of the invention and the combination partner) are administered to a patient as separate entities simultaneously, in parallel, or sequentially (without a specific time limit), wherein such administration provides therapeutically effective levels of both compounds in the patient. The latter also applies to cocktail therapies, such as the administration of three or more active ingredients.

[0273] The term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein, including but not limited to breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc. The terms "tumor" and "cancer" are used interchangeably herein, e.g., both terms cover solids and liquids, such as diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" includes pre-malignant as well as malignant cancers and tumors.

[0274] "Derivative of" (when the term is used herein) denotes the relationship between a first and a second molecule. It generally refers to the structural similarity between the first and second molecules and does not imply or include a limitation on the process or source of the first molecule that is derivative of the second molecule. For example, in the case of an intracellular signaling domain that is derivative of a CD3ζ molecule, the intracellular signaling domain retains sufficient CD3ζ structure such that it has the desired function, namely the ability to generate a signal under appropriate conditions. It does not imply or include a limitation on the particular process by which the intracellular signaling domain is generated; for example, it does not mean that in order to provide the intracellular signaling domain, one must start with the CD3ζ sequence and delete unwanted sequences or impose mutations to arrive at the intracellular signaling domain.

[0275] The phrase "diseases associated with B cell antigen expression" includes, but is not limited to, diseases associated with the expression of one or more of CD19, CD20, CD22 or ROR1, or conditions associated with cells that express or have at any time expressed one or more of CD19, CD20, CD22 or ROR1, including, for example, proliferative diseases (such as cancer or malignancy) or pre-cancerous conditions (such as myelodysplasia, myelodysplastic syndromes or pre-leukemia); or non-cancer-related indications associated with cells that express one or more of CD19, CD20, CD22 or ROR1. For the avoidance of doubt, diseases associated with B cell antigen expression may include conditions associated with cells that do not currently express B cell antigens (e.g., because antigen expression has been downregulated, e.g., due to treatment with a molecule that targets a B cell antigen, such as a CAR that targets B cells) but that have previously expressed the antigen. The phrase "diseases associated with B cell antigen expression" includes diseases associated with CD19 expression, as described herein.

[0276] The phrase "diseases associated with CD19 expression" includes, but is not limited to, diseases associated with CD19 expression, or conditions associated with cells that express or have expressed CD19 at any time, including, for example, proliferative diseases (such as cancer or malignancy) or pre-cancerous conditions (such as myelodysplasia, myelodysplastic syndrome or pre-leukemia); or non-cancer-related indications associated with cells that express CD19. For the avoidance of doubt, diseases associated with CD19 expression may include conditions associated with cells that do not currently express CD19 (e.g., because CD19 expression has been downregulated, e.g., due to treatment with a molecule that targets CD19 (such as a CD19 CAR)) but that have previously expressed CD19. In one aspect, the cancer associated with CD19 expression is a hematological cancer. In one aspect, the hematological cancer is leukemia or lymphoma. In one aspect, the cancer associated with CD19 expression includes cancers and malignancies that include, but are not limited to, for example, one or more acute leukemias (including, but not limited to, for example, B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), acute lymphoblastic leukemia (ALL)); one or more chronic leukemias (including, but not limited to, for example, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL)). Other cancers or hematological conditions associated with CD19 expression include, but are not limited to, for example, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma (MCL), marginal zone lymphoma, multiple myeloma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and "pre-leukemia" (which is a diverse collection of hematological conditions associated with ineffective production (or dysplasia) of myeloid blood cells) and the like. Additional diseases associated with CD19 expression include, but are not limited to, for example, atypical and / or non-classical cancers, malignancies, pre-cancerous conditions, or proliferative diseases associated with CD19 expression. Non-cancer-related indications associated with CD19 expression include, but are not limited to, for example, autoimmune diseases (such as lupus), inflammatory conditions (allergies and asthma), and transplantation. In some embodiments, cells that express a tumor antigen express or have expressed the mRNA encoding the tumor antigen at any time. In one embodiment, cells that express a tumor antigen produce a tumor antigen protein (e.g., wild-type or mutant), and the tumor antigen protein may be present at normal levels or reduced levels. In one embodiment, cells that express a tumor antigen produce a detectable level of the tumor antigen protein at a certain point and then substantially do not produce a detectable tumor antigen protein.

[0277] As used herein, the phrase "diseases associated with CD123 expression" includes, but is not limited to, diseases associated with CD123 expression or conditions associated with cells expressing CD123 (e.g., wild-type or mutant CD123), including, for example, proliferative diseases such as cancer or malignancy; pre-cancerous conditions such as myelodysplasia, myelodysplastic syndrome or pre-leukemia; or non-cancer related indications associated with cells expressing CD123 (e.g., wild-type or mutant CD123). In one aspect, the cancer associated with CD123 (e.g., wild-type or mutant CD123) expression is a hematological cancer. In one aspect, the diseases include AML, ALL, hairy cell leukemia, prolymphocytic leukemia, chronic myeloid leukemia (CML), Hodgkin lymphoma, blastic plasmacytoid dendritic cell neoplasm, lymphoblastic B cell leukemia (B cell acute lymphoblastic leukemia, BALL), acute lymphoblastic T cell leukemia (T cell acute lymphoblastic leukemia (TALL); myelodysplastic syndrome; myeloproliferative neoplasm; histiocytosis (e.g., mast cell disorder or blastic plasmacytoid dendritic cell neoplasm); mast cell disorder (e.g., systemic mastocytosis or mast cell leukemia), etc. Additional diseases associated with CD123 expression include, but are not limited to, for example, atypical and / or non-classical cancers, malignancies, pre-cancerous conditions or proliferative diseases associated with CD123 expression. Non-cancer related indications associated with CD123 expression may also be included.

[0278] As used herein, the phrase "disease associated with CD33 expression" includes, but is not limited to, diseases or conditions associated with cells expressing CD33 (e.g., wild-type or mutant CD33), including, for example, proliferative diseases (such as cancer or malignancy) or pre-cancerous conditions (such as myelodysplasia, myelodysplastic syndrome or pre-leukemia); or non-cancer-related indications associated with cells expressing CD33 (e.g., wild-type or mutant CD33). For the avoidance of doubt, diseases associated with CD33 expression may include conditions associated with cells that do not currently express CD33 (e.g., because CD33 expression has been downregulated, such as due to treatment with a molecule targeting CD33 (e.g., a CD33 inhibitor as described herein)) but that have expressed CD33 in the past. In one aspect, the cancer associated with CD33 (e.g., wild-type or mutant CD33) expression is a hematological cancer. In one aspect, hematological cancers include, but are not limited to, acute myeloid leukemia (AML), myelodysplasia and myelodysplastic syndrome, myelofibrosis and myeloproliferative neoplasms, acute lymphoblastic leukemia (ALL), hairy cell leukemia, prolymphocytic leukemia, chronic myeloid leukemia (CML), blastic plasmacytoid dendritic cell neoplasm, and the like. Additional diseases associated with CD33 (e.g., wild-type or mutant CD33) expression include, but are not limited to, for example, atypical and / or non-classical cancers, malignancies, pre-cancerous conditions or proliferative diseases associated with CD33 (e.g., wild-type or mutant CD33) expression. Non-cancer-related indications associated with CD33 (e.g., wild-type or mutant CD33) expression may also be included. In embodiments, non-cancer-related indications associated with CD33 expression include, but are not limited to, for example, autoimmune diseases (such as lupus), inflammatory conditions (allergies and asthma), and transplantation. In some embodiments, cells expressing a tumor antigen express or have at any time expressed the mRNA encoding the tumor antigen. In one embodiment, cells expressing a tumor antigen produce the tumor antigen protein (e.g., wild-type or mutant), and the tumor antigen protein may be present at normal levels or at reduced levels. In one embodiment, cells expressing a tumor antigen produce a detectable level of the tumor antigen protein at one point and then substantially do not produce a detectable tumor antigen protein.

[0279] The phrase "diseases associated with BCMA expression" includes, but is not limited to, diseases or disorders associated with cells that express BCMA (e.g., wild-type or mutant BCMA), including, for example, proliferative diseases (such as cancer or malignancy) or pre-cancerous conditions (such as myelodysplasia, myelodysplastic syndrome, or pre-leukemia); or non-cancer-related indications associated with cells that express BCMA (e.g., wild-type or mutant BCMA). For the avoidance of doubt, diseases associated with BCMA expression may include disorders associated with cells that do not currently express BCMA (e.g., because BCMA expression has been downregulated, such as due to treatment with a molecule that targets BCMA (e.g., a BCMA inhibitor described herein)) but that once expressed BCMA. In one aspect, the cancer associated with BCMA (e.g., wild-type or mutant BCMA) expression is a hematological cancer. In one aspect, the hematological cancer is leukemia or lymphoma. In one aspect, the cancer associated with BCMA (e.g., wild-type or mutant BCMA) expression is a malignancy of differentiated plasma B cells. In one aspect, the cancer associated with BCMA (e.g., wild-type or mutant BCMA) expression includes cancers and malignancies that include, but are not limited to, for example, one or more acute leukemias (including, but not limited to, for example, B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia (TALL), acute lymphoblastic leukemia (ALL)); one or more chronic leukemias (including, but not limited to, for example, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL)). Other cancers or hematological disorders associated with BMCA (e.g., wild-type or mutant BCMA) expression include, but are not limited to, for example, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and "pre-leukemia" (which is a diverse collection of hematological disorders associated with ineffective production (or dysplasia) of myeloid blood cells), etc. In some embodiments, the cancer is multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, or glioblastoma.In embodiments, diseases associated with BCMA expression include plasma cell proliferative disorders such as asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma), monoclonal gammopathy of undetermined significance (MGUS), Waldenström macroglobulinemia, plasmacytoma (e.g., plasma cell cachexia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytomas), systemic amyloid light chain amyloidosis, and POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease, and PEP syndrome). Other diseases associated with BCMA (e.g., wild-type or mutant BCMA) expression include, but are not limited to, for example, atypical and / or non-classical cancers, malignancies, pre-cancerous conditions, or proliferative diseases associated with BCMA (e.g., wild-type or mutant BCMA) expression such as cancers described herein, such as prostate cancer (e.g., castration-resistant or therapy-resistant prostate cancer, or metastatic prostate cancer), pancreatic cancer, or lung cancer.

[0280] Non-cancer-related conditions associated with BCMA (e.g., wild-type or mutant BCMA) include viral infections; e.g., HIV, fungal infections, e.g., Cryptococcus neoformans; autoimmune diseases; e.g., rheumatoid arthritis, systemic lupus erythematosus (SLE or lupus), pemphigus vulgaris, and Sjögren's syndrome; inflammatory bowel disease, ulcerative colitis; transplant-related allo-specific immune disorders associated with mucosal immunity; unwanted immune responses to biologics (such as factor VIII) in situations where humoral immunity is important. In embodiments, non-cancer-related indications associated with BCMA expression include, but are not limited to, for example, autoimmune diseases (e.g., lupus), inflammatory conditions (allergies and asthma), and transplantation. In some embodiments, cells expressing a tumor antigen express or have at any time expressed the mRNA encoding the tumor antigen. In one embodiment, cells expressing a tumor antigen produce a tumor antigen protein (e.g., wild-type or mutant), and the tumor antigen protein may be present at normal levels or reduced levels. In one embodiment, cells expressing a tumor antigen produce a detectable level of the tumor antigen protein at one point and then essentially no longer produce a detectable tumor antigen protein.

[0281] The phrase "diseases associated with CLL-1 expression" includes, but is not limited to, diseases or conditions associated with cells that express CLL-1, including, for example, proliferative diseases (such as cancer or malignancy) or pre-cancerous conditions (such as myelodysplasia, myelodysplastic syndrome or pre-leukemia); or non-cancer-related indications associated with cells that express CLL-1 (e.g., wild-type or mutant CLL-1). To avoid doubt, diseases associated with CLL-1 expression may include conditions associated with cells that do not currently express CLL-1 (e.g., because CLL-1 expression has been down-regulated, e.g., due to treatment with a molecule that targets CLL-1 (e.g., a CLL-1 inhibitor described herein)) but that have previously expressed CLL-1. In one aspect, the cancer associated with CLL-1 expression is a hematological cancer. In one aspect, hematological cancers include, but are not limited to, leukemia (e.g., acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and myelodysplastic syndrome) and malignant lymphoproliferative disorders (including lymphomas (such as multiple myeloma, non-Hodgkin lymphoma, Burkitt lymphoma, small and large cell follicular lymphoma)). Additional diseases associated with CLL-1 expression include, but are not limited to, for example, atypical and / or non-classical cancers, malignancies, pre-cancerous conditions or proliferative diseases associated with CLL-1 expression. Non-cancer-related indications associated with CLL-1 expression may also be included. In some embodiments, cells that express a tumor antigen express or have at any time expressed the mRNA encoding the tumor antigen. In one embodiment, cells that express a tumor antigen produce the tumor antigen protein (e.g., wild-type or mutant), and the tumor antigen protein may be present at normal levels or at reduced levels. In one embodiment, cells that express a tumor antigen produce a detectable level of the tumor antigen protein at a certain point and then substantially do not produce a detectable tumor antigen protein.

[0282] As used herein, the term "disease associated with EGFRvIII expression" includes, but is not limited to, diseases associated with EGFRvIII expression or conditions associated with cells expressing EGFRvIII, including tumor cells of various cancers, such as glioblastoma (including glioblastoma stem cells); breast cancer, ovarian cancer, and non-small cell lung cancer; head and neck squamous cell carcinoma; medulloblastoma, colorectal cancer, prostate cancer, and bladder cancer. Without being bound by a particular theory or mechanism, it is believed that by eliciting a specific response against the antigen of EGFRvIII, the CARs disclosed herein provide one or more of the following: targeting and destroying tumor cells expressing EGFRvIII, reducing or eliminating tumors, promoting infiltration of immune cells to the tumor site, and enhancing / prolonging the anti-tumor response. Because EGFRvIII is not expressed at detectable levels in normal (i.e., non-cancerous) tissues, it is expected that the CARs of the present invention advantageously substantially avoid targeting / destroying normal tissues and cells.

[0283] As used herein, the phrase "disease associated with mesothelin expression" includes, but is not limited to, diseases associated with mesothelin expression or conditions associated with cells expressing mesothelin, including, for example, proliferative diseases (such as cancer or malignancy) or pre-cancerous conditions (such as mesothelial cell hyperplasia); or non-cancer-related indications associated with cells expressing mesothelin. Examples of various cancers expressing mesothelin include, but are not limited to, mesothelioma, ovarian cancer, pancreatic cancer, and the like.

[0284] In some embodiments, cells expressing a tumor antigen (e.g., expressing CD123 or CD19) express or have at any time expressed mRNA encoding the tumor antigen. In one embodiment, cells expressing a tumor antigen (e.g., expressing CD123 or CD19) produce a tumor antigen protein (e.g., wild-type or mutant), and the tumor antigen protein may be present at normal levels or reduced levels. In one embodiment, cells expressing a tumor antigen (e.g., expressing CD123 or CD19) produce a detectable level of the tumor antigen protein at a certain point and then substantially do not produce a detectable tumor antigen protein.

[0285] The term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing an amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. The modifications can be introduced into the antibodies or antibody fragments of the invention by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). A conservative substitution is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having the following side chains: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Accordingly, one or more amino acid residues within the CARs of the invention can be replaced with other amino acid residues from the same side chain family, and the altered CARs can be tested using the functional assays described herein.

[0286] The term "stimulate" refers to inducing a primary response by the binding of a stimulatory molecule (e.g., the TCR / CD3 complex) to its cognate ligand, thereby mediating signal transduction events such as, but not limited to, signal transduction via the TCR / CD3 complex. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-β and / or reorganization of the cytoskeletal structure, etc.

[0287] The term "stimulatory molecule" refers to a molecule expressed by a T cell that provides one or more primary cytoplasmic signaling sequences that regulate, in a stimulatory manner, the primary activation of the TCR complex with respect to at least some aspects of the T cell signaling pathway. In one aspect, the primary signal is initiated, for example, by the binding of the TCR / CD3 complex to a peptide-loaded MHC molecule and results in the mediation of a T cell response (including but not limited to proliferation, activation, differentiation, etc.). The primary cytoplasmic signaling sequences (also referred to as "primary signaling domains") that act in a stimulatory manner may contain signaling motifs that are referred to as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAMs containing the primary cytoplasmic signaling sequences that are particularly used in the present invention include but are not limited to those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS"), FcεRI, CD66d, DAP10, and DAP12. In certain CARs of the present invention, the intracellular signaling domain in any one or more of the CARs of the present invention contains an intracellular signaling sequence, such as the primary signaling sequence of CD3-ζ. In certain CARs of the present invention, the primary signaling sequence of CD3-ζ is the sequence provided as SEQ ID NO:9, or an equivalent residue from a non-human species (such as a mouse, rodent, monkey, ape, etc.). In certain CARs of the present invention, the primary signaling sequence of CD3-ζ is the sequence provided in SEQ ID NO:10, or an equivalent residue from a non-human species (such as a mouse, rodent, monkey, ape, etc.).

[0288] The term "antigen-presenting cell" or "APC" refers to an immune system cell that displays a foreign antigen complexed with a major histocompatibility complex (MHC) on its surface, such as an accessory cell (e.g., B cell, dendritic cell, etc.). T cells can recognize these complexes using their T cell receptor (TCR). The APC processes the antigen and presents it to the T cells.

[0289] As used herein, the term "intracellular signaling domain" refers to the intracellular portion of a molecule. The intracellular signaling domain can generate signals that promote the immune effector functions of cells containing a CAR, such as CAR T cells or NK cells expressing a CAR. Examples of immune effector functions (e.g., in CAR T cells or NK cells expressing a CAR) include cytolytic activity and accessory activity, including the secretion of cytokines. In embodiments, the intracellular signaling domain transduces effector function signals and directs the cell to perform specialized functions. Although the entire intracellular signaling domain can be used, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the full chain, provided that it transduces effector function signals. Thus, the term intracellular signaling domain is intended to include any truncated portion of an intracellular signaling domain that is sufficient to transduce effector function signals.

