Treatment and prevention of cytokine release syndrome using chimeric antigen receptor in combination with kinase inhibitor

Abstract

To provide a composition and a method for treating a disease associated with antigen expression or for treating or preventing cytokine release syndrome.SOLUTION: The disclosure provides a composition comprising a JAK-STAT inhibitor (e.g., ruxolitinib), in combination with a CAR therapy (e.g., a CD123 CAR therapy), for use in preventing cytokine release syndrome (CRS) in a subject in need thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 362,659, filed July 15, 2016, U.S. Provisional Patent Application No. 62 / 366,997, filed July 26, 2016, and U.S. Provisional Patent Application No. 62 / 381,230, filed August 30, 2016, the contents of all of which are incorporated herein by reference in their entireties.

[0002] The present invention generally relates to the use of immune effector cells (e.g., T cells or NK cells) engineered to express a chimeric antigen receptor (CAR) in combination with a kinase inhibitor (e.g., a JAK-STAT or BTK inhibitor) to treat disease and / or prevent cytokine release syndrome (CRS). [Background technology]

[0003] Many patients with hematological malignancies (e.g., B-cell malignancies) are incurable with standard therapies. Additionally, conventional treatment options often have severe side effects. Recent advances in chimeric antigen receptor (CAR)-modified autologous T cell (CART) therapy, which relies on redirecting T cells to appropriate cell surface molecules on cancer cells, such as 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., Non-Patent Document 1). Clinical results with murine-derived CART19 (i.e., "CTL019") have shown promise for complete remission in patients with CLL and pediatric ALL (see, e.g., Non-Patent Documents 2, 3, and 4). In addition to the ability of the chimeric antigen receptor on genetically modified T cells to recognize and destroy target cells, successful therapeutic T cell therapy requires the ability to proliferate and persist for long periods of time, as well as the ability to monitor leukemic cell escape. Variations in T cell quality, whether the result of anergy, suppression, or exhaustion, can affect the performance of CAR-transformed T cells, over which those skilled in the art currently have limited control. To be effective, CAR-transformed patient T cells must persist and maintain the ability to proliferate in response to target antigens. ALL patient T cells have been shown to be able to do this with CART19, which contains a murine scFv (see, e.g., Non-Patent Document 4). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Sadelain et al., Cancer Discovery 3:388-398(2013) [Non-patent document 2] Kalos et al., Sci Transl Med 3:95ra73(2011) [Non-patent document 3] Porter et al.,NEJM 365:725-733(2011) [Non-patent document 4] Grupp et al.,NEJM 368:1509-1518(2013) Summary of the Invention [Problem to be solved by the invention]

[0005] Cytokine release syndrome (CRS) is a common and serious adverse side effect of immune cell-based therapies, such as CAR T cell therapy. Severe CRS is a potentially life-threatening toxicity. Deaths have been reported due to severe cases of CRS. Diagnosis and management of CRS in response to immune cell-based therapies are typically based on clinical parameters and symptoms. See, for example, the CRS rating scale described by Lee, D. et al. (2014) Blood 124(2):188-195. While the interleukin-6 receptor blocker tocilizumab and steroids can reverse CRS, concerns remain that these approaches may impair antitumor efficacy. Furthermore, preclinical models of CRS after human CART are lacking. Preclinical models of CRS after human CART administration are needed. There is also a need for modalities to prevent CRS, which would increase the clinical feasibility of CART therapy. [Means for solving the problem]

[0006] The present disclosure is based, at least in part, on the discovery that a JAK-STAT kinase inhibitor, such as ruxolitinib, can reduce the severity of or prevent cytokine release syndrome (CRS) after CART cell therapy for hematological cancers such as acute myeloid leukemia (AML) without significantly impairing the anti-tumor efficacy of CART therapy. The present disclosure is also based, at least in part, on the discovery that a BTK inhibitor, such as ibrutinib, can ameliorate or prevent CRS after CD19 CAR therapy for B-cell neoplasms. In addition, the present disclosure is based, at least in part, on the discovery that an IL-6 inhibitor (e.g., which can be used for CRS prevention / treatment) can be administered in combination with (e.g., prior to, concurrently with, or after) CAR therapy 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 described herein, e.g., a cancer described herein, with a combination therapy comprising a CAR-expressing cell and a JAK-STAT or BTK inhibitor results in improved inhibition or reduction of tumor progression and / or reduced adverse effects (e.g., reduced CRS) in the subject, compared to, e.g., treating a subject having the disease with a CAR-expressing cell or a JAK-STAT or BTK inhibitor alone.

[0008] Accordingly, the disclosure features, at least in part, compositions and methods for treating disorders such as cancer (e.g., hematological cancers or other B-cell malignancies) using immune effector cells (e.g., T cells or NK cells) expressing a chimeric antigen receptor (CAR) molecule (e.g., a CAR that binds to a B-cell antigen, such as CD123 or cluster of differentiation 19 protein (CD19) (e.g., OMIM Accession No. 107265, Swiss Prot Accession No. P15391)). The compositions include, and the methods include administering, immune effector cells (e.g., T cells or NK cells) expressing a CAR (e.g., a CAR that targets B cells) in combination with a kinase inhibitor (e.g., one or more JAK-STAT inhibitors and / or BTK inhibitors). In some embodiments, the combination maintains or has better clinical efficacy (e.g., due to prevention of CRS) and / or has lower toxicity when compared to either therapy alone. In some embodiments, the subject is at risk for or has CRS, or the subject has been identified as having CRS or at risk for developing CRS.

[0009] The present disclosure further relates to the use of cells, e.g., immune effector cells (e.g., T cells or NK cells), engineered to express a CAR molecule that binds to an antigen (e.g., a tumor antigen described herein, e.g., a B cell antigen, e.g., CD123 or CD19), in combination with a kinase inhibitor (e.g., at least one JAK-TAT inhibitor) to treat a disorder associated with expression of a B cell antigen, e.g., CD123 or CD19 (e.g., a cancer, e.g., a hematological cancer).

[0010] Also provided herein are compositions and methods for preventing CRS in a subject by using a combination of a JAK-STAT inhibitor and a CAR-expressing cell (e.g., a CAR-expressing cell that targets a B cell, e.g., a CD123 CAR-expressing cell).

[0011] Also provided are compositions and methods for preventing CRS in a subject, e.g., the subject is at risk for or has CRS, or the subject has been identified as having CRS or at risk for developing CRS, by using a combination of a BTK inhibitor and a CAR-expressing cell (e.g., a CAR-expressing cell that targets a B cell, e.g., a CD19 CAR-expressing cell).

[0012] In one aspect, provided herein is a method of treating a subject, e.g., a mammal, having a disease associated with expression of an antigen, e.g., a tumor antigen, e.g., a tumor antigen described herein. The method includes administering to the subject an effective amount 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, e.g., a tumor antigen, e.g., a B cell antigen), in combination with a JAK-STAT inhibitor, e.g., a JAK-STAT inhibitor described herein, e.g., ruxolitinib.

[0013] In another aspect, provided herein is a method of conferring anti-tumor immunity to a subject, e.g., a mammal, having a disease associated with expression of an antigen, e.g., a tumor antigen, e.g., a tumor antigen described herein. The method includes administering to the subject an effective amount 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, e.g., a tumor antigen, e.g., a B cell antigen), in combination with a JAK-STAT inhibitor, e.g., a JAK-STAT inhibitor described herein, e.g., ruxolitinib.

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

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

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

[0018] In another aspect, provided herein is a method of preventing cytokine release syndrome (CRS) (e.g., CRS associated with CAR therapy, e.g., B cell antigen CAR therapy, e.g., CD19 CAR therapy) in a subject in need thereof, the method comprising administering to the subject a BTK inhibitor (e.g., ibrutinib), alone or in combination with CAR therapy, 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 administered, is being administered, or will be administered a CAR therapy, such as a CAR therapy described herein. In embodiments, the subject is identified or has previously been identified as being at risk for CRS.

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

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

[0022] In yet another aspect, provided herein are methods of treating or preventing CRS associated with administration of a cell, e.g., a cell population, that expresses a CAR in a subject.

[0023] In yet another aspect, provided herein are methods of treating or preventing CRS associated with the administration of a T cell inhibitory therapy, e.g., a CD19-blocking or depleting therapy, e.g., a therapy comprising a CD19 inhibitor. In embodiments, the CD19-blocking or depleting therapy is associated with CRS.

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

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

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

[0027] CAR molecule 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 costimulatory domain and / or a primary signaling domain).

[0028] In embodiments, the CAR may be any of the following: CD19; CD123; CD22; CD30; CD171; CS-1 (CD2 subset 1, also referred to as 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 members B cell maturation ( BCMA); Tn antigen ((TnAg) 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 (testisin 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); prostase; prostatic acid phosphatase (PAP) elongation factor 2 mutated (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, 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein (bcr-abl) consisting of the breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl); tyrosinase; ephrin type A 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 β; 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); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); globoH glycoceramide hexasaccharide moiety (Glo boH); mammary differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cell receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K9 (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); 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; prostein; surviving; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or galectin-8), melanoma antigen 1 recognized by T cells (Melan-A or MART1); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (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) Sites), squamous cell carcinoma antigen 3 recognized by T cells (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A-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; heat shock protein 70-2 mutant (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); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); or immunoglobulin lambda-like polypeptide 1 (IGLL1);

[0029] In other embodiments, the CAR molecule is capable of binding to an antigen described herein, for example, an antigen described in the "Antigens" section below.

[0030] 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.

[0031] In an embodiment, the antigen is CD 123. In an embodiment, the antigen is CD19.

[0032] In other embodiments, the antigen is BCMA. In embodiments, the antigen is CLL.

[0033] Exemplary CAR Molecules In one embodiment, the CAR molecule comprises a CD123 CAR described herein, such as a CD123 CAR described in U.S. Patent Application Publication No. 2014 / 0322212A1 or U.S. Patent Application Publication No. 2016 / 0068601A1 (both of which are incorporated by reference herein). In an embodiment, the CD123 CAR comprises the amino acid or has the nucleotide sequence set forth in U.S. Patent Application Publication No. 2014 / 0322212A1 or U.S. Patent Application Publication No. 2016 / 0068601A1 (both of which are incorporated by reference herein).

[0034] In embodiments, the CAR molecule comprises a CD19 CAR molecule described herein, such as a CD19 CAR molecule described in U.S. Patent Application Publication No. 2015-0283178-A1, e.g., CTL019. In embodiments, the CD19 CAR comprises the amino acid or has the nucleotide sequence set forth in U.S. Patent Application Publication No. 2015-0283178-A1, which is incorporated herein by reference.

[0035] In one embodiment, the CAR molecule comprises a BCMA CAR molecule described herein, such as the BCMA CAR described in U.S. Patent Application Publication No. 2016-0046724-A1. In an embodiment, the BCMA CAR comprises the amino acid or has the nucleotide sequence set forth in U.S. Patent Application Publication No. 2016-0046724-A1, which is incorporated herein by reference.

[0036] In one embodiment, the CAR molecule comprises a CLL1 CAR described herein, such as the CLL1 CAR described in U.S. Patent Application Publication No. 2016 / 0051651A1, which is incorporated herein by reference. In an embodiment, the CLL1 CAR comprises the amino acid or has the nucleotide sequence set forth in U.S. Patent Application Publication No. 2016 / 0051651A1, which is incorporated herein by reference.

[0037] In one embodiment, the CAR molecule comprises a CD33 CAR described herein, such as the CD33 CAR described in U.S. Patent Application Publication No. 2016 / 0096892A1, which is incorporated herein by reference. In an embodiment, the CD33 CAR comprises the amino acid or has the nucleotide sequence set forth in U.S. Patent Application Publication No. 2016 / 0096892A1, which is incorporated herein by reference.

[0038] In some embodiments, the CAR molecule comprises an EGFRvIII CAR molecule described herein, such as the EGFRvIII CAR described in U.S. Patent Application Publication No. 2014 / 0322275A1, which is incorporated herein by reference. In embodiments, the EGFRvIII CAR comprises the amino acid or has the nucleotide sequence set forth in U.S. Patent Application Publication No. 2014 / 0322275A1, which is incorporated herein by reference.

[0039] In some embodiments, the CAR molecule comprises a mesothelin CAR described herein, such as the mesothelin CAR described in WO 2015 / 090230 (incorporated herein by reference). In embodiments, the mesothelin CAR comprises the amino acid or has the nucleotide sequence set forth in WO 2015 / 090230 (incorporated herein by reference).

[0040] CD123 CAR antigen-binding domain In embodiments, the CAR molecule is capable of binding to CD123 (e.g., wild-type or mutant CD123). In embodiments, the CAR molecule comprises one or more (e.g., all three) 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 an anti-CD123 binding domain described herein (e.g., as described in U.S. Patent Application Publication No. 2014 / 0322212A1 or U.S. Patent Application Publication No. 2016 / 0068601A1), and / or one or more (e.g., all three) 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 an anti-CD123 binding domain described herein (e.g., as described in U.S. Patent Application Publication No. 2014 / 0322212A1 or U.S. Patent Application Publication No. 2016 / 0068601A1). and one or more, such as all three LC CDRs and one or more, such as all three HC CDRs.

[0041] In one embodiment, the encoded CD123 binding domain comprises one or more (e.g., all three) 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 a CD123 binding domain described herein, and / or one or more (e.g., all three) 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 a CD123 binding domain described herein, e.g., including 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 a light chain variable region described herein (e.g., in Table 11A, 12A, or 12B) and / or a heavy chain variable region described herein (e.g., in Table 11A, 12A, or 12B). In one embodiment, the encoded CD123 binding domain is an scFv comprising light and heavy chain 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 comprising an amino acid sequence having at least one, two, or three alterations (e.g., substitutions, e.g., conservative substitutions) but not more than 30, 20, or 10 alterations (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequence of a light chain variable region provided in Table 11A, 12A, or 12B, or a sequence having at least 95%, e.g., 95-99%, identity to an amino acid sequence of Table 11A, 12A, or 12B; and / or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three alterations (e.g., substitutions, e.g., conservative substitutions) but not more than 30, 20, or 10 alterations (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequence of a heavy chain variable region provided in Table 11A, 12A, or 12B, or a sequence having at least 95% (e.g., 95-99%) identity to an amino acid sequence of Table 11A, 12A, or 12B.

[0042] In other embodiments, the encoded CD123 binding domain comprises the HC CDR1, HC CDR2, and HC CDR3 of any CD123 heavy chain binding domain amino acid sequence listed in Table 11A, 12A, or 12B. In embodiments, the CD33 binding domain further comprises an LC CDR1, LC CDR2, and LC CDR3. In embodiments, the CD123 binding domain comprises the LC CDR1, LC CDR2, and LC CDR3 of any CD123 light chain binding domain amino acid sequence listed in Table 11A, 12A, or 12B.

[0043] In some embodiments, the encoded CD123 binding domain comprises one, two, or all of the LC CDR1, LC CDR2, and LC CDR3 of any CD123 light chain binding domain amino acid sequence listed in Table 11A or 12B, and one, two, or all of the HC CDR1, HC CDR2, and HC CDR3 of any CD123 heavy chain binding domain amino acid sequence listed in Table 11A, 12A, or 12B.

[0044] In one embodiment, the encoded CD123-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 157-160, 184-215, 478, 480, 483, and 485. In certain embodiments, the encoded CD123-binding domain (e.g., scFv) comprises an amino acid sequence having at least one, two, or three alterations (e.g., substitutions, e.g., conservative substitutions) but not more than 30, 20, or 10 alterations (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequence of SEQ ID NOs: 157-160, 184-215, 478, 480, 483, and 485, or a sequence at least 95% identical to (e.g., 95-99% identity to) the amino acid sequence of SEQ ID NOs: 157-160, 184-215, 478, 480, 483, and 485.

[0045] In another embodiment, the encoded CD123-binding domain comprises a heavy chain variable region comprising 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 alterations (e.g., substitutions, e.g., conservative substitutions) but not more than 30, 20, or 10 alterations (e.g., substitutions, e.g., conservative substitutions) of SEQ ID NO: 216-219 or 243-274, or a sequence at least 95% identical to (e.g., 95-99% identity to) SEQ ID NO: 216-219 or 243-274. In another embodiment, the encoded CD123 binding domain comprises a heavy chain variable region comprising an amino acid sequence corresponding to the heavy chain variable region of SEQ ID NO: 478, 480, 483, or 485, or an amino acid sequence having at least one, two, or three alterations (e.g., substitutions, e.g., conservative substitutions) but not more than 30, 20, or 10 alterations (e.g., substitutions, e.g., conservative substitutions) of a corresponding portion of SEQ ID NO: 478, 480, 483, or 485, or a sequence at least 95% identical to (e.g., having 95-99% identity to) a corresponding portion of SEQ ID NO: 478, 480, 483, or 485.

[0046] 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 alterations (e.g., substitutions, e.g., conservative substitutions) but not more than 30, 20, or 10 alterations (e.g., substitutions, e.g., conservative substitutions) of SEQ ID NOs: 275-278 or 302-333, or a sequence at least 95% identical to (e.g., 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 an amino acid sequence having at least one, two, or three alterations (e.g., substitutions, e.g., conservative substitutions) but not more than 30, 20, or 10 alterations (e.g., substitutions, e.g., conservative substitutions) of a corresponding portion of SEQ ID NO: 478, 480, 483, or 485, or a sequence at least 95% identical to (e.g., having 95-99% identity to) a corresponding portion of SEQ ID NO: 478, 480, 483, or 485.

[0047] In one embodiment, the nucleic acid molecule encoding the scFv comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 479, 481, 482, and 484, or a sequence having at least 95% identity thereto, for example, 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 portion corresponding to the heavy chain variable region and / or the light chain variable region of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 479, 481, 482, and 484, or a sequence having at least 95% identity thereto, for example, 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 NOs: 157 to 160, or a sequence at least 95% identical thereto (for example, having 95-99% identity thereto). In one embodiment, the nucleic acid molecule encodes an scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 184 to 215, or a sequence having at least 95% identity, for example, 95 to 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, and the encoded amino acid sequence is selected from the group consisting of SEQ ID NOs: 184 to 215, or a sequence having at least 95% identity, for example, 95 to 99% identity thereto.

[0048] In one embodiment, the encoded 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 heavy chain variable region of the scFv can be in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region, for example.

[0049] CD19 CAR antigen-binding domain In embodiments, the CAR molecule is capable of binding to CD19 (e.g., wild-type or mutant CD19). In embodiments, the CAR molecule comprises an anti-CD19 binding domain comprising one or more (e.g., all three) 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 an anti-CD123 binding domain described herein, and / or one or more (e.g., all three) 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 an anti-CD19 binding domain described herein, e.g., comprising an anti-CD19 binding domain comprising one or more, e.g., all three LC CDRs, and one or more, e.g., all three HC CDRs.

[0050] In one embodiment, the anti-CD19 binding domain comprises one or more (e.g., all three) 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 an anti-CD19 binding domain described herein; for example, the anti-CD19 binding domain has two variable heavy chain regions, each comprising an HC CDR1, HC CDR2, and HC CDR3 described herein. In one embodiment, the anti-CD19 binding domain comprises a murine light chain variable region described herein (e.g., in Table 14A) and / or a murine heavy chain variable region described herein (e.g., in Table 14A). In one embodiment, the anti-CD19 binding domain is an scFv comprising a murine light chain and a murine heavy chain of the amino acid sequences in Table 14A. In one embodiment, the 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 alterations (e.g., substitutions), but not more than 30, 20, or 10 alterations (e.g., substitutions), of the amino acid sequence of a light chain variable region provided in Table 14A, or a sequence having at least 95% identity, e.g., 95-99% identity, to an amino acid sequence of Table 14A, and / or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three alterations (e.g., substitutions), but not more than 30, 20, or 10 alterations (e.g., substitutions), of the amino acid sequence of a heavy chain variable region provided in Table 14A, or a sequence having at least 95% identity, e.g., 95-99% identity, to an 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 thereof. In one embodiment, the anti-CD19 binding domain is an scFv, and a light chain variable region comprising an amino acid sequence described herein, e.g., in Table 14A, is attached to a heavy chain variable region comprising an amino acid sequence described herein, e.g., in Table 14A, via a linker, e.g., a linker described herein. 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, for example, in either of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0051] In one embodiment, the CAR molecule comprises a humanized anti-CD19 binding domain comprising one or more (e.g., all three) 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 a humanized anti-CD19 binding domain described herein, and one or more (e.g., all three) 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 a humanized anti-CD19 binding domain described herein, e.g., a humanized anti-CD19 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 anti-CD19 binding domain comprises at least HC CDR2. In one embodiment, the humanized anti-CD19 binding domain comprises one or more (e.g., all three) 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 a humanized anti-CD19 binding domain described herein; for example, the humanized anti-CD19 binding domain has two variable heavy chain regions, each comprising an HC CDR1, HC CDR2, and HC CDR3 described herein. In one embodiment, the humanized anti-CD19 binding domain comprises at least an 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 a modification (e.g., a substitution, e.g., a substitution of one or more amino acids found at the corresponding positions in the murine light chain variable region of SEQ ID NO: 773, e.g., a substitution 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 comprises a modification (e.g., a substitution, e.g., a substitution of one or more amino acids found at the corresponding positions in the murine heavy chain variable region of SEQ ID NO: 773, e.g., a substitution at one or more of positions 71, 73 and 78).In one embodiment, the humanized anti-CD19 binding domain comprises a light chain variable region described herein (e.g., in Table 13A) and / or a heavy chain variable region described herein (e.g., in Table 13A). In one embodiment, the humanized anti-CD19 binding domain is an scFv comprising light and heavy chains of the amino acid sequences in Table 13A. In one embodiment, the 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 alterations (e.g., substitutions), but not more than 30, 20, or 10 alterations (e.g., substitutions), of the amino acid sequence of a light chain variable region provided in Table 13A, or a sequence having at least 95% identity, e.g., 95-99% identity, to an 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 alterations (e.g., substitutions), but not more than 30, 20, or 10 alterations (e.g., substitutions), of the amino acid sequence of a heavy chain variable region provided in Table 13A, or a sequence having at least 95% identity, e.g., 95-99% identity, to an 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, e.g., 95-99% identity thereof. In one embodiment, the humanized anti-CD19 binding domain is an scFv, and a light chain variable region comprising an amino acid sequence described herein, e.g., in Table 13A, is attached to a heavy chain variable region comprising an amino acid sequence described herein, e.g., in Table 13A, via a linker, e.g., a linker described herein.

[0052] In embodiments, the antigen recognition domain binds to CD19. In embodiments, the CAR comprises the amino acid sequence of a CD19 CAR described herein. In embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 773.

[0053] In one embodiment, the humanized 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 of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region, for example.

[0054] Other CAR domains In one embodiment, the CAR molecule comprises a transmembrane domain of 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. 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 alterations (e.g., substitutions) but not more than 20, 10, or 5 alterations (e.g., substitutions) of the amino acid sequence of SEQ ID NO: 6, or a sequence having at least 95% identity, e.g., 95-99% identity, to the amino acid sequence of SEQ ID NO: 6.

[0055] In one embodiment, the antigen-binding domain (e.g., a CD123- or CD19-binding domain) is connected to the transmembrane domain by a hinge region, e.g., a 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 thereto, e.g., 95-99% identity thereto.

[0056] In one embodiment, the CAR molecule further comprises a sequence encoding a costimulatory domain, such as a costimulatory domain described herein. In one embodiment, the costimulatory 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 costimulatory domain comprises the sequence of SEQ ID NO: 7. In one embodiment, the costimulatory domain comprises the sequence of SEQ ID NO: 8. In one embodiment, the costimulatory domain comprises the sequence of SEQ ID NO: 43. In one embodiment, the costimulatory domain comprises the sequence of SEQ ID NO: 45. In one embodiment, the costimulatory domain comprises an amino acid sequence having at least one, two, or three alterations (e.g., substitutions) but not more than 20, 10, or 5 alterations (e.g., substitutions) of the amino acid sequence of SEQ ID NO: 7, 8, 43, or 45, or a sequence having at least 95% identity, e.g., 95-99% identity, to the amino acid sequence of SEQ ID NO: 7, 8, 43, or 45.

[0057] In one embodiment, the CAR molecule further comprises a sequence encoding an intracellular signaling domain, e.g., an intracellular signaling domain described herein. In one embodiment, the intracellular signaling domain comprises a functional signaling domain of 4-1BB and / or a 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 a functional signaling domain of CD27 and / or a 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 alterations (e.g., substitutions) but not more than 20, 10, or five alterations (e.g., 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, e.g., 95-99% identity, to 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, and the sequences comprising the intracellular signaling domain are expressed in the same frame and as a single polypeptide chain.

[0058] In one embodiment, the CAR molecule further comprises a leader sequence, e.g., a 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, e.g., 95-99% identity, to the amino acid sequence of SEQ ID NO: 1.