[0290] In one embodiment, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules responsible for primary stimulation or antigen-dependent stimulation. In one embodiment, the intracellular signaling domain can comprise a co-stimulatory intracellular domain. Exemplary co-stimulatory intracellular signaling domains include those derived from molecules responsible for co-stimulatory signals or antigen-independent stimulation. For example, in the case of immune effector cells expressing a CAR (e.g., CAR T cells or NK cells expressing a CAR), the primary intracellular signaling domain can comprise the cytoplasmic sequence of the T cell receptor, and the co-stimulatory intracellular domain can comprise the cytoplasmic sequence from a co-receptor or co-stimulatory molecule.

[0291] The primary intracellular signaling domain can comprise a signaling motif, which is referred to as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAMs containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 ("ICOS"), FcεRI, CD66d, DAP10, and DAP12.

[0292] The term "zeta" or alternatively "zeta chain", "CD3-zeta" or "TCR-zeta" is defined as the protein provided as GenBank accession number BAG36664.1, or equivalent residues from non-human species (such as mouse, rodent, monkey, ape, etc.), and "zeta stimulatory domain" or alternatively "CD3-zeta stimulatory domain" or "TCR-zeta stimulatory domain" is defined as the amino acid residues from the cytoplasmic domain of the zeta chain that are sufficient to functionally transmit the primary signal necessary for T cell activation. In one aspect, the cytoplasmic domain of zeta contains residues 52 to 164 of GenBank accession number BAG36664.1, or equivalent residues (which are functional orthologs) from non-human species (such as mouse, rodent, monkey, ape, etc.). In one aspect, the "zeta stimulatory domain" or "CD3-zeta stimulatory domain" is the sequence provided as SEQ ID NO:9. In one aspect, the "zeta stimulatory domain" or "CD3-zeta stimulatory domain" is the sequence provided as SEQ ID NO:10.

[0293] The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response (such as but not limited to proliferation) through the T cell. Costimulatory molecules are cell surface molecules other than the antigen receptor or its ligand required for an effective immune response. Costimulatory molecules include but are not limited to MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptors, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, and a ligand that specifically binds to CD83.

[0294] A costimulatory intracellular signaling domain refers to the intracellular portion of a costimulatory molecule. The intracellular signaling domain may comprise the entire intracellular portion of a molecule derived therefrom or the entire native intracellular signaling domain, or a functional fragment thereof.

[0295] The term "4-1BB" refers to a member of the TNFR superfamily that has the amino acid sequence provided as GenBank accession number AAA62478.2, or equivalent residues from non-human species (such as mouse, rodent, monkey, ape, etc.); and the "4-1BB co-stimulatory domain" is defined as amino acid residues 214-255 of GenBank accession number AAA62478.2, or equivalent residues from non-human species (such as mouse, rodent, monkey, ape, etc.). In one aspect, the "4-1BB co-stimulatory domain" is the sequence provided as SEQ ID NO:7, or equivalent residues from non-human species (such as mouse, rodent, monkey, ape, etc.).

[0296] "Immune effector cell" (when the term is used herein) refers to a cell that participates in an immune response, e.g., promotes an immune effector response. Examples of immune effector cells include T cells, such as α / β T cells and γ / δ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytic cells.

[0297] "Immune effector function or immune effector response" (when the term is used herein) refers to a function or response that enhances or promotes an immune attack on a target cell, e.g., the function or response of an immune effector cell. For example, an immune effector function or response refers to the property of a T cell or NK cell to promote the killing of a target cell or inhibit its growth or proliferation. In the case of a T cell, primary stimulation and co-stimulation are examples of immune effector functions or responses.

[0298] The term "effector function" refers to the specialized function of a cell. For example, the effector function of a T cell can be cytolytic activity or helper activity, including the secretion of cytokines.

[0299] The term "encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA, or mRNA) to serve as a template in a biological process for synthesizing other polymers and macromolecules having a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the resulting biological properties. Thus, if transcription and translation of the mRNA corresponding to a gene produce a protein in a cell or other biological system, the gene, cDNA, or RNA encodes that protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing) and the non-coding strand (which serves as the template for gene or cDNA transcription) can be said to encode the protein or other product of the gene or cDNA.

[0300] Unless otherwise specified, the term "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate to one another and encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may also include introns to the extent that the nucleotide sequence encoding the protein may contain one or more introns in certain forms.

[0301] The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, preparation, material, or composition as described herein that is effective to achieve a particular biological result.

[0302] The term "endogenous" refers to any material that is derived from or produced within an organism, cell, tissue, or system.

[0303] The term "exogenous" refers to any material that is introduced from or produced outside of an organism, cell, tissue, or system.

[0304] The term "expression" refers to the transcription and / or translation of a specific nucleotide sequence driven by a promoter.

[0305] The term "transfer vector" refers to a physical composition that contains a nucleic acid that has been isolated and is capable of being used to deliver the isolated nucleic acid into a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "transfer vector" includes autonomously replicating plasmids or viruses. The term should also be construed to further include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral transfer vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.

[0306] The term "expression vector" refers to a vector that contains a recombinant polynucleotide that contains an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system. Expression vectors include all expression vectors known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0307] As used herein, the term "vector" refers to any agent that can be used to deliver and / or express a nucleic acid molecule. It can be a transfer vector or an expression vector as described herein.

[0308] The term "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they are able to infect non-dividing cells; they can deliver large amounts of genetic information into the DNA of the host cell, and thus they are one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses.

[0309] The term "lentiviral vector" refers to a vector derived from at least a portion of the lentiviral genome, and particularly includes the self-inactivating lentiviral vectors provided as follows: Milone et al., Mol. Ther. [Molecular Therapy] 17(8):1453-1464 (2009). Other examples of lentiviral vectors that can be used clinically include, but are not limited to, for example, those from Oxford BioMedica gene delivery technology, LENTIMAX from Lentigen Corporation TM vector systems, etc. Non-clinical types of lentiviral vectors are also available and are known to those skilled in the art.

[0310] The term "homologous" or "identity" refers to subunit sequence identity between two polymeric molecules, such as between two nucleic acid molecules (such as two DNA molecules or two RNA molecules) or between two polypeptide molecules. When the subunit positions in these two molecules are occupied by the same monomeric subunit; for example, if the position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half of the positions in two sequences (e.g., five positions in a polymer of ten subunits) are homologous, then the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) are matching or homologous, then the two sequences are 90% homologous.

[0311] A "humanized" form of a non-human (e.g., murine) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequence of an antibody) that contains minimal sequence derived from a non-human immunoglobulin. In most instances, humanized antibodies and antibody fragments are human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementary determining region (CDR) of the recipient have been replaced by residues from a CDR of a non-human species (donor antibody) having the desired specificity, affinity, and capacity (such as a mouse, rat, or rabbit). In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Additionally, humanized antibodies / antibody fragments may contain residues that are not found in either the recipient antibody or the imported CDR or framework sequences. These modifications may further improve and optimize the performance of the antibody or antibody fragment. Typically, a humanized antibody or its antibody fragment will contain substantially all of at least one (typically two) variable domains, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or a significant portion of the FR regions are those of a human immunoglobulin sequence. A humanized antibody or antibody fragment may also contain at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. For additional details, see Jones et al., Nature, 321:522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2:593-596, 1992.

[0312] "Fully human" means an immunoglobulin, such as an antibody or antibody fragment, in which the entire molecule is of human origin or consists of the same amino acid sequence as a human form of an antibody or immunoglobulin.

[0313] The term "isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide that occurs naturally in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials in its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form or can exist in a non-natural environment (e.g., such as a host cell).

[0314] In the context of the present invention, the following abbreviations for common nucleic acid bases are used. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0315] The terms "operably linked" or "transcriptionally controlled" refer to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be contiguous with each other and, for example, in cases where two protein coding regions need to be joined, they are in the same reading frame.

[0316] The term "parenterally" administering an immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, intratumoral, or infusion techniques.

[0317] The terms "nucleic acid", "polynucleotide", or "nucleic acid molecule" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or combinations thereof, in single-stranded or double-stranded form, and polymers thereof. The term "nucleic acid" includes genes, cDNA, or mRNA. In one embodiment, the nucleic acid molecule is synthetic (e.g., chemically synthesized) or recombinant. Unless specifically limited, the term encompasses nucleic acids containing analogs or derivatives of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses variants, alleles, orthologs, SNPs, and complementary sequences with conservative modifications (e.g., degenerate codon substitutions) of the specified sequence. Specifically, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0318] The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds containing amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can make up the protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to short chains, such as those commonly referred to in the art as peptides, oligopeptides and oligomers, and also refers to longer chains, which are commonly referred to in the art as proteins, and there are many types of proteins. "Polypeptide" includes, for example, bioactive fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides or combinations thereof.

[0319] The term "promoter" refers to a DNA sequence recognized by the cellular synthetic machinery or introduced synthetic machinery required to initiate the specific transcription of a polynucleotide sequence.

[0320] The term "promoter / regulatory sequence" refers to a nucleic acid sequence required for the expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, the sequence may be a core promoter sequence, and in other cases, the sequence may also include enhancer sequences and other regulatory elements required for the expression of the gene product. The promoter / regulatory sequence may be, for example, a promoter / regulatory sequence that expresses a gene product in a tissue-specific manner.

[0321] The term "constitutive" promoter refers to a nucleotide sequence that causes a gene product to be produced in a cell under most or all physiological conditions of the cell when operably linked to a polynucleotide encoding or specifying the gene product.

[0322] The term "inducible" promoter refers to a nucleotide sequence that causes a gene product to be produced in a cell essentially only when an inducer corresponding to the promoter is present in the cell when operably linked to a polynucleotide encoding or specifying the gene product.

[0323] The term "tissue-specific" promoter refers to a nucleotide sequence that causes a gene product to be produced in a cell essentially only when the cell is a cell of the tissue type corresponding to the promoter when operably linked to a polynucleotide encoded or specified by a gene.

[0324] The term "cancer-associated antigen" or "tumor antigen" refers interchangeably to a molecule (typically a protein, carbohydrate, or lipid) that is expressed on the surface of cancer cells either fully or as a fragment (e.g., MHC / peptide), and that can be used to preferentially target pharmacological agents to cancer cells. In some embodiments, the tumor antigen is a marker expressed by both normal and cancer cells, such as a lineage marker, e.g., CD19 or CD123 on B cells. In some embodiments, the tumor antigen is a cell surface molecule that is overexpressed in cancer cells compared to normal cells, e.g., 1-fold overexpression, 2-fold overexpression, 3-fold overexpression, or more compared to normal cells. In some embodiments, the tumor antigen is a cell surface molecule that is inappropriately synthesized in cancer cells, e.g., a molecule that contains deletions, additions, or mutations compared to the molecule expressed on normal cells. In some embodiments, the tumor antigen will be expressed only fully or as a fragment (e.g., MHC / peptide) on the cell surface of cancer cells and will not be synthesized or expressed on the surface of normal cells. In some embodiments, the CARs of the invention include CARs that comprise an antigen-binding domain (e.g., an antibody or antibody fragment) that binds to a peptide presented by MHC. Typically, peptides derived from endogenous proteins fill the pockets of major histocompatibility complex (MHC) class I molecules and are recognized by T cell receptors (TCRs) on CD8+ T lymphocytes. MHC class I complexes are constitutively expressed by all nucleated cells. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes represent a unique class of cell surface targets for immunotherapy. TCR-like antibodies that target peptides derived from viral or tumor antigens in the context of human leukocyte antigen (HLA)-A1 or HLA-A2 have been described (see, e.g., Sastry et al., J Virol. 2011 85(5):1935-1942; Sergeeva et al., Blood 2011 117(16):4262-4272; Verma et al., J Immunol 2010 184(4):2156-2165; Willemsen et al., Gene Ther 2001 8(21):1601-1608; Dao et al., Sci Transl Med 2013 5(176):176ra33; Tassev et al., Cancer Gene Ther 2012 19(2):84-100). For example, TCR-like antibodies can be identified from screening libraries (e.g., human scFv phage display libraries).

[0325] The term "flexible polypeptide linker" or "linker" as used in the context of an scFv refers to a peptide linker composed of amino acid (such as glycine and / or serine) residues used alone or in combination to link the variable heavy chain region and the variable light chain region together. In one embodiment, the flexible polypeptide linker is a Gly / Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Ser)n (SEQ ID NO:38), where n is a positive integer equal to or greater than 1. For example, n = 1, n = 2, n = 3, n = 4, n = 5 and n = 6, n = 7, n = 8, n = 9 and n = 10. In one embodiment, the flexible polypeptide linker includes but is not limited to (Gly4Ser)4 (SEQ ID NO:27) or (Gly4Ser)3 (SEQ ID NO:28). In another embodiment, the linker includes multiple repeats of (Gly2Ser), (GlySer) or (Gly3Ser) (SEQ ID NO:29). The linkers described in WO 2012 / 138475 (which is incorporated herein by reference) are also included within the scope of the present invention.

[0326] As used herein, a 5' cap (also referred to as an RNA cap, RNA 7-methylguanosine cap or RNA m 7 G cap) is a modified guanine nucleotide that has been added to the "front" or 5' end of eukaryotic messenger RNA shortly after the start of transcription. The 5' cap consists of a terminal group linked to the first transcribed nucleotide. Its presence is crucial for recognition by ribosomes and protection from ribonucleases. Cap addition is coupled to transcription and occurs co-transcriptionally such that each affects the other. Shortly after the start of transcription, the 5' end of the synthesized mRNA is bound by a cap synthesis complex associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions required for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction. The capping moiety can be modified to regulate mRNA function, such as its stability or translation efficiency.

[0327] As used herein, "in vitro transcribed RNA" refers to RNA that has been synthesized in vitro, preferably mRNA. Typically, in vitro transcribed RNA is produced from an in vitro transcription vector. An in vitro transcription vector contains a template for producing in vitro transcribed RNA.

[0328] As used herein, "poly(A)" is a stretch of adenosines linked to mRNA by polyadenylation. In a preferred embodiment of a construct for transient expression, the poly A is between 50 and 5000 (SEQ ID NO:30), preferably greater than 64, more preferably greater than 100, and most preferably greater than 300 or 400. The poly(A) sequence can be chemically or enzymatically modified to regulate mRNA function, such as localization, stability or translation efficiency.

[0329] As used herein, "polyadenylation" refers to the covalent attachment of a polyadenyl moiety or a modified variant thereof to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end. The 3' poly(A) tail is a long sequence of adenine nucleotides (usually several hundred) that is added to the precursor mRNA by the action of an enzyme (polyadenylate polymerase). In higher eukaryotes, the poly(A) tail is added to transcripts containing a specific sequence (the polyadenylation signal). The poly(A) tail and the proteins that bind to it help protect the mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, export of mRNA from the nucleus, and translation. Polyadenylation occurs immediately in the nucleus after DNA is transcribed into RNA, but can also occur later in the cytoplasm. After transcription termination, the mRNA chain is cleaved by the action of an endonuclease complex associated with RNA polymerase. The cleavage site is typically characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA is cleaved, adenosine residues are added to the free 3' end at the cleavage site.

[0330] As used herein, "transient" refers to the expression of a non-integrated transgene that lasts for hours, days, or weeks, where the period of expression is less than the period of gene expression if integrated into the genome or contained within a stable plasmid replicon in a host cell.

[0331] As used herein, the terms "treat", "treatment", and "treating" refer to reducing or ameliorating the progression, severity, and / or duration of a proliferative disorder, or to ameliorating one or more symptoms (preferably, one or more distinguishable symptoms) of a proliferative disorder, which is caused by the administration of one or more therapies (e.g., one or more therapeutic agents such as a CAR of the present invention). In specific embodiments, the terms "treat", "treatment", and "treating" refer to ameliorating at least one measurable physical parameter of a proliferative disorder, such as tumor growth, which may not necessarily be distinguishable by the patient. In other embodiments, the terms "treat", "treatment", and "treating" refer to inhibiting the progression of a proliferative disorder physically, e.g., by stabilizing distinguishable symptoms, or physiologically, e.g., by stabilizing physical parameters, or by both. In other embodiments, the terms "treat", "treatment", and "treating" refer to reducing or stabilizing tumor size or cancer cell count.

[0332] A dosing regimen (e.g., a therapeutic dosing regimen) can include one or more treatment intervals. A dosing regimen can produce at least one beneficial or desired clinical outcome, including but not limited to alleviating symptoms, reducing the severity of a disease, stabilizing (i.e., not worsening) a disease state, delaying or slowing disease progression, improving or remitting (whether detectable or undetectable) a disease state.

[0333] As used herein, a "treatment interval" refers to a treatment cycle that can be repeated, for example, on a regular schedule, such as a process of administering a therapeutic agent. In an embodiment, a dosing regimen can have one or more periods during which no therapeutic agent is administered between treatment intervals. For example, a treatment interval can include a dose of a CAR molecule administered in combination with (prior to, concurrently with, or subsequent to) the administration of a second therapeutic agent (e.g., an inhibitor (e.g., a kinase inhibitor as described herein)).

[0334] The term "signal transduction pathway" refers to the biochemical relationship among a variety of signal transduction molecules that play a role in transmitting a signal from one part of a cell to another part of the cell. The phrase "cell surface receptor" includes molecules and molecular complexes that are capable of receiving a signal and transmitting the signal across the cell membrane.

[0335] The term "subject" is intended to include a living organism (e.g., a mammal, a human) in which an immune response can be elicited.

[0336] The term "substantially purified" cells refers to cells that are substantially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types that are normally associated with them in their native state. In some cases, a substantially purified cell population refers to a homogeneous cell population. In other cases, the term only refers to cells that have been separated from cells that are naturally associated with them in their native state. In some aspects, the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.

[0337] As used herein, the term "therapeutic agent" means treatment. A therapeutic effect is obtained by reducing, inhibiting, alleviating, or eradicating a disease state.

[0338] In an embodiment, the disease state to be treated includes CRS. In some embodiments, the treatment of CRS includes administering the compositions or combinations described herein after the onset of one or more CRS symptoms (e.g., after detection). In some embodiments, for example, compared to a subject who has not been administered the compositions or combinations described herein, the treatment of CRS results in a reduction in the severity of CRS. For example, a subject can reduce CRS to an undetectable level. In other embodiments, the treatment produces a less severe form of CRS, such as grade 1, grade 2, or grade 3 CRS.

[0339] As used herein, the term "prevention" refers to the prophylactic or protective treatment of a disease or disease state. Prevention of a disease or disease state can include, for example, reducing (e.g., alleviating) one or more symptoms of the disease or disease state relative to a reference level (e.g., one or more symptoms of a similar subject not given treatment). Prevention can also include, for example, delaying the onset of one or more symptoms of the disease or disease state relative to a reference level (e.g., the onset of one or more symptoms of a similar subject not given treatment). In embodiments, the disease is a disease described herein.

[0340] In embodiments, the disease state for prevention includes CRS. In some embodiments, prevention of CRS includes administering a composition or combination described herein, for example, prior to the detection or onset of one or more CRS symptoms. In some embodiments, administration of a JAK-STAT inhibitor or a BTK inhibitor occurs prior to CAR therapy. In some embodiments, for example, prevention of CRS results in a reduced likelihood or severity of CRS relative to a subject not administered the composition or combination described herein. For example, a subject may not develop CRS. In other embodiments, for example, relative to a subject not administered the composition or combination described herein, the subject develops a less severe form of CRS, such as grade 1, grade 2, or grade 3 CRS.

[0341] In the context of the present invention, "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with a hyperproliferative disorder" refers to an antigen common to a specific hyperproliferative disorder. In certain aspects, the hyperproliferative disorder antigens of the present invention are derived from: cancers, including but not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinomas (such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, etc.).

[0342] The terms "transfected" or "transformed" or "transduced" refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acid. Cells include primary subject cells and their progeny.

[0343] The term "specifically binds" refers to an antibody or ligand that recognizes and binds a cognate binding partner (e.g., a stimulatory and / or co-stimulatory molecule present on a T cell) protein present in a sample, but wherein the antibody or ligand substantially does not recognize or bind other molecules in the sample.

[0344] As used herein, an “adjustable chimeric antigen receptor (RCAR)” refers to a set (typically two in the simplest embodiments) of polypeptides that, when in an immune effector cell, confer on the cell specificity for a target cell (typically a cancer cell) and adjustable intracellular signaling. In some embodiments, the RCAR comprises at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an “intracellular signaling domain” and comprising functional signaling domains derived from a stimulatory molecule and / or a co-stimulatory molecule as defined herein in the context of a CAR molecule). In some embodiments, the set of polypeptides in the RCAR are not contiguous with one another, e.g., in different polypeptide chains. In some embodiments, the RCAR includes a dimerization switch that can couple the polypeptides to one another in the presence of a dimerizing molecule, e.g., can couple the antigen-binding domain to the intracellular signaling domain. In some embodiments, the RCAR is expressed in a cell as described herein (e.g., an immune effector cell), e.g., a cell expressing the RCAR (also referred to herein as an “RCARX cell”). In one embodiment, the RCARX cell is a T cell and is referred to as an RCART cell. In one embodiment, the RCARX cell is an NK cell and is referred to as an RCARN cell. The RCAR can confer on the cell expressing it specificity for a target cell (typically a cancer cell) and have adjustable intracellular signaling or proliferation, which can optimize the immune effector properties of the cell expressing the RCAR. In embodiments, the RCAR cell is at least partially dependent on the antigen-binding domain to provide specificity for a target cell comprising the antigen bound by the antigen-binding domain.

[0345] A “membrane anchor” or “membrane tethering domain” (when the term is used herein) refers to a polypeptide or moiety sufficient to anchor an extracellular or intracellular domain to the plasma membrane, e.g., a myristoyl group.

[0346] The term "switch domain" (when used herein), e.g., when referring to RCAR, refers to an entity that associates with another switch domain in the presence of a dimerizing molecule, typically a polypeptide-based entity. This association results in the functional coupling of a first entity linked to (e.g., fused to) the first switch domain and a second entity linked to (e.g., fused to) the second switch domain. The first and second switch domains are collectively referred to as the dimerizing switch. In embodiments, the first and second switch domains are identical to each other, e.g., they are polypeptides having the same primary amino acid sequence, and are collectively referred to as a homodimerizing switch. In embodiments, the first and second switch domains are different from each other, e.g., they are polypeptides having different primary amino acid sequences, and are collectively referred to as a heterodimerizing switch. In embodiments, the switch is intracellular. In embodiments, the switch is extracellular. In embodiments, the switch domain is a polypeptide-based entity (e.g., based on FKBP or FRB), and the dimerizing molecule is a small molecule, e.g., a rapalogue. In embodiments, the switch domain is a polypeptide-based entity, e.g., an scFv that binds to a myc peptide, and the dimerizing molecule is a polypeptide, a fragment thereof, or a multimer of a polypeptide, e.g., a myc ligand or a multimer of a myc ligand that binds to one or more myc scFvs. In embodiments, the switch domain is a polypeptide-based entity, e.g., a myc receptor, and the dimerizing molecule is an antibody or a fragment thereof, e.g., a myc antibody.

[0347] The term "dimerizing molecule" (when used herein), e.g., when referring to RCAR, refers to a molecule that promotes the association of the first switch domain with the second switch domain. In embodiments, the dimerizing molecule does not naturally occur in a subject, or does not occur at a concentration that results in significant dimerization. In embodiments, the dimerizing molecule is a small molecule, e.g., rapamycin or a rapalogue, e.g., RAD001.

[0348] The term "bioequivalent" refers to the amount of a reagent other than a reference compound (e.g., RAD001) that produces an effect equivalent to that produced by a reference dose or reference amount of the reference compound (e.g., RAD001). In one embodiment, the effect is the level of mTOR inhibition, e.g., as measured by P70 S6 kinase inhibition, e.g., as evaluated in an in vivo or in vitro assay, e.g., as measured by an assay described herein (e.g., the Boulay assay or by measuring the level of phosphorylated S6 by Western blotting). In one embodiment, the effect is a change in the ratio of PD-1 positive / PD-1 negative immune effector cells (e.g., T cells or NK cells) as measured by cell sorting. In one embodiment, the bioequivalent amount or dose of an mTOR inhibitor is the amount or dose that achieves the same level of P70 S6 kinase inhibition as the reference dose or reference amount of the reference compound. In one embodiment, the bioequivalent amount or dose of an mTOR inhibitor is the amount or dose that achieves the same level of change in the ratio of PD-1 positive / PD-1 negative immune effector cells (e.g., T cells or NK cells) as the reference dose or reference amount of the reference compound.

[0349] When used in combination with an mTOR inhibitor (e.g., an allosteric mTOR inhibitor such as RAD001 or rapamycin, or a catalytic mTOR inhibitor), the term "low immune enhancing dose" refers to a dose of the mTOR inhibitor that (partially but not completely) inhibits mTOR activity, e.g., as measured by inhibition of P70 S6 kinase activity. Methods for assessing mTOR activity are discussed herein, e.g., by inhibiting P70 S6 kinase. The dose is not sufficient to cause complete immunosuppression but is sufficient to enhance the immune response. In one embodiment, a low immune enhancing dose of an mTOR inhibitor results in a decrease in the number of PD-1 positive immune effector cells (e.g., T cells or NK cells) and / or an increase in the number of PD-1 negative immune effector cells (e.g., T cells or NK cells), or an increase in the ratio of PD-1 negative T cells / PD-1 positive immune effector cells (e.g., T cells or NK cells).

[0350] In one embodiment, a low immune enhancing dose of an mTOR inhibitor results in an increase in the number of naïve immune effector cells (e.g., T cells or NK cells). In one embodiment, a low immune enhancing dose of an mTOR inhibitor results in one or more of the following:

[0351] an increase in the expression of one or more of the following markers: CD62L 高 , CD127 高 , CD27 + and BCL2, e.g., on memory T cells, e.g., memory T cell precursors;

[0352] Reduced expression of KLRG1 on, for example, memory T cells (such as memory T cell precursors); and

[0353] An increased number of memory T cell precursors, such as cells having any one or combination of the following characteristics: increased CD62L 高 , increased CD127 高 , increased CD27 + , reduced KLRG1, increased BCL2;

[0354] wherein, for example, any of the above changes occur, at least transiently, compared to an untreated subject.

[0355] As used herein, "refractory" refers to a disease that does not respond to treatment, such as cancer. In an embodiment, a refractory cancer can be resistant to treatment before or at the start of treatment. In other embodiments, a refractory cancer can develop resistance during treatment. A refractory cancer is also referred to as a resistant cancer.

[0356] As used herein, "relapsed or relapse" refers to the return or recurrence of a disease (such as cancer) or the signs and symptoms of a disease (such as after a period of improvement or response, such as cancer after a previous treatment with a therapy (such as a cancer therapy)). Responsiveness in the initial phase can involve a decrease in the level of cancer cells below a certain threshold, such as below 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. The recurrence may involve an increase in the level of cancer cells above a certain threshold, such as above 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. For example, in the context of B-ALL, the return may involve, for example, the recurrence of blasts in the blood, bone marrow (>5%), or any extramedullary site after a complete response. In this context, a complete response may involve <5% BM blasts. More generally, in one embodiment, a response (such as a complete response or a partial response) may involve the absence of detectable MRD (minimal residual disease). In one embodiment, the responsive initial phase lasts for at least 1, 2, 3, 4, 5, or 6 days; at least 1, 2, 3, or 4 weeks; at least 1, 2, 3, 4, 6, 8, 10, or 12 months; or at least 1, 2, 3, 4, or 5 years.

[0357] In some embodiments, therapies that include a CD19 inhibitor (e.g., a CD19 CAR therapy) can be recurrent or refractory. Recurrence or resistance can be caused by CD19 loss (e.g., an antigen-loss mutation) or other CD19 alterations that reduce CD19 levels (e.g., caused by clonal selection of CD19-negative clones). Cancers having such CD19 loss or alteration are referred to herein as "CD19-negative cancers" or "CD19-negative recurrent cancers". It should be understood that CD19-negative cancers do not need to have 100% loss of CD19, but rather it is sufficient to reduce the effectiveness of CD19 therapy such that the cancer recurs or becomes refractory. In some embodiments, CD19-negative cancers are produced by CD19 CAR therapy.

[0358] As used herein, "JAK-STAT" refers to the JAK-STAT signaling pathway and / or one or more kinases in the JAK-STAT pathway. The JAK-STAT signaling pathway and its components are described in more detail herein.

[0359] Ranges: Throughout this disclosure, various aspects of the invention can be presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as the individual numerical values within that range. For example, a description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as the individual numbers within that range, for example 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95%-99% identity includes having 95%, 96%, 97%, 98%, or 99% identity, and includes subranges such as 96%-99%, 96%-98%, 96%-97%, 97%-99%, 97%-98%, and 98%-99% identity. This applies regardless of the width of the range.

[0360] Description

[0361] Provided herein are methods for preventing CRS in a subject. The method can include administering in combination the CARs described herein with a kinase inhibitor (e.g., an inhibitor of JAK-STAT or BTK).

[0362] Also provided herein are pharmaceutical compositions and methods of using a chimeric antigen receptor (CAR) in combination with a kinase inhibitor (e.g., an inhibitor of JAK-STAT or BTK) for treating or preventing a disease such as cancer.

[0363] Example 3 of the present disclosure describes that in CRS related to CAR T cells, IL-6 is produced by antigen-presenting cells (myeloid cells), and the presence or absence of IL-6 (e.g., measured by degranulation in the presence or absence of APCs) does not affect CART function. Thus, in some embodiments, the CARs described herein are administered in combination with an IL-6 inhibitor (e.g., tocilizumab). In an embodiment, the methods described herein provide for early administration of an IL-6 inhibitor (e.g., tocilizumab) to prevent CRS associated with CAR therapy. In an embodiment, early administration includes administration before CAR therapy, simultaneous administration with the CAR therapy dose, or administration up to the first sign of fever (e.g., after the CAR therapy dose). In some embodiments, the combination of the CARs and IL-6 inhibitors described herein may further comprise a kinase inhibitor (e.g., the kinase inhibitors described herein).

[0364] Chimeric antigen receptors (CARs) comprising antibodies or antibody fragments engineered to specifically bind an antigen (e.g., CD123 protein or CD19 protein or fragments thereof) can be used according to any of the methods or compositions described herein. In one aspect, the present disclosure provides cells (e.g., immune effector cells such as T cells or NK cells) engineered to express a CAR, wherein the cells expressing the CAR (e.g., "CART" or NK cells expressing the CAR) exhibit anti-tumor properties. In one aspect, cells are transformed with a CAR and at least a portion of the CAR is expressed on the cell surface. In some embodiments, cells (e.g., immune effector cells such as T cells or NK cells) are transduced with a viral vector encoding the CAR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cells can stably express the CAR. In another embodiment, cells (e.g., immune effector cells such as T cells or NK cells) are transfected with a nucleic acid encoding the CAR (e.g., mRNA, cDNA, DNA). In some such embodiments, the cells can transiently express the CAR.

[0365] In one aspect, the antigen-binding domain of the CAR (e.g., the CD123-binding domain or CD19-binding domain), such as the human or humanized CD123-binding domain or CD19-binding domain of the CAR, is an scFv antibody fragment. In one aspect, such antibody fragments are functional because they retain the same binding affinity, e.g., they bind the same antigen with comparable efficacy as an IgG antibody having the same heavy and light chain variable regions. In one aspect, as will be understood by those skilled in the art, such antibody fragments are functional because they provide a biological response (which may include, but is not limited to, activation of an immune response, inhibition of signal transduction originating from its target antigen, inhibition of kinase activity, etc.).

[0366] In some aspects, the antibodies of the invention are incorporated into a chimeric antigen receptor (CAR). In one aspect, the CAR is a CD123 CAR and comprises the polypeptide sequences provided herein, such as SEQ ID NOs: 98 - 101 and 125 - 156.

[0367] In one aspect, the antigen - binding domain of the CAR of the invention (CD123 or CD19 - binding domain, e.g., a humanized or human CD123 or CD19 - binding domain) is partially encoded by transgenes whose sequences have been codon - optimized for expression in mammalian cells. In one aspect, the entire CAR construct of the invention is encoded by a transgene whose entire sequence has been codon - optimized for expression in mammalian cells. Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons encoding the same amino acid) in coding DNA varies among different species. Such codon degeneracy allows the same polypeptide to be encoded by multiple nucleotide sequences. A variety of codon - optimization methods are known in the art and include, for example, the methods disclosed in at least U.S. Patent Nos. 5,786,464 and 6,114,148.

[0368] In one aspect, the antigen - binding domain of the CAR comprises a human CD123 antibody or antibody fragment, or a human CD19 antibody or antibody fragment. In one aspect, the antigen - binding domain of the CAR comprises a humanized CD123 or CD19 antibody or antibody fragment. In one aspect, the antigen - binding domain of the CAR comprises a human CD123 or CD19 antibody fragment that comprises an scFv. In one aspect, the antigen - binding domain of the CAR is a human CD123 scFv or a human CD19 scFv. In one aspect, the antigen - binding domain of the CAR comprises a humanized CD123 or CD19 antibody fragment that comprises an scFv. In one aspect, the antigen - binding domain of the CAR is a humanized CD123 scFv or a CD19 scFv.

[0369] In one aspect, the CAR123 - binding domain comprises the scFv portions provided in SEQ ID NOs: 157 - 160 and 184 - 215. In one aspect, the scFv portion is human. In one aspect, the human CAR123 - binding domain comprises the scFv portion provided in SEQ ID NOs: 157 - 160. In one aspect, the human CD123 - binding domain comprises the scFv portion provided in SEQ ID NO: 478, 480, 483, or 485.

[0370] In one aspect, the scFv portion is humanized. In one aspect, the humanized CAR123 binding domain comprises the scFv portion provided in SEQ ID NO: 184 - 215. In one aspect, the humanized CD123 binding domain comprises the scFv portion provided in SEQ ID NO: 556 - 587.

[0371] Furthermore, the present invention provides CD123 CAR compositions and their use in medicaments or methods for treating (among other diseases) cancer or any malignancy or autoimmune diseases involving cells or tissues expressing CD123.

[0372] In one aspect, the CAR of the present invention can be used to eradicate normal cells expressing CD123, and thus is suitable for cell conditioning therapy before cell transplantation. In one aspect, the normal cells expressing CD123 are myeloid progenitor cells expressing CD123, and the cell transplantation is stem cell transplantation.

[0373] In one aspect, the present invention provides cells (e.g., immune effector cells, such as T cells or NK cells) engineered to express the chimeric antigen receptor of the present invention (e.g., immune effector cells expressing CAR, such as CART or CAR-expressing NK cells), wherein the cells (e.g., "CART") exhibit anti-tumor properties. Accordingly, the present invention provides CD123-CAR, which comprises a CD123 binding domain and is engineered into immune effector cells (e.g., T cells or NK cells), and methods of using them in adoptive therapy.

[0374] In one aspect, the CD123-CAR comprises at least one intracellular domain, e.g., as described herein, e.g., selected from the group consisting of the CD137 (4-1BB) signaling domain, the CD28 signaling domain, the CD3ζ signaling domain, and any combination thereof. In one aspect, the CD123-CAR comprises at least one intracellular signaling domain (from one or more co-stimulatory molecules other than CD137 (4-1BB) or CD28).