[0059] CD123 CAR construct In embodiments, the CAR molecule comprises a leader sequence, such as a leader sequence described herein, such as the leader sequence of SEQ ID NO: 1, or one having at least 95% identity thereto, such as 95-99% identity thereto, a CD123 binding domain described herein, such as LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2 and HC CDR3 described herein. a CD123-binding domain comprising a CDR3, e.g., a CD123-binding domain described in Table 11A or 12A, or a sequence having at least 95% identity, e.g., 95-99% identity, thereof; a hinge region, e.g., a hinge region described herein, e.g., the hinge region of SEQ ID NO: 2, or one having at least 95% identity, e.g., 95-99% identity, thereof; a transmembrane domain, e.g., a transmembrane domain described herein, e.g., a transmembrane domain having the sequence of SEQ ID NO: 6, or a sequence having at least 95% identity, e.g., 95-99% identity, thereof; an intracellular signaling domain, e.g., an intracellular signaling domain described herein (e.g., an intracellular signaling domain comprising a costimulatory domain and / or a primary signaling domain). In one embodiment, the intracellular signaling domain comprises a costimulatory domain, e.g., a costimulatory domain described herein, e.g., a 4-1BB costimulatory domain having the sequence of SEQ ID NO: 7 or at least 95% identity, e.g., 95-99% identity, thereof, and / or a primary signaling domain, e.g., a primary signaling domain described herein, e.g., a CD3ζ stimulatory domain having the sequence of SEQ ID NO: 9 or SEQ ID NO: 10 or at least 95% identity, e.g., 95-99% identity, thereof. In one embodiment, the intracellular signaling domain comprises a costimulatory domain, e.g., a costimulatory domain described herein, e.g., a 4-1BB costimulatory domain having the sequence of SEQ ID NO: 7, and / or a primary signaling domain, e.g., a primary signaling domain described herein, e.g., a CD3ζ stimulatory domain having the sequence of SEQ ID NO: 9 or SEQ ID NO: 10.

[0060] CD19 CAR construct In one embodiment, the CAR molecule comprises a leader sequence, such as a leader sequence described herein, such as the leader sequence of SEQ ID NO: 1, or one having at least 95% identity thereto, such as 95-99% identity thereto; an anti-CD19 binding domain described herein, such as LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2 and HC CDR3 described herein; an anti-CD19 binding domain comprising a CDR3, such as a murine anti-CD19 binding domain described in Table 14A, a humanized anti-CD19 binding domain described in Table 13A, or a sequence with 95 to 99% identity thereof; a hinge region, such as a hinge region described herein, such as the hinge region of SEQ ID NO: 2, 3, or 4, or one with at least 95% identity, such as 95 to 99% identity thereof; a transmembrane domain, such as a transmembrane domain described herein, such as a transmembrane domain having the sequence of SEQ ID NO: 6, or a sequence with at least 95% identity, such as 95 to 99% identity thereof; an intracellular signaling domain, such as an intracellular signaling domain described herein (e.g., an intracellular signaling domain comprising a costimulatory domain and / or a primary signaling domain). In one embodiment, the intracellular signaling domain comprises a costimulatory domain, e.g., a costimulatory domain described herein, e.g., a 4-1BB costimulatory domain having the sequence of SEQ ID NO: 7, a CD28 costimulatory domain having the sequence of SEQ ID NO: 43, a CD27 costimulatory domain having the sequence of SEQ ID NO: 8, or an ICOS costimulatory domain having the sequence of SEQ ID NO: 45, or at least 95% identity, e.g., 95-99% identity, thereof, and / or a primary signaling domain, e.g., a primary signaling domain described herein, e.g., a CD3ζ stimulatory domain having the sequence of SEQ ID NO: 9 or SEQ ID NO: 10, or at least 95% identity, e.g., 95-99% identity, thereof.

[0061] Other Exemplary CAR Constructs In one embodiment, the CAR molecule is a CAR molecule described in U.S. Patent Application Publication No. 2015-0283178-A1, U.S. Patent Application Publication No. 2016-0046724-A1, U.S. Patent Application Publication No. 2014 / 0322212A1, U.S. Patent Application Publication No. 2016 / 0068601A1, U.S. Patent Application Publication No. 2016 / 0051651A1, U.S. Patent Application Publication No. 2016 / 0096892A1, U.S. Patent Application Publication No. 2014 / 0322275A1, or is an amino acid sequence described in WO 2015 / 090230; or U.S. Patent Application Publication Nos. 2015-0283178-A1, 2016-0046724-A1, 2014 / 0322212A1, 2016 / 0068601A1, 2016 / 0051651A1, 2016 / 0096892A1, an amino acid sequence having 1, 2, 3, 4, 5, 10, 15, 20, or 30 or more alterations (e.g., substitutions) but not more than 60, 50, or 40 alterations (e.g., substitutions) of an amino acid sequence described in U.S. Patent Application Publication No. 2014 / 0322275A1 or WO 2015 / 090230; or an amino acid sequence described in U.S. Patent Application Publication No. 2015-0283178-A1, U.S. Patent Application Publication No. 2016-0046724-A1, U.S. Patent Application Publication No. 2014 / 0322275A1, U.S. Patent Application Publication No. 2015 ... 12A1, U.S. Patent Application Publication No. 2016 / 0068601A1, U.S. Patent Application Publication No. 2016 / 0051651A1, U.S. Patent Application Publication No. 2016 / 0096892A1, U.S. Patent Application Publication No. 2014 / 0322275A1, or WO 2015 / 090230.

[0062] vector In one embodiment, the cell expressing the CAR molecule comprises a vector comprising a nucleic acid sequence encoding the CAR molecule. In one embodiment, the vector is selected from the group consisting of DNA, RNA, a plasmid, a lentiviral vector, an adenoviral vector, or a 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 transcription vector, e.g., a vector that transcribes RNA of a nucleic acid molecule described herein. In one embodiment, the nucleic acid sequence of the in vitro vector further comprises a poly(A) tail, e.g., a polyA tail comprising about 150 adenosine bases (SEQ ID NO: 30), e.g., as described herein. In one embodiment, the nucleic acid sequence of the in vitro vector further comprises a 3'UTR, e.g., a 3'UTR comprising at least one repeat of a 3'UTR derived from human β-globulin, e.g., as described herein. In one embodiment, the nucleic acid sequence of the in vitro vector further comprises a promoter, for example, a T2A promoter.

[0063] CAR-expressing cells In certain embodiments of the compositions and methods disclosed herein, the cell expressing a CAR molecule (also referred to herein as a "CAR-expressing cell") is a cell or cell population as described herein, e.g., a human immune effector cell or cell population (e.g., a human T cell or a human NK cell, e.g., a human T cell described herein or a human NK cell described herein). In one embodiment, the human T cell is a CD8+ T cell. 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, wherein the T cell is diacylglycerol kinase (DGK) deficient. In one embodiment, the cell is a T cell, wherein the T cell is Ikaros deficient. In one embodiment, the cell is a T cell, wherein the T cell is both DGK and Ikaros deficient. It should be understood that compositions and methods disclosed herein that describe the term "cell" encompass compositions and methods comprising one or more cells, e.g., cell populations.

[0064] In some embodiments, the administered CAR-expressing cells comprise a regulatable CAR (RCAR), e.g., an RCAR as described herein. The RCAR can comprise, e.g., 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, e.g., a B cell antigen, e.g., CD123 or CD19), and a second switch domain, and a transmembrane domain. The method can further comprise, e.g., administering a dimerization molecule in an amount sufficient to cause dimerization of the first switch domain and the second switch domain.

[0065] inhibitors In embodiments, the JAK-STAT inhibitor comprises / is an antibody molecule, a small molecule, a polypeptide, such as a fusion protein, or an inhibitory nucleic acid, such as an 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), decernotinib (V-509), INCB39110, GEN1, GEN2, GLPG0634, NS018, and N-(cyanomethyl)-4-[2-(4-morpholinoanilino)pyrimidin-4-yl]benzamide, or a pharmaceutically acceptable salt thereof. In an embodiment, the JAK-STAT inhibitor is ruxolitinib or a pharmaceutically acceptable salt thereof.

[0066] In embodiments, the BTK inhibitor comprises / is an antibody molecule, a small molecule, a polypeptide, e.g., a fusion protein, or an inhibitory nucleic acid, e.g., siRNA or shRNA. In embodiments, the BTK inhibitor is a small molecule, e.g., 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.

[0067] In embodiments, the IL-6 inhibitor, e.g., an IL-6 inhibitor used in any composition or method described herein, includes an inhibitor of IL-6 signaling, including, for example, an IL-6 inhibitor or an IL-6 receptor (IL-6R) inhibitor. Exemplary IL-6 inhibitors include tocilizumab, siltuximab, bazedoxifene, and soluble glycoprotein 130 (sgp130) blockers. Exemplary IL-6 inhibitors are described in International Publication No. 2014011984 (hereby incorporated by reference). Tocilizumab is described in further 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 such as siltuximab. In other embodiments, the inhibitor includes a soluble gp130 or a fragment thereof capable of blocking IL-6 signaling. In some embodiments, sgp130 or a fragment thereof is fused to a heterologous domain, e.g., an Fc domain, e.g., a gp130-Fc fusion protein such as FE301. In embodiments, the IL-6 inhibitor comprises an antibody against the IL-6 receptor, e.g., sarilumab, olokizumab (CDP6038), elcilimomab, sirukumab (CNTO 136), ALD518 / BMS-945429, ARGX-109, or FM101. In some embodiments, the IL-6 inhibitor comprises a small molecule, such as CPSI-2364.

[0068] disease In embodiments, the disease associated with expression of the antigen is a hyperproliferative disorder, such as cancer. In embodiments, the cancer is a solid cancer. In other embodiments, the cancer is a hematological cancer.

[0069] In embodiments, the blood cancer is leukemia. In embodiments, the blood cancer is acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), or chronic lymphocytic leukemia (CLL). In embodiments, the blood cancer is lymphoma, such as mantle cell lymphoma (MCL).

[0070] In embodiments, the hematological cancer is a B-cell malignancy, such as a B-cell leukemia or a B-cell lymphoma.

[0071] In embodiments, the hematological cancer is chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphocytic leukemia (ALL), Hodgkin's lymphoma, B-cell acute lymphocytic leukemia (BALL), T-cell acute lymphocytic leukemia (TALL), small lymphocytic leukemia (SLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, follicular lymphoma, childhood follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma (extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue), marginal zone lymphoma, myelodysplasia and myelodysplastic syndromes, non-small cell lung cancer, leukemia ... Selected from Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's 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, plasma cell myeloma, isolated bone plasmacytoma, extraskeletal plasmacytoma, nodal marginal zone lymphoma, childhood 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, B-cell lymphoma, or unclassifiable lymphoma.

[0072] 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 myelogenous leukemia (CML), hairy cell leukemia, Hodgkin's lymphoma, histiocytic disorders, mast cell disorders, myelodysplasia, myelodysplastic syndromes, myeloproliferative neoplasms, plasma cell myeloma, plasmacytoid dendritic cell neoplasms, or a combination thereof.

[0073] In embodiments, 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 embodiments, the disease associated with B cell antigen expression is selected from a proliferative disease, e.g., a cancer, a malignancy, or a precancerous condition, e.g., myelodysplasia, myelodysplastic syndrome, or preleukemia, or is a non-cancer-related indication associated with expression of one or more B cell antigens, e.g., CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, and / or CD79a. In certain embodiments, the disease associated with B cell antigen expression is a "preleukemia," which is a group of diverse hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells. In some embodiments, diseases associated with B cell antigen expression include, but are not limited to, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative disorders that express B cell antigens (e.g., one or more of CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, and / or CD79a). In embodiments, the disease associated with B cell antigen expression is a hematological cancer, leukemia, lymphoma, MCL, CLL, ALL, Hodgkin's lymphoma, or multiple myeloma. Any combination of diseases associated with B cell antigen expression described herein can be treated by the methods and compositions described herein.

[0074] CRS In embodiments, the CRS is severe CRS, e.g., grade 4 or 5 CRS. In embodiments, the CRS is less severe CRS, e.g., grade 1, 2, or 3 CRS. Further description of CRS is provided in the section entitled "Cytokine Release Syndrome."

[0075] In embodiments of any of the methods described herein, the CRS is CRS that is distinguished from sepsis, e.g., by a method described herein, e.g., a method of distinguishing CRS from sepsis in a subject as described herein. In embodiments, the method of distinguishing CRS from sepsis includes taking one or more of the following measurements: (i) the level or activity of one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, 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 higher level or activity than the reference is indicative of CRS; or (ii) the level or activity of one or more (e.g., two, three, four, five, six, or all) of CD163, IL-1β, sCD30, sIL-4R, sRAGE, sVEGFR-1, and sVEGFR-2, where a level or activity higher than the reference is indicative of sepsis. Further embodiments of methods for distinguishing between CRS and sepsis in a subject are described herein.

[0076] Dosing regimen In some embodiments, the CAR-expressing cells and the inhibitor (e.g., a JAK-STAT or BTK inhibitor) are administered sequentially, concurrently, or within a therapeutic interval, e.g., as described herein.

[0077] In one embodiment, the CAR-expressing cells and the 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 prior to administration of the CAR-expressing cells. In one embodiment, the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered after administration of the CAR-expressing cells.

[0078] In one embodiment, the inhibitor (e.g., a JAK-STAT or BTK inhibitor) and the CAR-expressing cells are administered simultaneously or concurrently.

[0079] In embodiments, the CAR-expressing cells and the inhibitor (e.g., a JAK-STAT or BTK inhibitor) are administered in a treatment interval. In one embodiment, the treatment 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 treatment interval comprises multiple doses (e.g., a first and a second dose) of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) and a dose of the CAR-expressing cells (e.g., in any order).

[0080] Where a treatment interval comprises a single dose of an inhibitor (e.g., a JAK-STAT or BTK inhibitor) and a single dose of 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 concurrently. 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 embodiments, a treatment interval begins with administration of the first administered dose and ends with administration of the last administered dose.

[0081] Where a treatment interval comprises a single dose of an inhibitor (e.g., a JAK-STAT or BTK inhibitor) and a single dose of 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 embodiments, 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 with the administration of the dose of the CAR-expressing cells and ends with the administration of the dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor). 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 with the administration of the dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) and ends with the administration of the dose of the CAR-expressing cells. 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 an inhibitor (e.g., a JAK-STAT or BTK inhibitor). In such an embodiment, the treatment interval comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses of an inhibitor (e.g., a JAK-STAT or BTK inhibitor) and a dose of a CAR-expressing cell. In one embodiment, the dose of the CAR-expressing cell 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 embodiments in which more than one dose of an inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered, the dose of 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 start of a treatment interval.In embodiments, when more than one dose of an inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered, the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered about 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 24 hours, 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.

[0082] Where a treatment interval comprises multiple doses (e.g., first and second and optionally subsequent doses) of an inhibitor (e.g., a JAK-STAT or BTK inhibitor) and a dose of CAR-expressing cells, 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, for example, within 2 days of each other (e.g., within 2 days, 1 day, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, or less). In embodiments, the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered after the later of (i) the dose of CAR-expressing cells or (ii) the first dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor). In embodiments, the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered at least 8 hours (e.g., at least 8, 9, 10, 12, 14, 16, 18, 20, 24 hours, 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 more) after (i) or (ii). In embodiments, a subsequent dose (e.g., a third, fourth, or fifth dose, etc.) of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered after the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor). In embodiments, the subsequent dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered at least 8 hours (e.g., at least 8, 9, 10, 12, 14, 16, 18, 20, 24 hours, 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 more) after the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor). In such embodiments, the treatment interval begins with administration of the initial dose and ends with administration of the second (or subsequent) dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor).In embodiments, the dose of inhibitor (e.g., JAK-STAT or BTK inhibitor) is administered once daily (QD) or twice daily (BID) over a treatment interval 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. Any of the treatment intervals described herein can include one or more doses of CAR-expressing cells.

[0083] In other embodiments, in which the treatment interval comprises multiple doses (e.g., first and second, and optionally subsequent doses) of an inhibitor (e.g., a JAK-STAT or BTK inhibitor) and a dose of a CAR-expressing cell, the dose of the CAR-expressing cell and the first dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) are administered sequentially. In embodiments, the dose of the CAR-expressing cell is administered after the administration of the first dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) but before the administration of the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor). In embodiments, a subsequent dose (e.g., a third, fourth, or fifth dose, etc.) of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered after the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor). In such embodiments, a treatment interval begins with administration of a first dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) and ends with administration of a second, third, fourth, fifth, or sixth dose (or subsequent doses) of the inhibitor (e.g., a JAK-STAT or BTK inhibitor). In one embodiment, the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered at least 8 hours (e.g., at least 8, 9, 10, 12, 14, 16, 18, 20, 24 hours, 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 more) after administration of the first dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor). In one embodiment, the subsequent dose (e.g., the third, fourth, or fifth dose) of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered at least 8 hours (e.g., at least 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 24 hours, 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 more) after the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor).In one embodiment, the dose of CAR-expressing cells is administered at least one 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 more) after administration of the first dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor). In one embodiment, the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered within one day (e.g., within 24 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, or less) of administration of the dose of CAR-expressing cells. In an embodiment, the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered concurrently with the dose of CAR-expressing cells. In one embodiment, the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered at least one day (e.g., at least 1, 2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4, 5, or more weeks) after administration of the dose of CAR-expressing cells. In an embodiment, the treatment interval comprises continuous administration of the inhibitor (e.g., a JAK-STAT or BTK inhibitor), e.g., once daily, twice daily, three times daily, every two days, every three days, or every four days. In embodiments in which the inhibitor is administered continuously, the dose of CAR-expressing cells (e.g., a first dose) is administered after the first dose of the inhibitor, e.g., at least one day, e.g., at least 1, 2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4, 5, 6 weeks, 1, 2, 3, 4, 5, 6 months or more. In other embodiments in which the inhibitor is administered continuously, the dose of CAR-expressing cells (e.g., the first dose) is administered concurrently (e.g., within one day (e.g., within 24 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, or less) with the administration of the first dose of the inhibitor. In embodiments in which the inhibitor is administered continuously, the inhibitor is administered at least one day, e.g., at least 1, 2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4, 5, 6 weeks, 1, 2, 3, 4, 5, 6 months or more after the administration of the first dose of the CAR-expressing cells.In other embodiments, the dose of CAR-expressing cells is administered prior to administration of the first dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor). In such embodiments, the treatment interval begins with administration of the CAR-expressing cells and ends with administration of the second dose (or subsequent dose) of the inhibitor (e.g., a JAK-STAT or BTK inhibitor). In embodiments, the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered at least 8 hours (e.g., at least 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 24 hours, 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 more) after administration of the first dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor). In embodiments, the subsequent dose (e.g., the third, fourth, or fifth dose, etc.) of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered at least 8 hours (e.g., at least 8, 9, 10, 12, 14, 16, 18, 20, 24 hours, 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 more) after the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor). In embodiments, the 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 more) after administration of the CAR-expressing cells. In embodiments, the dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered once daily (QD) or twice daily (BID) over a treatment interval 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.

[0084] In one embodiment, any of the treatment intervals described herein can be repeated one or more additional times, for example, 1, 2, 3, 4, or 5 times. In one embodiment, the treatment interval is repeated once, resulting in a treatment regimen including two treatment intervals. In some embodiments, the repeat treatment interval is administered at least one day, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 2 weeks or more, after the completion of the first or previous treatment interval. In some embodiments, the repeat treatment interval is administered at least three days after the completion of the first or previous treatment interval.

[0085] In one embodiment, any of the treatment intervals described herein is followed by one or more subsequent treatment intervals, e.g., 1, 2, 3, 4, or 5 subsequent treatment intervals. The one or more subsequent treatment intervals are different from the first or previous treatment interval. For 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., 2, 3, 4, or more) of an 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, e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 2 weeks after the completion of the first or previous treatment interval.

[0086] In any of the methods described herein, one or more subsequent doses, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 additional doses of the inhibitor (e.g., a JAK-STAT or BTK inhibitor), are administered after completion of one or more treatment intervals. In embodiments in which treatment intervals are repeated or more than one treatment interval is administered, one or more subsequent doses, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 additional doses of the 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, a dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered every 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 20 hours, 24 hours, 1 day, 1.5 days, 2 days, 3 days, 4 days, 5 days, 7 days, 2 weeks, 3 weeks, or 4 weeks after the 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 the completion of one or more or each treatment interval.

[0087] In any of the methods described herein, one or more subsequent doses of CAR-expressing cells are administered after completion of one or more treatment intervals, e.g., 1, 2, 3, 4, 5, or more doses. In embodiments in which treatment intervals are repeated or more than one treatment interval is administered, one or more subsequent doses, e.g., 1, 2, 3, 4, or 5 doses, or more, of CAR-expressing cells are administered after completion of one treatment interval and before the start of another treatment interval. In one embodiment, doses of CAR-expressing cells are administered every 2, 3, 4, 5, 7 days, 2 weeks, 3 weeks, or 4 weeks after completion of one or more or each treatment interval.

[0088] In one embodiment, the treatment interval comprises a single dose of a CAR-expressing cell (e.g., a CD123 CAR-expressing cell or a CD19 CAR-expressing cell) administered concurrently (e.g., within two days (e.g., 2 days, 1 day, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, or less) with a first dose of an inhibitor (e.g., a JAK-STAT inhibitor, e.g., ruxolitinib, or a BTK inhibitor, e.g., ibrutinib). In an embodiment, the JAK-STAT inhibitor (e.g., ruxolitinib) or the BTK inhibitor (e.g., ibrutinib) is administered twice daily (BID) for the duration of the treatment interval. In an embodiment, the JAK-STAT inhibitor (e.g., ruxolitinib) or the BTK inhibitor (e.g., ibrutinib) is administered once daily (QD) for the duration of the treatment interval.

[0089] In other embodiments, the treatment interval comprises a single dose of CAR-expressing cells (e.g., CD123 CAR-expressing cells or CD19 CAR-expressing cells) administered (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more after) administration of a first dose of an inhibitor (e.g., a JAK-STAT inhibitor, e.g., ruxolitinib, or a BTK inhibitor, e.g., ibrutinib). In embodiments, a second dose of an inhibitor (e.g., a JAK-STAT inhibitor, e.g., ruxolitinib, or a BTK inhibitor, e.g., ibrutinib) is administered after administration of the first dose of the inhibitor (e.g., a JAK-STAT inhibitor, e.g., ruxolitinib, or a BTK inhibitor, e.g., ibrutinib). In embodiments, a subsequent dose of the inhibitor (e.g., a JAK-STAT inhibitor, e.g., ruxolitinib, or a BTK inhibitor, e.g., ibrutinib) is administered. In embodiments, the dose of the inhibitor (e.g., a JAK-STAT inhibitor, e.g., ruxolitinib, or a BTK inhibitor, e.g., ibrutinib) is administered twice daily (BID). In embodiments, the dose of the inhibitor (e.g., a JAK-STAT inhibitor, e.g., ruxolitinib, or a BTK inhibitor, e.g., ibrutinib) is administered once daily (QD). In embodiments, the treatment interval comprises at least 5 doses (e.g., at least 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20 doses, or more) of the inhibitor (e.g., a JAK-STAT inhibitor, e.g., ruxolitinib, or a BTK inhibitor, e.g., ibrutinib). In embodiments, the treatment interval comprises continuous administration of the inhibitor (e.g., QD or BID). In embodiments, the treatment interval ranges in duration from 1 to 7 days, 1 to 5 weeks, or 1 to 12 months.

[0090] In any of the methods described herein, the subject is administered a single dose of CAR-expressing cells and a single dose of an inhibitor (e.g., a JAK-STAT inhibitor, e.g., ruxolitinib, or a BTK inhibitor, e.g., ibrutinib). 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 more, after administration of the single dose of the inhibitor (e.g., a JAK-STAT inhibitor, e.g., ruxolitinib, or a BTK inhibitor, e.g., ibrutinib).

[0091] In one embodiment, after the initial dose of CAR-expressing cells, one or more subsequent doses of CAR-expressing cells, for example, 1, 2, 3, 4, or 5 doses, are administered to the subject. In one embodiment, the one or more subsequent doses of CAR-expressing cells are administered at least 2 days, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 20, 25, 30, 35, 40 days, or 2 weeks, 3 weeks, 4 weeks, or more, after the previous dose of CAR-expressing cells. In one embodiment, the one or more subsequent doses of CAR-expressing cells are administered at least 5 days after the previous dose of CAR-expressing cells. In one embodiment, the subject is administered 3 doses per week or 1 dose every 2 days of CAR-expressing cells.