[0375] Chimeric Antigen Receptor (CAR)

[0376] According to any method or composition described herein, in embodiments, the CAR molecule comprises a CD123 CAR as described herein, such as the CD123 CAR described in US2014 / 0322212 A1 or US2016 / 0068601 A1 (both incorporated herein by reference). In embodiments, the CD123 CAR comprises amino acids or has the nucleotide sequence shown in US2014 / 0322212 A1 or US2016 / 0068601 A1 (both incorporated herein by reference). In other embodiments, the CAR molecule comprises a CD19 CAR molecule as described herein, such as the CD19 CAR molecule described in US-2015-0283178-A1, such as CTL019. In embodiments, the CD19 CAR comprises amino acids or has the nucleotide sequence shown in US-2015-0283178-A1 (incorporated herein by reference). In one embodiment, the CAR molecule comprises a BCMACAR molecule as described herein, such as the BCMACAR described in US-2016-0046724-A1. In embodiments, the BCMACAR comprises amino acids or has the nucleotide sequence shown in US-2016-0046724-A1 (incorporated herein by reference). In one embodiment, the CAR molecule comprises a CLL1 CAR as described herein, such as the CLL1 CAR described in US2016 / 0051651 A1 (incorporated herein by reference). In embodiments, the CLL1 CAR comprises amino acids or has the nucleotide sequence shown in US 2016 / 0051651A1 (incorporated herein by reference). In one embodiment, the CAR molecule comprises a CD33 CAR as described herein, such as the CD33 CAR described in US2016 / 0096892A1 (incorporated herein by reference). In embodiments, the CD33 CAR comprises amino acids or has the nucleotide sequence shown in US2016 / 0096892 A1 (incorporated herein by reference). In one embodiment, the CAR molecule comprises an EGFRvIII CAR molecule as described herein, such as the EGFRvIIICAR described in US2014 / 0322275 A1 (incorporated herein by reference). In embodiments, the EGFRvIIICAR comprises amino acids or has the nucleotide sequence shown in US 2014 / 0322275A1 (incorporated herein by reference). In embodiments, the CAR molecule comprises a mesothelin CAR as described herein, such as the mesothelin CAR described in WO2015 / 090230 (incorporated herein by reference). In embodiments, the mesothelin CAR comprises amino acids or has the nucleotide sequence shown in WO 2015 / 090230 (incorporated herein by reference).

[0377] CAR123

[0378] The present invention encompasses recombinant DNA constructs comprising a sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain (e.g., an antibody, an antibody fragment) that specifically binds to CD123 or a fragment thereof, such as human CD123, wherein the sequence of the CD123-binding domain (e.g., an antibody or an antibody fragment) is adjacent to and in the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain. The intracellular signaling domain can comprise a co-stimulatory signaling domain and / or a primary signaling domain, such as the ζ chain. The co-stimulatory signaling domain refers to the part of the CAR that comprises at least a portion of the intracellular domain of a co-stimulatory molecule.

[0379] In a particular aspect, the CAR constructs of the present invention comprise an scFv domain selected from the group consisting of SEQ ID NOs: 157-160, 184-215, 478, 480, 483, 485, and 556-587, wherein the scFv can optionally be preceded by a leader sequence (such as that provided in SEQ ID NO: 1), and followed by an optional hinge sequence (such as those provided in SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5), a transmembrane region (such as that shown in SEQ ID NO: 6), an intracellular signaling domain comprising SEQ ID NO: 7 or SEQ ID NO: 8, and a CD3ζ sequence comprising SEQ ID NO: 9 or SEQ ID NO: 10, e.g., wherein these domains are adjacent and in the same reading frame to form a single fusion protein. In some embodiments, the scFv domain is a human scFv domain selected from the group consisting of SEQ ID NOs: 157-160, 478, 480, 483, and 485. In some embodiments, the scFv domain is a humanized scFv domain selected from the group consisting of SEQ ID NOs: 184-215 and 556-587. The present invention also includes nucleotide sequences encoding each of the polypeptides of the scFv fragments selected from the group consisting of SEQ ID NOs: 157-160, 184-215, 478, 480, 483, 485, and 556-587. The present invention also includes nucleotide sequences encoding each of the polypeptides of the scFv fragments selected from the group consisting of SEQ ID NOs: 157-160, 184-215, 478, 480, 483, 485, and 556-587, and each of the domains of SEQ ID NOs: 1, 2, and 6-9, plus the encoded CD123 CAR of the present invention.

[0380] In one aspect, an exemplary CD123 CAR construct comprises an optional leader sequence, an extracellular antigen-binding domain, a hinge, a transmembrane domain, and an intracellular stimulatory domain. In one aspect, an exemplary CD123 CAR construct comprises an optional leader sequence, an extracellular antigen-binding domain, a hinge, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular stimulatory domain.

[0381] In some embodiments, the full-length CD123 CAR sequences are also provided herein as SEQ ID NOs: 98 - 101 and 125 - 156, as shown in Tables 11A or 12A.

[0382] Exemplary leader sequences are provided as SEQ ID NO: 1. Exemplary hinge / spacer sequences are provided as SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5. Exemplary transmembrane domain sequences are provided as SEQ ID NO: 6. Exemplary sequences of the intracellular signaling domain of the 4 - 1BB protein are provided as SEQ ID NO: 7. Exemplary sequences of the intracellular signaling domain of CD27 are provided as SEQ ID NO: 8. Exemplary CD3ζ domain sequences are provided as SEQ ID NO: 9 or SEQ ID NO: 10. Exemplary sequences of the intracellular signaling domain of CD28 are provided as SEQ ID NO: 43. Exemplary sequences of the intracellular signaling domain of ICOS are provided as SEQ ID NO: 45.

[0383] In one aspect, the present invention encompasses a recombinant nucleic acid construct comprising a nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding a CD123-binding domain, such as described herein, for example, which is adjacent and in the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain. In one aspect, the CD123-binding domain is selected from one or more of SEQ ID NOs: 157 - 160, 184 - 215, 478, 480, 483, 485, and 556 - 587. In some embodiments, the CD123-binding domain is a human CD123-binding domain selected from the group consisting of SEQ ID NOs: 157 - 160, 478, 480, 483, and 485. In some embodiments, the CD123-binding domain is a humanized CD123-binding domain selected from the group consisting of SEQ ID NOs: 184 - 215 and 556 - 587.

[0384] In one aspect, the present invention encompasses recombinant nucleic acid constructs comprising a nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding a CD123 binding domain, such as wherein the sequence is contiguous with and in the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain. Exemplary intracellular signaling domains that can be used in the CAR include, but are not limited to, one or more intracellular signaling domains such as CD3-ζ, CD28, 4-1BB, ICOS, etc. In some cases, the CAR can comprise any combination of CD3-ζ, CD28, 4-1BB, ICOS, etc.

[0385] In one aspect, the nucleic acid sequence of the CAR construct of the present invention is selected from one or more of SEQ ID NO: 39-42 and 66-97. The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, such as by screening a library from cells expressing the gene using standard techniques, by obtaining the gene from a vector known to include the gene, or by directly isolating it from cells and tissues containing the gene. Alternatively, the nucleic acid of interest can be produced synthetically rather than cloned.

[0386] CAR19 (or CD19 CAR)

[0387] This disclosure encompasses immune effector cells (e.g., T cells or NK cells) that comprise a CAR molecule that targets (e.g., specifically binds) CD19 (CD19 CAR). In one embodiment, the immune effector cells are engineered to express CD19 CAR. In one embodiment, the immune effector cells comprise a recombinant nucleic acid construct that comprises a nucleic acid sequence encoding CD19 CAR.

[0388] In embodiments, the CD19 CAR comprises an antigen-binding domain that specifically binds CD19 (e.g., a CD19 binding domain), a transmembrane domain, and an intracellular signaling domain. In one embodiment, the sequence of the antigen-binding domain is contiguous with and in the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain. The intracellular signaling domain can comprise a co-stimulatory signaling domain and / or a primary signaling domain, such as the ζ chain. A co-stimulatory signaling domain refers to the portion of the CAR that comprises at least a part of the intracellular domain of a co-stimulatory molecule.

[0389] In one aspect, an exemplary CAR construct comprises an optional leader sequence (e.g., a leader sequence as described herein), an extracellular antigen-binding domain (e.g., an antigen-binding domain as described herein), a hinge (e.g., a hinge region as described herein), a transmembrane domain (e.g., a transmembrane domain as described herein), and an intracellular stimulatory domain (e.g., an intracellular stimulatory domain as described herein). In one aspect, an exemplary CAR construct comprises an optional leader sequence (e.g., a leader sequence as described herein), an extracellular antigen-binding domain (e.g., an antigen-binding domain as described herein), a hinge (e.g., a hinge region as described herein), a transmembrane domain (e.g., a transmembrane domain as described herein), an intracellular co-stimulatory signaling domain (e.g., a co-stimulatory signaling domain as described herein), and / or an intracellular primary signaling domain (e.g., a primary signaling domain as described herein).

[0390] In one aspect, the CD19 CAR of the invention comprises at least one signaling domain selected from the group consisting of a CD137 (4-1BB) signaling domain, a CD28 signaling domain, a CD27 signaling domain, an ICOS signaling domain, a CD3ζ signaling domain, and any combination thereof. In one aspect, the CAR of the invention comprises at least one intracellular signaling domain (from one or more co-stimulatory molecules selected from CD137 (4-1BB), CD28, CD27 or ICOS).

[0391] Vectors and RNA constructs

[0392] The invention includes retroviral and lentiviral vector constructs expressing a CAR that can be directly transfected into cells.

[0393] The invention also includes RNA constructs that can be directly transfected into cells. The method for generating mRNA for transfection involves in vitro transcription (IVT) of a template with specially designed primers, followed by addition of polyA to generate a construct containing 3' and 5' untranslated sequences ("UTRs"), a 5' cap and / or an internal ribosome entry site (IRES), the nucleic acid to be expressed, and a polyA tail, typically 50 - 2000 bases in length (SEQ ID NO:35). The RNA so produced can efficiently transfect different types of cells. In one embodiment, the template comprises the sequence of the CAR. In one embodiment, the RNA CAR vector is transfected into T cells by electroporation.

[0394] Antigen-binding domain

[0395] In one aspect, the CAR of the present invention comprises a target-specific binding element, also referred to as an antigen-binding domain. The selection of the moiety depends on the type and number of ligands that define the surface of the target cell. For example, an antigen-binding domain can be selected to recognize a ligand that is a cell surface marker on a target cell associated with a particular disease state. Thus, examples of cell surface markers that can serve as ligands for the antigen-binding domain in the CAR of the present invention include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells.

[0396] In one aspect, by engineering the antigen-binding domain of the CAR to specifically bind the desired antigen, the CAR-mediated T cell response can be directed to the antigen of interest.

[0397] In one aspect, the CAR moiety comprising the antigen-binding domain comprises an antigen-binding domain that targets a tumor antigen (such as the tumor antigens described herein).

[0398] In one aspect, the CAR moiety comprising the antigen-binding domain comprises an antigen-binding domain that targets CD123 or a fragment thereof. In embodiments, the antigen-binding domain targets human CD123 or a fragment thereof. In other embodiments, the antigen-binding domain targets B cell antigens (such as, B cell surface antigens), such as CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a.

[0399] The antigen-binding domain can be any domain that binds an antigen, including but not limited to monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and functional fragments thereof, including but not limited to single-domain antibodies (such as the heavy chain variable domain (VH), light chain variable domain (VL), and variable domain (VHH) of camelid-derived nanobodies), and alternative scaffolds known in the art that function as antigen-binding domains (such as recombinant fibronectin domains, etc.). In some cases, it is beneficial for the antigen-binding domain to be derived from the same species in which the CAR will ultimately be used. For example, for use in humans, it can be beneficial for the antigen-binding domain of the CAR to comprise human or humanized residues of the antigen-binding domain of an antibody or antibody fragment.

[0400] In one embodiment, the antigen-binding domain comprises one, two, three (e.g., all three) heavy-chain CDRs (HC CDR1, HC CDR2, and HC CDR3 (e.g., the antibodies described in WO 2015 / 142675, US-2015-0283178-A1, US-2016-0046724-A1, US2014 / 0322212 A1, US2016 / 0068601 A1, US 2016 / 0051651A1, US2016 / 0096892 A1, US2014 / 0322275 A1, or WO 2015 / 090230, which are incorporated herein by reference)), and / or one, two, three (e.g., all three) light-chain CDRs (LC CDR1, LC CDR2, and LC CDR3 (e.g., the antibodies described in WO 2015 / 142675, US-2015-0283178-A1, US-2016-0046724-A1, US2014 / 0322212 A1, US2016 / 0068601 A1, US 2016 / 0051651A1, US2016 / 0096892 A1, US2014 / 0322275 A1, or WO 2015 / 090230, which are incorporated herein by reference)) from the antibodies described herein. In one embodiment, the antigen-binding domain comprises the heavy-chain variable region and / or the variable light-chain region of the antibodies listed above.

[0401] In an embodiment, the antigen-binding domain is the antigen-binding domain described in WO 2015 / 142675, US-2015-0283178-A1, US-2016-0046724-A1, US2014 / 0322212 A1, US 2016 / 0068601A1, US2016 / 0051651 A1, US2016 / 0096892 A1, US 2014 / 0322275A1, or WO 2015 / 090230, which are incorporated herein by reference.

[0402] In an embodiment, the antigen-binding domain targets BCMA and is described in US-2016-0046724-A1.

[0403] In an embodiment, the antigen-binding domain targets CD19 and is described in US-2015-0283178-A1.

[0404] In an embodiment, the antigen-binding domain targets CD123 and is described in US 2014 / 0322212A1, US2016 / 0068601 A1.

[0405] In an embodiment, the antigen-binding domain targets CLL and is described in US2016 / 0051651A1.

[0406] In an embodiment, the antigen-binding domain targets CD33 and is described in US2016 / 0096892A1.

[0407] Exemplary target antigens that can be targeted by CAR-expressing cells include, but are not limited to, CD19, CD123, EGFRvIII, CD33, mesothelin, BCMA, and GFR ALPHA-4, etc., as described in, for example, WO 2014 / 153270, WO 2014 / 130635, WO 2016 / 028896, WO 2014 / 130657, WO 2016 / 014576, WO 2015 / 090230, WO 2016 / 014565, WO 2016 / 014535, and WO 2016 / 025880 (each of which is incorporated herein by reference in its entirety).

[0408] In other embodiments, the CAR-expressing cells can specifically bind to humanized CD19. For example, it can include a CAR molecule, or an antigen-binding domain according to Table 3 of WO 2014 / 153270 (incorporated herein by reference) (e.g., a humanized antigen-binding domain). The amino acid and nucleotide sequences encoding the CD19 CAR molecule and the antigen-binding domain (e.g., including one, two, three VH CDRs according to Kabat or Chothia; one, two, three VL CDRs) are detailed in WO 2014 / 153270.

[0409] In other embodiments, the CAR-expressing cells can specifically bind to CD123. For example, it can include a CAR molecule (e.g., any one of CAR1 to CAR8), or an antigen-binding domain according to Tables 1-2 of WO2014 / 130635 (incorporated herein by reference). The amino acid and nucleotide sequences encoding the CD123 CAR molecule and the antigen-binding domain (e.g., including one, two, three VH CDRs according to Kabat or Chothia; one, two, three VL CDRs) are detailed in WO 2014 / 130635.

[0410] In other embodiments, the CAR-expressing cells can specifically bind to CD123. For example, they can include a CAR molecule (e.g., any one of CAR123-1 to CAR123-4 and hzCAR123-1 to hzCAR123-32), or an antigen-binding domain according to Tables 2, 6, and 9 of WO 2016 / 028896 (incorporated herein by reference). The amino acid and nucleotide sequences encoding the CD123 CAR molecule and the antigen-binding domain (e.g., including one, two, three VH CDRs; one, two, three VL CDRs according to Kabat or Chothia) are detailed in WO 2016 / 028896.

[0411] In other embodiments, the CAR-expressing cells can specifically bind to EGFRvIII. For example, they can include a CAR molecule, or an antigen-binding domain according to Table 2 of WO 2014 / 130657 (incorporated herein by reference) or SEQ ID NO:11. The amino acid and nucleotide sequences encoding the EGFRvIII CAR molecule and the antigen-binding domain (e.g., including one, two, three VH CDRs; one, two, three VL CDRs according to Kabat or Chothia) are detailed in WO 2014 / 130657.

[0412] In other embodiments, the CAR-expressing cells can specifically bind to CD33. For example, they can include a CAR molecule (e.g., any one of CAR33-1 to CAR-33-9), or an antigen-binding domain according to Table 2 or 9 of WO 2016 / 014576 (incorporated herein by reference). The amino acid and nucleotide sequences encoding the CD33 CAR molecule and the antigen-binding domain (e.g., including one, two, three VH CDRs; one, two, three VL CDRs according to Kabat or Chothia) are detailed in WO 2016 / 014576.

[0413] In other embodiments, the CAR-expressing cells can specifically bind to mesothelin. For example, they can include a CAR molecule, or an antigen-binding domain according to Tables 2-3 of WO 2015 / 090230 (incorporated herein by reference). The amino acid and nucleotide sequences encoding the mesothelin CAR molecule and the antigen-binding domain (e.g., including one, two, three VH CDRs; one, two, three VL CDRs according to Kabat or Chothia) are detailed in WO 2015 / 090230.

[0414] In other embodiments, the cells expressing the CAR can specifically bind to BCMA, and for example, can include a CAR molecule, or an antigen-binding domain according to Table 1 or 16 of WO 2016 / 014565 (incorporated herein by reference), SEQ ID NO: 271 or SEQ ID NO: 273. The amino acid and nucleotide sequences encoding the BCMA CAR molecule and antigen-binding domains (for example, including one, two, three VH CDRs; one, two, three VL CDRs according to Kabat or Chothia) are detailed in WO 2016 / 014565.