[0092] In one embodiment, after administration of a single dose of an inhibitor (e.g., a JAK-STAT inhibitor, e.g., ruxolitinib, or a BTK inhibitor, e.g., ibrutinib), one or more subsequent doses of the inhibitor (e.g., a JAK-STAT inhibitor, e.g., ruxolitinib, or a BTK inhibitor, e.g., ibrutinib) are administered, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more subsequent doses of the inhibitor (e.g., a JAK-STAT inhibitor, e.g., ruxolitinib, or a BTK inhibitor, e.g., ibrutinib). In one embodiment, the one or more subsequent doses of the inhibitor (e.g., a JAK-STAT inhibitor, e.g., ruxolitinib, or a BTK inhibitor, e.g., 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 the previous dose of the inhibitor (e.g., a JAK-STAT inhibitor, e.g., ruxolitinib, or a BTK inhibitor, e.g., ibrutinib). In other embodiments, one or more subsequent doses of the inhibitor (e.g., a JAK-STAT inhibitor, e.g., ruxolitinib, or a BTK inhibitor, e.g., ibrutinib) are administered every other day, once daily, or twice daily after the previous dose of the inhibitor (e.g., a JAK-STAT inhibitor, e.g., ruxolitinib, or a BTK inhibitor, e.g., ibrutinib).

[0093] In one embodiment, one or more subsequent doses of an inhibitor (e.g., a JAK-STAT inhibitor, e.g., ruxolitinib, or a BTK inhibitor, e.g., ibrutinib) are administered at least 1, 2, 3, 4, 5, 6, or 7 days after the dose of CAR-expressing cells, e.g., the initial dose of CAR-expressing cells.

[0094] In one embodiment, one or more doses, e.g., 1, 2, 3, 4, or 5 doses of an inhibitor (e.g., a JAK-STAT inhibitor, e.g., ruxolitinib, or a BTK inhibitor, e.g., ibrutinib) are administered prior to the first dose of CAR-expressing cells.

[0095] In one embodiment, the administration of one or more doses of CAR-expressing cells and one or more doses of an inhibitor (e.g., a JAK-STAT inhibitor, e.g., ruxolitinib, or a BTK inhibitor, e.g., ibrutinib) is repeated, e.g., 1, 2, 3, 4, or 5 additional times.

[0096] Dosages and treatment regimens for the therapeutic agents disclosed herein can be determined by one of skill in the art.

[0097] In any of the dosing regimens or treatment intervals described herein, in some embodiments, the dose of CAR-expressing cells (e.g., CD19 CAR-expressing or CD123 CAR-expressing cells) is at least about 1 x 10 5 , 5×10 6 , 1×10 7 , 1.5×10 7 , 2 × 10 7 , 2.5×10 7 , 3×10 7 , 3.5×10 7 , 4×10 7 , 5×10 7 , 1×10 8 , 1.5×10 8 , 2 × 10 8 , 2.5×10 8 , 3×10 8 , 3.5×10 8 , 4×10 8 , 5×10 8 , 1×10 9 , 2 × 10 9 , or 5 x 10 9 In some embodiments, the dose of CAR-expressing cells comprises at least about 1-5 x 10 cells. 7 ~1~5×10 8 In some embodiments, the subject comprises about 1-5 x 10 7 In other embodiments, the subject is administered about 1-5 x 10 CAR-expressing cells. 8 The patient will be administered CAR-expressing cells.

[0098] In embodiments, the CAR-expressing cells are 1.5 x 10 per kg 7 ~5×10 9 cells (e.g., 0.3 × 10 per kg) 6 ~1×10 8 In embodiments, the total dose is 1.5 x 10 cells. 10cells / kg, e.g., administered in multiple doses over time, e.g., 1.5 x 10 9 Not exceeding 1.5 x 10 cells / kg 8 Do not exceed cells / kg.

[0099] In one embodiment, up to 10, 9, 8, 7, 6, 5, 4, 3, or 2 doses of cells are administered. In other embodiments, 1, 2, 3, 4, 5, or 6 doses of cells are administered to the mammal, for example, with a treatment interval of 1, 2, 3, 4, or more weeks. In one embodiment, up to 6 doses are administered over a 2-week period. The doses can be the same or different. In one embodiment, a lower dose is administered first, followed by one or more higher doses. In one exemplary embodiment, the lower dose is about 1 x 10 5 ~1×10 9 cells / kg, or 1 x 10 6 ~1×10 8 cells / kg, and higher doses are approximately 2 x 10 5 ~2×10 9 cells / kg or 2 x 10 6 ~2×10 8 cells / kg, followed by approximately 4 × 10 5 ~4×10 9 cells / kg, or 4 x 10 6 ~4×10 8 Followed by 3–6 doses of cells / kg.

[0100] In embodiments, CAR-expressing cells are administered to a subject according to a dosing regimen that includes a total dose of cells administered to the subject in divided doses, e.g., one, two, three, or more divided 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 a subsequent day of treatment (e.g., day 2, 3, 4, 5, 6, or 7 or later), and optionally a third percentage (e.g., the remaining percentage) of the total dose is administered on a further subsequent day of treatment (e.g., day 3, 4, 5, 6, 7, 8, 9, 10 or later). For example, 10% of the total dose of cells is delivered on day 1 of treatment, 30% of the total dose of cells is delivered on day 2, and the remaining 60% of the total dose of cells is delivered on day 3. For example, the total cell dose is 1-5×10 7 or 1 to 5 x 10 8 Contains CAR-expressing cells.

[0101] In embodiments, the total dose is administered in multiple doses (eg, a first dose, a second dose, and optionally a third dose, etc.).

[0102] In embodiments, the first dose is about 10% (e.g., about 1 x 10) of the total dose, e.g., administered on the first day. 7 In embodiments, the second dose comprises about 30% (e.g., about 3 x 10 cells / kg) of the total dose, administered, for example, on a subsequent day (e.g., 1, 2, 3, 4, 5, 6, or 7 days after the first dose). 7cells / kg). In embodiments, the second dose is administered when the subject is clinically stable after the first dose. In embodiments, subsequent doses (e.g., a third, optionally a fourth, etc. dose) are administered to the subject, e.g., the sum of the first dose, the second dose, and the subsequent doses is the total dose. In embodiments, 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 more). In embodiments, the time between the second and third doses, and / or between the third and fourth doses, and / or between the fourth and fifth doses is at least 1 week (e.g., at least 1, 2, 3, 4 weeks, or more).

[0103] In embodiments, in any of the dosing regimens described herein, the dose of the 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.

[0104] In embodiments, the JAK-STAT inhibitor, e.g., ruxolitinib, is administered (e.g., orally) at a dose of 2.5 mg to 50 mg (e.g., 2.5 to 5 mg, 5 to 10 mg, 10 to 15 mg, 15 to 20 mg, 20 to 25 mg, 25 to 30 mg, 30 to 35 mg, 35 to 40 mg, 40 to 45 mg, or 45 to 50 mg) twice daily (e.g., a total of 5 mg to 100 mg per day).

[0105] In an embodiment, a BTK inhibitor, e.g., ibrutinib (PCI-32765), is administered daily (e.g., 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) for a period of time, e.g., daily for 21 day cycles, or daily for 28 cycles. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of a BTK inhibitor, e.g., ibrutinib, are administered.

[0106] In some embodiments of any of the methods disclosed herein, the method includes administering an inhibitor (e.g., a BTK inhibitor, e.g., ibrutinib, or a JAK-STAT inhibitor, e.g., ruxolitinib) to a subject, reducing the dose of the inhibitor (e.g., discontinuing its administration), and subsequently administering a CAR-expressing cell (e.g., a CAR19- or CAR123-expressing cell) to the subject.

[0107] In some embodiments, the method includes administering an inhibitor (e.g., a BTK inhibitor, e.g., ibrutinib, or a JAK-STAT inhibitor, e.g., ruxolitinib) to the subject, and subsequently administering a combination of the inhibitor and a CAR-expressing cell (e.g., a CAR19- or CAR123-expressing cell) to the subject.

[0108] In some embodiments, the method includes administering an inhibitor (e.g., a BTK inhibitor, e.g., ibrutinib, or a JAK-STAT inhibitor, e.g., ruxolitinib) to a subject, reducing the inhibitor (e.g., ceasing or discontinuing its administration), and subsequently administering to the subject a combination of a CAR-expressing cell (e.g., a CAR19- or CAR123-expressing cell) and a second inhibitor (e.g., a second inhibitor other than the first inhibitor). 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., a BTK inhibitor 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., a JAK-STAT inhibitor 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.

[0109] In one embodiment, cells expressing a CAR molecule, e.g., a CAR molecule described herein, are administered at a dose and / or administration schedule described herein.

[0110] In certain embodiments, any method described herein further comprises administering a therapy to prevent or treat CRS. In embodiments, the therapy comprises an IL-6 inhibitor (e.g., an anti-IL6 receptor inhibitor, e.g., an anti-IL6 receptor inhibitor, e.g., tocilizumab). In other embodiments, the therapy comprises an IL-6 inhibitor in combination with one or more (or all) of a vasoactive agent, an immunosuppressant, a corticosteroid, or mechanical ventilation. In embodiments, the method comprises administering an IL-6 inhibitor (e.g., tocilizumab) prior to (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days or 1, 2, 3, or 4 weeks prior to) administration of a dose (e.g., a first dose) of a CAR-expressing cell (e.g., a CAR-expressing cell described herein). In embodiments, the methods involve administering an IL-6 inhibitor (e.g., tocilizumab) concurrently with administration of a dose (e.g., a first dose) of a CAR-expressing cell (e.g., a CAR-expressing cell described herein). In embodiments, the methods involve administering the IL-6 inhibitor (e.g., tocilizumab), e.g., after administration of a dose (e.g., a first dose) of a CAR-expressing cell (e.g., a CAR-expressing cell described herein), but before or within one week (e.g., within one week, seven, six, five, four, three, two, one day, or less) of the first sign of fever in the subject. In embodiments, the method comprises administering an IL-6 inhibitor (e.g., tocilizumab) after administration of a dose (e.g., a first dose) of CAR-expressing cells (e.g., a CAR-expressing cell described herein) and within one week (e.g., within one week, seven, six, five, four, three, two, one day, or less) of the onset of a temperature of at least 38°C (e.g., at least 38.5°C) in the subject, e.g., for two consecutive measurements in a 24-hour period (e.g., at least four hours apart). In embodiments, the subject has (e.g., is diagnosed with or identified as having) a high tumor burden prior to treatment with the CAR-expressing cells. In embodiments, the high tumor burden comprises at least 40% blasts (e.g., at least 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or more blasts) in the subject's bone marrow prior to administration of the CAR-expressing cells (e.g., about 1-5 days prior to administration of the CAR-expressing cells).

[0111] In embodiments, the method includes administering tocilizumab at a dose of about 5-15 mg / kg, e.g., 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).

[0112] In one embodiment, the CAR molecule is introduced into T cells using, for example, in vitro transcription, and the subject (e.g., human) receives an initial administration of cells comprising the CAR molecule and one or more subsequent administrations of cells comprising the CAR molecule, where the one or more subsequent administrations are administered less than 15 days after the previous administration, for example, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days. In one embodiment, two or more administrations of cells comprising the CAR molecule are administered to the subject (e.g., human) per week, for example, two, three, or four administrations of cells comprising the CAR molecule are administered per week. In one embodiment, the subject (e.g., human subject) receives two or more administrations of cells comprising the CAR molecule per week (e.g., two, three, or four administrations per week) (also referred to herein as a cycle), followed by a week without administration of cells comprising the CAR molecule, and then one or more further administrations of cells comprising the CAR molecule (e.g., two or more administrations of cells comprising the CAR molecule per week). In another embodiment, a subject (e.g., a human subject) receives two or more cycles of cells comprising a CAR molecule, with the time between each cycle being less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, cells comprising a CAR molecule are administered every other day for three doses per week. In one embodiment, cells comprising a CAR molecule are administered for at least 2, 3, 4, 5, 6, 7, 8 weeks, or more.

[0113] In one embodiment, the combination of a kinase inhibitor and cells expressing a CAR molecule, e.g., a CAR molecule described herein, is administered as a first line treatment for a disease, e.g., cancer, e.g., a cancer described herein. In another embodiment, the combination of a kinase inhibitor and cells expressing a CAR molecule, e.g., a CAR molecule described herein, is administered as a second, third, or fourth line treatment for a disease, e.g., cancer, e.g., a cancer described herein.

[0114] In embodiments, any of the methods described herein further include performing lymphodepletion on the subject prior to administering one or more cells expressing a CAR molecule described herein, e.g., a CAR molecule that binds CD19 or CD123. Lymphodepletion can include, for example, administering one or more of melphalan, cytoxan, cyclophosphamide, and fludarabine.

[0115] subject In embodiments, the subject is at risk of developing CRS (eg, identified as being at risk), has CRS, or is diagnosed with CRS.

[0116] In embodiments, the subject has been administered, is being administered, or will be administered a CAR therapy, such as a CAR therapy described herein. In embodiments, the subject has been administered, is being administered, or will be administered a CAR123-expressing cell or a CAR19-expressing cell.

[0117] In an embodiment, the method comprises identifying (and optionally selecting) a subject i) at risk of developing CRS or ii) having CRS.

[0118] In embodiments, the method includes selecting a subject for administration of an inhibitor (e.g., a JAK-STAT inhibitor or a BTK inhibitor). In embodiments, the subject is selected based on (i) the subject's risk of developing CRS, (ii) the subject's diagnosis of CRS, and / or (iii) whether the subject has received, is receiving, or will receive CAR therapy (e.g., a CAR therapy described herein, e.g., a CAR19 therapy, e.g., CTL019, or CD123 CAR therapy). In embodiments, if the subject is diagnosed with CRS, e.g., severe or non-severe CRS, the subject is selected for administration of a JAK-STAT or BTK inhibitor. In embodiments, if the subject is at risk (e.g., identified as at risk) of developing CRS, the subject is selected for administration of a JAK-STAT or BTK inhibitor. In embodiments, a subject is selected for administration of a JAK-STAT or BTK inhibitor if the subject has been administered, is being administered, or will be administered a CAR therapy (e.g., a CAR therapy described herein, e.g., a CAR19 therapy, e.g., CTL019, or CAR123 therapy).

[0119] Subjects at risk for CRS In embodiments, a subject is identified as being at risk for CRS if the subject has a high tumor burden, for example, prior to administration of a CAR therapy (e.g., a CAR therapy described herein).

[0120] In embodiments, a subject is identified as being at risk for CRS by obtaining the subject's CRS risk status, wherein said CRS risk status comprises measurements of one, two, three, four, five, six, seven, eight, nine, ten, or more (all) of the following: (i) 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), e.g., wherein the subject is an adult or pediatric subject; (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., in a sample (e.g., a blood sample), for example, where the subject is an adult or pediatric subject; (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 bone marrow disease in the subject, e.g., where the subject is a pediatric subject; (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., in a sample (e.g., a blood sample), for example, where the subject is a pediatric subject; (v) the level or activity of sgp130, MCP1, or eotaxin, or a combination thereof (e.g., a combination of any two or all three of sgp130, MCP1, or eotaxin), in a subject, e.g., in a sample (e.g., a blood sample), for example, where the subject is an adult or pediatric subject; (vi) the level or activity of IL-2, eotaxin, or sgp130, or a combination thereof (e.g., a combination of any two or all three of IL-2, eotaxin, 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; (vii) the level or activity of IFN-γ, IL-2, or eotaxin, or a combination thereof (e.g., a combination of any two or all three of IFN-γ, IL-2, or eotaxin), in a subject, e.g., in a sample (e.g., a blood sample), for example, where the subject is a pediatric subject; (viii) the level or activity of IL-10 in a subject, e.g., in a sample (e.g., a blood sample), and the level of disease burden in the subject, or a combination thereof, e.g., where the subject is a pediatric subject; (ix) the level or activity of IFN-γ or IL-13, or a combination thereof, in a subject, for example, where the subject is a pediatric subject; or (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), for example, where the subject is a pediatric subject; or (xi) the level or activity of IFN-γ or MIP1-α, or a combination thereof, in a sample (e.g., a blood sample), for example, wherein the subject is a pediatric subject; Here, CRS risk status is indicative of a subject's risk of developing CRS, for example, severe CRS.

[0121] Any of the foregoing methods, responsive to the determination of CRS risk status, identifying a subject as being at high risk for developing severe CRS or at low risk for developing severe CRS; administering a BTK inhibitor (e.g., ibrutinib) or a JAK-STAT inhibitor (e.g., ruxolitinib); administering a modified dose of a CAR-expressing cell therapy; altering the schedule or time course of a CAR-expressing cell therapy; treating CRS by administering a therapy selected from one or more of an IL-6 inhibitor (e.g., an anti-IL6 receptor inhibitor, e.g., tocilizumab), a vasoactive agent, an immunosuppressant, a corticosteroid, or mechanical ventilation; and / or For example, administering alternative therapies, such as standard treatments for certain cancer types, to subjects at high risk of developing severe CRS. It may further include performing one, two, or more (all) of the following:

[0122] In some embodiments of the method, the CRS risk status includes measuring 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 a subject, e.g., in a sample (e.g., a blood sample), e.g., wherein the subject is an adult or pediatric subject.

[0123] In some embodiments of the methods, the CRS risk status is an indication of whether the subject is at high or low risk of developing severe CRS, e.g., CRS can be clinical grade 1-3 or severe CRS, clinical grade 4-5.

[0124] In some embodiments, the method is performed on a subject who does not have one or more symptoms (e.g., clinical symptoms) of CRS, such as hypotension or fever, or severe CRS, such as grade 4 organ toxicity or need for mechanical ventilation.

[0125] In some embodiments of the method, a high level or activity of IFN-γ, sgp130, MCP1, IL-10, or disease burden, or any combination thereof, is an indicator of an increased risk of severe CRS, hi some embodiments, a low level or activity of IL13, IL1Ra, MIP1a, or eotaxin, or any combination thereof, is an indicator of an increased risk of severe CRS.

[0126] In some embodiments of the methods, subjects at high risk of severe CRS have, or are identified as having, a higher level or activity of sgp130 or IFN-γ, or a combination thereof, e.g., compared to a reference (e.g., in a sample, e.g., in a blood sample).

[0127] In other embodiments of the method, a subject at high risk of severe CRS has or is identified as having, e.g., a higher level or activity of sgp130, a higher level or activity of IFN-γ, or a lower level or activity of IL1Ra, or a combination thereof, compared to a reference standard (e.g., in a sample, e.g., a blood sample). In one embodiment, a subject at high risk of severe CRS is identified as having, e.g., 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, a higher level or activity of IFN-γ, and a lower level or activity of IL1Ra, compared to a reference standard. In some embodiments, the reference standard is a subject at low risk of severe CRS or a control level or activity. The subject may be a human, e.g., an adult or pediatric subject.

[0128] In some embodiments of the methods, 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, and a higher level of bone marrow disease in the subject (e.g., in a sample, e.g., a blood sample), e.g., compared to a reference, e.g., compared to 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, e.g., higher levels of sgp130 and IFN-γ, sgp130 and bone marrow disease, IFN-γ and bone marrow disease, or sgp130, IFN-γ, and bone marrow disease, e.g., compared to a reference, e.g., a subject at low risk of severe CRS, or a control level or activity. The subject may be a human, e.g., a pediatric subject.

[0129] In some embodiments of the methods, subjects (e.g., pediatric subjects) at high risk of severe CRS are 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, subjects at high risk of severe CRS are identified as having, for example, 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 compared to a reference, e.g., a subject at low risk of severe CRS, or compared to a control level or activity.

[0130] In some embodiments of the 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 eotaxin, or a combination thereof (e.g., in a sample, e.g., a blood sample) compared to a reference standard, 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 eotaxin, a higher level or activity of MCP1 and a lower level or activity of eotaxin, a higher level or activity of sgp130, a higher level or activity of MCP1, and a lower level or activity of eotaxin compared to a reference standard, e.g., a subject at low risk of severe CRS, or compared to a control level or activity.

[0131] In some embodiments of the 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 eotaxin, or a higher level or activity of sgp130, 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 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 eotaxin, 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 eotaxin 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 eotaxin, 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.

[0132] In some embodiments of the 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 eotaxin, 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 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 eotaxin, an altered (e.g., higher) level or activity of IL-2 and a lower level or activity of eotaxin, 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 eotaxin compared to a reference, e.g., a subject at low risk of severe CRS, or compared to a control level or activity.

[0133] In some embodiments of the methods, subjects at high risk of severe CRS are identified as having a higher level or activity of IL-10, or a higher level of disease burden, 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 (e.g., in a sample, e.g., in a blood sample). In some embodiments, the subject is a pediatric subject.

[0134] In some embodiments of the 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, compared to a reference, e.g., a subject at low risk of severe CRS, or compared to a control level or activity (e.g., in a sample, e.g., in a blood sample). In some embodiments, the subject is a pediatric subject.

[0135] In some embodiments of the methods, subjects at high risk of severe CRS are identified as having a higher level or activity of IFN-γ, a lower level or activity of IL-13, 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 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 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.

[0136] In some embodiments of the methods, subjects at high risk of severe CRS are 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, e.g., in 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.

[0137] In some embodiments, for example, in a three biomarker panel including, for example, IL2, eotaxin, and sgp130 (e.g., in a pediatric patient), or in a three biomarker panel including IFN-γ, IL2, and eotaxin, a higher level or activity of IL2 indicates a higher risk of severe CRS in a subject. In other embodiments, for example, in a two biomarker panel, e.g., for a pediatric patient, a higher level or activity of IL2 indicates a lower risk of severe CRS in a subject.

[0138] In some embodiments of the methods, higher levels of a marker described herein are levels of 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 or greater. In some embodiments, higher levels of sgp130 are levels of 150,000, 200,000, 210,000, 215,000, 218,000, 218,179, 220,000, 225,000, 230,000, or 250,000 pg / ml or greater. In some embodiments, the higher level of IFN-γ is 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 or more. In some embodiments, a higher level of IL-10 is 5, 6, 7, 8, 9, 10, 11, 11.7457, 12, 13, 14, 15, 16, 17, 18, 19, or 20 pg / ml or more. In some embodiments, a higher tumor burden is 25, 30, 35, 40, 45, 50, 51.9, 55, 60, 65, 70, or 75% or more. In some embodiments, a lower level of sgp130, IFN-γ, IL-10, or tumor burden is a level equal to or less than any value in this paragraph.

[0139] In some embodiments of the methods, a lower level of a marker described herein is 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 or greater. In some embodiments, a lower level of IL1Ra is 550, 575, 600, 625, 650, 657,987, 675, 700, 720, or 750 pg / ml or less. In some embodiments, a lower level of MCP1 is 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, a lower level of eotaxin is 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, a lower level of MIP1a is 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 or less. In some embodiments, a higher level of IL1Ra, MCP1, eotaxin, or MIP1a is a level equal to or greater than any value in this paragraph.

[0140] In some embodiments of the method, 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.

[0141] In some embodiments of the method, measuring eotaxin includes measuring one or more (e.g., two or all) of eotaxin-1, eotaxin-2, and eotaxin-3. In some embodiments, measuring eotaxin includes measuring eotaxin-1 and eotaxin-2, eotaxin-1 and eotaxin-3, or eotaxin-2 and eotaxin-3.

[0142] Any of the methods disclosed herein may further include obtaining a measurement of the level or activity of one, two, three, four, five, ten, twenty or more cytokines 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 in the subject, for example, in a sample (e.g., a blood sample) from the subject, or a combination thereof. In some embodiments, a subject having or at high risk of having 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 cytokines 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 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.

[0143] Any of the methods disclosed herein may further include obtaining measurements of the level or activity of one, two, three, four, five, six, seven, eight, or all of the cytokines selected from IFN-γ, IL10, IL6, IL8, IP10, MCP1, M1G, sIL2Rα, GM-CSF, or TNFα, or a combination thereof, in the subject, e.g., in a sample (e.g., a blood sample) from the subject. In some embodiments, a subject having or at high risk of having 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 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.

[0144] Any of the methods disclosed herein may further include obtaining a measurement of the level or activity of one, two, three, four, five, six, or all of the cytokines selected from IFN-γ, IL10, IL6, IL8, IP10, MCP1, M1G, or sIL2Rα, or a combination thereof, in the subject, e.g., in a sample (e.g., a blood sample) from the subject. In some embodiments, a subject having or at high risk of having 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 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.

[0145] In some embodiments, any method disclosed herein may further comprise determining the level of C-reactive protein (CRP) in a sample (e.g., a blood sample) from the 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 higher) compared to a subject at low risk of severe CRS or compared to a control level or activity.

[0146] In other embodiments, the methods disclosed herein further comprise selecting or altering a therapy, e.g., a CAR-expressing cell therapy, for a subject based on the acquired CRS risk status. In embodiments where the acquired CRS risk status is that the subject is at high risk for severe CRS, the therapy is discontinued or altered such that a subsequent (e.g., second, third, or fourth) dose of therapy (e.g., CAR-expressing cells) is a lower dose than the previous dose. In other embodiments, the subsequent (e.g., second, third, or fourth) dose of CAR-expressing cells comprises a different CAR or a different cell type than the CAR-expressing cell therapy previously administered to the subject.

[0147] In other embodiments of the method, one or more measurements of biomarkers (e.g., one or more biomarkers (i)-(xi)) are obtained from a sample (e.g., a blood sample) obtained from the subject. In some embodiments, the subject, e.g., a sample from the subject, is assessed while receiving a CAR-expressing cell therapy. In other embodiments, the subject, e.g., a sample from the subject, is assessed after receiving a CAR-expressing cell therapy. For example, the subject, e.g., a sample from the subject, is assessed 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 of the 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 1, 3, 5, 10, 12, 15, 20 hours or more after infusion of the CAR-expressing therapy). In other embodiments, measuring one or more biomarkers comprises detecting one or more of nucleic acid (e.g., mRNA) levels or protein levels.

[0148] In an embodiment, the method includes determining whether a subject has severe CRS. The method includes, for example, obtaining a CRS risk status in response to an immune cell-based therapy, such as a CAR-expressing cell therapy (e.g., a CAR19-expressing cell therapy or a CAR123-expressing cell therapy), for the subject, wherein the CRS risk status is determined based on one of the following: (i) the level or activity of one or more (e.g., 3, 4, 5, 10, 15, 20, or more) cytokines 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 an 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, or a combination thereof, in a sample (e.g., a blood sample); (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 (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); The method includes measuring one, two, or more (all) of the following, which values ​​are indicative of a severe CRS condition in the subject:

[0149] In embodiments, assessed levels of cytokines (i)-(iii), or all analytes except fibrinogen, are indicative of severe CRS. In embodiments, low fibrinogen is indicative of severe CRS.

[0150] Compositions and compositions for use In another aspect, the disclosure features a composition (e.g., one or more dosage formulations, combinations, or one or more pharmaceutical compositions) including cells expressing a CAR described herein (e.g., a CD123 CAR) and an inhibitor described herein (e.g., a JAK-STAT inhibitor, e.g., ruxolitinib). The CAR-expressing cells and the inhibitor (e.g., a JAK-STAT inhibitor) can be in the same or different formulations or pharmaceutical compositions. The CAR-expressing cells and the one or more kinase inhibitors can be present in a single dose form or in two or more dose forms.

[0151] In embodiments, the compositions disclosed herein are for use as a medicament.

[0152] In embodiments, the compositions disclosed herein are used to treat a disease associated with expression of an antigen described herein, such as a B cell antigen (eg, CD123 or CD19).

[0153] In another aspect, the disclosure features compositions (e.g., one or more dosage formulations, combinations, or one or more pharmaceutical compositions) including cells expressing a CAR described herein (e.g., a CD123 CAR) and an inhibitor described herein (e.g., a JAK-STAT inhibitor), for use in a method of treating (or preparing a medicament for treating) a disease associated with expression of an antigen (e.g., a B cell antigen, e.g., CD123 or CD19), e.g., a cancer described herein.

[0154] In another aspect, the disclosure features compositions (e.g., one or more dosage formulations, combinations, or one or more pharmaceutical compositions) including a cell expressing a CAR described herein (e.g., a CD123 CAR or a CD19 CAR) and an inhibitor described herein (e.g., a JAK-STAT inhibitor or a BTK inhibitor), for use in a method of preventing CRS in a subject.

[0155] In another aspect, the invention relates to a cell expressing a CAR molecule described herein for use as a medicament in combination with a kinase inhibitor, e.g., a kinase inhibitor described herein (e.g., a BTK inhibitor such as ibrutinib, or a JAK-STAT inhibitor such as ruxolitinib), e.g., to prevent CRS in a subject. In another aspect, the invention relates to a kinase inhibitor described herein (e.g., a BTK inhibitor such as ibrutinib, or a JAK-STAT inhibitor such as ruxolitinib), for use as a medicament in combination with a cell expressing a CAR molecule described herein, e.g., to prevent CRS in a subject.

[0156] In another aspect, the invention relates to a cell expressing a CAR molecule described herein for use in combination with a kinase inhibitor, e.g., a kinase inhibitor described herein (e.g., a BTK inhibitor such as ibrutinib, or a JAK-STAT inhibitor such as ruxolitinib), in the treatment of a disease expressing a B cell antigen (e.g., CD19 or CD123).

[0157] In another aspect, the invention pertains to a kinase inhibitor described herein (e.g., a BTK inhibitor such as ibrutinib, or a JAK-STAT inhibitor such as ruxolitinib) for use in combination with cells expressing a CAR molecule described herein in the treatment of a disease expressing a B-cell antigen (e.g., CD19 or CD123).

[0158] In another aspect, the invention relates to a kinase inhibitor described herein (e.g., a BTK inhibitor such as ibrutinib, or a JAK-STAT inhibitor such as ruxolitinib) for use in combination with a cell expressing a CAR molecule described herein in reducing one or more side effects of a CAR therapy described herein.

[0159] In another aspect, the invention relates to cells expressing a CAR molecule described herein for use in combination (e.g., as a medicament) with a cytokine as described herein, e.g., IL-7, IL-15, and / or IL-21. In another aspect, the invention relates to cytokines described herein for use in combination (e.g., as a medicament) with cells expressing a CAR molecule described herein.

[0160] In another aspect, the invention relates to cells expressing a CAR molecule described herein for use in combination (e.g., as a medicament) with a cytokine as described herein, e.g., IL-7, IL-15 and / or IL-21, in the treatment of a disease expressing a B cell antigen, e.g., CD123 or CD19. In another aspect, the invention relates to cytokines described herein for use in combination (e.g., as a medicament) with cells expressing a CAR molecule described herein, in the treatment of a disease expressing a B cell antigen, e.g., CD123 or CD19.

[0161] In some embodiments, the present disclosure provides a method of distinguishing between CRS and sepsis in a subject, the method comprising obtaining one or more measurements of: (i) the level or activity of one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, 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 higher level or activity than the reference is indicative of CRS; or (ii) the level or activity of one or more (e.g., two, three, four, five, six, or all) of CD163, IL-1β, sCD30, sIL-4R, sRAGE, sVEGFR-1, and sVEGFR-2, wherein a higher level or activity than baseline is indicative of sepsis.

[0162] In embodiments, the method includes administering a therapy (e.g., a therapy described herein) to treat CRS if the measurements indicate CRS. In embodiments, the method includes administering a therapy to treat sepsis if the measurements indicate sepsis.

[0163] The present disclosure also provides, in some embodiments, a kit for distinguishing between CRS and sepsis in a patient, the kit comprising a set of reagents that specifically detect the level or activity of one or more genes or proteins (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, two, twenty-two, or all of them) selected from the following: 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 Instructions for use of said kit Including, The instructions may include determining that the subject is likely to have CRS if the detected level or activity of one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, 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 higher than a reference value; and / or If the detected level or activity of one or more (e.g., two, three, four, five, six, or all) of CD163, IL-1β, sCD30, sIL-4R, sRAGE, sVEGFR-1, or sVEGFR-2 is higher than the reference value, the subject is provided with a high likelihood of having sepsis.

[0164] The present disclosure provides, in some aspects, a set of reagents that specifically detect the level or activity of one or more 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 (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, two, twenty-two, twenty-three, or all thereof); and a biological sample, such as a blood sample Also provided is a reaction mixture comprising:

[0165] In embodiments, the biological sample is from a subject being treated with a CAR-expressing cell therapy and / or a subject with symptoms of CRS and / or sepsis.

[0166] The present disclosure also provides, in certain embodiments, a method of identifying sepsis in a subject, the method comprising obtaining one or more measurements of: (i) the level or activity of one or more 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 (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, or all thereof), wherein an elevated level or activity relative to a reference is indicative of sepsis; (ii) the level or activity of one or more (e.g., both) of IL13 and RANTES, where a low level or activity compared to baseline is indicative of sepsis.

[0167] In some aspects, the present disclosure provides a method of treating one or more of neurotoxicity, CRS, or posterior reversible encephalopathy syndrome (PRES), comprising administering a therapeutically effective amount of cyclophosphamide to a subject in need thereof. In a related aspect, the present disclosure provides cyclophosphamide for use in treating neurotoxicity, CRS, or posterior reversible encephalopathy syndrome (PRES). In embodiments, administration of cyclophosphamide follows cell-based therapy, such as cell-based therapy for cancer, CD19 inhibitory therapy, or CD19 depletion therapy, or the subject has previously been treated with cell-based therapy, such as cell-based therapy for cancer, CD19 inhibitory therapy, or CD19 depletion therapy. In embodiments, administration of cyclophosphamide is before, at the same time as, or after cell-based therapy.

[0168] In embodiments, the patient has or is identified as having CRS, PRES, or both. In some embodiments, the subject has been treated with CD19-blocking or depleting therapy. In some embodiments, the CD19 inhibitor is a CD19 antibody, e.g., a CD19 bispecific antibody (e.g., a bispecific T-cell engager targeting CD19, e.g., blinatumomab). In some embodiments, the therapy comprises a CAR-expressing cell, e.g., an anti-BCMA CAR or an anti-CD19 CAR. In embodiments, the subject is suffering from neurotoxicity, e.g., focal deficits (e.g., cranial nerve palsy or hemiparesis) or global abnormalities (e.g., generalized seizures, confusion), or status epilepticus. In embodiments, the subject does not have any clinical symptoms of CRS. In embodiments, the subject has one or more clinical symptoms of CRS. In embodiments, the subject has or is identified as having elevated IL-6 compared to a baseline, e.g., the subject's IL-6 levels prior to therapy with CAR-expressing cells. In embodiments, the subject has or is identified as having elevated serum levels of CRS-associated cytokines (e.g., IL-6 and / or IL-8) compared to baseline. In embodiments, the subject has or is identified as having elevated levels of CRS-associated cytokines (e.g., CSF IL-6 and / or IL-8) compared to baseline. In embodiments, the subject is treated or has been treated with a therapy for CRS, such as tocilizumab or a corticosteroid (e.g., methylprednisolone, hydrocortisone, or both). In embodiments, the subject has or is identified as having an increase in activated CAR-expressing cells in the circulation. In embodiments, the subject has or is identified as having CAR-expressing cells in the CSF.

[0169] Unless otherwise defined, all scientific and technical 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, suitable methods and materials are described below. 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, e.g., (a), (b), (i), etc., are provided merely for ease of reading. The use of headings or numbered or lettered elements herein does not require the steps or elements to be performed in alphabetical order or that the steps or elements are necessarily distinct from one another. Other features, objects, and advantages of the present invention will be apparent from the description and drawings, and from the claims.

[0170] The following detailed description of the preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Brief explanation of the drawings]

[0171] [Figure 1-1] Figure 1A is a schematic diagram illustrating experiments performed, e.g., as described in Example 1, to generate a mouse model of CRS after CART. Figure 1B is a graph showing the expansion of CART cells after AML injection. [Figure 1-2] FIG. 1C is a survival curve showing the survival rate of mice after high doses of CART123. [Figure 1-3] Figure 1D is a panel of graphs showing the levels of various cytokines in mice treated with high-dose CART123. [Figure 2-1]FIG. 2A is a schematic diagram illustrating an experiment performed as described in Example 1, for example, to determine the effect of ruxolitinib on CRS following CART therapy. [Figure 2-2] Figure 2B is a graph showing the change in mouse body weight as measured by % change from baseline plotted on the y-axis against time on the x-axis, and Figure 2C is a graph showing disease burden as measured by leukemic cells / ul (huCD45 dim cells) from serial retro-orbital bleeds plotted on the y-axis against time on the x-axis. [Figure 2-3] Figure 2D is a graph showing the change in body weight of mice upon treatment with ruxolitinib. Body weight, as measured by percent change from baseline, is plotted on the y-axis against time on the x-axis. Figure 2E is a graph showing absolute CD3+ cell counts from serial retro-orbital bleeds from mice. Serial retro-orbital bleeds were performed at the time points indicated on the x-axis. Absolute CD3+ cell counts are plotted on the y-axis. [Figure 2-4] Figure 2F is a set of graphs showing levels of inflammatory cytokines from mouse serum obtained by retroorbital bleeding of mice 1 week after CAR123 injection. [Figure 2-5] Figure 2G is a survival plot showing the survival of mice treated with 60 mg / kg ruxolitinib in combination with CART123, and Figure 2H is a flow cytometry plot showing analysis of peripheral blood from surviving mice treated with ruxolitinib 70 days after AML injection (gated on live human CD45-positive cells). [Figure 3-1] Figure 3A is a schematic representation of the experiment described in Example 2, specifically the creation of a CRS model following CART19 treatment in B cell neoplasms. Figure 3B is an image of the spleen from a representative mouse sacrificed prior to T cell therapy, showing high tumor burden. [Figure 3-2]Figure 3C is a flow cytometry plot showing high levels of circulating neoplastic B cells present in peripheral blood (PB) at the time of randomization (gating strategy: time gate, lymphocytes, single cells, live gate, huCD45+ muCD45-). Figure 3D is a survival curve showing that mice treated with CART19 experience a significant decrease in overall survival. [Figure 3-3] Figure 3E is a panel of graphs showing Luminex analysis of serum human cytokines, which revealed a significant increase in cytokines in the PB of mice receiving CART19 compared to untreated mice. For Figures 3C–E, all graphs are representative of two independent experiments (five mice per group). Two groups were compared using a Student's t-test. Survival curves were compared using the log-rank test. Asterisks represent p values ​​(*=<0.05, **=<0.01, ***=<0.001, ****=<0.0001), and "ns" means "not significant" (p>0.05). [Figure 4-1] FIG. 4A is a schematic diagram showing the experiments of Example 2, e.g., administration of CART19 in combination with ibrutinib or vehicle in the mouse model generated in Example 2. [Figure 4-2] Figure 4B is a survival curve showing that mice treated with CART19+ ibrutinib experienced a significant increase in overall survival. Figure 4C is a graph showing CD19+ cell counts in peripheral blood after vehicle or ibrutinib treatment. Figure 4D is a graph showing that T cell expansion was not adversely affected by ibrutinib treatment (rather, T cell expansion was enhanced by ibrutinib treatment). [Figure 4-3] Figure 4E is a panel of graphs showing serum cytokines from mice treated with CART19 or CART19 plus ibrutinib analyzed by Luminex. Significant reductions in all cytokines implicated in CRS were observed. [Figure 4-4]Figure 4F is a panel of graphs showing significant dose-dependent cytokine production in primary MCL cells incubated with ibrutinib for 24 hours. All graphs in Figures 4B–4F are representative of two independent experiments (five mice per group). Two groups were compared using a Student's t-test. For analyses comparing multiple groups, one-way analysis of variance (ANOVA) with Holm-Sidak correction for multiple comparisons was performed. Survival curves were compared using the log-rank test. Asterisks represent p values ​​(*=<0.05, **=<0.01, ***=<0.001, ****=<0.0001), and "ns" means "not significant" (p>0.05). [Figure 4-5] Same as above. [Figure 5] Figure 5 is a graph showing serum cytokine concentrations in xenograft mice bearing primary pediatric ALL treated with CD19 CAR T cells. NSG mice were administered 10 primary ALL and 7 days later, 5 x 10 autologous CD19 CAR T cells. Serum was collected 3 days after T cell delivery, and subgroups of animals received tocilizumab on days 1 and 3 post-T cell. Cytokine concentrations were measured in pg / mL. [Figure 6] Figure 6 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 106 Nalm-6 ALL cells and 7 days later received 5x106 CD19 CAR T cells derived from a normal donor. Serum was collected 3 days after T cell delivery, and subgroups of animals received tocilizumab on days 1 and 3 post-T cell. Cytokine concentrations are measured in pg / mL. [Figure 7] Figures 7A-7J are graphs 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 18 hours after co-culture. Cytokine levels are measured in pg / mL. Significant differences are indicated by either * or **, representing a p-value of <0.05. [Figure 8]Figures 8A-8E are graphs showing cytokine secretion from co-culture experiments combining monocyte-lineage cells with T cells and targets. Monocyte-lineage cells were differentiated in vitro and combined with T cells, targets, and APCs 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. [Figure 9] Figures 9A-9C are graphs showing transcriptional analysis of isolated cell populations. T cells and targets were separated from APCs using transwell inserts and co-cultured for 18 hours. 697 RNA transcripts were quantified from each cell population, and the logarithmic counts for each are displayed. (A) Transcriptional profiles of CD19 CAR T cells combined with targets and with targets and pooled monocytes, (B) APCs combined with targets and with targets and non-targeting T cells, and (C) APCs combined with targets and non-targeting T cells and with targets and targeting T cells. [Figure 10] Figure 10 is a graph showing the transcript profile of activated CD19 CAR T cells and monocyte-lineage APCs. Cells were harvested 18 hours after transwell co-culture of CD19 CAR T cells, Nalm-6 leukemia, and pooled monocytes. Transcript counts from T cells are displayed in blue, and counts from APCs in red. [Figure 11] 11A-11C are graphs showing T cell degranulation in the presence of APC. T cells expressing either (A) no CAR molecule expression, (B) a GD2-targeted CAR, or (C) a CD19-targeted CAR were combined with the CD19+ target ALL cell line Nalm-6. Degranulation was measured by quantification of CD107a surface expression. [Figure 12] Figure 12 shows 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. The first seven patients had detectable T cells in their peripheral blood but no detectable ALL, while the last three patients had only ALL cells and no detectable T cells. [Figure 13]Figure 13 is a set of images showing microscopic analysis of peripheral blood T cells collected at the time of first fever after CD19 CAR T cell infusion in a patient with acute lymphoblastic leukemia. Images were taken at 1000x magnification. DETAILED DESCRIPTION OF THE INVENTION

[0172] definition 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 pertains.

[0173] The terms "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical referent of the article. By way of example, "an element" means one element or more than one element.

[0174] The term "about," when referring to a measurable value, such as an amount, a temporal duration, or the like, means that 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 are encompassed, as such variations are appropriate for the practice of the methods of the present disclosure.

[0175] The term "chimeric antigen receptor" or alternatively "CAR" refers to a recombinant polypeptide construct comprising at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") that comprises a functional signaling domain derived from a stimulatory molecule, as defined below. In some embodiments, the domains of a CAR polypeptide construct are on the same polypeptide chain, e.g., comprising a chimeric fusion protein. In some embodiments, the domains of a CAR polypeptide construct are not contiguous with one another, e.g., are on different polypeptide chains, e.g., as provided in an RCAR as described herein.

[0176] In one embodiment, the stimulatory molecule of the CAR is the zeta chain associated with the T cell receptor complex. In one embodiment, the cytoplasmic signaling domain comprises a primary signaling domain (e.g., the primary signaling domain of CD3-zeta). In one embodiment, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule, as defined below. In one embodiment, the costimulatory molecule is selected from 4-1BB (i.e., CD137), CD27, ICOS, and / or CD28. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In one embodiment, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, which is optionally cleaved from the antigen recognition domain (e.g., scFv) during cellular processing and cell membrane localization of the CAR.

[0177] A CAR comprising an antigen-binding domain (e.g., an scFv, a single-domain antibody, or a TCR (e.g., a TCRα-binding domain or a TCRβ-binding domain)) that specifically binds to a specific tumor marker X (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 CD123 CAR 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. A CAR can be expressed in any cell as described herein, for example, an immune effector cell (e.g., a T cell or an NK cell).

[0178] Therapies that include CAR-expressing cells are referred to herein as CAR therapy. For example, therapies that include CD123 CAR-expressing cells, or CD19 CARs, are referred to herein as CD123 CAR therapy or CD19 CAR therapy, respectively.

[0179] The term "signaling domain" refers to a functional portion of a protein that functions by transmitting information within the cell to regulate cellular activity through a defined signaling pathway, either by generating second messengers or by functioning as an effector in response to such messengers.

[0180] As used herein, the terms "IL-3 receptor alpha subunit," "IL3Rα," "CD123," "IL3Rα chain," and "IL3Rα subunit" synonymously refer to antigenic determinants known to be detectable on leukemia progenitor cells. Human and mouse 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 embodiment, the antigen-binding portion of the CAR recognizes and binds to an epitope within the extracellular domain of the CD123 protein. In one embodiment, the CD123 protein is expressed on cancer cells. As used herein, "CD123" includes proteins containing mutations of full-length wild-type CD123, such as point mutations, fragments, insertions, deletions, and splice variants.

[0181] As used herein, the term "CD19" refers to the cluster of differentiation 19 protein, an antigenic determinant detectable on leukemia progenitor cells. Human and mouse 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 CD19 can be found under UniProt / Swiss-Prot accession number P15391, and the nucleotide sequence encoding human CD19 can be found under accession number NM_001178098. As used herein, "CD19" includes proteins containing mutations of full-length wild-type CD19, such as point mutations, fragments, insertions, deletions, and splice variants. CD19 is expressed in most B-cell lineage cancers, including acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma. Other cells that express CD19 are provided below in the definition of "diseases associated with CD19 expression." It is also an early marker of B-cell progenitor cells. See, e.g., Nicholson et al. Mol. Immun. 34(16-17):1157-1165 (1997). In one embodiment, the antigen-binding portion of the CART recognizes and binds to an antigen within the extracellular domain of the CD19 protein. In one embodiment, the CD19 protein is expressed on cancer cells.

[0182] As used herein, the term "CD20" refers to an antigenic determinant known to be detectable on B cells. Human CD20 is also referred to as transmembrane 4-domain, subfamily A, member 1 (MS4A1). Human and mouse 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 CD20 can be found under accession numbers NP_690605.1 and NP_068769.2, and nucleotide sequences encoding human CD20 transcriptional variants 1 and 3 can be found under accession numbers NM_152866.2 and NM_021950.3, respectively. In one embodiment, the antigen-binding portion of the CAR recognizes and binds to an antigen within the extracellular domain of the CD20 protein. In one embodiment, the CD20 protein is expressed on cancer cells.

[0183] As used herein, the term "CD22" refers to an antigenic determinant known to be detectable on leukemia progenitor cells. Human and mouse 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 human CD22 isoforms 1-5 can be found under accession numbers NP 001762.2, NP 001172028.1, NP 001172029.1, NP 001172030.1, and NP 001265346.1, respectively, and the nucleotide sequences encoding human CD22 variants 1-5 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 embodiment, the antigen binding portion of the CAR recognizes and binds to an antigen within the extracellular domain of the CD22 protein. In one embodiment, the CD22 protein is expressed on cancer cells.

[0184] As used herein, the term "ROR1" refers to an antigenic determinant known to be detectable on leukemia progenitor cells. Human and mouse 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 human ROR1 isoforms 1 and 2 precursors 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, respectively. In one embodiment, the antigen-binding portion of the CAR recognizes and binds to an antigen within the extracellular domain of the ROR1 protein. In one embodiment, the ROR1 protein is expressed on cancer cells.

[0185] As used herein, the term "CD33" refers to the cluster of differentiation 33 protein, an antigenic determinant detectable on leukemia cells and normal progenitor cells of the myeloid lineage. Human and mouse 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 under UniProt / Swiss-Prot accession number P20138, and the nucleotide sequence encoding human CD33 can be found under accession number NM_001772.3. In one embodiment, the antigen-binding portion of the CAR recognizes and binds to an epitope within the extracellular domain of the CD33 protein or a fragment thereof. In one embodiment, the CD33 protein is expressed on cancer cells. As used herein, "CD33" includes proteins containing mutations of full-length wild-type CD33, such as point mutations, fragments, insertions, deletions, and splice variants.

[0186] As used herein, the term "BCMA" refers to a 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 activator of the TNF family (BAFF) and proliferation-inducing ligand (APRIL). BCMA is involved in mediating plasma cell survival to maintain long-term humoral immunity. The BCMA gene is encoded on chromosome 16 and produces a 994-nucleotide primary mRNA transcript (NCBI accession number NM_001192.2) that encodes a 184-amino acid protein (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., 1994, 22:1147-1154). Additional transcript variants have been described, but the significance is unknown (Smirnova AS et al. Mol Immunol., 2008, 45(4):1179-1183). A second isoform, also known as TV4, has been identified (Uniprot identifier Q02223-2). As used herein, "BCMA" includes proteins containing mutations of full-length wild-type BCMA, such as point mutations, fragments, insertions, deletions, and splice variants.

[0187] As used herein, the term "CLL-1" refers to C-type lectin-like molecule-1, an antigenic determinant detectable on leukemia 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 mouse 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 under UniProt / Swiss-Prot accession number Q5QGZ9, and the nucleotide sequence encoding human CLL-1 can be found under accession numbers NM 001207010.1, NM 138337.5, 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.

[0188] 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 in GenBank Accession No. AF125253 and SwissProt Accession No. P00533-2.

[0189] The term "EGFRvIII" refers to epidermal growth factor receptor variant III. EGFRvIII is the most common variant of EGFR observed in human tumors but rarely in normal tissues. This protein arises from an in-frame deletion of exons 2-7 within the extracellular domain of EGFR and the generation of a novel glycine residue at the junction of exons 1 and 8, thereby creating a tumor-specific epitope. EGFRvIII is expressed in 24%-67% of GBMs but not in normal tissues. EGFRvIII, also known as type III mutant, Δ-EGFR, EGFRde2-7, and EGFR, 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. EGFRvIII expression can also result from chromosomal deletions or aberrant alternative splicing. See Sugawa et al., 1990, Proc. Natl. Acad. Sci. 87:8602-8606.