[0415] In other embodiments, the cells expressing the CAR can specifically bind to CLL-1, and for example, can include a CAR molecule, or an antigen-binding domain according to Table 2 of WO 2016 / 014535 (incorporated herein by reference). The amino acid and nucleotide sequences encoding the CLL-1 CAR molecule and antigen-binding domains (for example, including one, two, three VH CDRs; one, two, three VL CDRs according to Kabat or Chothia) are detailed in WO 2016 / 014535.

[0416] In other embodiments, the cells expressing the CAR can specifically bind to GFR ALPHA-4, and for example, can include a CAR molecule, or an antigen-binding domain according to Table 2 of WO 2016 / 025880 (incorporated herein by reference). The amino acid and nucleotide sequences encoding the GFR ALPHA-4 CAR molecule and antigen-binding domains (for example, including one, two, three VH CDRs; one, two, three VL CDRs according to Kabat or Chothia) are detailed in WO 2016 / 025880.

[0417] In one embodiment, the antigen-binding domain of any of the CAR molecules described herein (for example, any of CD19, CD123, EGFRvIII, CD33, mesothelin, BCMA, and GFR ALPHA-4) comprises one, two, three (for example, all three) heavy chain CDRs (HC CDR1, HC CDR2, and HC CDR3) from the antibodies listed above and / or one, two, three (for example, all three) light chain CDRs (LC CDR1, LC CDR2, and LC CDR3) from the antigen-binding domains listed above. In one embodiment, the antigen-binding domain comprises the heavy chain variable region and / or the variable light chain region of the antibodies listed or described above.

[0418] In another aspect, the antigen-binding domain comprises a humanized antibody or antibody fragment. In some aspects, a non-human antibody is humanized, wherein specific sequences or regions of the antibody are modified to increase similarity to antibodies or fragments thereof that are naturally produced in humans. In one aspect, the antigen-binding domain is humanized.

[0419] In some cases, it is beneficial for the antigen-binding domain of the CAR to be derived from the same species in which the CAR will ultimately be used. For example, for use in humans, it may be beneficial for the antigen-binding domain of the CAR to comprise human or humanized residues of the antigen-binding domain of an antibody or antibody fragment. Thus, in one aspect, the antigen-binding domain comprises a human antibody or antibody fragment.

[0420] CD123 binding domain

[0421] In one embodiment, the human CD123 binding domain comprises one or more (e.g., all three) of the light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of the human CD123 binding domain described herein, and / or one or more (e.g., all three) of the heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of the human CD123 binding domain described herein, e.g., a human CD123 binding domain comprising one or more (e.g., all three) LC CDRs and one or more (e.g., all three) HC CDRs. In one embodiment, the human CD123 binding domain comprises one or more (e.g., all three) of the heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of the human CD123 binding domain described herein, e.g., the human CD123 binding domain has two variable heavy chain regions, each variable heavy chain region comprising HC CDR1, HC CDR2, and HC CDR3 described herein. In one embodiment, the human CD123 binding domain comprises the human light chain variable region (e.g., in Table 11A or 12B) and / or the human heavy chain variable region (e.g., in 11A or 12B) described herein. In one embodiment, the human CD123 binding domain comprises the human heavy chain variable region (e.g., in Table 11A or 12B 9), e.g., at least two human heavy chain variable regions (e.g., in Table 11A or 12B) described herein. In one embodiment, the CD123 binding domain is a scFv that comprises the light chain and heavy chain of the amino acid sequence of Table 11A or 12B. In one embodiment, the CD123 binding domain (e.g., scFv) comprises: a light chain variable region that comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence of the light chain variable region provided in Table 11A or 12B but not more than 30, 20, or 10 modifications (e.g., substitutions), or a sequence having at least 95% identity, e.g., 95%-99% identity, to the amino acid sequence of Table 11A; and / or a heavy chain variable region that comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence of the heavy chain variable region provided in Table 11A or 12B but not more than 30, 20, or 10 modifications (e.g., substitutions), or a sequence having at least 95% identity, e.g., 95%-99% identity, to the amino acid sequence of Table 11A or 12B.In one embodiment, the human CD123 binding domain comprises a sequence selected from the group consisting of SEQ ID NOs: 157-160, 478, 480, 483, and 485, or a sequence having at least 95% identity thereto, such as 95%-99% identity. In one embodiment, the human CD123 binding domain is a scFv and comprises a light chain variable region (e.g., in Table 11A or 12B) of the amino acid sequence described herein attached via a linker (e.g., a linker described herein) to a heavy chain variable region (e.g., in Table 11A) of the amino acid sequence described herein. In one embodiment, the human CD123 binding domain includes a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO: 26). The light chain variable region and the heavy chain variable region of the scFv can be in any orientation, for example: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0422] In some aspects, the non-human antibodies are humanized, where specific sequences or regions of the antibody are modified to increase similarity to antibodies or fragments thereof that occur naturally in humans. Thus, in one aspect, the antigen-binding domain comprises a humanized antibody or antibody fragment. In one embodiment, the humanized CD123-binding domain comprises one or more (e.g., all three) light chain complementarity determining regions 1 (LC CDR1), light chain complementarity determining regions 2 (LC CDR2), and light chain complementarity determining regions 3 (LC CDR3) of the humanized CD123-binding domain described herein, and / or one or more (e.g., all three) heavy chain complementarity determining regions 1 (HC CDR1), heavy chain complementarity determining regions 2 (HC CDR2), and heavy chain complementarity determining regions 3 (HC CDR3) of the humanized CD123-binding domain described herein, e.g., a humanized CD123-binding domain comprising one or more (e.g., all three) LC CDRs and one or more (e.g., all three) HC CDRs. In one embodiment, the humanized CD123-binding domain comprises one or more (e.g., all three) heavy chain complementarity determining regions 1 (HC CDR1), heavy chain complementarity determining regions 2 (HC CDR2), and heavy chain complementarity determining regions 3 (HC CDR3) of the humanized CD123-binding domain described herein, e.g., the humanized CD123-binding domain has two variable heavy chain regions, each variable heavy chain region comprising HC CDR1, HCCDR2, and HC CDR3 described herein. In one embodiment, the humanized CD123-binding domain comprises the humanized light chain variable region (e.g., in Table 12A) and / or the humanized heavy chain variable region (e.g., in Table 12B) described herein. In one embodiment, the humanized CD123-binding domain comprises the humanized heavy chain variable region (e.g., in Table 12A) described herein, e.g., at least two humanized heavy chain variable regions (e.g., in Table 12A) described herein. In one embodiment, the CD123-binding domain is an scFv, and the scFv comprises the light chain and heavy chain of the amino acid sequence of Table 12A. In one embodiment, the CD123-binding domain (e.g., scFv) comprises: a light chain variable region that comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence of the light chain variable region provided in Table 4 but no more than 30, 20, or 10 modifications (e.g., substitutions), or a sequence having at least 95% identity, e.g., 95%-99% identity, to the amino acid sequence of Table 12A; and / or a heavy chain variable region that comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence of the heavy chain variable region provided in Table 12A but no more than 30, 20, or 10 modifications (e.g., substitutions), or a sequence having at least 95% identity, e.g., 95%-99% identity, to the amino acid sequence of Table 12A.In one embodiment, the humanized CD123 binding domain comprises a sequence selected from the group consisting of SEQ ID NOs: 184 - 215 and 302 - 333, or a sequence having at least 95% identity thereto, such as 95% - 99% identity. In one embodiment, the humanized CD123 binding domain is a scFv and comprises a light chain variable region (e.g., in Table 12A) of the amino acid sequence described herein attached via a linker (e.g., a linker described herein) to a heavy chain variable region (e.g., in Table 12A) of the amino acid sequence described herein. In one embodiment, the humanized CD123 binding domain comprises a (Gly4 - Ser)n linker, where n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO: 26). The light chain variable region and the heavy chain variable region of the scFv can be in any of the following orientations, for example: light chain variable region - linker - heavy chain variable region or heavy chain variable region - linker - light chain variable region.

[0423] Humanized antibody

[0424] A variety of techniques known in the art can be used to generate humanized antibodies, including but not limited to CDR-grafting (see, for example, European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated herein by reference in its entirety), veneering or resurfacing (see, for example, European Patents EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1994, PNAS, 91:969-973, each of which is incorporated herein by reference in its entirety), chain shuffling (see, for example, U.S. Patent No. 5,565,332, which is incorporated herein by reference in its entirety), and methods disclosed in, for example, U.S. Patent Application Publication No. US2005 / 0042664, U.S. Patent Application Publication No. US2005 / 0048617, U.S. Patent No. 6,407,213, U.S. Patent No. 5,766,886, International Publication No. WO 9317105, Tan et al., J. Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16):10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55(23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994), each of which is incorporated herein by reference in its entirety. Generally, framework residues in the framework regions will be replaced with the corresponding residues from the CDR donor antibody to alter (e.g., improve) antigen binding.These framework replacements are identified by methods well known in the art, such as by modeling the interactions of CDR and framework residues to identify framework residues important for antigen binding, and by sequence comparison to identify uncommon framework residues at specific positions. (See, e.g., Queen et al., U.S. Patent No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, which are incorporated herein by reference in their entirety.)

[0425] A humanized antibody or antibody fragment has one or more amino acid residues from a non-human source retained therein. These non-human amino acid residues are generally referred to as "import" residues, and they are typically taken from an "import" variable domain. As provided herein, a humanized antibody or antibody fragment comprises one or more CDRs from a non-human immunoglobulin molecule and a framework region, wherein the amino acid residues comprising the framework are derived entirely or predominantly from human germline. A variety of techniques for humanizing antibodies or antibody fragments are well known in the art and can generally be carried out according to the methods of Winter and colleagues (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by replacing the corresponding sequences of a human antibody with rodent CDR or CDR sequences, i.e., CDR grafting (EP 239,400; PCT Publication No. WO 91 / 09967; and U.S. Patent Nos. 4,816,567, 6,331,415, 5,225,539, 5,530,101, 5,585,089, 6,548,640, the contents of which are incorporated herein by reference in their entirety). In such humanized antibodies and antibody fragments, substantially less than the complete human variable domain has been replaced by the corresponding sequences from a non-human species. A humanized antibody is generally a human antibody in which some CDR residues and possibly some framework (FR) residues are replaced by residues from similar sites in a rodent antibody. Humanization of antibodies and antibody fragments can also be achieved by veneering or surface resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., Protein Engineering, 7(6):805-814 (1994); and Roguska et al., PNAS, 91:969-973 (1994)) or chain shuffling (U.S. Patent No. 5,565,332), the contents of which are incorporated herein by reference in their entirety).

[0426] The human light and heavy chain variable domains selected for the preparation of the humanized antibody are selected to reduce antigenicity. According to the so-called "best-fit" method, the variable domain sequences of rodent antibodies are screened against the entire library of known human variable domain sequences. The human sequence that is closest to the rodent sequence is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol. 151:2296 (1993); Chothia et al., J. Mol. Biol. 196:901 (1987), the contents of which are incorporated herein by reference in their entirety). Another method uses a specific framework derived from the consensus sequence of all human antibodies having a particular light or heavy chain subgroup. The same framework can be used for several different humanized antibodies (see, for example, Nicholson et al. Mol. Immunol. 34(16-17):1157-1165 (1997); Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), the contents of which are incorporated herein by reference in their entirety). In some embodiments, the framework regions of the heavy chain variable region (e.g., all four framework regions) are derived from the VH4_4-59 germline sequence. In one embodiment, the framework region can contain, for example, one, two, three, four, or five modifications (e.g., substitutions) of amino acids from the corresponding murine sequence. In one embodiment, the framework region (e.g., all four framework regions of the light chain variable region) is derived from the VK3_1.25 germline sequence. In one embodiment, the framework region can contain, for example, one, two, three, four, or five modifications (e.g., substitutions) of amino acids from the corresponding murine sequence.

[0427] In some aspects, portions of the CAR compositions of the invention that include antibody fragments are humanized and retain high affinity for the target antigen and other favorable biological properties. According to one aspect of the invention, humanized antibodies and antibody fragments are prepared by a method of analyzing the parental sequence and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs that illustrate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences are available. Examination of these displays permits analysis of the likely role of residues in the function of the candidate immunoglobulin sequence, such as residues that affect the ability of the candidate immunoglobulin to bind the target antigen. In such a manner, FR residues can be selected and combined from the recipient as well as the input sequences such that desired antibody or antibody fragment characteristics, such as increased affinity for the target antigen, are achieved. Generally, CDR residues are directly and most importantly involved in affecting antigen binding.

[0428] A humanized antibody or antibody fragment can retain antigen specificity similar to the original antibody, e.g., in the present invention, the ability to bind an antigen described herein (e.g., a tumor antigen, e.g., a B cell antigen, e.g., human CD123, CD19) or a fragment thereof. In some embodiments, the humanized antibody or antibody fragment can have improved affinity and / or specificity for binding an antigen (e.g., a tumor antigen, e.g., a B cell antigen, e.g., human CD123, CD19) or a fragment thereof.

[0429] In one aspect, the antigen-binding domain portion comprises one or more sequences selected from SEQ ID NOs: 157-160, 184-215, 478, 480, 483, 485, and 556-587. In one aspect, the CD123 CAR comprising a human CD123-binding domain is selected from one or more sequences selected from SEQ ID NOs: 157-160, 478, 480, 483, and 485. In one aspect, the CD123 CAR comprising a humanized CD123-binding domain is selected from one or more sequences selected from SEQ ID NOs: 184-215 and 556-587.

[0430] In one aspect, an antigen-binding domain (e.g., a tumor antigen-binding domain, e.g., a B cell antigen-binding domain, e.g., a CD123-binding domain or a CD19-binding domain) is characterized by specific functional features or properties of an antibody or antibody fragment. For example, in one aspect, the portion of the CAR composition of the invention comprising the antigen-binding domain specifically binds an antigen (e.g., a tumor antigen, e.g., a B cell antigen, e.g., human CD123, CD19) or a fragment thereof. In one aspect, the invention relates to an antigen-binding domain comprising an antibody or antibody fragment, wherein the antibody-binding domain specifically binds to the CD123 protein or a fragment thereof, and wherein the antibody or antibody fragment comprises a variable light chain and / or a variable heavy chain, and the variable light chain and / or variable heavy chain comprises the amino acid sequences of SEQ ID NOs: 157-160, 184-215, 478, 480, 483, 485 and 556-587. In one aspect, the antigen-binding domain comprises the amino acid sequence of an scFv selected from SEQ ID NOs: 157-160, 184-215, 478, 480, 483, 485 and 556-587. In certain aspects, the scFv is contiguous with a leader sequence and in the same reading frame. In one aspect, the leader sequence is the polypeptide sequence provided as SEQ ID NO: 1.

[0431] Antigen-binding domain - additional embodiments

[0432] In one aspect, the antigen-binding domain (e.g., a tumor antigen-binding domain, e.g., a B cell antigen-binding domain, e.g., a CD123-binding domain or a CD19-binding domain) is a fragment, such as a single-chain variable fragment (scFv). In one aspect, the antigen-binding domain (e.g., a tumor antigen-binding domain, e.g., a B cell antigen-binding domain, e.g., a CD123-binding domain or a CD19-binding domain) is an Fv, Fab, (Fab')2 or a bifunctional (e.g., bispecific) hybrid antibody (e.g., Lanzavecchia et al., Eur. J. Immunol. [European Journal of Immunology] 17, 105 (1987)). In one aspect, the antibodies and fragments thereof of the invention bind an antigen (e.g., a tumor antigen, e.g., a B cell antigen, e.g., the CD123 or CD19 protein) or a fragment thereof with wild-type or enhanced affinity.

[0433] In some cases, human scFvs can be derived from display libraries. A display library is a collection of entities; each entity includes an accessible polypeptide component and a retrievable component that encodes or identifies the polypeptide component. The polypeptide component is altered to represent different amino acid sequences. The polypeptide component can be of any length, for example from three amino acids to over 300 amino acids. A display library entity can include more than one polypeptide component, such as the two polypeptide chains of a Fab. In one exemplary embodiment, a display library can be used to identify human CD123 binding domains. In the selection, the polypeptide component of each member of the library is probed with CD123 or a fragment thereof, and if the polypeptide component binds to CD123, the display library member is typically identified by retention on a support.

[0434] The retained display library members are recovered from the support and analyzed. The analysis can include amplification and subsequent selection under similar or dissimilar conditions. For example, positive and negative selections can be alternated. The analysis can also include determining the amino acid sequence of the polypeptide component (i.e., the anti-CD123 binding domain), and purifying the polypeptide component for detailed characterization.

[0435] A variety of formats can be used for display libraries. Examples include phage display. In phage display, the protein component is typically covalently linked to a phage coat protein. The linkage is produced by translation of a nucleic acid encoding the protein component fused to the coat protein. The linkage can include a flexible peptide linker, a protease site, or an amino acid incorporated due to suppression of a stop codon. Phage display is described, for example, in U.S. 5,223,409; Smith (1985) Science 228:1315-1317; WO92 / 18619; WO 91 / 17271; WO 92 / 20791; WO 92 / 15679; WO 93 / 01288; WO 92 / 01047; WO 92 / 09690; WO 90 / 02809; de Haard et al. (1999) J. Biol. Chem. 274:18218-30; Hoogenboom et al. (1998) Immunotechnology 4:1-20; Hoogenboom et al. (2000) Immunol Today 2:371-8 and Hoet et al. (2005) Nat Biotechnol. 23(3) 344-8. Phages displaying the protein component can be grown and harvested using standard phage preparation methods (e.g., PEG precipitation from growth medium). After selection of individual display phages, the nucleic acid encoding the selected protein component can be isolated from cells infected with the selected phage or from the phage itself after amplification. Individual colonies or plaques can be picked, the nucleic acid isolated and sequenced.

[0436] Other display formats include cell-based display (see, e.g., WO 03 / 029456), protein-nucleic acid fusions (see, e.g., US 6,207,446), ribosome display (see, e.g., Mattheakis et al. (1994) Proc. Natl. Acad. Sci. USA 91:9022 and Hanes et al. (2000) Nat Biotechnol. 18:1287-92; Hanes et al. (2000) Methods Enzymol. 328:404-30; and Schaffitzel et al. (1999) J Immunol Methods. 231(1-2):119-35), and Escherichia coli periplasmic display (November 22, 2005; PMID: 16337958).