[0190] As used herein, the term "mesothelin" refers to mesothelin, a 40 kDa protein anchored to the cell membrane by a glycosylphosphatidylinositol (GPI) linkage and an amino-terminal 31 kDa shedding fragment, termed megakaryocyte potentiating factor (MPF). Both fragments contain N-glycosylation sites. The term also refers to a soluble splice variant of the 40 kDa carboxyl-terminal fragment, also termed "soluble mesothelin / MPF-related." Preferably, the term refers to human mesothelin, GenBank Accession No. AAH03512.1, as expressed on cell membranes, e.g., cancer cell membranes, and naturally cleaved portions thereof.

[0191] The term "antibody," as used herein, refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and can be derived from natural or recombinant sources. An antibody can be a tetramer of immunoglobulin molecules.

[0192] The term "antibody fragment" refers to at least a portion of an intact antibody or a recombinant variant thereof, and refers to the antigen-binding domain of an intact antibody, e.g., the antigen-determining variable region, sufficient to result in recognition and specific binding of the antibody fragment to a target such as an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab'), and Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies (either VL or VH) such as sdAbs, camelid VHH domains, and multispecific antibodies formed from antibody fragments such as bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, and isolated CDRs or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be grafted onto polypeptide-based scaffolds, such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies).

[0193] The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light and heavy chain variable regions are closely linked by a short flexible polypeptide linker, capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein, an scFv can have the VL and VH variable regions in either order, e.g., relative to the N- and C-terminal ends of the polypeptide, and can comprise a VL-linker-VH or a VH-linker-VL.

[0194] The term "complementarity-determining region" or "CDR," as used herein, refers to a sequence of amino acids within an antibody variable region that confers antigen specificity and binding affinity. For example, there are typically 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 several well-known schemes, including 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 a combination thereof. Based on the Kabat numbering scheme, in some embodiments, the CDR amino acid residues of the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues of the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Based on the Chothia numbering scheme, in some embodiments, the CDR amino acid residues of the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues of the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In a combination of the Kabat and Chothia numbering schemes, in some embodiments, the CDRs correspond to amino acid residues that are part of a Kabat CDR, a Chothia CDR, or both.For example, in some embodiments, the CDRs correspond to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) of a VH, e.g., a mammalian VH, e.g., a human VH, and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) of a VL, e.g., a mammalian VL, e.g., a human VL.

[0195] The portion of the CAR composition of the present invention comprising an antibody or antibody fragment thereof can exist in various forms, with the antigen-binding domain expressed as part of a contiguous polypeptide chain, including, for example, single-domain antibody fragments (sdAbs), single-chain antibodies (scFvs), and humanized or human antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, 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 embodiment, the antigen-binding domain of the CAR composition of the present invention comprises an antibody fragment. In a further embodiment, the CAR comprises an antibody fragment comprising an scFv.

[0196] 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, e.g., an immunoglobulin chain or fragment thereof, that comprises at least one immunoglobulin variable domain sequence. The term "binding domain" or "antibody molecule" encompasses antibodies and antibody fragments. In certain embodiments, an antibody molecule is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In certain embodiments, a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity 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.

[0197] The term "antibody heavy chain" refers to the larger of the two polypeptide chains present in antibody molecules in their naturally occurring conformation, and which usually determines the class to which the antibody belongs.

[0198] The term "antibody light chain" refers to the smaller of the two polypeptide chains present in antibody molecules in their naturally occurring conformation. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

[0199] The term "recombinant antibody" refers to an antibody made using recombinant DNA techniques, such as, for example, an antibody expressed in a bacteriophage or yeast expression system. The term should also be construed to mean an antibody made by synthesis of a DNA molecule encoding the antibody and which expresses the antibody protein, or an amino acid sequence specifying that antibody, where the DNA or amino acid sequence was obtained using well-known recombinant DNA or amino acid sequence techniques available in the art.

[0200] The term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response may include either or both antibody production and activation of specific immunologically competent cells. Those skilled in the art will understand that any macromolecule can be an antigen, including virtually any protein or peptide. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will therefore understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response encodes an "antigen" as that term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of two or more genes, and that these nucleotide sequences are arranged in various combinations to encode a polypeptide that elicits a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be synthetically produced, obtained from a biological sample, or can be a macromolecule other than a polypeptide. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells or bodily fluids, along with other biological components.

[0201] The term "anti-tumor effect" refers to a biological effect that can be manifested by various means, including, but not limited to, a reduction in tumor volume, a reduction in tumor cell number, a reduction in the number of metastases, an increase in life expectancy, a reduction in tumor cell proliferation, a reduction in tumor cell survival, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the invention in preventing the development of tumors in the first place.

[0202] The term "anti-cancer effect" refers to a biological effect that can be manifested by various means, including, but not limited to, a reduction in tumor volume, a reduction in the number of cancer cells, a reduction in the number of metastases, an increase in life expectancy, a reduction in cancer cell proliferation, a reduction in cancer cell survival, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-cancer effect" can also be manifested by the ability of peptides, polynucleotides, cells, and antibodies in preventing the development of cancer in the first place.

[0203] The term "anti-tumor effect" refers to a biological effect that may be manifested by various means, including, but not limited to, a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0204] The term "autologous" refers to any material originating from the same individual that is later reintroduced into that individual.

[0205] The term "allogeneic" refers to any material derived from a different animal of the same species as the individual into which 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 embodiments, allogeneic materials from individuals of the same species may be genetically different enough to interact antigenically.

[0206] The term "xenogeneic" refers to a graft derived from an animal of a different species.

[0207] The term "apheresis," as used herein, refers to the art-recognized extracorporeal process in which a donor's or patient's blood is removed from the donor or patient, passed through a device to remove one or more selected specific components, and the remainder is returned to the donor's or patient's circulation, e.g., by transfusion. Thus, reference to an "apheresis sample" refers to a sample obtained using apheresis.

[0208] The term "combination" refers to either a fixed combination in a single dosage unit form, or a combination administration in which a compound of the present invention and a combination partner (e.g., another drug, as described below, also referred to as a "therapeutic agent" or "co-drug") can be independently administered separately at the same time or within a time interval, particularly where these time intervals allow the combination partners to exhibit a cooperative effect, e.g., a synergistic effect. Single components can be packaged in a kit or separately. One or both of the components (e.g., powder or liquid) can be reconstituted or diluted to the desired dose before administration. The terms "co-administration" or "co-administration," etc., as used herein, are meant to encompass the administration of selected combination partners to a single subject (e.g., patient) in need thereof, and are intended to include therapeutic regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The term "pharmaceutical combination," as used herein, refers to a product obtained by mixing or combining two or more active ingredients, and includes both fixed and non-fixed combinations of active ingredients. The term "fixed combination" means that the active ingredients, e.g., a compound of the present invention and a combination partner, are both 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., a compound of the present invention and a combination partner, are both administered to a patient simultaneously, in parallel, or alternatively sequentially as separate entities without any specific time limit, such administration providing therapeutically effective levels of the two compounds in the patient's body. The latter also applies to cocktail therapy, e.g., the administration of three or more active ingredients.

[0209] The term "cancer" refers to a disease characterized by the rapid, uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Various examples of cancer are described herein and include, but are 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 "carcinoma" are used interchangeably herein; for example, both terms encompass solid and liquid tumors, e.g., diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" includes precancerous as well as malignant cancers and tumors.

[0210] "Derived from," as the term is used herein, refers to the relationship between a first molecule and a second molecule. This generally refers to the structural similarity between the first and second molecules and does not imply or include a limitation on the manner or source by which the first molecule is derived from the second molecule. For example, in the case of an intracellular signaling domain derived from the CD3ζ molecule, the intracellular signaling domain retains sufficient CD3ζ structure such that it has the desired function, i.e., the ability to generate a signal under appropriate conditions. This does not imply or include a limitation to a particular method of making the intracellular signaling domain, e.g., it does not mean that one must start with the CD3ζ sequence and delete unnecessary sequence or make mutations to arrive at the intracellular signaling domain in order to provide the intracellular signaling domain.

[0211] The phrase "disease associated with B cell antigen expression" includes diseases associated with 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, but not limited to, proliferative diseases such as cancer or malignant tumors, or precancerous conditions such as myelodysplasia, myelodysplastic syndrome, or preleukemia, 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 once expressed the B cell antigen but no longer express it, e.g., because antigen expression has been downregulated by, e.g., treatment with a molecule that targets the B cell antigen, e.g., a B cell-targeted CAR. The phrase "disease associated with B cell antigen expression" includes diseases associated with expression of CD19, as described herein.

[0212] The phrase "disease associated with CD19 expression" includes diseases associated with CD19 expression, or conditions associated with cells that express, or have at any time expressed, CD19, including, but not limited to, proliferative diseases such as cancer or malignant tumors, or precancerous conditions such as myelodysplasia, myelodysplastic syndrome, or preleukemia, or non-cancer-related symptoms associated with cells that express CD19. For the avoidance of doubt, diseases associated with CD19 expression may include conditions associated with cells that once expressed CD19 but no longer express CD19, for example, due to downregulation of CD19 expression, for example, by treatment with a molecule that targets CD19, e.g., a CD19 CAR. In one embodiment, the cancer associated with CD19 expression is a blood cancer. In one embodiment, the blood cancer is leukemia or lymphoma. In one aspect, cancers associated with expression of CD19 include cancers and malignancies including, but not limited to, one or more acute leukemias, for example, but not limited to, B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), acute lymphoblastic leukemia (ALL), one or more chronic leukemias, for example, but not limited to, chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL). Additional cancers or hematological conditions associated with expression of CD19 include, but are not limited to, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma (MCL), marginal zone lymphoma, multiple myeloma, myelodysplastic and myelodysplastic syndromes, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, and "preleukemias," a group of diverse hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells. Additionally, diseases associated with the expression of CD19 expression include, but are not limited to, atypical and / or non-classical cancers, malignancies, precancerous conditions or proliferative disorders associated with the expression of CD19.Non-cancer-related indications associated with CD19 expression include, but are not limited to, autoimmune diseases (e.g., lupus), inflammatory disorders (allergies and asthma), and transplantation. In some embodiments, tumor antigen-expressing cells express, or have at some time expressed, mRNA encoding a tumor antigen. In certain embodiments, tumor antigen-expressing cells produce tumor antigen proteins (e.g., wild-type or mutant), which may be present at normal or reduced levels. In certain embodiments, tumor antigen-expressing cells transiently produce detectable levels of tumor antigen proteins, but subsequently cease to produce substantially detectable tumor antigen proteins.

[0213] As used herein, the phrase "disease associated with expression of CD123" includes a disease associated with expression of CD123 or a condition associated with cells expressing CD123 (e.g., wild-type or mutant CD123), or a non-cancer-related indication associated with cells expressing CD123 (e.g., wild-type or mutant CD123), including, but not limited to, a proliferative disease such as a cancer or malignant tumor, a precancerous condition such as myelodysplasia, myelodysplastic syndrome, or preleukemia. In one embodiment, the cancer associated with expression of CD123 (e.g., wild-type or mutant CD123) is a hematological cancer. In one aspect, the disease includes AML, ALL, hairy cell leukemia, prolymphocytic leukemia, chronic myeloid leukemia (CML), Hodgkin's 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, histiocytic disorder (e.g., mast cell disorder or blastic plasmacytoid dendritic cell neoplasm), mast cell disorder, such as systemic mastocytosis or mast cell leukemia, etc. Additional diseases associated with the expression of CD123 expression include, but are not limited to, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative disorders associated with the expression of CD123, for example. Non-cancer related indications associated with the expression of CD123 may also be included.

[0214] As used herein, the phrase "disease associated with CD33 expression" includes a disease associated with cells expressing CD33 (e.g., wild-type or mutant CD33), or a condition associated with cells expressing CD33 (e.g., wild-type or mutant CD33), including, but not limited to, a proliferative disease such as a cancer or malignant tumor, or a precancerous condition such as myelodysplasia, myelodysplastic syndrome, or preleukemia, or a non-cancer-related indication associated with cells expressing CD33 (e.g., wild-type or mutant CD33). For the avoidance of doubt, a disease associated with CD33 expression may include a condition associated with cells that previously expressed CD33 but no longer express CD33, e.g., due to downregulation of CD33 expression, e.g., by treatment with a molecule that targets CD33, e.g., a CD33 inhibitor described herein. In one embodiment, 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), myelodysplastic and myelodysplastic syndromes, myelofibrosis and myeloproliferative neoplasms, acute lymphocytic leukemia (ALL), hairy cell leukemia, prolymphocytic leukemia, chronic myelogenous leukemia (CML), blastic plasmacytoid dendritic cell neoplasms, etc. Additional diseases associated with the expression of CD33 (e.g., wild-type or mutant CD33) include, but are not limited to, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative disorders associated with the expression of CD33 (e.g., wild-type or mutant CD33). Non-cancer-related indications associated with the expression of CD33 (e.g., wild-type or mutant CD33) may also be included. In embodiments, non-cancer-related indications associated with the expression of CD33 include, but are not limited to, autoimmune diseases (e.g., lupus), inflammatory disorders (allergies and asthma), and transplantation. In some embodiments, the tumor antigen-expressing cells express, or at some time expressed, mRNA encoding the tumor antigen. In certain embodiments, the tumor antigen-expressing cells produce a tumor antigen protein (e.g., wild-type or mutant), which may be present at normal or reduced levels.In certain embodiments, the tumor antigen-expressing cells transiently produced detectable levels of the tumor antigen protein, but subsequently ceased to produce substantially detectable tumor antigen protein.

[0215]

[0013] The phrase "disease associated with BCMA expression" includes a disease or condition associated with cells expressing BCMA (e.g., wild-type or mutant BCMA), or a non-cancer related indication associated with cells expressing BCMA (e.g., wild-type or mutant BCMA), including, but not limited to, a proliferative disorder such as a cancer or malignancy, or a precancerous condition such as myelodysplasia, myelodysplastic syndrome, or preleukemia. For the avoidance of doubt, a disease associated with BCMA expression may include a condition associated with cells that previously expressed BCMA, but no longer express BCMA due to downregulation of BCMA expression, for example, by treatment with a molecule that targets BCMA, such as a BCMA inhibitor described herein. In one embodiment, the cancer associated with BCMA (e.g., wild-type or mutant BCMA) expression is a hematological cancer. In one embodiment, the hematological cancer is leukemia or lymphoma. In one embodiment, the cancer associated with expression of BCMA (e.g., wild-type or mutant BCMA) is a differentiated plasmacytic B-cell malignancy. In one embodiment, the cancer associated with expression of BCMA (e.g., wild-type or mutant BCMA) includes cancers and malignancies including, but not limited to, one or more acute leukemias, for example, but not limited to, B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), acute lymphoblastic leukemia (ALL), and one or more chronic leukemias, for example, but not limited to, chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL).Additional cancers or hematological conditions associated with expression of BMCA (e.g., wild-type or mutant BCMA) include, but are not limited to, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic and myelodysplastic syndromes, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, and "preleukemias," a group of diverse hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells. In some embodiments, the cancer is multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's 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), Waldenstrom's macroglobulinemia, plasmacytomas (e.g., dysplasmocytosis, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytoma), systemic amyloid light-chain amyloidosis, and POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease, and PEP syndrome). Further diseases associated with expression of BCMA (e.g., wild-type or mutant BCMA) expression include, but are not limited to, atypical and / or non-classical cancers, malignancies, pre-cancerous conditions or proliferative disorders associated with expression of BCMA (e.g., wild-type or mutant BCMA), such as those cancers described herein, for example, prostate cancer (e.g., castration-resistant or therapy-resistant prostate cancer, or metastatic prostate cancer), pancreatic cancer, or lung cancer.

[0216] Non-cancer-related conditions associated with BCMA (e.g., wild-type or mutant BCMA) include viral infections, such as HIV; fungal infections, such as C. neoformans; autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus (SLE or lupus), pemphigus vulgaris, and Sjogren's syndrome; inflammatory bowel disease; ulcerative colitis; transplant-associated allospecific immune disorders associated with mucosal immunity; and undesired immune responses to biologics (e.g., factor VIII) where humoral immunity is important. In embodiments, non-cancer-related indications associated with BCMA expression include, but are not limited to, autoimmune diseases (e.g., lupus), inflammatory disorders (allergies and asthma), and transplantation. In some embodiments, tumor antigen-expressing cells express, or have at some time expressed, mRNA encoding a tumor antigen. In certain embodiments, tumor antigen-expressing cells produce a tumor antigen protein (e.g., wild-type or mutant), which may be present at normal or reduced levels. In certain embodiments, the tumor antigen-expressing cells transiently produced detectable levels of the tumor antigen protein, but subsequently ceased to produce substantially detectable tumor antigen protein.

[0217] The phrase "disease associated with CLL-1 expression" includes diseases or conditions associated with cells expressing CLL-1, including, but not limited to, proliferative diseases such as cancer or malignant tumors, or precancerous conditions such as myelodysplasia, myelodysplastic syndrome, or preleukemia, or non-cancer-related indications associated with cells expressing CLL-1 (e.g., wild-type or mutant CLL-1). For the avoidance of doubt, diseases associated with CLL-1 expression may include conditions associated with cells that formerly expressed CLL-1 but no longer express CLL-1, e.g., due to downregulation of CLL-1 expression, e.g., by treatment with a molecule that targets CLL-1, e.g., a CLL-1 inhibitor described herein. In one embodiment, the cancer associated with CLL-1 expression is a hematological cancer. In one aspect, hematological cancers include malignant lymphoproliferative conditions, including, but not limited to, leukemias (such as acute myeloid leukemia, chronic myelocytic leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, and myelodysplastic syndromes) and lymphomas (such as multiple myeloma, non-Hodgkin's lymphoma, Burkitt's lymphoma, and small cell and large cell follicular lymphoma). Additional diseases associated with the expression of CLL-1 expression include, but are not limited to, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative disorders associated with the expression of CLL-1. Non-cancer-related indications associated with the expression of CLL-1 may also be included. In some embodiments, tumor antigen-expressing cells express, or have at some time expressed, mRNA encoding a tumor antigen. In certain embodiments, tumor antigen-expressing cells produce a tumor antigen protein (e.g., wild-type or mutant), which may be present at normal or reduced levels. In certain embodiments, the tumor antigen-expressing cells transiently produced detectable levels of the tumor antigen protein, but subsequently ceased to produce substantially detectable tumor antigen protein.

[0218] As used herein, the term "diseases associated with EGFRvIII expression" includes diseases associated with EGFRvIII expression or pathological conditions associated with cells expressing EGFRvIII, including, but not limited to, 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 the CARs disclosed herein induce an antigen-specific response to EGFRvIII, thereby providing one or more of the following: targeting and destruction of EGFRvIII-expressing tumor cells, tumor reduction or elimination, enhanced infiltration of immune cells to the tumor site, and enhanced / prolonged anti-tumor responses. Because EGFRvIII is not expressed at detectable levels in normal (i.e., non-cancerous) tissues, it is contemplated that the CARs of the present invention advantageously substantially avoid targeting / destruction of normal tissues and cells.

[0219] As used herein, the phrase "disease associated with mesothelin expression" includes diseases associated with mesothelin expression or conditions associated with cells that express mesothelin, including, but not limited to, proliferative diseases such as cancer or malignant tumors or precancerous conditions such as mesothelin hyperplasia, or non-cancer-related indications associated with cells that express mesothelin. Examples of various cancers that express mesothelin include, but are not limited to, mesothelioma, ovarian cancer, pancreatic cancer, etc.

[0220] In some embodiments, tumor antigen (e.g., CD123 or CD19)-expressing cells express, or have at some time expressed, mRNA encoding the tumor antigen. In certain embodiments, tumor antigen (e.g., CD123 or CD19)-expressing cells produce tumor antigen protein (e.g., wild-type or mutant), which may be present at normal or reduced levels. In certain embodiments, tumor antigen (e.g., CD123 or CD19)-expressing cells transiently produce detectable levels of tumor antigen protein, but subsequently cease to produce substantially detectable tumor antigen protein.

[0221] The term "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing that amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into an antibody or antibody fragment of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative substitutions are those 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 with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a CAR of the invention can be replaced with other amino acid residues from the same side chain family, and the altered CAR can be tested using the functional assays described herein.

[0222] The term "stimulation" refers to a primary response resulting from the binding of a stimulatory molecule (e.g., a TCR / CD3 complex) to its cognate ligand, thereby mediating a signal transduction event, such as, but not limited to, signal transduction through the TCR / CD3 complex. Stimulation can mediate changes in the expression of certain molecules, such as downregulation of TGF-β, and / or recognition of cytoskeletal structures.

[0223] The term "stimulatory molecule" refers to a molecule expressed by a T cell that provides one or more primary cytoplasmic signaling sequences that regulate primary activation of the TCR complex in a stimulatory manner for at least some aspect of the T cell signaling pathway. In one embodiment, the primary signal is initiated, for example, by binding of the TCR / CD3 complex to a peptide-loaded MHC molecule, which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, etc. Primary cytoplasmic signaling sequences (also referred to as "primary signaling domains") that function in a stimulatory manner can contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs. Examples of ITAM-containing primary cytoplasmic signaling sequences that are particularly useful 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 known as "ICOS"), FcεRI, CD66d, DAP10, and DAP12. In specific CARs of the invention, the intracellular signaling domain of any one or more CARs of the invention comprises an intracellular signaling sequence, e.g., the primary signaling sequence of CD3-zeta. In specific CARs of the invention, the primary signaling sequence of CD3-zeta is the sequence provided as SEQ ID NO: 9, or equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc. In specific CARs of the invention, the primary signaling sequence of CD3-zeta is the sequence as provided in SEQ ID NO: 10, or equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc.

[0224] The term "antigen-presenting cell" or "APC" refers to a cell of the immune system, such as an accessory cell (e.g., a B cell, a dendritic cell, etc.), that presents foreign antigens complexed with major histocompatibility complexes (MHC) on its surface. T cells can recognize such complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.

[0225] As used herein, the term "intracellular signaling domain" refers to the intracellular portion of a molecule. The intracellular signaling domain can generate a signal that promotes immune effector function of a CAR-containing cell, such as a CART cell or a CAR-expressing NK cell. For example, examples of immune effector function in a CART cell or a CAR-expressing NK cell include helper activity, including cytolytic activity and cytokine secretion. In embodiments, the intracellular signaling domain transmits an effector function signal, directing the cell to perform a specialized function. While the entire intracellular signaling domain can be used, it is often not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, it can be used in place of the intact chain, so long as the truncated portion transmits the effector function signal. Thus, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0226] In certain embodiments, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules involved in primary stimulation or antigen-dependent stimulation. In certain embodiments, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules involved in costimulatory signals or antigen-independent stimulation. For example, in the case of CAR-expressing immune effector cells, such as CART cells or CAR-expressing NK cells, the primary intracellular signaling domain can comprise a cytoplasmic sequence of a T cell receptor, and the costimulatory intracellular signaling domain can comprise a cytoplasmic sequence from a co-receptor or costimulatory molecule.

[0227] A primary intracellular signaling domain can contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. Examples of primary cytoplasmic signaling sequences containing ITAMs include, but are not limited to, those derived from CD3zeta, FcRgamma, FcRbeta, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, CD278 ("ICOS"), FcεRI, CD66d, DAP10, and DAP12.

[0228] The term "ζ" or alternatively "ζ chain," "CD3-ζ," or "TCR-ζ" is defined as the protein provided under GenBank Accession No. BAG36664.1, or equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc., and "ζ stimulatory domain" or alternatively "CD3-ζ stimulatory domain" or "TCR-ζ stimulatory domain" is defined as the amino acid residues from the cytoplasmic domain of the ζ chain sufficient to functionally transmit the initial signal required for T cell activation. In one embodiment, the cytoplasmic domain of ζ comprises residues 52-164 of GenBank Accession No. BAG36664.1, or equivalent residues from a non-human species that is a functional ortholog thereof, e.g., mouse, rodent, monkey, ape, etc. In one embodiment, the "ζ stimulatory domain" or "CD3-ζ stimulatory domain" is the sequence provided as SEQ ID NO:9. In one embodiment, the "zeta stimulatory domain" or "CD3-zeta stimulatory domain" is the sequence provided as SEQ ID NO:10.

[0229] 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 by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient 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 molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), and 4-1BB. (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NK p44, 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), CEA These include ligands that specifically bind to CAM1, 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 CD83.

[0230] A costimulatory intracellular signaling domain refers to the intracellular portion of a costimulatory molecule. The intracellular signaling domain can include the entire intracellular portion of the molecule from which it is derived, or the entire native intracellular signaling domain, or a functional fragment thereof.

[0231] The term "4-1BB" refers to a member of the TNFR superfamily having the amino acid sequence provided as GenBank Accession No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc., and a "4-1BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank Accession No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc. In one embodiment, the "4-1BB costimulatory domain" is the sequence provided as SEQ ID NO: 7, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc.

[0232] "Immune effector cells," as the term is used herein, refer to cells that are involved in an immune response, e.g., promoting an immune effector response. Examples of immune effector cells include T cells, e.g., α / β T cells and γ / δ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes.

[0233] "Immune effector function or immune effector response," as the term is used herein, refers to a function or response, e.g., of an immune effector cell, that enhances or promotes an immune attack of a target cell. For example, an immune effector function or response refers to a property of a T cell or NK cell that promotes the killing or growth or proliferation inhibition of a target cell. In the case of T cells, primary stimulation and costimulation are examples of immune effector functions or responses.

[0234] The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell can be cytolytic activity or helper activity, including secretion of cytokines.

[0235] The term "encoding" refers to the inherent property of a specific nucleotide sequence within a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have either a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a defined amino acid sequence. Thus, a gene, cDNA, or RNA encodes a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. 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 is used as a transcription template for a gene or cDNA, can be said to encode the protein or other product of that gene's cDNA.

[0236] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns, to the extent that the nucleotide sequence encoding the protein may, in some versions, contain one or more introns.

[0237] The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material, or composition as described herein that is effective to achieve a particular biological result.

[0238] The term "endogenous" refers to any material that is derived from or produced within an organism, cell, tissue, or system.

[0239] The term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0240] The term "expression" refers to the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0241] The term "transfer vector" refers to a composition containing an isolated nucleic acid that can be 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 self-replicating plasmids or viruses. This term should be construed to further include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.

[0242] The term "expression vector" refers to a vector containing a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those 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.

[0243] The term "vector," as used herein, refers to any vehicle that can be used to deliver and / or express a nucleic acid molecule. This may be a transfer vector or an expression vector, as described herein.

[0244] The term "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells, and they can deliver large amounts of genetic information into the DNA of host cells, making them one of the most efficient gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses.

[0245] The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, including, inter alia, self-inactivating lentiviral vectors as provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that may be used clinically include, but are not limited to, the LENTIVECTOR® gene delivery technology from Oxford BioMedica and the LENTIMAX™ vector system from Lentigen. Non-clinical types of lentiviral vectors are also available and will be known to those skilled in the art.

[0246] The term "homologous" or "identity" refers to the subunit sequence identity between two polymer molecules, e.g., between two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomer subunit, e.g., if a position in each of the two DNA molecules is occupied by adenine, 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 are homologous (e.g., 5 positions in a polymer 10 subunits long), the two sequences are 50% homologous. If 90% of the positions (e.g., 9 out of 10) are matched or homologous, the two sequences are 90% homologous.

[0247] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies and antibody fragments thereof are those in which residues from a recipient complementarity-determining region (CDR) of a human immunoglobulin (recipient antibody or antibody fragment) are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies / antibody fragments can comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications can further refine and optimize antibody or antibody fragment performance. Generally, a humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin and all or most of the FR regions being those of a human immunoglobulin sequence. The humanized antibody or antibody fragment can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further 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.

[0248] "Fully human" refers to an immunoglobulin, such as an antibody or antibody fragment, whose entire molecule is of human origin or consists of an amino acid sequence identical to a human form of an antibody or immunoglobulin.

[0249] The term "isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that has been partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0250] In the context of the present invention, the following abbreviations are used for commonly occurring nucleobases: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0251] The term "operably linked" or "transcriptional control" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in 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 it affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, for example, in the same reading frame, as necessary to join two protein-coding regions.

[0252] The term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, intratumoral, or infusion techniques.

[0253] The terms "nucleic acid," "polynucleotide," or "nucleic acid molecule" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or combinations of DNA or RNA, and polymers thereof, in either single- or double-stranded form. The term "nucleic acid" includes genes, cDNA, or mRNA. In one embodiment, a 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 specified nucleic acid sequence implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly indicated sequence. Specifically, degenerate codon substitutions can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (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)).

[0254] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, with no limit on the maximum number of amino acids that can comprise a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and to the numerous types of longer chains generally referred to in the art as proteins. "Polypeptide" specifically includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins. A polypeptide includes natural peptides, recombinant peptides, or combinations thereof.

[0255] The term "promoter" refers to a DNA sequence recognized by the synthetic machinery of a cell or introduced synthetic machinery necessary to initiate the specific transcription of a polynucleotide sequence.

[0256] The term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to that promoter / regulatory sequence. In some instances, this sequence may be a core promoter sequence, and in other instances, this sequence may also include an enhancer sequence and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that confers tissue-specific expression of the gene product.

[0257] The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the production of the gene product in a cell under most or all physiological conditions of the cell.

[0258] The term "inducible" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes production of the gene product in a cell substantially only when an inducer corresponding to the promoter is present in the cell.

[0259] The term "tissue-specific" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specified by a gene, causes the production of a gene product in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0260] The terms "cancer-associated antigen" or "tumor antigen" refer interchangeably to a molecule (typically a protein, carbohydrate, or lipid) that is expressed on the surface of cancer cells, either entirely or alternatively as fragments (e.g., MHC / peptides), and that is useful for preferential targeting of pharmacological agents to cancer cells. In some embodiments, a 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, a tumor antigen is a cell surface molecule that is overexpressed on 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, a tumor antigen is a cell surface molecule that is inappropriately synthesized on cancer cells, e.g., a molecule that contains deletions, additions, or mutations compared to the molecule expressed on normal cells. In some embodiments, a tumor antigen will be expressed exclusively on the cell surface of cancer cells, either entirely or alternatively as fragments (e.g., MHC / peptides), and not synthesized or expressed on the surface of normal cells. In some embodiments, the CARs of the present invention include CARs comprising an antigen-binding domain (e.g., an antibody or antibody fragment) that binds to an MHC-presented peptide. Typically, peptides derived from endogenous proteins fit into a pocket of a major histocompatibility complex (MHC) class I molecule 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 by screening a library, such as a human scFv phage display library.

[0261] The term "flexible polypeptide linker" or "linker," when used in the context of an scFv, refers to a peptide linker composed of amino acids such as glycine and / or serine residues used alone or in combination to link the variable heavy and variable light chain regions 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, e.g., 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, flexible polypeptide linkers include, but are not limited to, (Gly4Ser)4 (SEQ ID NO:27) or (Gly4Ser)3 (SEQ ID NO:28). In another embodiment, the linker comprises multiple repeats of (Gly2Ser), (GlySer), or (Gly3Ser) (SEQ ID NO:29). Also included within the scope of the present invention are the linkers described in WO 2012 / 138475, which is incorporated herein by reference.

[0262] As used herein, a 5' cap (RNA cap, RNA 7-methylguanosine cap or RNA m 7 A 5' cap (also called a G-cap) is a modified guanine nucleotide added to the "pre" or 5' end of eukaryotic messenger RNA immediately after transcription initiation. The 5' cap consists of a terminal group attached to the first transcribed nucleotide. Its presence is important for ribosome recognition and protection from RNases. Capping is coupled to transcription and occurs cotranscriptionally, with each cap affecting the other. Shortly after transcription initiation, a cap-synthesizing complex associated with RNA polymerase binds to the 5' end of the mRNA being synthesized. This enzyme complex catalyzes the chemical reactions required for mRNA capping. Synthesis proceeds as a multistep biochemical reaction. The capping moiety can be modified to adjust mRNA functions such as its stability or translation efficiency.

[0263] As used herein, "in vitro transcribed RNA" refers to in vitro synthesized RNA, preferably mRNA. Generally, in vitro transcribed RNA is generated from an in vitro transcription vector. The in vitro transcription vector contains a template used to generate the in vitro transcribed RNA.

[0264] As used herein, "poly(A)" refers to a series of adenosines added to mRNA by polyadenylation. In a preferred embodiment of a construct for transient expression, the poly(A) is 50 to 5,000 (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 modified chemically or enzymatically to adjust mRNA function, such as localization, stability, or translation efficiency.

[0265] As used herein, "polyadenylation" refers to the covalent attachment of a polyadenylyl moiety, or modified variants 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 (often several hundred) of adenine nucleotides added to pre-mRNA by the action of the 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 its associated proteins help protect the mRNA from exonuclease degradation. Polyadenylation is also important for transcription termination, mRNA nuclear export, and translation. Polyadenylation occurs in the nucleus immediately after DNA-to-RNA transcription, but can also occur later in the cytoplasm. After transcription is terminated, the mRNA strand is cleaved by the action of an endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA is cleaved, an adenosine residue is added to the free 3' end of the cleavage site.

[0266] As used herein, "transient" refers to expression of a non-integrated transgene for a period of hours, days, or weeks, which is shorter than the period of expression of the gene when integrated into the genome or contained in a stable plasmid replicon in the host cell.

[0267] As used herein, the terms "treat," "treatment," and "treating" refer to a reduction or amelioration of the progression, severity, and / or duration of a proliferative disorder, or an amelioration of one or more symptoms (preferably one or more discernible symptoms) of a proliferative disorder, brought about by the administration of one or more therapies (e.g., one or more therapeutic agents, such as a CAR of the invention). In specific embodiments, the terms "treat," "treatment," and "treating" refer to an improvement in at least one measurable physical parameter of a proliferative disorder, such as tumor growth, which may not necessarily be discernible to the patient. In other embodiments, the terms "treat," "treatment," and "treating" refer to either or both of a physical inhibition of the progression of a proliferative disorder, e.g., by stabilization of a discernible symptom, or a physiological inhibition, e.g., by stabilization of a physical parameter. In other embodiments, the terms "treat," "treatment," and "treating" refer to a reduction or stabilization of tumor size or cancerous cell number.

[0268] A dosage regimen, e.g., a therapeutic dosage regimen, can include one or more treatment intervals. A dosage regimen can produce at least one beneficial or desired clinical result, whether detectable or undetectable, including, but not limited to, alleviation of symptoms, reduction in the extent of the disease, stabilization of (i.e., not worsening of) the disease state, delay or slowing of disease progression, or improvement or palliation of the disease state.

[0269] As used herein, "treatment interval" refers to a treatment cycle, e.g., a course of administration of a therapeutic agent, which may be repeated, for example, on a regular schedule. In embodiments, a dosage regimen may have one or more periods during which no therapeutic agent is administered over the treatment interval. For example, a treatment interval may include a dose of a CAR molecule administered in combination with (before, concurrently with, or after) the administration of a second therapeutic agent, e.g., an inhibitor (e.g., a kinase inhibitor as described herein).

[0270] The term "signal transduction pathway" refers to the biochemical relationships between various signaling 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 have the ability to receive a signal and transmit the signal across the membrane of a cell.

[0271] The term "subject" is intended to include living organisms (eg, mammals, humans) in which an immune response can be generated.

[0272] The term "substantially purified" cells refers to cells that are essentially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types with which they are normally associated in their naturally occurring state. In some instances, a substantially purified cell population refers to a homogenous cell population. In other instances, the term simply refers to cells that have been separated from the cells with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0273] The term "therapeutic" as used herein means treatment. A therapeutic effect is achieved by the reduction, suppression, amelioration, or eradication of a disease state.

[0274] In embodiments, the disease state to be treated includes CRS. In some embodiments, treating CRS involves administering a composition or combination described herein after onset, e.g., after detection of one or more CRS symptoms. In some embodiments, treating CRS results in a reduction in the severity of CRS, e.g., compared to a subject not administered a composition or combination described herein. For example, a subject may experience a reduction in CRS to an undetectable level. In other embodiments, treatment may result in a less severe form of CRS, e.g., grade 1, 2, or 3 CRS.

[0275] The term "prevention," as used herein, refers to the prevention of or prophylactic treatment for a disease or disease state. Prevention of a disease or disease state can include, for example, a reduction (e.g., alleviation) of one or more symptoms of a disease or disease state compared to a baseline level (e.g., one or more symptoms thereof in a similar subject not receiving the treatment). Prevention can also include a delay in the onset of one or more symptoms of a disease or disease state compared to a baseline level (e.g., the onset of one or more symptoms thereof in a similar subject not receiving the treatment). In an embodiment, the disease is a disease described herein.

[0276] In embodiments, the disease state to be prevented includes CRS. In some embodiments, preventing CRS involves administering a composition or combination described herein before one or more symptoms of CRS, e.g., before their detection or occurrence. In some embodiments, administering a JAK-STAT inhibitor or a BTK inhibitor occurs before CAR therapy. In some embodiments, preventing CRS results in a reduced likelihood or severity of CRS, e.g., compared to a subject not administered a composition or combination described herein. For example, the subject may not develop CRS. In other embodiments, the subject develops a less severe form of CRS, e.g., grade 1, 2, or 3 CRS, e.g., compared to a subject not administered a composition or combination described herein.

[0277] In the context of the present invention, a "tumor antigen," or a "hyperproliferative disorder antigen," or an "antigen associated with a hyperproliferative disorder" refers to an antigen common to a specific hyperproliferative disorder. In certain embodiments, the hyperproliferative disorder antigen of the present invention is derived from a cancer, including, but not limited to, primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, renal cancer, and adenocarcinoma, such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, etc.

[0278] 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 one that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0279] The term "specifically binds" refers to an antibody or ligand that recognizes and binds to a cognate binding partner (e.g., a stimulatory and / or costimulatory molecule present on a T cell) protein present in a sample, but does not substantially recognize or bind to other molecules in the sample.

[0280] As used herein, a "regulatable chimeric antigen receptor (RCAR)" refers to a set of polypeptides, typically two polypeptides in the simplest embodiment, that, when present in an immune effector cell, confers specificity for a target cell, typically a cancer cell, and regulatable intracellular signal generation to the cell. In some embodiments, an 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") that comprises a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule as defined herein in the context of the CAR molecule. In some embodiments, the set of polypeptides of an RCAR are not contiguous with each other, e.g., on different polypeptide chains. In some embodiments, an RCAR comprises a dimerization switch that can couple the polypeptides to each other upon the presence of a dimerization molecule, e.g., can couple an antigen-binding domain to an intracellular signaling domain. In some embodiments, an RCAR is expressed in a cell (e.g., an immune effector cell) as described herein, e.g., an RCAR-expressing cell (also referred to herein as an "RCARX cell"). In some embodiments, the RCARX cells are T cells and are referred to as RCAR cells. In some embodiments, the RCARX cells are NK cells and are referred to as RCARN cells. The RCAR can confer specificity to the RCAR-expressing cells for target cells, typically cancer cells, and regulatable intracellular signal generation or proliferation, which can optimize the immune effector properties of the RCAR-expressing cells. In embodiments, the RCAR cells rely, at least in part, on the antigen-binding domain to confer specificity for target cells that contain the antigen bound by the antigen-binding domain.

[0281] "Membrane anchor" or "membrane tethering domain," as that term is used herein, refers to a polypeptide or moiety, e.g., a myristoyl group, sufficient to anchor an extracellular or intracellular domain to a cell membrane.

[0282] A "switch domain," as the term is used herein, e.g., in reference to an RCAR, refers to an entity, typically a polypeptide-based entity, that associates with another switch domain in the presence of a dimerization molecule. This association results in functional coupling between a first entity linked to, e.g., fused to, a first switch domain and a second entity linked to, e.g., fused to, a second switch domain. The first and second switch domains are collectively referred to as a dimerization switch. In embodiments, the first and second switch domains are identical to one another, e.g., they are polypeptides having the same primary amino acid sequence, and are collectively referred to as a homodimerization switch. In embodiments, the first and second switch domains are different from one another, e.g., they are polypeptides having different primary amino acid sequences, and are collectively referred to as a heterodimerization 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., FKBP- or FRB-based, and the dimerization molecule is a small molecule, e.g., a rapalog. In embodiments, the switch domain is a polypeptide-based entity, e.g., an scFv that binds a myc peptide, and the dimerization molecule is a polypeptide, fragment thereof, or multimer of a polypeptide, e.g., a myc ligand or multimer of a myc ligand that binds one or more myc scFvs. In embodiments, the switch domain is a polypeptide-based entity, e.g., a myc receptor, and the dimerization molecule is an antibody or fragment thereof, e.g., a myc antibody.

[0283] "Dimerization molecule," as the term is used herein, e.g., in reference to an RCAR, refers to a molecule that promotes association of a first switch domain with a second switch domain. In embodiments, the dimerization molecule is not naturally present in a subject or is not present at concentrations that would result in significant dimerization. In embodiments, the dimerization molecule is a small molecule, e.g., rapamycin or a rapalog, e.g., RAD001.

[0284] The term "bioequivalent" refers to an amount of an agent other than a reference compound (e.g., RAD001) required to produce an effect equivalent to that produced by a reference dose or amount of a reference compound (e.g., RAD001). In some embodiments, the effect is the level of mTOR inhibition, e.g., as measured by P70 S6 kinase inhibition, e.g., as assessed in an in vivo or in vitro assay, e.g., an assay described herein, e.g., a Boulay assay, or by measuring phosphorylated S6 levels by Western blot. In some embodiments, 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 some embodiments, a bioequivalent amount or dose of an mTOR inhibitor is an amount or dose that achieves the same level of P70 S6 kinase inhibition as a reference dose or amount of a reference compound. In one embodiment, a bioequivalent amount or dose of an mTOR inhibitor is an 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 a reference dose or reference amount of a reference compound.

[0285] The term "low immune-enhancing dose," when used in conjunction with an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., RAD001 or rapamycin, or a catalytic mTOR inhibitor, 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, e.g., by inhibition of P70 S6 kinase, are discussed herein. The dose is insufficient to result in complete immune suppression, but sufficient to enhance the immune response. In certain embodiments, the low immune-enhancing dose of the 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 to PD-1-positive immune effector cells, e.g., T cells or NK cells.

[0286] In certain embodiments, the low immune enhancing dose of an mTOR inhibitor results in an increase in the number of naive immune effector cells, such as T cells or NK cells. In certain embodiments, the low immune enhancing dose of an mTOR inhibitor results in one or more of the following: For example, the following markers on memory T cells, e.g., on memory T cell precursors: CD62L high , CD127 high , CD27 + and increased expression of one or more of BCL2; For example, a decrease in the expression of KLRG1 on memory T cells, e.g., on memory T cell precursors, and Memory T cell precursors, e.g., those with the following characteristics: CD62L high Increased CD127 high Increased CD27 + an increase in the number of cells with any one or combination of increased KLRG1, decreased KLRG1, and increased BCL2. wherein any of the above-described changes occur, eg, at least transiently, eg, when compared to an untreated subject.

[0287] "Refractory," as used herein, refers to a disease, e.g., cancer, that does not respond to treatment. In embodiments, a refractory cancer may be resistant to treatment before or at the time of initiation of treatment. In other embodiments, a refractory cancer may become resistant during treatment. A refractory cancer is also referred to as a resistant cancer.

[0288] "Relapsed" or "recurring," as used herein, refers to the return or reappearance of a disease (e.g., cancer) or signs and symptoms of a disease, such as cancer, after a period of improvement or response, e.g., after prior treatment with a therapy, e.g., a cancer therapy. The initial period of response may involve levels of cancer cells below a certain threshold, e.g., below 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. Reappearance may include levels of cancer cells above a certain threshold, e.g., above 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. For example, in the context of B-ALL, reappearance may include the reappearance of blasts in the blood, bone marrow (>5%), or any extramedullary site, e.g., after a complete response. In this context, a complete response may include <5% BM blasts. More generally, in certain embodiments, a response (e.g., complete response or partial response) may include the absence of detectable MRD (minimal residual disease). In certain embodiments, the initial response period 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.

[0289] In some embodiments, a therapy involving a CD19 inhibitor, such as a CD19 CAR therapy, may relapse or become refractory to treatment. Relapse or resistance may be caused by CD19 deficiency (e.g., antigen-deficient mutation) or other CD19 alterations that reduce CD19 levels (e.g., caused by clonal selection of CD19-negative clones). Cancers with such CD19 deficiency or alterations are referred to herein as "CD19-negative cancers" or "CD19-negative recurrent cancers." It should be understood that a CD19-negative cancer need not have 100% CD19 deficiency, but rather a sufficient reduction to reduce the effectiveness of CD19 therapy such that the cancer relapses or becomes refractory. In some embodiments, the CD19-negative cancer is caused by CD19 CAR therapy.

[0290] 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 further detail herein.

[0291] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a 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 construed to include all the possible subranges specifically disclosed as well as individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be construed to include specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity includes those with 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.

[0292] explanation Provided herein are methods for preventing CRS in a subject, which can include administering a CAR described herein in combination with a kinase inhibitor, such as an inhibitor of JAK-STAT or BTK.

[0293] Also provided herein are compositions of matter and methods of use for the treatment or prevention of diseases such as cancer using chimeric antigen receptors (CARs) in combination with kinase inhibitors, such as inhibitors of JAK-STAT or BTK.

[0294] Example 3 herein describes that IL-6 is produced by antigen-presenting cells (myeloid cells) in CAR T cell-associated CRS, and that the presence or absence of IL-6 (e.g., as measured by degranulation in the presence or absence of APCs) did not affect CART function. Thus, in some embodiments, a CAR described herein is administered in combination with an IL-6 inhibitor, e.g., tocilizumab. In embodiments, the methods described herein provide for early administration of an IL-6 inhibitor, e.g., tocilizumab, to prevent CRS associated with CAR therapy. In embodiments, early administration includes administration before CAR therapy, administration at the same time as CAR therapy administration, or administration until the first sign of fever (e.g., after CAR therapy administration). In some embodiments, the combination of a CAR with an IL-6 inhibitor described herein may further include a kinase inhibitor, e.g., a kinase inhibitor as described herein.

[0295] A chimeric antigen receptor (CAR) comprising an antibody or antibody fragment engineered to specifically bind to an antigen (e.g., CD123 protein or CD19 protein or fragment thereof) can be used in any of the methods or compositions described herein. In one aspect, the invention provides cells (e.g., immune effector cells, such as T cells or NK cells) engineered to express a CAR, and the CAR-expressing cells (e.g., "CART" or CAR-expressing NK cells) exhibit anti-tumor properties. In one aspect, a cell is transformed with a CAR, and at least a portion of the CAR is expressed on the cell surface. In some embodiments, a cell (e.g., an immune effector cell, such as a T cell or NK cell) is transduced with a viral vector encoding a 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 cell can stably express the CAR. In another embodiment, a cell (e.g., an immune effector cell, such as a T cell or NK cell) is transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding a CAR. In some such embodiments, the cells may transiently express the CAR.

[0296] In one embodiment, the antigen-binding domain (e.g., CD123-binding domain or CD19-binding domain) of the CAR, e.g., a human or humanized CD123-binding domain or CD19-binding domain, is an scFv antibody fragment. In one embodiment, such an antibody fragment is functional in that it retains equivalent binding affinity to an IgG antibody having the same heavy and light chain variable regions, e.g., it binds to the same antigen with equivalent efficacy. In one embodiment, such an antibody fragment is functional in that it produces a biological response, which may include, but is not limited to, activation of an immune response, inhibition of signaling resulting from its target antigen, inhibition of kinase activity, etc., as will be understood by one of skill in the art.

[0297] In some embodiments, the antibodies of the present invention are incorporated into a chimeric antigen receptor (CAR). In one embodiment, the CAR is a CD123 CAR and comprises the polypeptide sequences provided herein as SEQ ID NOs: 98-101, and 125-156.

[0298] In one embodiment, the antigen-binding domain (CD123 or CD19 binding domain, e.g., a humanized or human CD123 or CD19 binding domain) portion of the CAR of the invention is encoded by a transgene whose sequence has been codon-optimized for expression in mammalian cells. In one embodiment, 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 synonymous codons (i.e., codons that encode the same amino acid) in coding DNA is biased across different species. Such codon degeneracy allows the same polypeptide to be encoded by a variety of nucleotide sequences. Various codon optimization methods are known in the art, including, for example, those disclosed in at least U.S. Pat. Nos. 5,786,464 and 6,114,148.

[0299] In one embodiment, 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 embodiment, the antigen binding domain of the CAR comprises a humanized CD123 or CD19 antibody or antibody fragment. In one embodiment, the antigen binding domain of the CAR comprises a human CD123 or CD19 antibody fragment comprising an scFv. In one embodiment, the antigen binding domain of the CAR is a human CD123 scFv or a human CD19 scFv. In one embodiment, the antigen binding domain of the CAR comprises a humanized CD123 or CD19 antibody fragment comprising an scFv. In one embodiment, the antigen binding domain of the CAR is a humanized CD123 scFv or CD19 scFv.

[0300] In one embodiment, the CAR123 binding domain comprises an scFv portion provided in SEQ ID NOs: 157-160 and 184-215. In one embodiment, the scFv portion is human. In one embodiment, the human CAR123 binding domain comprises an scFv portion provided in SEQ ID NOs: 157-160. In one embodiment, the human CD123 binding domain comprises an scFv portion provided in SEQ ID NOs: 478, 480, 483, or 485.

[0301] In one embodiment, the scFv portion is humanized. In one embodiment, the humanized CAR123 binding domain comprises the scFv portion provided in SEQ ID NOs: 184-215. In one embodiment, the humanized CD123 binding domain comprises the scFv portion provided in SEQ ID NOs: 556-587.

[0302] Additionally, 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 disease involving cells or tissues that express CD123.