[0437] In some cases, scFv can be prepared according to methods known in the art (see, e.g., Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFv molecules can be generated by linking the VH and VL regions together using a flexible polypeptide linker. The scFv molecule contains a linker with an optimized length and / or amino acid composition (e.g., a Ser-Gly linker). The linker length can greatly affect the folding and interaction of the variable regions of the scFv. In fact, if a short polypeptide linker is used (e.g., between 5-10 amino acids), then in-chain folding is blocked. Inter-chain folding is also required to combine the two variable regions together to form a functional epitope-binding site. For examples of linker orientation and size, see, e.g., Hollinger et al. 1993 Proc Natl Acad Sci. U.S.A. 90:6444-6448, U.S. Patent Application Publication Nos. 2005 / 0100543, 2005 / 0175606, 2007 / 0014794, and PCT Publication Nos. WO 2006 / 020258 and WO2007 / 024715 which are incorporated herein by reference.

[0438] The scFv may contain a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more amino acid residues between its VL and VH regions. The linker sequence may contain any naturally occurring amino acid. In some embodiments, the linker sequence contains the amino acids glycine and serine. In another embodiment, the linker sequence contains multiple sets of glycine and serine repeats, such as (Gly4Ser)n, where n is a positive integer equal to or greater than 1 (SEQ ID NO:25). In one embodiment, the linker can be (Gly4Ser)4 (SEQ ID NO:27) or (Gly4Ser)3 (SEQ ID NO:28). Variations in linker length may preserve or enhance activity, resulting in excellent efficacy in activity studies.

[0439] Exemplary CD123 CAR constructs and antigen-binding domains

[0440] Exemplary CD123 CAR constructs disclosed herein comprise an scFv (e.g., the human scFvs disclosed in Tables 11A, 12A, and 12B herein, optionally preceded by an optional leader sequence (e.g., SEQ ID NO:1 and SEQ ID NO:12 are exemplary leader amino acid and nucleotide sequences, respectively)). Sequences of the human scFv fragments are provided in Table 11A herein (amino acid sequences of SEQ ID NO:157 - 160). Sequences of the human scFv fragments without a leader sequence are provided in Table 12B herein (nucleotide sequences are SEQ ID NO:479, 481, 482, and 484, and amino acid sequences are SEQ ID NO:478, 480, 483, and 485). The CD123 CAR construct can further comprise an optional hinge domain, such as a CD8 hinge domain (e.g., comprising the amino acid sequence of SEQ ID NO:2 or the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO:13); a transmembrane domain, such as a CD8 transmembrane domain (e.g., comprising the amino acid sequence of SEQ ID NO:6 or the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:17); an intracellular domain, such as a 4 - 1BB intracellular domain (e.g., comprising the amino acid sequence of SEQ ID NO:7 or the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:18); a functional signaling domain, such as a CD3ζ domain (e.g., comprising the amino acid sequence of SEQ ID NO:9 or 10 or the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:20 or 21). In certain embodiments, these domains are adjacent and in the same reading frame to form a single fusion protein. In other embodiments, the domains are separate polypeptides, e.g., an RCAR molecule as described herein.

[0441] In certain embodiments, the full-length CD123 CAR molecule comprises the amino acid sequence of CD123-1, CD123-2, CD123-3, CD123-4, hzCD123-1, hzCD123-2, hzCD123-3, hzCD123-4, hzCD123-5, hzCD123-6, hzCD123-7, hzCD123-8, hzCD123-9, hzCD123-10, hzCD123-11, hzCD123-12, hzCD123-13, hzCD123-14, hzCD123-15, hzCD123-16, hzCD123-17, hzCD123-18, hzCD123-19, hzCD123-20, hzCD123-21, hzCD123-22, hzCD123-23, hzCD123-24, hzCD123-25, hzCD123-26, hzCD123-27, hzCD123-28, hzCD123-29, hzCD123-30, hzCD123-31, or hzCD123-32 provided in Table 11A, 12A, or 12B, or is encoded by the nucleotide sequence of CD123-1, CD123-2, CD123-3, CD123-4, hzCD123-1, hzCD123-2, hzCD123-3, hzCD123-4, hzCD123-5, hzCD123-6, hzCD123-7, hzCD123-8, hzCD123-9, hzCD123-10, hzCD123-11, hzCD123-12, hzCD123-13, hzCD123-14, hzCD123-15, hzCD123-16, hzCD123-17, hzCD123-18, hzCD123-19, hzCD123-20, hzCD123-21, hzCD123-22, hzCD123-23, hzCD123-24, hzCD123-25, hzCD123-26, hzCD123-27, hzCD123-28, hzCD123-29, hzCD123-30, hzCD123-31, or hzCD123-32, or a sequence that is substantially the same thereto (e.g., having at least 95% identity, such as 95%-99% identity).

[0442] In certain embodiments, the CD123 CAR molecule or CD123 antigen-binding domain comprises the amino acid sequence of the scFv of CD123-1, CD123-2, CD123-3, CD123-4, hzCD123-1, hzCD123-2, hzCD123-3, hzCD123-4, hzCD123-5, hzCD123-6, hzCD123-7, hzCD123-8, hzCD123-9, hzCD123-10, hzCD123-11, hzCD123-12, hzCD123-13, hzCD123-14, hzCD123-15, hzCD123-16, hzCD123-17, hzCD123-18, hzCD123-19, hzCD123-20, hzCD123-21, hzCD123-22, hzCD123-23, hzCD123-24, hzCD123-25, hzCD123-26, hzCD123-27, hzCD123-28, hzCD123-29, hzCD123-30, hzCD123-31, or hzCD123-32 provided in Table 11A, 12A or 12B;or comprising the scFv amino acid sequence of CD123-1, CD123-2, CD123-3, CD123-4, hzCD123-1, hzCD123-2, hzCD123-3, hzCD123-4, hzCD123-5, hzCD123-6, hzCD123-7, hzCD123-8, hzCD123-9, hzCD123-10, hzCD123-11, hzCD123-12, hzCD123-13, hzCD123-14, hzCD123-15, hzCD123-16, hzCD123-17, hzCD123-18, hzCD123-19, hzCD123-20, hzCD123-21, hzCD123-22, hzCD123-23, hzCD123-24, hzCD123-25, hzCD123-26, hzCD123-27, hzCD123-28, hzCD123-29, hzCD123-30, hzCD123-31, or hzCD123-32, or encoded by the nucleotide sequence of CD123-1, CD123-2, CD123-3, CD123-4, hzCD123-1, hzCD123-2, hzCD123-3, hzCD123-4, hzCD123-5, hzCD123-6, hzCD123-7, hzCD123-8, hzCD123-9, hzCD123-10, hzCD123-11, hzCD123-12, hzCD123-13, hzCD123-14, hzCD123-15, hzCD123-16, hzCD123-17, hzCD123-18, hzCD123-19, hzCD123-20, hzCD123-21, hzCD123-22, hzCD123-23, hzCD123-24, hzCD123-25, hzCD123-26, hzCD123-27, hzCD123-28, hzCD123-29, hzCD123-30, hzCD123-31, or hzCD123-32; or a sequence that is substantially identical to any of the foregoing sequences (e.g., having at least 95% identity, such as 95%-99% identity, or up to 20, 15, 10, 8, 6, 5, 4, 3, 2, or 1 amino acid changes).;

[0443] In certain embodiments, the CD123 CAR molecule, or the CD123 antigen-binding domain, comprises the heavy chain variable region and / or the light chain variable region of CD123-1, CD123-2, CD123-3, CD123-4, hzCD123-1, hzCD123-2, hzCD123-3, hzCD123-4, hzCD123-5, hzCD123-6, hzCD123-7, hzCD123-8, hzCD123-9, hzCD123-10, hzCD123-11, hzCD123-12, hzCD123-13, hzCD123-14, hzCD123-15, hzCD123-16, hzCD123-17, hzCD123-18, hzCD123-19, hzCD123-20, hzCD123-21, hzCD123-22, hzCD123-23, hzCD123-24, hzCD123-25, hzCD123-26, hzCD123-27, hzCD123-28, hzCD123-29, hzCD123-30, hzCD123-31, or hzCD123-32 provided in Table 11A or 12A, or a sequence that is substantially identical to any of the foregoing sequences (e.g., having at least 95% identity, such as 95%-99% identity, or up to 20, 15, 10, 8, 6, 5, 4, 3, 2, or 1 amino acid alteration).

[0444] In certain embodiments, the CD123 CAR molecule or CD123 antigen-binding domain comprises one, two, or three CDRs from the heavy chain variable regions provided in Table 1A or 3A (e.g., HCDR1, HCDR2, and / or HCDR3); and / or one, two, or three CDRs from the light chain variable regions of CD123-1, CD123-2, CD123-3, CD123-4, hzCD123-1, hzCD123-2, hzCD123-3, hzCD123-4, hzCD123-5, hzCD123-6, hzCD123-7, hzCD123-8, hzCD123-9, hzCD123-10, hzCD123-11, hzCD123-12, hzCD123-13, hzCD123-14, hzCD123-15, hzCD123-16, hzCD123-17, hzCD123-18, hzCD123-19, hzCD123-20, hzCD123-21, hzCD123-22, hzCD123-23, hzCD123-24, hzCD123-25, hzCD123-26, hzCD123-27, hzCD123-28, hzCD123-29, hzCD123-30, hzCD123-31, or hzCD123-32 provided in Table 2A or 4A (e.g., LCDR1, LCDR2, and / or LCDR3); or a sequence that is substantially identical to any of the foregoing sequences (e.g., at least 95% identity, such as 95%-99% identity, or up to 5, 4, 3, 2, or 1 amino acid alterations).

[0445] In certain embodiments, the CD123 CAR molecule or CD123 antigen-binding domain comprises one, two, or three CDRs from the heavy chain variable regions provided in Table 5A (e.g., HCDR1, HCDR2, and / or HCDR3); and / or one, two, or three CDRs from the light chain variable regions of CD123-1, CD123-2, CD123-3, CD123-4, hzCD123-1, hzCD123-2, hzCD123-3, hzCD123-4, hzCD123-5, hzCD123-6, hzCD123-7, hzCD123-8, hzCD123-9, hzCD123-10, hzCD123-11, hzCD123-12, hzCD123-13, hzCD123-14, hzCD123-15, hzCD123-16, hzCD123-17, hzCD123-18, hzCD123-19, hzCD123-20, hzCD123-21, hzCD123-22, hzCD123-23, hzCD123-24, hzCD123-25, hzCD123-26, hzCD123-27, hzCD123-28, hzCD123-29, hzCD123-30, hzCD123-31, or hzCD123-32 provided in Table 6A (e.g., LCDR1, LCDR2, and / or LCDR3); or a sequence that is substantially identical to any of the foregoing sequences (e.g., at least 95% identity, such as 95%-99% identity, or up to 5, 4, 3, 2, or 1 amino acid alterations).

[0446] In certain embodiments, the CD123 molecule or CD123 antigen-binding domain comprises one, two, or three CDRs from the heavy chain variable regions provided in Table 7A (e.g., HCDR1, HCDR2, and / or HCDR3); and / or one, two, or three CDRs from the light chain variable regions of CD123-1, CD123-2, CD123-3, CD123-4, hzCD123-1, hzCD123-2, hzCD123-3, hzCD123-4, hzCD123-5, hzCD123-6, hzCD123-7, hzCD123-8, hzCD123-9, hzCD123-10, hzCD123-11, hzCD123-12, hzCD123-13, hzCD123-14, hzCD123-15, hzCD123-16, hzCD123-17, hzCD123-18, hzCD123-19, hzCD123-20, hzCD123-21, hzCD123-22, hzCD123-23, hzCD123-24, hzCD123-25, hzCD123-26, hzCD123-27, hzCD123-28, hzCD123-29, hzCD123-30, hzCD123-31, or hzCD123-32 provided in Table 8A (e.g., LCDR1, LCDR2, and / or LCDR3); or a sequence that is substantially identical to any of the foregoing sequences (e.g., at least 95% identity, such as 95%-99% identity, or up to 5, 4, 3, 2, or 1 amino acid alterations).

[0447] For the sequences of the CDR sequences of the scFv domain, the heavy chain variable domain is shown in Tables 3A, 5A, and 7A, and the light chain variable domain is shown in Tables 2A, 4A, 6A, and 8A. "ID" represents the corresponding SEQ ID NO of each CDR.

[0448] The CDRs provided in Tables 1A, 2A, 3A, and 4A are a combination according to the Kabat and Chothia numbering schemes.

[0449] Table 1A. CDRs of Heavy Chain Variable Domain

[0450] Candidate HCDR1 ID HCDR2 ID HCDR3 ID CAR123 - 2 GYTFTGYYMH 335 WINPNSG...

Claims

1. A composition comprising a JAK-STAT inhibitor (e.g., ruxolitinib), the composition for use in combination with CAR therapy (e.g., CD123 CAR therapy) for preventing cytokine release syndrome (CRS) in a subject in need thereof.

2. A method of preventing cytokine release syndrome (CRS) of CAR therapy (e.g., CD123 CAR therapy) in a subject in need thereof, the method comprising administering a JAK-STAT inhibitor (e.g., ruxolitinib) in combination with CAR therapy to the subject, thereby preventing CRS in the subject.

3. A composition comprising: (i) cells expressing a chimeric antigen receptor (CAR), such as a population of immune effector cells, wherein the CAR comprises a CD123 binding domain, a transmembrane domain, and an intracellular signaling domain; and (ii) a JAK-STAT inhibitor, such as ruxolitinib, for use in treating a subject having a disease associated with CD123 expression.

4. A method of treating a subject having a disease associated with CD123 expression, the method comprising administering to the subject: (i) cells expressing a chimeric antigen receptor (CAR), such as a population of immune effector cells, wherein the CAR comprises a CD123 binding domain, a transmembrane domain, and an intracellular signaling domain; and (ii) a JAK-STAT inhibitor, such as ruxolitinib.

5. The method or composition for use according to any one of the preceding claims, wherein the subject (i) is at risk of developing CRS, has CRS, or is diagnosed with CRS; (ii) is identified or has previously been identified as being at risk of CRS; and / or (iii) has been, is being, or will be administered CAR therapy, such as cells expressing CD123 CAR.

6. The method or composition for use according to any one of claims 1-2 or 5-6, wherein the JAK-STAT inhibitor is selected from: ruxolitinib, AG490, AZD1480, tofacitinib (tasocitinib or CP-690550), CYT387, fedratinib, baricitinib (INCB039110), lestaurtinib (CEP701), pacritinib (SB1518), XL019, gandotinib (LY2784544), BMS911543, fedratinib (SAR302503), decemotinib (V-509), INCB39110, GEN1, GEN2, GLPG0634, NS018, and N-(cyanomethyl)-4-[2-(4-morpholinylanilino)pyrimidin-4-yl]benzamide, or a pharmaceutically acceptable salt thereof, for example, wherein the JAK-STAT inhibitor is ruxolitinib or a pharmaceutically acceptable salt thereof.

7. The method or composition for use according to any one of claims 1-2 or 5-6, wherein the CAR therapy comprises cells expressing CD123 CAR.

8. The method or composition for use as described in any one of the preceding claims, the method or composition further comprising selecting a subject for administration of a JAK-STAT inhibitor (e.g., ruxolitinib).

9. The method or composition for use as described in any one of the preceding claims, wherein the subject is selected based on (i) his or her risk of developing CRS, (ii) his or her diagnosis of CRS, and / or (iii) whether he or she has been, is being, or will be administered CAR therapy (e.g., cells expressing a CD123 CAR).

10. The method or composition for use as described in any one of the preceding claims, wherein the subject is selected for administration of a JAK-STAT inhibitor (e.g., ruxolitinib) if the subject is diagnosed with CRS, such as severe or non-severe CRS.

11. The method or composition for use as described in any one of the preceding claims, wherein the subject is selected for administration of a JAK-STAT inhibitor (e.g., ruxolitinib) if the subject is at risk of developing CRS.

12. The method or composition for use as described in any one of the preceding claims, wherein the subject is selected for administration of a JAK-STAT inhibitor (e.g., ruxolitinib) if the subject has been, is being, or will be administered CAR therapy (e.g., cells expressing a CD123 CAR).

13. The method or composition for use as described in any one of the preceding claims, wherein the JAK-STAT inhibitor is ruxolitinib and the CAR therapy is cells expressing a CD123 CAR.

14. The method or composition for use as described in any one of the preceding claims, wherein the CAR therapy (e.g., cells expressing a CD123 CAR) and the JAK-STAT inhibitor (e.g., ruxolitinib) are administered sequentially.

15. The method or composition for use as described in any one of the preceding claims, wherein the JAK-STAT inhibitor (e.g., ruxolitinib) is administered before the CAR therapy (e.g., cells expressing a CD123 CAR).

16. The method or composition for use as described in any one of claims 1-12, wherein the JAK-STAT inhibitor (e.g., ruxolitinib) and the CAR therapy (e.g., cells expressing a CD123 CAR) are administered simultaneously or in parallel.

17. The method or composition for use as described in any one of the preceding claims, wherein the CAR therapy (e.g., cells expressing a CD123 CAR) and the JAK-STAT inhibitor (e.g., ruxolitinib) are administered at a treatment interval, and wherein the treatment interval comprises a single dose of the CAR therapy and multiple doses of the JAK-STAT inhibitor (e.g., first and second, and optionally subsequent doses).

18. The method or composition for use as described in any one of claims 1-15 or 17, wherein the dose of the CAR therapy is administered after the first dose of the JAK-STAT inhibitor (e.g., at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more) and, for example, before the second dose of the inhibitor.

19. The method or composition for use as described in any one of claims 1-13 and 16-17, wherein the dose of the CAR therapy is administered in parallel with the first dose of the JAK-STAT inhibitor (e.g., within 2 days (e.g., within 2 days, 1 day, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours or less)).