[0303] In one embodiment, the CAR of the present invention can be used to eradicate CD123-expressing normal cells and is therefore applicable for use as a cell conditioning therapy prior to cell transplantation. In one embodiment, the CD123-expressing normal cells are CD123-expressing myeloid progenitor cells, and the cell transplantation is stem cell transplantation.

[0304] In one aspect, the invention provides cells (e.g., immune effector cells, e.g., T cells or NK cells) engineered to express a chimeric antigen receptor of the invention (e.g., a CAR-expressing immune effector cell, e.g., a CART or a CAR-expressing NK cell), wherein the cells (e.g., a "CART") exhibit anti-tumor properties. Thus, the invention provides CD123-CARs that comprise a CD123 binding domain and are engineered into immune effector cells, e.g., T cells or NK cells, and methods of their use in adoptive therapy.

[0305] In one embodiment, the CD123-CAR comprises at least one intracellular domain, e.g., as described herein, e.g., selected from the group of a CD137(4-1BB) signaling domain, a CD28 signaling domain, a CD3ζ signal domain, and any combination thereof. In one embodiment, the CD123-CAR comprises at least one intracellular signaling domain, which is from one or more costimulatory molecules other than CD137(4-1BB) or CD28.

[0306] Chimeric antigen receptor (CAR) In any of the methods or compositions described herein, in embodiments, the CAR molecule comprises a CD123 CAR described herein, such as the CD123 CAR described in U.S. Patent Application Publication No. 2014 / 0322212A1 or U.S. Patent Application Publication No. 2016 / 0068601A1 (both of which are incorporated herein by reference). In embodiments, the CD123 CAR comprises the amino acid or has the nucleotide sequence set forth in U.S. Patent Application Publication No. 2014 / 0322212A1 or U.S. Patent Application Publication No. 2016 / 0068601A1 (both of which are incorporated herein by reference). In other embodiments, the CAR molecule comprises a CD19 CAR molecule described herein, such as the CD19 CAR molecule described in U.S. Patent Application Publication No. 2015-0283178-A1, such as CTL019. In embodiments, the CD19 CAR comprises the amino acid or has the nucleotide sequence set forth in U.S. Patent Application Publication No. 2015-0283178-A1 (incorporated herein by reference). In one embodiment, the CAR molecule comprises a BCMA CAR molecule described herein, for example, the BCMA CAR described in U.S. Patent Application Publication No. 2016-0046724-A1. In embodiments, the BCMA CAR comprises the amino acid or has the nucleotide sequence set forth in U.S. Patent Application Publication No. 2016-0046724-A1 (incorporated herein by reference). In an embodiment, the CAR molecule comprises a CLL1 CAR described herein, for example, the CLL1 CAR described in U.S. Patent Application Publication No. 2016 / 0051651A1 (incorporated herein by reference). In embodiments, the CLL1 CAR comprises the amino acid or has the nucleotide sequence set forth in U.S. Patent Application Publication No. 2016 / 0051651A1, which is incorporated herein by reference. In some embodiments, the CAR molecule comprises a CD33 CAR described herein, such as the CD33 CAR described in U.S. Patent Application Publication No. 2016 / 0096892A1, which is incorporated herein by reference.In embodiments, the CD33 CAR comprises the amino acid sequence or has the nucleotide sequence set forth in U.S. Patent Application Publication No. 2016 / 0096892A1 (incorporated herein by reference). In some embodiments, the CAR molecule comprises an EGFRvIII CAR molecule described herein, such as the EGFRvIII CAR described in U.S. Patent Application Publication No. 2014 / 0322275A1 (incorporated herein by reference). In some embodiments, the EGFRvIII CAR comprises the amino acid sequence or has the nucleotide sequence set forth in U.S. Patent Application Publication No. 2014 / 0322275A1 (incorporated herein by reference). In some embodiments, the CAR molecule comprises a mesothelin CAR described herein, such as the mesothelin CAR described in WO 2015 / 090230 (incorporated herein by reference). In embodiments, the mesothelin CAR comprises the amino acid or has the nucleotide sequence set forth in WO 2015 / 090230, which is incorporated herein by reference.

[0307] CAR123 The present invention encompasses recombinant DNA constructs comprising sequences encoding a CAR, wherein the CAR comprises an antigen-binding domain (e.g., an antibody, antibody fragment) that specifically binds to CD123 or a fragment thereof, e.g., human CD123, wherein the sequence of the CD123-binding domain (e.g., antibody or antibody fragment) is contiguous to and in the same reading frame as, for example, a nucleic acid sequence encoding an intracellular signaling domain. The intracellular signaling domain can comprise a costimulatory signaling domain and / or a primary signaling domain, e.g., the zeta chain. A costimulatory signaling domain refers to a portion of the CAR that comprises at least a portion of the intracellular domain of a costimulatory molecule.

[0308] In specific aspects, the CAR construct of the present invention comprises an scFv domain selected from the group consisting of SEQ ID NOs: 157-160, 184-215, 478, 480, 483, 485, and 556-587, where the scFv can be preceded by an optional leader sequence such as provided in SEQ ID NO: 1 and followed by an optional hinge sequence such as 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 provided 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., where these domains are contiguous 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 polypeptide of an scFv fragment selected from the group consisting of SEQ ID NOs: 157 to 160, 184 to 215, 478, 480, 483, 485, and 556 to 587. The present invention also includes nucleotide sequences encoding each of the scFv fragments selected from the group consisting of SEQ ID NOs: 157 to 160, 184 to 215, 478, 480, 483, 485, and 556 to 587, and each polypeptide of the domains of SEQ ID NOs: 1, 2, and 6 to 9, as well as the CD123 CAR of the present invention encoded therewith.

[0309] In one embodiment, an exemplary CD123CAR construct comprises an optional leader sequence, an extracellular antigen binding domain, a hinge, a transmembrane domain, and an intracellular stimulatory domain. In one embodiment, an exemplary CD123CAR construct comprises an optional leader sequence, an extracellular antigen binding domain, a hinge, a transmembrane domain, an intracellular costimulatory domain, and an intracellular stimulatory domain.

[0310] In some embodiments, full-length CD123 CAR sequences are also provided herein as SEQ ID NOs: 98-101 and 125-156, as shown in Table 11A or Table 12A.

[0311] An exemplary leader sequence is provided as SEQ ID NO: 1. An exemplary hinge / spacer sequence is provided as SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5. An exemplary transmembrane domain sequence is provided as SEQ ID NO: 6. An exemplary sequence of the intracellular signaling domain of 4-1BB protein is provided as SEQ ID NO: 7. An exemplary sequence of the intracellular signaling domain of CD27 is provided as SEQ ID NO: 8. An exemplary CD3 zeta domain sequence is provided as SEQ ID NO: 9 or SEQ ID NO: 10. An exemplary sequence of the intracellular signaling domain of CD28 is provided as SEQ ID NO: 43. An exemplary sequence of the intracellular signaling domain of ICOS is provided as SEQ ID NO: 45.

[0312] In one aspect, the 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, e.g., as described herein, contiguous to 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.

[0313] In one embodiment, 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, for example, this sequence is contiguous to 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 CARs include, but are not limited to, one or more intracellular signaling domains such as CD3-zeta, CD28, 4-1BB, ICOS, etc. In some examples, CARs can include any combination of CD3-zeta, CD28, 4-1BB, ICOS, etc.

[0314] In one embodiment, the nucleic acid sequence of a CAR construct of the invention is selected from one or more of SEQ ID NOs: 39-42 and 66-97. Nucleic acid sequences encoding the desired molecules can be obtained using standard techniques, e.g., recombinant methods known in the art, by screening libraries from cells expressing the gene, by deriving the gene from a vector known to contain it, or by isolating it directly from cells and tissues containing it. Alternatively, the nucleic acid of interest can be produced synthetically rather than cloned.

[0315] CAR19 (or CD19 CAR) The present 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 cell is engineered to express the CD19 CAR. In one embodiment, the immune effector cell comprises a recombinant nucleic acid construct that includes a nucleic acid sequence encoding the CD19 CAR.

[0316] In one embodiment, the CD19 CAR comprises an antigen-binding domain that specifically binds to 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 to and in the same reading frame as the nucleic acid sequence encoding the intracellular signaling domain. The intracellular signaling domain may comprise a costimulatory signaling domain and / or a primary signaling domain, e.g., the zeta chain. A costimulatory signaling domain refers to a portion of the CAR that comprises at least a portion of the intracellular domain of a costimulatory molecule.

[0317] In one embodiment, an exemplary CAR construct comprises an optional leader sequence (e.g., a leader sequence described herein), an extracellular antigen binding domain (e.g., an antigen binding domain described herein), a hinge (e.g., a hinge region described herein), a transmembrane domain (e.g., a transmembrane domain described herein), and an intracellular stimulatory domain (e.g., an intracellular stimulatory domain described herein). In one embodiment, an exemplary CAR construct comprises an optional leader sequence (e.g., a leader sequence described herein), an extracellular antigen binding domain (e.g., an antigen binding domain described herein), a hinge (e.g., a hinge region described herein), a transmembrane domain (e.g., a transmembrane domain described herein), an intracellular costimulatory signaling domain (e.g., a costimulatory signaling domain described herein), and / or an intracellular primary signaling domain (e.g., a primary signaling domain described herein).

[0318] In one embodiment, the CD19 CAR of the present invention comprises at least one intracellular 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 signal domain, and any combination thereof. In one embodiment, the CAR of the present invention comprises at least one intracellular signaling domain from one or more costimulatory molecules selected from CD137 (4-1BB), CD28, CD27, or ICOS.

[0319] Vectors and RNA constructs The present invention includes retroviral and lentiviral vector constructs expressing CARs that can be directly transduced into cells.

[0320] The present invention also includes RNA constructs that can be directly transfected into cells. Methods for generating mRNA for use in transfection include in vitro transcription (IVT) of a template with specifically designed primers, followed by poly(A) addition, resulting in a construct, typically 50-2000 bases in length (SEQ ID NO: 35), containing 3' and 5' untranslated sequences ("UTRs"), a 5' cap and / or internal ribosome entry site (IRES), the nucleic acid to be expressed, and a poly(A) tail. RNA generated in this manner can efficiently transfect a variety of cell types. In one embodiment, the template contains the sequence of the CAR. In some embodiments, the RNA CAR vector is transduced into T cells by electroporation.

[0321] antigen-binding domain In one embodiment, the CAR of the present invention comprises a target-specific binding element, otherwise referred to as an antigen-binding domain. The selection of the portion depends on the type and number of ligands that define the surface of the target cell. For example, the antigen-binding domain can be selected to recognize a ligand that serves as a cell surface marker on the 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.

[0322] In one embodiment, CAR-mediated T cell responses can be directed to an antigen of interest by engineering an antigen-binding domain into the CAR that specifically binds to the desired antigen.

[0323] In one embodiment, the portion of the CAR comprising the antigen binding domain comprises an antigen binding domain that targets a tumor antigen, e.g., a tumor antigen described herein.

[0324] In one aspect, the portion of the CAR 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 a B-cell antigen (e.g., a B-cell surface antigen), such as CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a.

[0325] The antigen-binding domain can be any domain that binds to an antigen, including, but not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and functional fragments thereof, such as, but not limited to, single-domain antibodies, such as heavy chain variable domains (VH), light chain variable domains (VL), and variable domains of camelid-derived nanobodies (VHH), and alternative scaffolds known in the art to function as antigen-binding domains, such as recombinant fibronectin domains. 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 human use, it may be beneficial for the antigen-binding domain of the CAR to comprise human or humanized residues in the antigen-binding domain of an antibody or antibody fragment.

[0326] In one embodiment, the antigen-binding domain comprises one, two, three (e.g., all three) heavy chain CDRs, HC CDR1, HC CDR2, HC CDR3, HC CDR4, HC CDR5, HC CDR6, HC CDR7, HC CDR8, HC CDR9, HC CDR10, HC CDR11, HC CDR12, HC CDR13, HC CDR14, HC CDR15, HC CDR16, HC CDR17, HC CDR18, HC CDR19 ... and / or one, two, three (e.g., all three) light chain CDRs, LC CDR2 and HC CDR3, from an antibody described herein (e.g., an antibody described in WO 2015 / 142675, U.S. Patent Application Publication No. 2015-0283178-A1, U.S. Patent Application Publication No. 2016-0046724-A1, U.S. Patent Application Publication No. 2014 / 0322212A1, U.S. Patent Application Publication No. 2016 / 0068601A1, U.S. Patent Application Publication No. 2016 / 0051651A1, U.S. Patent Application Publication No. 2016 / 0096892A1, U.S. Patent Application Publication No. 2014 / 0322275A1, or WO 2015 / 090230, which are incorporated by reference herein). In one embodiment, the antigen-binding domain comprises a heavy chain variable region and / or a variable light chain region of an antibody listed above.

[0327] In embodiments, the antigen binding domain is an antigen binding domain described in WO 2015 / 142675, US 2015-0283178-A1, US 2016-0046724-A1, US 2014 / 0322212A1, US 2016 / 0068601A1, US 2016 / 0051651A1, US 2016 / 0096892A1, US 2014 / 0322275A1, or WO 2015 / 090230 (incorporated herein by reference).

[0328] In embodiments, the antigen binding domain targets BCMA and is described in U.S. Patent Application Publication No. 2016-0046724-A1.

[0329] In embodiments, the antigen binding domain targets CD19 and is described in US Patent Application Publication No. 2015-0283178-A1.

[0330] In embodiments, the antigen binding domain targets CD123 and is described in US Patent Application Publication No. 2014 / 0322212A1, US Patent Application Publication No. 2016 / 0068601A1.

[0331] In embodiments, the antigen binding domain targets CLL and is described in US Patent Application Publication No. 2016 / 0051651A1.

[0332] In embodiments, the antigen binding domain targets CD33 and is described in US Patent Application Publication No. 2016 / 0096892A1.

[0333] Exemplary target antigens that can be targeted using CAR-expressing cells include, but are not limited to, CD19, CD123, EGFRvIII, CD33, mesothelin, BCMA, and GFR ALPHA-4, among others, as described, for example, in 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).

[0334] In other embodiments, the CAR-expressing cells can specifically bind humanized CD19 and may comprise, for example, a CAR molecule or antigen-binding domain (e.g., a humanized antigen-binding domain) according to Table 3 of WO 2014 / 153270 (incorporated herein by reference). Amino acid and nucleotide sequences encoding CD19 CAR molecules and antigen-binding domains (e.g., comprising one, two, or three VH CDRs and one, two, or three VL CDRs according to Kabat or Chothia) are specified in WO 2014 / 153270.

[0335] In other embodiments, the CAR-expressing cells can specifically bind to CD123 and can comprise, for example, a CAR molecule (e.g., any of CAR1-CAR8) or antigen-binding domain according to Tables 1-2 of WO 2014 / 130635 (incorporated herein by reference). Amino acid and nucleotide sequences encoding CD123 CAR molecules and antigen-binding domains (e.g., comprising one, two, or three VH CDRs and one, two, or three VL CDRs according to Kabat or Chothia) are specified in WO 2014 / 130635.

[0336] In other embodiments, the CAR-expressing cells can specifically bind CD123 and can comprise, for example, a CAR molecule (e.g., any of CAR123-1 through CAR123-4 and hzCAR123-1 through hzCAR123-32) or antigen-binding domain according to Tables 2, 6, and 9 of WO 2016 / 028896 (incorporated herein by reference). Amino acid and nucleotide sequences encoding CD123 CAR molecules and antigen-binding domains (e.g., comprising one, two, or three VH CDRs and one, two, or three VL CDRs according to Kabat or Chothia) are specified in WO 2016 / 028896.

[0337] In other embodiments, the CAR-expressing cells can specifically bind EGFRvIII and may comprise, for example, a CAR molecule or antigen-binding domain according to Table 2 or SEQ ID NO: 11 of WO 2014 / 130657 (incorporated herein by reference). Amino acid and nucleotide sequences encoding EGFRvIII CAR molecules and antigen-binding domains (e.g., comprising one, two, or three VH CDRs and one, two, or three VL CDRs according to Kabat or Chothia) are specified in WO 2014 / 130657.

[0338] In other embodiments, the CAR-expressing cells can specifically bind CD33 and can comprise, for example, a CAR molecule (e.g., any of CAR33-1 through CAR-33-9) or antigen-binding domain according to Table 2 or Table 9 of WO 2016 / 014576 (incorporated herein by reference). Amino acid and nucleotide sequences encoding CD33 CAR molecules and antigen-binding domains (e.g., comprising one, two, or three VH CDRs and one, two, or three VL CDRs according to Kabat or Chothia) are specified in WO 2016 / 014576.

[0339] In other embodiments, the CAR-expressing cells can specifically bind mesothelin and can comprise, for example, a CAR molecule or antigen-binding domain according to Tables 2-3 of WO 2015 / 090230 (incorporated herein by reference). Amino acid and nucleotide sequences encoding mesothelin CAR molecules and antigen-binding domains (e.g., comprising one, two, or three VH CDRs and one, two, or three VL CDRs according to Kabat or Chothia) are specified in WO 2015 / 090230.

[0340] In other embodiments, the CAR-expressing cells can specifically bind BCMA and may comprise, for example, a CAR molecule or antigen-binding domain according to Table 1 or Table 16, SEQ ID NO: 271 or SEQ ID NO: 273 of WO 2016 / 014565 (incorporated herein by reference). Amino acid and nucleotide sequences encoding BCMA CAR molecules and antigen-binding domains (e.g., comprising one, two, or three VH CDRs and one, two, or three VL CDRs according to Kabat or Chothia) are specified in WO 2016 / 014565.

[0341] In other embodiments, the CAR-expressing cells can specifically bind to CLL-1 and may comprise, for example, a CAR molecule or antigen-binding domain according to Table 2 of WO 2016 / 014535 (incorporated herein by reference). Amino acid and nucleotide sequences encoding CLL-1 CAR molecules and antigen-binding domains (e.g., comprising one, two, or three VH CDRs and one, two, or three VL CDRs according to Kabat or Chothia) are specified in WO 2016 / 014535.

[0342] In other embodiments, the CAR-expressing cells can specifically bind GFR ALPHA-4 and may comprise, for example, a CAR molecule or antigen binding domain according to Table 2 of WO 2016 / 025880 (incorporated herein by reference). Amino acid and nucleotide sequences encoding GFR ALPHA-4 CAR molecules and antigen binding domains (e.g., comprising one, two, or three VH CDRs and one, two, or three VL CDRs according to Kabat or Chothia) are specified in WO 2016 / 025880.

[0343] In one embodiment, the antigen binding domain of any of the CAR molecules described herein (e.g., any of CD19, CD123, EGFRvIII, CD33, mesothelin, BCMA, and GFR ALPHA-4) comprises one, two, three (e.g., all three) heavy chain CDRs, HC CDR1, HC CDR2, and HC CDR3, from an antibody listed above, and / or one, two, three (e.g., all three) light chain CDRs, LC CDR1, LC CDR2, and LC CDR3, from an antigen binding domain listed above. In one embodiment, the antigen binding domain comprises the heavy chain variable region and / or the variable light chain region of an antibody listed or described above.

[0344] In another embodiment, the antigen-binding domain comprises a humanized antibody or antibody fragment. In some embodiments, a non-human antibody is humanized, where certain sequences or regions of the antibody are modified to increase similarity to antibodies or fragments thereof that are naturally produced in humans. In one embodiment, the antigen-binding domain is humanized.

[0345] 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 may be beneficial for the antigen-binding domain of the CAR to comprise human or humanized residues in the antigen-binding domain of an antibody or antibody fragment. Thus, in one embodiment, the antigen-binding domain comprises a human antibody or antibody fragment.

[0346] CD123-binding domain In one embodiment, the human CD123 binding domain comprises one or more (e.g., all three) of 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 a human CD123 binding domain described herein, and / or one or more (e.g., all three) of 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 a human CD123 binding domain described herein, e.g., a human CD123 binding domain comprising one or more, e.g., all three, of the LC CDRs and one or more, e.g., all three, of the HC CDRs. In one embodiment, the human CD123 binding domain comprises one or more (e.g., all three) of 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 a human CD123 binding domain described herein, e.g., the human CD123 binding domain comprises two variable heavy chain regions, each comprising an HC CDR1, HC CDR2, and HC CDR3 described herein. In one embodiment, the human CD123 binding domain comprises a human light chain variable region described herein (e.g., in Table 11A or 12B) and / or a human heavy chain variable region described herein (e.g., in Table 11A or 12B). In one embodiment, the human CD123 binding domain comprises a human heavy chain variable region described herein (e.g., in Table 11A or 12B9), e.g., at least two human heavy chain variable regions described herein (e.g., in Table 11A or 12B). In one embodiment, the CD123 binding domain is an scFv comprising a light chain and a heavy chain of the amino acid sequences of Table 11A or 12B.In one embodiment, the CD123 binding domain (e.g., scFv) comprises a light chain variable region comprising an amino acid sequence having at least one, two, or three alterations (e.g., substitutions), but not more than 30, 20, or 10 alterations (e.g., substitutions), of an amino acid sequence of a light chain variable region provided in Table 11A or 12B, or a sequence having at least 95% identity, e.g., 95-99% identity, to an amino acid sequence of Table 11A, and / or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three alterations (e.g., substitutions), but not more than 30, 20, or 10 alterations (e.g., substitutions), of an amino acid sequence of a heavy chain variable region provided in Table 11A or 12B, or a sequence having at least 95% identity, e.g., 95-99% identity, to an 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, e.g., 95-99% identity thereto. In one embodiment, the human CD123-binding domain is an scFv, in which a light chain variable region comprising an amino acid sequence described herein, e.g., in Table 11A or 12B, is linked to a heavy chain variable region comprising an amino acid sequence described herein, e.g., in Table 11A, via a linker, e.g., a linker described herein. In one embodiment, the human CD123-binding domain comprises a (Gly4-Ser)n linker (SEQ ID NO: 26) (wherein n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4). The light chain variable region and heavy chain variable region of the scFv can be oriented, for example, in any of the following ways: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0347] In some embodiments, a non-human antibody is humanized, and specific sequences or regions of the antibody are modified to increase similarity to antibodies or fragments thereof naturally produced in humans. Thus, in one embodiment, 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) 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 a humanized 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 a humanized CD123 binding domain described herein, such as all three and one or more LC CDRs, and such as all three and one or more HC CDRs. In one embodiment, the humanized CD123 binding domain comprises one or more (e.g., all three) of 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 a humanized CD123 binding domain described herein, e.g., the humanized CD123 binding domain has two variable heavy chain regions, each comprising an HC CDR1, HC CDR2, and HC CDR3 described herein. In one embodiment, the humanized CD123 binding domain comprises a humanized light chain variable region described herein (e.g., in Table 12A) and / or a humanized heavy chain variable region described herein (e.g., in Table 12A). In one embodiment, the humanized CD123 binding domain comprises a humanized heavy chain variable region described herein (e.g., in Table 12A), e.g., at least two humanized heavy chain variable regions described herein (e.g., in Table 12A). In one embodiment, the CD123 binding domain is an scFv comprising light and heavy chains of the amino acid sequences in Table 12A.In one embodiment, the CD123-binding domain (e.g., scFv) comprises a light chain variable region comprising an amino acid sequence having at least one, two, or three, but not more than 30, 20, or 10, amino acid modifications (e.g., substitutions) of a light chain variable region provided in Table 4, or a sequence having at least 95% identity, e.g., 95-99% identity, to an amino acid sequence of Table 12A, and / or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three, but not more than 30, 20, or 10, amino acid modifications (e.g., substitutions) of a heavy chain variable region provided in Table 12A, or a sequence having at least 95% identity, e.g., 95-99% identity, to an 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, e.g., 95-99% identity thereto. In one embodiment, the humanized CD123-binding domain is an scFv, and a light chain variable region comprising an amino acid sequence described herein, e.g., in Table 12A, is joined to a heavy chain variable region comprising an amino acid sequence described herein, e.g., in Table 12A, by a linker, e.g., a linker described herein. In one embodiment, the humanized CD123-binding domain comprises a (Gly4-Ser)n linker (SEQ ID NO: 26), where n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4. The light chain variable region and heavy chain variable region of the scFv can be, for example, in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0348] humanized antibodies Humanized antibodies can be prepared by a variety of techniques, including CDR-grafting (see, e.g., EP 239,400; WO 91 / 09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated by reference in its entirety), veneering, or resurfacing (see, e.g., EP 592,106 and 519,596, each of which is incorporated by reference in its entirety; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1994, Protein Engineering, 7(6):805-814). al., 1994, PNAS, 91:969-973), chain shuffling (see, e.g., U.S. Pat. No. 5,565,332, which is incorporated herein by reference in its entirety), as well as methods such as those described in U.S. Patent Application Publication Nos. 2005 / 0042664, 2005 / 0048617, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, 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. 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 JS, Gene, 150(2):409-10(1994) and Pedersen et al., J.Mol.Biol.CDRs can be generated using a variety of methods known in the art, including, but not limited to, the techniques described in [PubMed], 235(3):959-73 (1994). Often, framework residues in the framework regions are substituted with corresponding residues from the CDR donor antibody to alter, e.g., improve, antigen binding. These framework substitutions are identified by methods well known in the art, such as modeling the interactions between CDRs and framework residues to identify framework residues important for antigen binding, and sequence comparison to identify unusual framework residues at specific positions (see, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, which are incorporated herein by reference).