20. The method or composition for use as described in any one of claims 17-19, wherein one or more subsequent doses of the JAK-STAT inhibitor are administered after the second dose of the JAK-STAT inhibitor.

21. The method or composition for use as described in any one of claims 17-20, wherein the dose of the JAK-STAT inhibitor is administered twice a day (BID).

22. The method or composition for use as described in any of the preceding claims, wherein the treatment interval comprises at least 7 days, for example a duration of at least 7 days, 8 days, 9 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months or more.

23. The method or composition for use as described in any one of claims 17-22, wherein the treatment interval is repeated, for example, one or more times, such as 1, 2, 3, 4, 5 times or more, for example, after the treatment interval there is one or more subsequent treatment intervals, such as 1, 2, 3, 4 or 5 subsequent treatment intervals.

24. The method or composition for use as described in any of the preceding claims, wherein the CD123 binding domain comprises: heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of any CD123 heavy chain binding domain amino acid sequence listed in Table 12B, Table 11A or Table 12A; and light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of any CD19 light chain binding domain amino acid sequence listed in Table 12B, Table 11A or Table 12A.

25. The method or composition for use as described in any one of the preceding claims, wherein the CD123 binding domain comprises HC CDR1, HC CDR2, and HC CDR3 according to the HC CDR amino acid sequences in Table 5A, 7A, 1A, or 3A, and LC CDR1, LC CDR2, and LC CDR3 according to the LC CDR amino acid sequences in Table 6A, 8A, 2A, or 4A.

26. The method or composition for use as described in any one of the preceding claims, wherein the CD123 binding domain comprises: i) the amino acid sequence of any heavy chain variable region of the CD123 binding domain listed in Table 12B or 11A; ii) an amino acid sequence having at least one, two, or three modifications but not more than 30, 20, or 10 modifications to the amino acid sequence of any heavy chain variable region of the CD123 binding domain provided in Table 12B or 11A; or iii) an amino acid sequence having at least 95% identity to the amino acid sequence of any heavy chain variable region of the CD123 binding domain provided in Table 12B or 11A.

27. The method or composition for use as described in any one of the preceding claims, wherein the CD123 binding domain comprises: (i) the amino acid sequence of any heavy chain of the CD123 binding domain provided in Table 12B, Table 11A, or Table 12A; (ii) an amino acid sequence having at least one, two, or three modifications but not more than 30, 20, or 10 modifications to any heavy chain of the CD123 binding domain provided in Table 12B, Table 11A, or Table 12A; or (iii) an amino acid sequence having at least 95% identity to the amino acid sequence of any heavy chain of the CD123 binding domain provided in Table 12B, Table 11A, or Table 12A.

28. The method or composition for use as described in any one of the preceding claims, wherein the CD123 binding domain comprises: (i) the amino acid sequence of any light chain variable region of the CD123 binding domain provided in Table 12B, Table 11A, or Table 12A; (ii) an amino acid sequence having at least one, two, or three modifications but not more than 30, 20, or 10 modifications to the amino acid sequence of any light chain variable region of the CD123 binding domain provided in Table 12B, Table 11A, or Table 12A; or (iii) an amino acid sequence having at least 95% identity to the amino acid sequence of any light chain variable region of the CD123 binding domain provided in Table 12B, Table 11A, or Table 12A.

29. The method or composition for use as described in any one of the preceding claims, wherein the CD123 binding domain comprises: (i) the amino acid sequence of any light chain of the CD123 binding domain provided in Table 12B, Table 11A, or Table 12A; (ii) an amino acid sequence having at least one, two, or three modifications but not more than 30, 20, or 10 modifications to any light chain of the CD123 binding domain provided in Table 12B, Table 11A, or Table 12A; or (iii) An amino acid sequence having at least 95% identity with the amino acid sequence of any light chain of the CD123 binding domain provided in Table 12B, Table 11A or Table 12A.

30. The method or composition for use according to any one of the preceding claims, wherein the CD123 binding domain comprises the amino acid sequence of any heavy chain variable region listed in Table 12B or 11A, and the amino acid sequence of any light chain variable region listed in Table 12B or 11A.

31. The method or composition for use according to any one of the preceding claims, wherein the CD123 binding domain comprises: (i) An amino acid sequence selected from the group consisting of: SEQ ID NO: 480, 483, 485, 478, 158, 159, 160, 157, 217, 218, 219, 216, 276, 277, 278, or 275; (ii) An amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to any one of SEQ ID NO: 480, 483, 485, 478, 158, 159, 160, 157, 217, 218, 219, 216, 276, 277, 278, or 275; or (iii) An amino acid sequence having at least 95% identity with any one of SEQ ID NO: 480, 483, 485, 478, 158, 159, 160, 157, 217, 218, 219, 216, 276, 277, 278 or 275.

32. The method or composition for use as described in any one of the preceding claims, wherein the transmembrane domain comprises a transmembrane domain from a protein selected from the group consisting of: The α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

33. The method or composition for use according to any one of the preceding claims, wherein the transmembrane domain comprises (i) The amino acid sequence of SEQ ID NO: 6, (ii) An amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of the amino acid sequence comprising SEQ ID NO: 6, or (iii) A sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:

6.

34. The method or composition for use according to any one of the preceding claims, wherein the CD123 binding domain is linked to the transmembrane domain by a hinge region.

35. The method or composition for use according to any one of the preceding claims, wherein the hinge region comprises SEQ ID NO: 2, or a sequence having at least 95% identity therewith.

36. The method or composition for use as described in any one of the preceding claims, wherein the intracellular signaling domain comprises a co-stimulatory signaling domain, and the co-stimulatory signaling domain comprises a functional signaling domain obtained from a protein selected from the group consisting of: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a and a ligand that specifically binds to CD83.

37. The method or composition for use according to any one of the preceding claims, wherein the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having at least one, two or three modifications but not more than 20, 10, or 5 modifications of the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

7.

38. The method or composition for use according to any one of the preceding claims, wherein the intracellular signaling domain comprises the functional signaling domain of 4-1BB and / or the functional signaling domain of CD3ζ.

39. The method or composition for use as described in any one of the preceding claims, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:7 and / or the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10; or an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO:7 and / or the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10; or an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:7 and / or the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:

10.

40. The method or composition for use as described in any one of the preceding claims, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:7 and the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10, wherein the amino acid sequence comprising the intracellular signaling domain is expressed in the same reading frame and expressed as a single polypeptide chain.

41. The method or composition for use as described in any one of the preceding claims, wherein the CAR further comprises a leader sequence comprising the amino acid sequence of SEQ ID NO:

1.

42. The method or composition for use as described in any one of the preceding claims, wherein the CAR comprises: (i) the amino acid sequence of any one of SEQ ID NO:99, 100, 101 or 98; (ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to any one of SEQ ID NO:99, 100, 101 or 98; or (iii) an amino acid sequence having at least 95% identity to any one of SEQ ID NO:99, 100, 101 or 98.

43. The method or composition for use as described in any one of the preceding claims, wherein the cell comprising the CAR comprises a nucleic acid encoding the CAR.

44. The method or composition for use as described in claim 43, wherein the nucleic acid encoding the CAR is a lentiviral vector.

45. The method or composition for use as described in claim 43 or 44, wherein the nucleic acid encoding the CAR is introduced into these cells by lentiviral transduction.

46. The method or composition for use as described in any one of claims 43-45, wherein the nucleic acid encoding the CAR is RNA, such as in vitro transcribed RNA.

47. The method or composition for use as described in any one of claims 43-46, wherein the nucleic acid encoding the CAR is introduced into these cells by electroporation.

48. The method or composition for use as described in any one of the preceding claims, wherein the cell is a T cell or an NK cell.

49. The method or composition for use according to claim 48, wherein the T cells are autologous or allogeneic T cells.

50. The method or composition for use according to any one of the preceding claims, wherein the CRS is severe CRS, such as grade 4 or 5 CRS.

51. The method or composition for use according to any one of claims 1-49, wherein the CRS is lower than severe CRS, such as grade 1, 2 or 3 CRS.

52. The method or composition for use according to any one of the preceding claims, wherein the subject is a mammal, such as a human.

53. The method or composition for use according to any one of the preceding claims, wherein the subject has or has been diagnosed with a disease associated with a B cell antigen, such as CD123, such as a hematological cancer, such as lymphoma or leukemia, such as acute myeloid leukemia (AML).

54. The method or composition for use as described in any one of the preceding claims, wherein the dose of the CAR therapy (e.g., CD123 CAR therapy) comprises at least about 1 x 10 5 , 5 x 10 6 , 1 x 10 7 , 1.5 x 10 7 , 2 x 10 7 , 2.5 x 10 7 , 3 x 10 7 , 3.5 x 10 7 , 4 x 10 7 , 5 x 10 7 , 1 x 10 8 , 1.5 x 10 8 , 2 x 10 8 , 2.5 x 10 8 , 3 x 10 8 , 3.5 x 10 8 , 4 x 10 8 , 5 x 10 8 , 1 x 10 9 , 2 x 10 9 , or 5 x 10 9 cells (e.g., cells expressing CD123 CAR).

55. The method or composition for use according to any one of the preceding claims, wherein the dose (e.g., each dose) of the JAK-STAT inhibitor (e.g., ruxolitinib) comprises 2.5 mg to 50 mg (e.g., 2.5-5 mg, 5-10 mg, 10-15 mg, 15-20 mg, 20-25 mg, 25-30 mg, 30-35 mg, 35-40 mg, 40-45 mg, or 45-50 mg) of the JAK-STAT inhibitor.

56. A composition comprising a BTK inhibitor (e.g., ibrutinib), the composition for use alone or in combination with a CAR therapy (e.g., CD19 CAR therapy, such as CTL019 therapy) for preventing cytokine release syndrome (CRS) associated with CAR therapy in a subject in need thereof, wherein the subject is identified as or has previously been identified as being at risk of CRS, thereby preventing CRS in the subject.

57. A method for preventing cytokine release syndrome (CRS), such as CRS associated with a CAR therapy (e.g., CD19 CAR therapy, such as CTL019 therapy), in a subject in need thereof, the method comprising administering a BTK inhibitor (e.g., ibrutinib) alone or in combination with a CAR therapy to the subject, wherein the subject is identified as or has previously been identified as being at risk of CRS, thereby preventing CRS in the subject.

58. The composition for use according to claim 56 or the method according to claim 57, wherein the subject has been, is being, or will be administered a CAR therapy, such as CD19 CAR therapy, such as CTL019.

59. The composition for use or the method according to claim 56 or 58, or the method according to claims 57-58, the method comprising selecting a subject for administration of a BTK inhibitor, such as ibrutinib.

60. The composition or method for use according to claim 59, wherein the subject is selected based on (i) his or her risk of developing CRS, (ii) his or her CRS diagnosis, and / or (iii) whether he or she has been, is, or will be administered CAR therapy (e.g., CAR19 therapy, e.g., CTL019 therapy).

61. The composition or method for use according to claim 59 or 60, wherein: (i) if the subject is diagnosed with CRS, such as severe or non-severe CRS, the subject is selected for administration of a BTK inhibitor (e.g., ibrutinib); (ii) if the subject is at risk of developing CRS (e.g., is identified as being at risk of developing CRS), the subject is selected for administration of a BTK inhibitor (e.g., ibrutinib); or (iii) if the subject has been, is, or will be administered CAR therapy (e.g., CAR19 therapy, e.g., CTL019 therapy), the subject is selected for administration of a BTK inhibitor (e.g., ibrutinib).

62. The composition or method for use according to any one of claims 57-61, wherein the BTK inhibitor is selected from ibrutinib, GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774, or LFM-A13 or a pharmaceutically acceptable salt thereof, such as wherein the BTK inhibitor is ibrutinib or a pharmaceutically acceptable salt thereof.

63. The composition or method for use according to any one of claims 57-62, wherein the CAR therapy is CAR19 therapy, e.g., CTL019 therapy.

64. The composition or method for use according to any one of claims 57-63, wherein the CAR therapy (e.g., CAR19 therapy) and the BTK inhibitor (e.g., ibrutinib) are administered at a treatment interval, and wherein the treatment interval comprises a single dose of the CAR therapy and multiple doses of the BTK inhibitor (e.g., first and second, and optionally subsequent doses).

65. The composition or method for use according to any one of claims 57-64, wherein the dose of the CAR therapy is administered after the first dose of the BTK inhibitor (e.g., at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more) and, for example, but before the second dose of the inhibitor.

66. The composition or method for use according to any one of claims 57-64, wherein the dose of the CAR therapy is administered in parallel with the first dose of the BTK inhibitor (e.g., within 2 days (e.g., within 2 days, 1 day, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours or less)).

67. The composition or method for use according to any one of claims 62-66, wherein one or more subsequent doses of the BTK inhibitor are administered after the second dose of the BTK inhibitor.

68. A composition or method for use as described in any one of claims 57 - 67, wherein the dose of the BTK inhibitor is administered once daily (QD).

69. A composition or method for use as described in claims 64 - 68, wherein the treatment interval comprises a duration of at least 7 days, such as at least 7 days, 8 days, 9 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months or longer.

70. A composition or method for use as described in any one of claims 64 - 69, wherein the treatment interval is repeated, for example, one or more times, such as 1, 2, 3, 4, 5 times or more.

71. A composition or method for use as described in any one of claims 64 - 70, wherein after the treatment interval there is one or more, such as 1, 2, 3, 4 or 5 subsequent treatment intervals.

72. The composition or method for use as described in any one of claims 64 - 71, wherein the dose of the CAR therapy (e.g., CAR19 therapy) comprises at least about 1x10 5 , 5x10 6 , 1x10 7 , 1.5x10 7 , 2x10 7 , 2.5x10 7 , 3x10 7 , 3.5x10 7 , 4x10 7 , 5x10 7 , 1x10 8 , 1.5x10 8 , 2x10 8 , 2.5x10 8 , 3x10 8 , 3.5x10 8 , 4x10 8 , 5x10 8 , 1x10 9 , 2x10 9 , or 5x10 9 cells (e.g., cells expressing CD19 CAR).

73. A composition or method for use as described in any one of claims 64 - 72, wherein the dose of the BTK inhibitor (such as ibrutinib (PCI - 32765)), for example, each dose, comprises about 250 mg, 300 mg, 350 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600 mg (such as 250 mg, 420 mg or 560 mg) of ibrutinib.

74. A composition or method for use as described in any one of claims 57 - 73, wherein the CD19 - binding domain comprises heavy - chain complementarity - determining region 1 (HCCDR1), heavy - chain complementarity - determining region 2 (HC CDR2) and heavy - chain complementarity - determining region 3 (HC CDR3) of any CD19 heavy - chain - binding domain amino - acid sequence listed in Table 13A or 14A; and light - chain complementarity - determining region 1 (LC CDR1), light - chain complementarity - determining region 2 (LC CDR2) and light - chain complementarity - determining region 3 (LC CDR3) of any CD19 light - chain - binding domain amino - acid sequence listed in Table 13A or 14A.

75. A composition or method for use as described in any one of claims 57 - 73, wherein the CD19 - binding domain comprises HC CDR1, HC CDR2 and HC CDR3 according to the HC CDR amino - acid sequences in Table 15A, and LC CDR1, LC CDR2 and LC CDR3 according to the LC CDR amino - acid sequences in Table 16A.

76. A composition or method for use as described in any one of claims 57 - 75, wherein the CD19 - binding domain comprises: (i) the amino - acid sequence of any heavy - chain variable region of the CD19 - binding domain listed in Table 13A or 14A; (ii) an amino acid sequence having at least one, two, or three modifications but not more than 30, 20, or 10 modifications to the amino acid sequence of any heavy chain variable region of the CD19-binding domain provided in Table 13A or 14A; or (iii) an amino acid sequence having at least 95% identity to the amino acid sequence of any heavy chain variable region of the CD19-binding domain provided in Table 13A or 14A.

77. The composition or method for use according to any one of claims 57-76, wherein the CD19-binding domain comprises: (i) the amino acid sequence of any heavy chain of the CD19-binding domain provided in Table 13A or 14A; (ii) an amino acid sequence having at least one, two, or three modifications but not more than 30, 20, or 10 modifications to any heavy chain of the CD19-binding domain provided in Table 13A or 14A; or (iii) an amino acid sequence having at least 95% identity to the amino acid sequence of any heavy chain of the CD19-binding domain provided in Table 13A or 14A.

78. The composition or method for use according to any one of claims 57-77, wherein the CD19-binding domain comprises: (i) the amino acid sequence of any light chain variable region of the CD19-binding domain provided in Table 13A or 14A; (ii) an amino acid sequence having at least one, two, or three modifications but not more than 30, 20, or 10 modifications to the amino acid sequence of any light chain variable region of the CD19-binding domain provided in Table 13A or 14A; or (iii) an amino acid sequence having at least 95% identity to the amino acid sequence of any light chain variable region of the CD19-binding domain provided in Table 13A or 14A.

79. The composition or method for use according to any one of claims 57-78, wherein the CD19-binding domain comprises: (i) the amino acid sequence of any light chain of the CD19-binding domain provided in Table 13A or 14A; (ii) an amino acid sequence having at least one, two, or three modifications but not more than 30, 20, or 10 modifications to any light chain of the CD19-binding domain provided in Table 13A or 14A; or (iii) an amino acid sequence having at least 95% identity to the amino acid sequence of any light chain of the CD19-binding domain provided in Table 13A or 14A.

80. The composition or method for use according to any one of claims 57-79, wherein the CD19-binding domain comprises the amino acid sequence of any heavy chain variable region listed in Table 13A or 14A and the amino acid sequence of any light chain variable region listed in Table 13A or 14A.