[0349] A humanized antibody or antibody fragment has one or more amino acid residues remaining from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, 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 framework regions, where the framework is derived entirely or predominantly from human germline. Numerous techniques for humanizing antibodies or antibody fragments are known in the art, essentially as described by Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Nature, 332:323-327 (1988)). This can be achieved by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody, i.e., CDR-grafting, according to the method of (European Patent No. 239,400; International 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; and 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 entire human variable domain is replaced by the corresponding sequence from a non-human species. Humanized antibodies are often human antibodies in which some CDR residues and possibly some framework (FR) residues are substituted by residues from analogous sites in rodent antibodies.Humanization of antibodies and antibody fragments can also be achieved by veneering or 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. Pat. No. 5,565,332), the contents of which are incorporated herein by reference in their entirety.

[0350] The selection of human variable domains, both light and heavy, used to produce humanized antibodies is intended to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence that most closely resembles the rodent is then accepted as the human framework (FR) for the humanized antibody (see Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987), the entire contents of which are incorporated herein by reference). Another method uses a specific framework derived from the consensus sequence of all light or heavy chain antibodies of a particular subgroup. The same framework can be used for several different humanized antibodies (see, e.g., Nicholson et al. Mol. Immun. 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 one embodiment, 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 regions can include, for example, one, two, three, four, or five modifications, e.g., substitutions, from the amino acids of the corresponding murine sequence. In one embodiment, the framework regions of the light chain variable region, e.g., all four framework regions, are derived from the VK3_1.25 germline sequence. In one embodiment, the framework regions can include, for example, one, two, three, four, or five modifications, eg, substitutions, from the amino acids of the corresponding murine sequence.

[0351] In some embodiments, portions of the CAR compositions of the present invention, including antibody fragments, are humanized while maintaining high affinity for the target antigen and other favorable biological properties. According to one aspect of the present invention, humanized antibodies and antibody fragments are produced by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the potential role of residues in the function of the candidate immunoglobulin sequence, for example, analysis of residues that affect the ability of the candidate immunoglobulin to bind to the target antigen. In this manner, FR residues can be selected and combined from the recipient and import sequences to achieve the desired antibody or antibody fragment characteristics, such as increased affinity for the target antigen. Generally, CDR residues are directly and most substantially involved in influencing antigen binding.

[0352] A humanized antibody or antibody fragment may, for example, maintain a similar antigenic specificity as the original antibody of the invention, the ability to bind to 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 one embodiment, the humanized antibody or antibody fragment may have improved binding affinity and / or specificity to an antigen, e.g., a tumor antigen, e.g., a B cell antigen, e.g., human CD123, CD19, or a fragment thereof.

[0353] In one embodiment, the antigen-binding domain portion comprises one or more sequences selected from SEQ ID NOs: 157 to 160, 184 to 215, 478, 480, 483, 485, and 556 to 587. In one embodiment, a CD123 CAR comprising a human CD123-binding domain is selected from one or more sequences selected from the group consisting of SEQ ID NOs: 157 to 160, 478, 480, 483, and 485. In one embodiment, a CD123 CAR comprising a humanized CD123-binding domain is selected from one or more sequences selected from SEQ ID NOs: 184 to 215, and 556 to 587.

[0354] In one embodiment, 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 characterized by a particular functional property or property of an antibody or antibody fragment. For example, in one embodiment, a portion of the CAR composition of the present invention comprising an antigen-binding domain specifically binds to an antigen (e.g., a tumor antigen, e.g., a B cell antigen, e.g., human CD123, CD19, or a fragment thereof). In one embodiment, the present invention relates to an antigen-binding domain constituting an antibody or antibody fragment, wherein the antibody-binding domain specifically binds to CD123 protein or a fragment thereof, and wherein the antibody or antibody fragment comprises a variable light chain and / or a variable heavy chain comprising the amino acid sequence of SEQ ID NOs: 157-160, 184-215, 478, 480, 483, 485, and 556-587. In one embodiment, 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 a particular embodiment, the scFv is contiguous with and in the same reading frame as a leader sequence. In one embodiment, the leader sequence is the polypeptide sequence provided as SEQ ID NO: 1.

[0355] Antigen Binding Domains—Further Embodiments In one embodiment, 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, e.g., a single-chain variable fragment (scFv). In one embodiment, 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. 17, 105 (1987)). In one embodiment, the antibodies and fragments thereof of the present invention bind to an antigen (e.g., a tumor antigen, e.g., a B cell antigen, e.g., a CD123 or CD19 protein) or a fragment thereof with wild-type or enhanced affinity.

[0356] In one example, the human scFv can be derived from a display library. A display library is a collection of entities. Each entity includes an accessible polypeptide component and a recoverable component that encodes or specifies the polypeptide component. The polypeptide components vary so that different amino acid sequences are represented. The polypeptide components can be of any length, for example, from 3 amino acids to over 300 amino acids. A display library entity can include two or more polypeptide components of a Fab, for example, two polypeptide chains. In one exemplary embodiment, a display library can be used to identify a human CD123-binding domain. During selection, the polypeptide component of each library member is probed with CD123 or a fragment thereof, and if the polypeptide component binds to CD123, the display library member is identified, typically by retention on a support.

[0357] The retained display library members are recovered from the support and analyzed. Analysis can include amplification followed by selection under similar or dissimilar conditions. For example, positive and negative selection can be alternating. Analysis can also include purification of the polypeptide components for determination and detailed characterization of the amino acid sequence of the polypeptide components, i.e., the anti-CD123 binding domain.

[0358] A variety of formats can be used for the display library. Examples include phage display. In phage display, protein components are typically covalently linked to a bacteriophage coat protein. Linkage results from translation of a nucleic acid encoding the protein component fused to the coat protein. Linkages include flexible peptide linkers, protease sites, or amino acids incorporated as a result of suppression of a stop codon. Phage display has been described, for example, in U.S. Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317; WO 92 / 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. Bacteriophage displaying protein components can be grown and harvested using standard phage sorting techniques, such as PEG precipitation from the growth medium. After selection of individual display phage, nucleic acid encoding the selected protein component can be isolated from cells infected with the selected phage after amplification or from the phage itself. Individual colonies or plaques can be picked, and the nucleic acid isolated and sequenced.

[0359] Other display formats include cell-based display (see, e.g., WO 03 / 029456), protein-nucleic acid fusions (see, e.g., U.S. Pat. No. 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 E. coli periplasmic display (2005 Nov 22; PMID: 16337958).

[0360] In some instances, scFvs can be produced by 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 produced by linking the VH and VL domains together using a flexible polypeptide linker. The scFv molecule contains a linker (e.g., a Ser-Gly linker) of optimal length and / or amino acid composition. Linker length can significantly affect how the variable regions of an scFv fold and interact. Indeed, when short polypeptide linkers are used (e.g., 5-10 amino acids), intrachain folding is prevented. Intrachain folding is also required for the two variable regions to unite and form a functional epitope-binding site. For examples of linker orientations and sizes, see, e.g., Hollinger et al. 1993 Proc Natl Acad. Sci. USA 90:6444-6448, U.S. Patent Application Publication Nos. 2005 / 0100543, 2005 / 0175606, 2007 / 0014794, and WO 2006 / 020258 and WO 2007 / 024715, which are incorporated herein by reference.

[0361] An scFv can include 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 can include any naturally occurring amino acid. In one embodiment, the linker sequence includes the amino acids glycine and serine. In another embodiment, the linker sequence includes a series 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). Varying the linker length can maintain or enhance activity, resulting in superior efficacy in activity tests.

[0362] Exemplary CD123 CAR Constructs and Antigen Binding Domains Exemplary CD123 CAR constructs disclosed herein comprise an scFv (e.g., a human scFv as 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 as exemplary leader amino acid and nucleotide sequences, respectively). The sequences of the human scFv fragments (amino acid sequences of SEQ ID NOs:157-160) are provided herein in Table 11A. The sequences of the human scFv fragments without the leader sequence are provided herein in Table 12B (SEQ ID NOs:479, 481, 482, and 484 for the nucleotide sequence, and SEQ ID NOs:478, 480, 483, and 485 for the amino acid sequence). The CD123 CAR construct further comprises an optional hinge domain, e.g., a CD8 hinge domain (e.g., comprising the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 2 or SEQ ID NO: 13); a transmembrane domain, e.g., a CD8 transmembrane domain (e.g., comprising the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 17); an intracellular domain, e.g., a 4-1BB intracellular domain (e.g., comprising the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 18), and a functional signaling domain, e.g., a CD3 zeta domain (e.g., comprising the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 9 or 10 or SEQ ID NO: 20 or 21). In one embodiment, the domains are adjacent or in the same reading frame to form a single fusion protein. In other embodiments, the domains are in separate polypeptides, such as, for example, in an RCAR molecule as described herein.

[0363] In one embodiment, the full-length CD123 CAR molecule is a 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, hzCD123-32, hzCD123-33, hzCD123-34, hzCD123-35, hzCD123-36, hzCD123-37, hzCD123-38, hzCD123-39, hzCD123-40, hzCD123-41, hzCD123-42, hzCD123-43, hzCD123-44, hzCD123-45, hzCD123-46, hzCD123-47, hzCD hzCD123-31, hzCD123-32, hzCD123-33, hzCD123-34, hzCD123-35, hzCD123-36, hzCD123-37, hzCD123-38, hzCD123-39, hzCD123-40, hzCD123-41, hzCD123-42, hzCD123-43, hzCD123-44, hzCD123-45, hzCD123-46, hzCD123-47, hzCD123-48, hzCD123-49, hzCD123-50, hzCD123-51, hzCD123-52, hzCD123-53, hzCD123-54, hzCD123-55, hzCD123-56, hzCD123-57, hzCD123-58, hzCD123-59, hzCD123-60, hzCD123-61, hzCD123-62, hzCD123-63, hzCD123-64, hzCD123-65, hzCD123-66, hzCD123-67, hzCD123-68, hzCD123-69, hzCD123-70, hzCD123-71, hzCD123-72, hzCD123-73, hzCD123-74, hzCD123-75, hzCD123-76, hzCD123-77, hzCD123-78, hzCD123-79, hzCD123-80, hzCD123-81, h

[0364] In one embodiment, the CD123 CAR molecule or CD123 antigen binding domain is selected from the group consisting 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, hzCD123-32, hzCD123-33, hzCD123-34, hzCD123-35, hzCD123-36, hzCD123-37, hzCD123-38, hzCD123-39, hzCD123-40, hzCD123-41, hzCD123-42, hzCD123-43, hzCD123-44, hzCD123-45, hzCD123-46, hzCD12 , 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 comprising the scFv amino acid sequence of CD123-1, CD123-2, CD123-3, CD123-4, hzCD123-1, hzCD123-2 zCD123-2, 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, hzCD1 hzCD123-23, hzCD123-24, hzCD123-25, hzCD123-26, hzCD123-27, hzCD123-28, hzCD123-29, hzCD123-30, hzCD123-31 or hzCD123-32, or a sequence substantially identical (e.g., having at least 95% identity, e.g., 95-99% identity, or having up to 20, 15, 10, 8, 6, 5, 4, 3, 2, or 1 amino acid change) to any of the foregoing sequences.

[0365] In one embodiment, the CD123 CAR molecule or CD123 antigen binding domain is selected from the group consisting 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, hzCD123-32, hzCD123-33, hzCD123-34, hzCD123-35, hzCD123-36, hzCD123-37, hzCD123-38, hzCD123-39, hzCD123-40, hzCD123-41, hzCD123-42, hzCD123-43, hzCD123-44, hzCD123-45, hzCD123-46, hzCD12 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 substantially identical (e.g., having at least 95% identity, e.g., 95-99% identity, or having up to 20, 15, 10, 8, 6, 5, 4, 3, 2, or 1 amino acid change) to any of the foregoing sequences.

[0366] In one embodiment, the CD123 CAR molecule or CD123 antigen binding domain comprises one, two, or three CDRs (e.g., HCDR1, HCDR2, and / or HCDR3) from the heavy chain variable region provided in Table 1A or 3A, and / or CD123-1, CD123-2, CD123-3, CD123-4, hzCD123-1, hzCD123-1, hzCD123-3, hzCD123-4, hzCD123-1, hzCD123-3 ... -2, hzCD123-3, hzCD123-4, hzCD123-5, hzCD123-6, hzCD123-7, hzCD123-8, hzCD123-9, hzCD1 23-10, hzCD123-11, hzCD123-12, hzCD123-13, hzCD123-14, hzCD123-15, hzCD123-16, hzCD12 hzCD123-31 or hzCD123-32, or a sequence substantially identical (e.g., at least 95% identical, e.g., 95-99% identical, or up to 5, 4, 3, 2, or 1 amino acid change) to any of the foregoing sequences.

[0367] In one embodiment, the CD123 CAR molecule or CD123 antigen binding domain comprises one, two, or three CDRs (e.g., HCDR1, HCDR2, and / or HCDR3) from the heavy chain variable region provided in Table 5A, and / or CD123-1, CD123-2, CD123-3, CD123-4, hzCD123-1, hzCD123-2, hzCD123-1 provided in Table 6A. 23-3, 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 substantially identical (e.g., at least 95% identical, e.g., 95-99% identical, or up to five, four, three, two or one amino acid changes) to any of the foregoing sequences.

[0368] In one embodiment, the CD123 molecule or CD123 antigen binding domain comprises one, two, or three CDRs (e.g., HCDR1, HCDR2, and / or HCDR3) from the heavy chain variable region provided in Table 7A, and / or CD123-1, CD123-2, CD123-3, CD123-4, hzCD123-1, hzCD123-2, hzCD123-3, hzCD123-4, hzCD123-1, hzCD123-2, hzCD123-3, hzCD123-4, hzCD123-1, hzCD123-2, hzCD123-3, hzCD123-4, hzCD123-2 ... 3-2, hzCD123-3, hzCD123-4, hzCD123-5, hzCD123-6, hzCD123-7, hzCD123-8, hzCD123-9, hzC D123-10, hzCD123-11, hzCD123-12, hzCD123-13, hzCD123-14, hzCD123-15, hzCD123-16, hzCD 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 substantially identical (e.g., at least 95% identical, e.g., 95-99% identical, or up to five, four, three, two or one amino acid changes) to any of the foregoing sequences.

[0369] The sequences of the CDR sequences of the scFv domains are shown in Tables 3A, 5A and 7A for the heavy chain variable domain and in Tables 2A, 4A, 6A and 8A for the light chain variable domain. "ID" refers to the respective SEQ ID NO of each CDR. ...

Claims

1. 1. A pharmaceutical composition comprising a BTK inhibitor (e.g., ibrutinib) for use in combination with a population of immune effector cells expressing a CAR in the treatment or prevention of cytokine release syndrome (CRS) in a subject in need thereof.

2. 1. A pharmaceutical composition comprising a population of immune effector cells expressing a CAR for use in combination with a BTK inhibitor (e.g., ibrutinib) in the treatment or prevention of cytokine release syndrome (CRS) in a subject in need thereof.

3. 1. Use of a BTK inhibitor (e.g., ibrutinib) in combination with a population of immune effector cells expressing a CAR in the manufacture of a medicament for treating or preventing cytokine release syndrome (CRS) in a subject in need thereof.

4. 1. A pharmaceutical composition comprising a BTK inhibitor (e.g., ibrutinib) for use in treating or preventing cytokine release syndrome (CRS) in a subject in need thereof, wherein the subject has been administered a population of immune effector cells that express a CAR, and the CRS is associated with said administration of the population of immune effector cells that express the CAR.

5. 1. Use of a BTK inhibitor (e.g., ibrutinib) in the manufacture of a medicament for treating cytokine release syndrome (CRS) in a subject in need thereof, wherein the subject has been administered a population of immune effector cells that express a CAR, and the CRS is associated with the administration of the population of immune effector cells that express the CAR.

6. 10. The pharmaceutical composition of any one of claims 1, 2, or 4, wherein the subject (i) is at risk of developing, has, or has been diagnosed with CRS, (ii) is identified or has previously been identified as being at risk for CRS, and / or (iii) has been administered, is being administered, or will be administered immune effector cells expressing a CAR.

7. 10. The pharmaceutical composition of claim 1, further comprising selecting a subject to administer the BTK inhibitor, e.g., ibrutinib.

8. 5. The pharmaceutical composition of any one of claims 1, 2, or 4, 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.

9. 10. The pharmaceutical composition of claim 1, 2, or 4, wherein the BTK inhibitor is ibrutinib or a pharmaceutically acceptable salt thereof.

10. (a) the CAR-expressing immune effector cells are administered after (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) administration of a first dose of the BTK inhibitor, e.g., but before administration of a second dose of the BTK inhibitor; (b) the dose of immune effector cells expressing a CAR is administered concurrently with the administration of the first dose of the BTK inhibitor (e.g., within 2 days (e.g., 2 days, 1 day, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, or less) of the administration of the first dose of the BTK inhibitor); (c) the dose of the CAR-expressing immune effector cell population (e.g., CD19 CAR) is at least about 1 x 10 ５ , 5 x 10 ６ , 1×10 ７ , 1.5 × 10 ７ , 2 × 10 ７ , 2.5 × 10 ７ , 3 x 10 ７ , 3.5 × 10 ７ , 4 x 10 ７ , 5 x 10 ７ , 1×10 ８ , 1.5 × 10 ８ , 2 × 10 ８ , 2.5 × 10 ８ , 3 x 10 ８ , 3.5 × 10 ８ , 4 x 10 ８ , 5 x 10 ８ , 1×10 ９ , 2 × 10 ９ , or 5 x 10 ９ Contains cells; (d) one or more subsequent doses of the BTK inhibitor are administered after the second dose of the BTK inhibitor; (e) the dose of the BTK inhibitor is administered once daily (QD); and / or (f) the dose (e.g., each dose) of the BTK inhibitor, e.g., ibrutinib, 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 (e.g., 250 mg, 420 mg, or 560 mg) of the BTK inhibitor; 10. The pharmaceutical composition of claim 1, 2 or 4.

11. (a) the treatment interval comprises a duration of at least 7 days, e.g., at least 7, 8, 9, 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; (b) the treatment interval is repeated, e.g., one or more times, e.g., 1, 2, 3, 4, or 5 times; and / or (c) a treatment interval is followed by one or more, e.g., 1, 2, 3, 4, or 5, subsequent treatment intervals; 10. The pharmaceutical composition of claim 1, 2 or 4.

12. The pharmaceutical composition of claim 1 , wherein the CAR comprises a transmembrane domain, an intracellular signaling domain, and an antigen-binding domain.

13. The pharmaceutical composition of claim 12, wherein the CAR comprises a domain that binds to CD123, CD20, CD22, BCMA, CD34, CLL1, CD33, EGFRvIII, or mesothelin.

14. The pharmaceutical composition of claim 12, wherein the CAR comprises a CD19-binding domain.

15. (a) the CD19-binding domain is (i) a HC CDR1 of SEQ ID NO: 782, a HC CDR2 of SEQ ID NO: 783, a HC CDR3 of SEQ ID NO: 787, a LC CDR1 of SEQ ID NO: 788, a LC CDR2 of SEQ ID NO: 789, and a LC CDR3 of SEQ ID NO: 790; or (ii) comprising a HC CDR1 of SEQ ID NO:782, a HC CDR2 of SEQ ID NO:785, a HC CDR3 of SEQ ID NO:787, a LC CDR1 of SEQ ID NO:788, a LC CDR2 of SEQ ID NO:789, and a LC CDR3 of SEQ ID NO:790; (b) the CD19-binding domain is (i) an amino acid 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; (ii) an amino acid sequence having at least one, two, or three modifications but no more than 30, 20, or 10 modifications relative to any 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) comprising an amino acid sequence having at least 95% identity to any 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; and / or (c) the CAR is (i) the amino acid sequence of any 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 no more than 30, 20, or 10 modifications relative to any 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 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.

16. (a) 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; and / or (b) the transmembrane domain is (i) the amino acid sequence of SEQ ID NO: 6; (ii) an amino acid sequence containing at least one, two, or three, but not more than 20, 10, or 5, modifications of the amino acid sequence of SEQ ID NO: 6; or 15. The pharmaceutical composition of claim 14, comprising (iii) a sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

6.

17. 15. The pharmaceutical composition of claim 14, wherein the CD19 binding domain is connected to the transmembrane domain by a hinge region, optionally the hinge region comprising SEQ ID NO: 2 or a sequence having at least 95% identity thereto.

18. (a) Intracellular signaling domains of MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), 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, I TGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TR ANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL 1, comprising a costimulatory signaling domain comprising a functional signaling domain obtained from a protein selected from the group consisting of 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; (b) the costimulatory domain comprises an amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least one, two, or three 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; (c) the intracellular signaling domain comprises a functional signaling domain of 4-1BB and / or a functional signaling domain of CD3ζ; (d) 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 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; (e) 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, and the amino acid sequences comprising the intracellular signaling domain are expressed in the same frame and as a single polypeptide chain; and / or (f) the CAR further comprises a leader sequence comprising the amino acid sequence of SEQ ID NO:

1.

15. The pharmaceutical composition of claim 14.

19. CAR is, (i) the amino acid sequence of any of SEQ ID NOs: 99, 100, 101, or 98; (ii) an amino acid sequence having at least one, two, or three alterations but no more than 30, 20, or 10 alterations relative to any of SEQ ID NOs: 99, 100, 101, or 98; or 10. The pharmaceutical composition of claim 1, 2, or 4, comprising: (iii) an amino acid sequence having at least 95% identity to any of SEQ ID NOs: 99, 100, 101, or 98.

20. the CAR comprises a nucleic acid encoding said CAR, optionally wherein: (a) the nucleic acid encoding the CAR is a lentiviral vector; (b) the nucleic acid encoding the CAR is introduced into the cell by lentiviral transduction; (c) the nucleic acid encoding the CAR is RNA, e.g., in vitro transcribed RNA; and / or (d) the nucleic acid encoding the CAR is introduced into the cell by electroporation.

10. The pharmaceutical composition of claim 1, 2 or 4.

21. 5. The pharmaceutical composition of any one of claims 1, 2, or 4, wherein the CAR-expressing immune effector cell is a T cell or an NK cell, optionally wherein the T cell is an autologous T cell or an allogeneic T cell.

22. (a) CRS is severe CRS, e.g., grade 4 or 5 CRS; (b) the CRS is less than severe, e.g., grade 1, 2, or 3 CRS; or 10. The pharmaceutical composition of claim 1, wherein (c) CRS is grade 3 or grade 4 CRS.

23. The target is, (a) is a mammal, such as a human; and / or (b) having or diagnosed with a B-cell antigen-associated disease, such as a blood cancer, such as lymphoma or leukemia, such as acute myeloid leukemia (AML), according to any one of claims 1, 2 or 4.

24. 5. The pharmaceutical composition of any one of claims 1, 2, or 4, wherein the use further comprises administering an IL-6 inhibitor (e.g., an anti-IL6 receptor inhibitor, e.g., tocilizumab) to the subject, optionally wherein the IL-6 inhibitor is administered before, concurrently with, or after a dose (e.g., a first dose) of immune effector cells expressing a CAR.

25. (a) the IL-6 inhibitor is administered within two 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 or after the first sign of symptoms of CRS in the subject (e.g., fever, e.g., fever characterized by a temperature of at least 38°C (e.g., at least 38.5°C), e.g., fever characterized by a temperature of at least 38°C (e.g., at least 38.5°C), e.g., over two consecutive measurements over a 24-hour period (e.g., at least 4, 5, 6, 7, 8 hours or more apart)); and / or (b) The pharmaceutical composition of claim 24, wherein the IL-6 inhibitor is administered after administration of a dose (e.g., a first dose) of immune effector cells expressing the CAR, and optionally the IL-6 inhibitor is administered 1 hour to 10 days (e.g., 1 to 24 hours, 1 to 2 hours, 2 to 4 hours, 4 to 8 hours, 8 to 12 hours, 12 to 24 hours, 1 to 2 days, 2 to 3 days, 3 to 4 days, 4 to 5 days, 5 to 7 days, or 7 to 10 days) after administration of the dose of immune effector cells expressing the CAR.

26. (a) the IL-6 inhibitor is tocilizumab, and the use further comprises administering tocilizumab at a dose of 5 to 15 mg / kg, e.g., 8 to 12 mg / kg (e.g., 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, or 12 mg / kg); (b) the subject has (e.g., is diagnosed with or identified as having) a high tumor burden prior to treatment with the CAR-expressing immune effector cells, e.g., 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 subject's bone marrow prior to administration of the CAR-expressing immune effector cells (e.g., 1-5 days prior to administration of the CAR-expressing immune effector cells); and / or 25. The pharmaceutical composition of claim 24, wherein (c) the CAR comprises a CD19 CAR, such as a CTL-019 CAR.