81. The composition or method for use according to any one of claims 57-80, wherein the CD19-binding domain comprises: (i) A nucleotide sequence selected from the group consisting of: SEQ ID NO:774, SEQ ID NO:710, SEQ ID NO:711, SEQ ID NO:712, SEQ ID NO:713, SEQ ID NO:714, SEQ ID NO:715, SEQ ID NO:716, SEQ ID NO:717, SEQ ID NO:718, SEQ ID NO:719, SEQ ID NO:720, SEQ ID NO:721, SEQ ID NO:775, SEQ ID NO:777, or SEQ ID NO:780; (i) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to any one of SEQ ID NO:774, SEQ ID NO:710, SEQ ID NO:711, SEQ ID NO:712, SEQ ID NO:713, SEQ ID NO:714, SEQ ID NO:715, SEQ ID NO:716, SEQ ID NO:717, SEQ ID NO:718, SEQ ID NO:719, SEQ ID NO:720, SEQ ID NO:721, SEQ ID NO:775, SEQ ID NO:777, or SEQ ID NO:780; or (iii) an amino acid sequence having at least 95% identity to the amino acid sequence of any one of SEQ ID NO:774, SEQ ID NO:710, SEQ ID NO:711, SEQ ID NO:712, SEQ ID NO:713, SEQ ID NO:714, SEQ ID NO:715, SEQ ID NO:716, SEQ ID NO:717, SEQ ID NO:718, SEQ ID NO:719, SEQ ID NO:720, SEQ ID NO:721, SEQ ID NO:775, SEQ ID NO:777, or SEQ ID NO:

780.

82. A composition or method for use as described in any one of claims 57 - 81, wherein the transmembrane domain comprises a transmembrane domain from a protein selected from the group consisting of: The α, β or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

83. The composition or method for use according to any one of claims 57 - 82, wherein the transmembrane domain comprises (i) the amino acid sequence of SEQ ID NO:6, (ii) an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications that comprises the amino acid sequence of SEQ ID NO:6, or (iii) A sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

6.

84. The composition or method for use according to any one of claims 57 - 83, wherein the CD19 binding domain is linked to the transmembrane domain via a hinge region.

85. The composition or method for use according to any one of claims 57 - 84, wherein the hinge region comprises SEQ ID NO: 2, or a sequence having at least 95% identity thereto.

86. A composition or method for use as described in any one of claims 57 - 85, wherein the intracellular signaling domain comprises a co - stimulatory signaling domain, and the co - stimulatory signaling domain comprises a functional signaling domain obtained from a protein selected from the group consisting of: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a and ligands that specifically bind to CD83.

87. The composition or method for use as described in claim 86, wherein the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having at least one, two, or three modifications but not more than 20, 10, or 5 modifications of the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

7.

88. The composition or method for use as described in claim 86, wherein the intracellular signaling domain comprises the functional signaling domain of 4-1BB and / or the functional signaling domain of CD3ζ.

89. The composition or method for use as described in any one of claims 86-88, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:7 and / or the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10; or an amino acid sequence having at least one, two, or three modifications but not more than 20, 10, or 5 modifications of the amino acid sequence of SEQ ID NO:7 and / or the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10; or an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:7 and / or the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:

10.

90. The composition or method for use as described in any one of claims 86-89, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:7 and the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10, wherein the amino acid sequence comprising the intracellular signaling domain is expressed in the same reading frame and expressed as a single polypeptide chain.

91. The composition or method for use as described in any one of claims 57-90, wherein the CAR further comprises a leader sequence comprising the amino acid sequence of SEQ ID NO:

1.

92. The composition or method for use as described in any one of claims 57-91, wherein the CAR comprises: (i) the amino acid sequence of any one of SEQ ID NO:773; SEQ ID NO:758; SEQ ID NO:759, SEQ ID NO:760, SEQ ID NO:761, SEQ ID NO:762, SEQ ID NO:763, SEQ ID NO:764, SEQ ID NO:765, SEQ ID NO:766, SEQ ID NO:767, SEQ ID NO:768, SEQ ID NO:769, SEQ ID NO:776, SEQ ID NO:779, or SEQ ID NO:781; (ii) An amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to any one of SEQ ID NO:773; SEQ ID NO:758; SEQ ID NO:759, SEQ ID NO:760, SEQ ID NO:761, SEQ ID NO:762, SEQ ID NO:763, SEQ ID NO:764, SEQ ID NO:765, SEQ ID NO:766, SEQ ID NO:767, SEQ ID NO:768, SEQ ID NO:769, SEQ ID NO:776, SEQ ID NO:779, or SEQ ID NO:781; or (iii) An amino acid sequence having at least 95% identity to any one of SEQ ID NO:773; SEQ ID NO:758; SEQ ID NO:759, SEQ ID NO:760, SEQ ID NO:761, SEQ ID NO:762, SEQ ID NO:763, SEQ ID NO:764, SEQ ID NO:765, SEQ ID NO:766, SEQ ID NO:767, SEQ ID NO:768, SEQ ID NO:769, SEQ ID NO:776, SEQ ID NO:779, or SEQ ID NO:

781.

93. The composition or method for use according to any one of claims 57 - 92, wherein the cell comprising the CAR comprises a nucleic acid encoding the CAR.

94. The composition or method for use according to claim 93, wherein the nucleic acid encoding the CAR is a lentiviral vector.

95. The composition or method for use according to claim 93 or 94, wherein the nucleic acid encoding the CAR is introduced into these cells by lentiviral transduction.

96. The composition or method for use according to any one of claims 93 - 95, wherein the nucleic acid encoding the CAR is RNA, such as in vitro transcribed RNA.

97. The composition or method for use according to any one of claims 93 - 96, wherein the nucleic acid encoding the CAR is introduced into these cells by electroporation.

98. The composition or method for use according to claims 57 - 97, wherein the cell is a T cell or an NK cell.

99. The composition or method for use according to claim 98, wherein the T cell is an autologous or allogeneic T cell.

100. The composition or method for use according to any one of claims 57 - 99, wherein the CD19 - binding domain is the amino acid sequence of SEQ ID NO:774, or wherein the CAR comprises the amino acid sequence of SEQ ID NO:

773.

101. A composition or method for use as described in any one of claims 57 - 100, wherein the CRS is severe CRS, such as grade 4 or 5 CRS.

102. A composition or method for use as described in any one of claims 57 - 100, wherein the CRS is lower than severe CRS, such as grade 1, 2, or 3 CRS.

103. A composition or method for use as described in any one of claims 57 - 102, wherein the subject has a disease associated with the expression of a B - cell antigen such as CD19, such as cancer, such as hematological cancer, such as lymphoma or leukemia, such as acute lymphoblastic leukemia (ALL).

104. A composition or method for use as described in any one of claims 57 - 103, wherein the subject is a mammal, such as a human.

105. A composition or method for use as described in any of the preceding claims, further comprising administering to the subject an IL - 6 inhibitor (e.g., an anti - IL6 receptor inhibitor, such as an anti - IL6 receptor inhibitor, such as tocilizumab).

106. The composition or method for use according to claim 105, wherein the IL - 6 inhibitor is administered before, concurrently with, or after the dose of the CAR therapy (e.g., the first dose).

107. The composition or method for use according to any one of claims 105 - 106, wherein the IL - 6 inhibitor is administered within or 2 weeks (e.g., 2 weeks, 1.5 weeks, 1 week, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 24 hours, 20 hours, 15 hours, 10 hours, 5 hours, 2 hours, 1 hour, or less) before the first sign of CRS symptoms (e.g., fever, such as characterized by: e.g., two consecutive measurements (e.g., at least 4, 5, 6, 7, 8 hours, or more apart) within 24 hours, temperature of at least 38°C (e.g., at least 38.5°C)) in the subject.

108. The composition or method for use according to claim 107, wherein the IL - 6 inhibitor is administered after the dose of the CAR therapy (e.g., the first dose).

109. The composition or method for use according to claim 108, wherein the IL - 6 inhibitor is administered 1 hour to 10 days (e.g., 1 - 24 hours, 1 - 2 hours, 2 - 4 hours, 4 - 8 hours, 8 - 12 hours, 12 - 24 hours, 1 - 2 days, 2 - 3 days, 3 - 4 days, 4 - 5 days, 5 - 7 days, or 7 - 10 days) after the dose of the CAR therapy.

110. The composition or method for use according to any one of claims 105 - 109, comprising administering a dose of tocilizumab of about 5 - 15 mg / kg, such as 8 - 12 mg / kg (e.g., about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 11 mg / kg, or about 12 mg / kg).

111. A composition or method for use as described in any one of claims 105 - 110, wherein the subject has (e.g., is diagnosed as or identified as having) a high tumor burden prior to treatment with CAR therapy, such as wherein the high tumor burden is characterized by: at least 40% blasts (e.g., at least 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95% or more blasts) in the bone marrow of the subject prior to administration of CAR therapy (e.g., about 1 - 5 days prior to administration of CAR therapy).

112. A composition or method for use as described in any one of claims 105 - 111, wherein the CAR therapy comprises cells expressing a CD19 CAR, such as cells expressing CTL - 019.

113. An IL - 6 inhibitor (e.g., an anti - IL6 receptor inhibitor, such as tocilizumab), for use in treating or preventing cytokine release syndrome (CRS) associated with the use of chimeric antigen receptor (CAR) therapy (e.g., a population of cells expressing CAR in a subject), wherein the IL - 6 inhibitor is used 1 day before, simultaneously with, or within (e.g., within 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour or less) the dose of the CAR therapy (e.g., the first dose).

114. A method of treating or preventing cytokine release syndrome (CRS) associated with the administration of chimeric antigen receptor (CAR) therapy (e.g., a population of cells expressing CAR) in a subject, the method comprising administering to the subject an IL - 6 inhibitor (e.g., an anti - IL6 receptor inhibitor, such as tocilizumab) 1 day before, simultaneously with, or within (e.g., within 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour or less) the dose of the CAR therapy (e.g., the first dose).

115. A composition for use as described in claim 113 or a method as described in claim 114, wherein the IL - 6 inhibitor (e.g., tocilizumab) is administered to the subject (e.g., within 1 hour, 30 minutes, 20 minutes, 15 minutes or less) after the first sign of CRS symptoms (e.g., fever, such as characterized by: for example, two consecutive measurements (e.g., taken at least 4, 5, 6, 7, 8 hours or more apart) within 24 hours, temperature of at least 38°C).

116. The composition or method for use according to any one of claims 113 - 115, wherein the CAR comprises an antigen - binding domain that binds to one or more of the following: CD19; CD123; CD22; CD30; CD171; CS - 1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C - type lectin - like molecule - 1 (CLL - 1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2 - 8)aNeu5Ac(2 - 3)bDGalp(1 - 4)bDGlcp(1 - 1)Cer); TNF receptor family member B - cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAcα - Ser / Thr)); prostate - specific membrane antigen (PSMA); receptor tyrosine kinase - like orphan receptor 1 (ROR1); Fms - like tyrosine kinase 3 (FLT3); tumor - associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin - 13 receptor subunit α - 2 (IL - 13Ra2 or CD213A2); mesothelin; interleukin 11 receptor α (IL - 11Ra); prostate stem cell antigen (PSCA); protease serine 21 (testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet - derived growth factor receptor β (PDGFR - β); stage - specific embryonic antigen - 4 (SSEA - 4); CD20; folate receptor α; receptor tyrosine protein kinase ERBB2 (Her2 / neu); mucin 1, cell - surface - associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostate enzyme; Prostate Acid Phosphatase (PAP); Mutated Elongation Factor 2 (ELF2M); Ephrin B2; Fibroblast Activation Protein Alpha (FAP); Insulin-like Growth Factor 1 Receptor (IGF-I Receptor), Carbonic Anhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2); Glycoprotein 100 (gp100); Oncogenic Fusion Protein Composed of Breakpoint Cluster Region (BCR) and Abelson Murine Leukemia Viral Oncogene Homolog 1 (Abl) (bcr-abl); Tyrosinase; Ephrin A Type Receptor 2 (EphA2); Fucosyl GM1; Sialyl Lewis Adhesion Molecule (sLe); Ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); Transglutaminase 5 (TGS5); High Molecular Weight - Melanoma Associated Antigen (HMWMAA); o-Acetyl-GD2 Ganglioside (OAcGD2); Folate Receptor Beta; Tumor Endothelial Marker 1 (TEM1 / CD248); Tumor Endothelial Marker 7 Related (TEM7R); Claudin 6 (CLDN6); Thyroid Stimulating Hormone Receptor (TSHR); G Protein-Coupled Receptor Class C Group 5, Member D (GPRC5D); Chromosome X Open Reading Frame 61 (CXORF61); CD97; CD179a; Anaplastic Lymphoma Kinase (ALK); Polysialic Acid; Placenta Specific 1 (PLAC1); Hexasaccharide Portion of Globoside H Glycosphingolipid (GloboH); Breast Differentiation Antigen (NY-BR-1); Uroplakin 2 (UPK2); Hepatitis A Virus Cellular Receptor 1 (HAVCR1); Adrenergic Receptor Beta 3 (ADRB3); Pannexin 3 (PANX3); G Protein-Coupled Receptor 20 (GPR20); Lymphocyte Antigen 6 Complex, Locus K 9 (LY6K); Olfactory Receptor 51E2 (OR51E2); TCRγ Alternative Reading Frame Protein (TARP); Wilms Tumor Protein (WT1); Cancer / Testis Antigen 1 (NY-ESO-1); Cancer / Testis Antigen 2 (LAGE-1a); Melanoma Associated Antigen 1 (MAGE-A1); ETS Translocation Variant Gene 6, Located on Chromosome 12p (ETV6-AML); Spermatid Protein 17 (SPA17); X Antigen Family, Member 1A (XAGE1); Angiopoietin Binding Cell Surface Receptor 2 (Tie 2); Melanoma Cancer Testis Antigen-1 (MAD-CT-1); Melanoma Cancer Testis Antigen-2 (MAD-CT-2); Fos-Related Antigen 1; Tumor Protein p53 (p53); p53 Mutant; Prostein; Survivin; Telomerase; Prostate cancer tumor antigen-1 (PCTA-1 or Galectin-8); Melanoma antigen recognized by T cells 1 (MelanA or MART1); Rat sarcoma (Ras) mutant; Human telomerase reverse transcriptase (hTERT); Sarcoma translocation breakpoint; Melanoma apoptosis inhibitor (ML-IAP); ERG (Transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); Paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of Imprinted Sites-like protein); Squamous cell carcinoma antigen recognized by T cells 3 (SART3); Paired box protein Pax-5 (PAX5); Proacrosomal protein-binding protein sp32 (OY-TES1); Lymphocyte-specific protein tyrosine kinase (LCK); Kinase anchor protein 4 (AKAP-4); Synovial sarcoma, X breakpoint 2 (SSX2); Receptor for advanced glycation end products (RAGE-1); Renal ubiquitin 1 (RU1); Renal ubiquitin 2 (RU2); Podoplanin; Human papillomavirus E6 (HPV E6); Human papillomavirus E7 (HPV E7); Intestinal carboxylesterase; Mutated heat shock protein 70-2 (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); Bone marrow stromal cell antigen 2 (BST2); Mucin-like hormone receptor-like 2 containing EGF-like modules (EMR2); Lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); or Immunoglobulin lambda-like polypeptide 1 (IGLL1).

117. The composition or method for use according to any one of claims 113-115, wherein the antigen recognition domain binds to CD19.

118. The composition or method for use according to claim 116, wherein the CAR comprises the amino acid sequence of SEQ ID NO:

773.

119. A composition or method for use as described in any one of the preceding claims, wherein the CAR-expressing cells are administered at a dose (e.g., a total dose) of 1.5 x 10 7 to 5 x 10 9 cells / kg (e.g., 0.3 x 10 6 to 1 x 10 8 cells / kg), e.g., wherein the total dose is administered in multiple doses (e.g., a first dose, a second dose, and optionally a third dose).

120. The composition or method for use as described in claim 119, wherein, for example, the first dose administered on the first day comprises 10% of the total dose (e.g., about 1 x 10 7 cells / kg).

121. The composition or method for use as claimed in claim 120, wherein a second dose administered, for example, on a subsequent day (e.g., 1, 2, 3, 4, 5, 6, or 7 days after the first dose) comprises 30% of the total dose (e.g., about 3 x 10 7 cells / kg).

122. A composition or method for use as described in any one of claims 113-121, wherein the IL-6 inhibitor (such as tocilizumab) is administered at a dose of about 5-15 mg / kg, such as 8-12 mg / kg (for example, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 11 mg / kg, or about 12 mg / kg).

123. A pharmaceutical composition comprising: (i) a population of immune effector cells expressing a chimeric antigen receptor (CAR), wherein the CAR comprises a CD123 binding domain, a transmembrane domain, and an intracellular signaling domain; and (ii) a JAK-STAT inhibitor, such as ruxolitinib.

124. The pharmaceutical composition according to claim 123, wherein the composition further comprises an IL-6 inhibitor (such as an anti-IL6 receptor inhibitor, such as tocilizumab).

125. A pharmaceutical composition comprising (i) a CD123 chimeric antigen receptor (CAR) therapy (such as a population of immune effector cells expressing the CAR, wherein the CAR comprises a CD123 binding domain, a transmembrane domain, and an intracellular signaling domain); and (ii) a JAK-STAT inhibitor, such as ruxolitinib, the pharmaceutical composition being for the treatment of cancer or for the prevention of cytokine release syndrome (CRS).

126. The pharmaceutical composition according to claim 125, wherein the composition for use further comprises an IL-6 inhibitor (such as an anti-IL6 receptor inhibitor, such as tocilizumab).

127. A pharmaceutical composition comprising (i) a BTK inhibitor (such as ibrutinib); and (ii) a chimeric antigen receptor (CAR) therapy (such as a CD19 CAR therapy, such as CTL019 therapy); the pharmaceutical composition being for the prevention of CRS, for example, in a subject identified as or previously identified as being at risk of cytokine release syndrome (CRS).

128. The pharmaceutical composition according to claim 127, wherein the composition further comprises an IL-6 inhibitor (such as an anti-IL6 receptor inhibitor, such as tocilizumab).

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